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Chronische Entzündungen und die daraus resultierenden Morbiditäten gehören zu den häufigsten Ursachen für einen frühen Tod beim Menschen. Einer der Hauptfaktoren für die Verschlechterung des Gesundheitszustands bei Patienten mit chronischen-entzündlichen Erkrankungen ist die pathologische Infiltration von Leukozyten in gesundes Gewebe, die zu Gewebeschäden und dem Fortschreiten der Krankheit führt. Das vaskuläre Endothel, das die Innenseite der Blutgefäße auskleidet, spielt eine entscheidende Rolle bei der Entzündungsreaktion, da es als Schnittstelle für die Interaktion mit Leukozyten fungiert, um die Extravasation von Leukozyten aus dem Blutstrom in das Gewebe zu ermöglichen. Die Adhäsion von Leukozyten an die Zellen des Endothels wird dabei hauptsächlich durch die von Zytokinen ausgelösten pro-inflammatorischen NFκB- und AP-1-Signalkaskaden ermöglicht, die die Hochregulierung der wichtigsten endothelialen Adhäsionsmoleküle – ICAM-1, VCAM-1 und E-Selektin – bewirken. Eine Klasse von Wirkstoffen, die für ihre entzündungshemmenden Eigenschaften und ihren Nutzen bei der Behandlung chronischer Entzündungskrankheiten bekannt sind, sind die Mikrotubuli-bindenden-Substanzen (microtubule-targeting-agents; MTAs), die nachweislich auch den Entzündungszustand in den Zellen des Endothels und die Leukozyten-Adhäsionskaskade beeinflussen können. MTAs lassen sich in Mikrotubuli-Destabilisatoren, die eine Depolymerisation des Mikrotubuli-Zytoskeletts bewirken, und Mikrotubuli-Stabilisatoren, die die Depolymerisation der Mikrotubuli verhindern, unterteilen. Die zugrundeliegenden biomolekularen Vorgänge und Wirkungen, die die MTAs auf die Zellen des Gefäßendothels haben, und wie sie die Adhäsionskaskade der Leukozyten beeinflussen, sind jedoch weitgehend unbekannt.
Ziel dieser Studie war es, die Auswirkungen des neuartigen Mikrotubuli-Destabilisators Prätubulysin, eines Vorläufers der Tubulysine, die ursprünglich in Stämmen des Myxobakteriums Angiococcus disciformis entdeckt wurden, auf die entzündlichen Prozesse zu untersuchen, die die Leukozyten-adhäsion in TNF-aktivierten primären Endothelzellen aus der menschlichen Nabelschnurvene (HUVECs) ermöglichen. Zusätzlich wurden auch die Auswirkungen der bereits klinisch etablierten Mikrotubuli-Destabilisatoren Colchicin und Vincristin sowie des Mikrotubuli-Stabilisators Paclitaxel untersucht.
Das entzündungshemmende Potenzial von Prätubulysin wurde daher zunächst in vivo in einem Imiquimod-induzierten psoriasiformen Dermatitis-Mausmodell getestet, wobei sich zeigte, dass Prätubulysin den Entzündungszustand deutlich verringert. Um zu beweisen, dass der entzündungshemmende Effekt mit einer verringerten Interaktion von Leukozyten mit dem Endothel zusammenhängt, wurde die Wirkung von Prätubulysin in vivo mittels Intravitalmikroskopie des TNF-aktivierten Kremaster-Muskels der Maus untersucht. Dabei zeigte sich, dass die Behandlung mit Prätubulysin zu einer signifikant verringerten Adhäsion von Leukozyten an die Zellen des Gefäßendothels führte. Die verringerte Adhäsion von Leukozyten an Endothelzellen wurde auch in der in vitro Umgebung bestätigt, indem die Adhäsion von Leukozyten unter Flussbedingungen getestet wurde. Mittels Durchflusszytometrie, Western-Blot-Analyse, sowie qRT-PCR-Analyse der jeweiligen mRNA-Level konnte gezeigt werden, dass die verringerten Leukozyten-Interaktionen auf der verringerten Expression der Zelladhäsionsmoleküle ICAM-1 und VCAM-1 sowie teilweise von E-Selektin nach Behandlung mit Prätubulysin, Vincristin und Colchicin beruhen, wobei Paclitaxel keine signifikanten hemmenden Auswirkungen hatte. Weitere Untersuchungen des Einflusses von Prätubulysin auf die NFκB- und AP-1-Signalübertragung zeigten, dass diese intrazellulären Signalkaskaden durch Prätubulysin nicht behindert werden, wobei NFκB und AP-1 weitgehend in den Promotoren der Zelladhäsionsmoleküle angereichert waren, wie durch Chromatin-Immunpräzipitation nachgewiesen wurde. Darüber hinaus induzierte die Behandlung mit Prätubulysin die Aktivität der NFκB-induzierenden Kinase IKK und führte zu einem signifikanten Anstieg der Aktivität der AP-1 Upstream-Kinase JNK, wie eine Western Blot Analyse ergab. Die Prüfung der Transkriptionsaktivität von NFκB und AP-1 in Reportergen Assays zeigte, dass insbesondere die Mikrotubuli-Destabilisatoren die Promotoraktivität dieser Transkriptionsfaktoren in einer konzentrationsabhängigen Weise verringerten. Weitere Tests zur Abhängigkeit der durch Prätubulysin induzierten Hemmung der Zelladhäsionsmoleküle von der Aktivität der JNK zeigten, dass die Hemmung empfindlich auf die Aktivität dieser Kinase reagiert. Es konnte gezeigt werden, dass die Inhibition der Aktivität der JNK die Expression der Zelladhäsionsmoleküle durch die Behandlung mit Prätubulysin auf mRNA und Proteinebene wiederherstellt. Mit Hilfe der Chromatin-Immunpräzipitation konnte weiterhin gezeigt werden, dass die Behandlung mit Prätubulysin zunächst die Assoziation des Bromodomänen-enthaltenden Proteins 4 mit den Promotoren/Genen von ICAM-1 und VCAM-1 erhöhte, aber zu einem behandlungszeitabhängigen Rückgang der Anreicherung führte. Darüber hinaus wurde durch die Behandlung mit Prätubulysin auch der Abbau dieses Proteins leicht erhöht. Durch den Einsatz eines JNK Inhibitors konnte gezeigt werden, dass die Verdrängung des Bromodomänen-enthaltenden Proteins 4 von icam-1 und vcam-1, sowie der erhöhte Abbau dieses Faktors auch von der Aktivität der JNK abhängig sind. Die Verdrängung des Bromodomänen-enthaltenden Proteins 4 induzierte auch das Vorhandensein von repressiven Chromatinmarkierungen in den Genen von ICAM-1 und VCAM-1. Die Prüfung der Anreicherung der RNA-Polymerase II an den Promotoren/Genen von ICAM-1 und VCAM-1 zeigte jedoch auch eine behandlungszeitabhängige differentielle Anreicherung dieser Polymerase, wobei die Anreicherung nach kurzen Behandlungszeiten reduziert war, sich nach mittleren Behandlungszeiten erholte und nach längeren Behandlungszeiten wieder stark reduziert war. Die anschließende Prüfung der Bedeutung des Bromodomänen-enthaltenden Proteins 4 für die Expression von ICAM-1 und VCAM-1 durch Knock-down-Experimente ergab, dass das vcam-1 Gen durch Knock-down dieses Proteins unterdrückt, das icam-1 Gen jedoch induziert wird. Dies deutet auf das Vorhandensein zusätzlicher Faktoren hin, die auch auf die Aktivität der JNK reagieren und neben dem Bromodomänen-enthaltenden Proteins 4 die Transkriptionsverlängerung des icam-1 Gens bewirken.
Lifestyle factors—such as diet, physical activity (PA), smoking, and alcohol consumption—have a significant impact on mortality as well as healthcare costs. Moreover, they play a crucial role in the development of type 2 diabetes mellitus (DM2). There also seems to be a link between lifestyle behaviours and insulin resistance, which is often a precursor of DM2. This study uses an enhanced Healthy Living Index (HLI) integrating accelerometric data and an Ecological Momentary Assessment (EMA) to explore differences in lifestyle between insulin-sensitive (IS) and insulin-resistant (IR) individuals. Moreover, it explores the association between lifestyle behaviours and inflammation. Analysing data from 99 participants of the mPRIME study (57 women and 42 men; mean age 49.8 years), we calculated HLI scores—ranging from 0 to 4— based on adherence to specific low-risk lifestyle behaviours, including non-smoking, adhering to a healthy diet, maximally moderate alcohol consumption, and meeting World Health Organization (WHO) PA guidelines. Insulin sensitivity was assessed using a Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and C-reactive protein (CRP) levels were used as a proxy for inflammation. Lifestyle behaviours, represented by HLI scores, were significantly different between IS and IR individuals (U = 1529.0; p = 0.023). The difference in the HLI score between IR and IS individuals was mainly driven by lower adherence to PA recommendations in the IR group. Moreover, reduced PA was linked to increased CRP levels in the IR group (r = −0.368, p = 0.014). Our findings suggest that enhancing PA, especially among individuals with impaired insulin resistance, holds significant promise as a preventive strategy.
In this comprehensive review, we will dissect the impact of research on proteoglycans focusing on recent developments involved in their synthesis, degradation, and interactions, while critically assessing their usefulness in various biological processes. The emerging roles of proteoglycans in global infections, specifically the SARS-CoV-2 pandemic, and their rising functions in regenerative medicine and biomaterial science have significantly affected our current view of proteoglycans and related compounds. The roles of proteoglycans in cancer biology and their potential use as a next-generation protein-based adjuvant therapy to combat cancer is also emerging as a constructive and potentially beneficial therapeutic strategy. We will discuss the role of proteoglycans in selected and emerging areas of proteoglycan science, such as neurodegenerative diseases, autophagy, angiogenesis, cancer, infections and their impact on mammalian diseases.
Chronic kidney disease (CKD) represents an independent risk factor for cardiovascular diseases (CVD). Accordingly, CKD patients show a substantial increased risk of cardiovascular mortality. Inflammation represents an important link between CKD and CVD. The interaction between endothelial cells and effector cells of the innate immune system plays a central role in the development and progression of inflammation. Vascular injury causes endothelial dysfunction, leading to augmented oxidative stress, increased expression of leukocyte adhesion molecules and chronic inflammation. CKD induces numerous metabolic changes, creating a uremic milieu resulting in the accumulation of various uremic toxins. These toxins lead to vascular injury, endothelial dysfunction and activation of the innate immune system. Recent studies describe CKD-dependent changes in monocytes that promote endothelial dysfunction and thus CKD progression and CKD-associated CVD. The NLR family pyrin domain containing 3–interleukin-1β–interleukin-6 (NLRP3–IL-1β–IL-6) signaling pathway plays a pivotal role in the development and progression of CVD and CKD alike. Several clinical trials are investigating targeted inhibition of this pathway indicating that anti-inflammatory therapeutic strategies may emerge as novel approaches in patients at high cardiovascular risk and nonresolving inflammation. CKD patients in particular would benefit from targeted anti-inflammatory therapy, since conventional therapeutic regimens have limited efficacy in this population.
Essentials
• The role of platelet IL-1β release in chronic inflammation is currently unclear.
• Platelets from 65 patients with varying degrees of chronic inflammation were studied.
• Chronic inflammation linked to reduced levels of intracellular IL-1β and IL-1β release.
• Chronic inflammation induces a phenotype that indicates chronic IL-1β release from platelets.
Abstract
Background: Chronic inflammation is a cardiovascular risk factor, and interleukin-1β (IL-1β) is central to the inflammatory host response. Platelets contain the NLRP3 inflammasome and are able to translate IL-1β messenger RNA (mRNA) and secrete mature IL-1β upon activation. However, the role of a chronic inflammatory environment in platelet IL-1β mRNA and protein content remains unclear.
Objectives: The aim of the current study was to investigate intracellular platelet IL-1β and IL-1β mRNA in a chronic inflammatory state.
Methods: Sixty-five patients with stable inflammation (ie, high-sensitivity C-reactive protein within predefined margins in 2 separate measurements) were stratified according to high-sensitivity C-reactive protein levels in low (0.0-0.9 mg/L), medium (1.0-2.9 mg/L), and high (3.0-9.9 mg/L) risk groups. Platelet reactivity as well as platelet IL-1β protein synthesis were studied.
Results: The highest risk group was characterized by a distinct cardiovascular risk profile and approximately 20% higher platelet counts. While platelet reactivity was not different, a reduction in intracellular platelet IL-1β mRNA and IL-1β protein levels was observed in the highest risk group and was linked to decreased platelet size and granularity. This signature suggests a phenotype of chronic IL-1β secretion and could be experimentally phenocopied by stimulation of platelets from healthy volunteers with either TRAP-6 or collagen related peptide (CRP-XL).
Conclusion: Our data suggest a phenotype of chronic IL-1β secretion by platelets in patients with chronic sterile inflammation.
Epigenetic regulation of inflammation by microRNAs in post-infectious bronchiolitis obliterans
(2022)
Objectives: Post-infectious bronchiolitis obliterans (PiBO) is a rare, chronic disease initiated by severe infection and followed by perpetuating inflammation and obliteration of the small airways. MicroRNAs (miRNAs) have been proposed to play a central role as epigenetic regulators, which control resolution and prevent the uncontrolled progress of inflammation. The aim of this study was to define biomarkers on the level of post-transcriptional gene regulation in order to characterise PiBO.
Methods: A total of 39 patients with well-defined PiBO and 31 controls from two centres, Barcelona, Spain, and Frankfurt, Germany, were analysed by next-generation sequencing (NGS). The evaluation of the biological targets of the miRNAs was performed by pathway enrichment analysis and protein–protein interaction network analysis respectively.
Results: Patients with PiBO had significantly lower lung function values and increased airway inflammation in induced sputum as indicated by total cell counts, neutrophils, IL-1β, IL-6, IL-8 and TGF-β compared to controls.
Next-generation sequencing analysis revealed a total of 22 dysregulated miRNAs, which passed significance threshold for Padj ≤ 0.001 with 17 being upregulated and 5 being downregulated. Of these dysregulated miRNAs, miR-335-5p, miR-186-5p, miR-30b-5p and miR-30c-5p were further validated using qRT-PCR. Interestingly, these miRNAs are functionally implicated in cytokine–cytokine receptor interaction, TGF-β signalling and FoxO signalling pathway and significantly correlated with lung function values (FEV1).
Conclusion: Our results demonstrate an aberrant miRNA expression profile in PiBO, which impacts pathways responsible for the regulation of inflammation and fibrosis. The defined miRNAs are useful biomarkers and should be assessed as potential target in the field of miRNA therapeutics.
5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E2 (PGE2) release into the supernatants of cytokine-stimulated (TNFα/IL-1β) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC50 values ranging from 0.1–9.1 µM). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH2 and PGE2 formation were not impaired by the compounds. However, accumulation of intracellular PGE2 was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE2 inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE2 export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.
Inflammation or injury to the somatosensory nervous system may result in chronic pain conditions, which affect millions of people and often cause major health problems. Emerging lines of evidence indicate that reactive oxygen species (ROS), such as superoxide anion or hydrogen peroxide, are produced in the nociceptive system during chronic inflammatory and neuropathic pain and act as specific signaling molecules in pain processing. Among potential ROS sources in the somatosensory system are NADPH oxidases, a group of electron-transporting transmembrane enzymes whose sole function seems to be the generation of ROS. Interestingly, the expression and relevant function of the Nox family members Nox1, Nox2, and Nox4 in various cells of the nociceptive system have been demonstrated. Studies using knockout mice or specific knockdown of these isoforms indicate that Nox1, Nox2, and Nox4 specifically contribute to distinct signaling pathways in chronic inflammatory and/or neuropathic pain states. As selective Nox inhibitors are currently being developed and investigated in various physiological and pathophysiological settings, targeting Nox1, Nox2, and/or Nox4 could be a novel strategy for the treatment of chronic pain. Here, we summarize the distinct roles of Nox1, Nox2, and Nox4 in inflammatory and neuropathic processing and discuss the effectiveness of currently available Nox inhibitors in the treatment of chronic pain conditions.
(1) AlphαSynuclein (αSyn) is a synaptic protein which is expressed in the nervous system and has been linked to neurodegenerative diseases, in particular Parkinson’s disease (PD). Symptoms of PD are mainly due to overexpression and aggregation of αSyn and include pain. However, the interconnection of αSyn and pain has not been clarified so far. (2) We investigated the potential effects of a αSyn knock-out on the nociceptive behaviour in mouse models of acute, inflammatory and neuropathic pain. Furthermore, we assessed the impact of αSyn deletion on pain-related cellular and molecular mechanisms in the spinal cord in these models. (3) Our results showed a reduction of acute cold nociception in αSyn knock-out mice while responses to acute heat and mechanical noxious stimulation were similar in wild type and knock-out mice. Inflammatory nociception was not affected by αSyn knock-out which is also mirrored by unaltered inflammatory gene expression. In contrast, in the SNI model of neuropathic pain, αSyn knock-out mice showed decreased mechanical allodynia as compared to wild type mice. This effect was associated with reduced proinflammatory mechanisms and suppressed activation of MAP kinase signalling in the spinal cord while endogenous antinociceptive mechanisms are not inhibited. (4) Our data indicate that αSyn plays a role in neuropathy and its inhibition might be useful to ameliorate pain symptoms after nerve injury.
Hepatic cells are sensitive to internal and external signals. Ethanol is one of the oldest and most widely used drugs in the world. The focus on the mechanistic engine of the alcohol-induced injury has been in the liver, which is responsible for the pathways of alcohol metabolism. Ethanol undergoes a phase I type of reaction, mainly catalyzed by the cytoplasmic enzyme, alcohol dehydrogenase (ADH), and by the microsomal ethanol-oxidizing system (MEOS). Reactive oxygen species (ROS) generated by cytochrome (CYP) 2E1 activity and MEOS contribute to ethanol-induced toxicity. We aimed to: (1) Describe the cellular, pathophysiological and clinical effects of alcohol misuse on the liver; (2) Select the biomarkers and analytical methods utilized by the clinical laboratory to assess alcohol exposure; (3) Provide therapeutic ideas to prevent/reduce alcohol-induced liver injury; (4) Provide up-to-date knowledge regarding the Corona virus and its affect on the liver; (5) Link rare diseases with alcohol consumption. The current review contributes to risk identification of patients with alcoholic, as well as non-alcoholic, liver disease and metabolic syndrome. Additional prevalence of ethnic, genetic, and viral vulnerabilities are presented.
In the basal, proliferative layer of healthy skin, the mTOR complex 1 (mTORC1) is activated, thus regulating proliferation while preventing differentiation. When cells leave the proliferative, basal compartment, mTORC1 signaling is turned off, which allows differentiation. Under inflammatory conditions, this switch is hijacked by cytokines and prevents proper differentiation. It is currently unknown how mTORC1 is regulated to mediate these effects on keratinocyte differentiation. In other tissues, mTORC1 activity is controlled through various pathways via the tuberous sclerosis complex (TSC). Thus, we investigated whether the TS complex is regulated by proinflammatory cytokines and contributes to the pathogenesis of psoriasis. TNF-α as well as IL-1β induced the phosphorylation of TSC2, especially on S939 via the PI3-K/AKT and MAPK pathway. Surprisingly, increased TSC2 phosphorylation could not be detected in psoriasis patients. Instead, TSC2 was strongly downregulated in lesional psoriatic skin compared to non-lesional skin of the same patients or healthy skin. In vitro inflammatory cytokines induced dissociation of TSC2 from the lysosome, followed by destabilization of the TS complex and degradation. Thus, we assume that in psoriasis, inflammatory cytokines induce strong TSC2 phosphorylation, which in turn leads to its degradation. Consequently, chronic mTORC1 activity impairs ordered keratinocyte differentiation and contributes to the phenotypical changes seen in the psoriatic epidermis.
Rhizomes from Zingiber officinale Roscoe are traditionally used for the treatment of a plethora of pathophysiological conditions such as diarrhea, nausea, or rheumatoid arthritis. While 6-gingerol is the pungent principle in fresh ginger, in dried rhizomes, 6-gingerol is dehydrated to 6-shogaol. 6-Shogaol has been demonstrated to exhibit anticancer, antioxidative, and anti-inflammatory actions more effectively than 6-gingerol due to the presence of an electrophilic Michael acceptor moiety. In vitro, 6-shogaol exhibits anti-inflammatory actions in a variety of cell types, including leukocytes. Our study focused on the effects of 6-shogaol on activated endothelial cells. We found that 6-shogaol significantly reduced the adhesion of leukocytes onto lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs), resulting in a significantly reduced transmigration of THP-1 cells through an endothelial cell monolayer. Analyzing the mediators of endothelial cell–leukocyte interactions, we found that 30 µM of 6-shogaol blocked the LPS-triggered mRNA and protein expression of cell adhesion molecules. In concert with this, our study demonstrates that the LPS-induced nuclear factor κB (NFκB) promoter activity was significantly reduced upon treatment with 6-shogaol. Interestingly, the nuclear translocation of p65 was slightly decreased, and protein levels of the LPS receptor Toll-like receptor 4 remained unimpaired. Analyzing the impact of 6-shogaol on angiogenesis-related cell functions in vitro, we found that 6-shogaol attenuated the proliferation as well as the directed and undirected migration of HUVECs. Of note, 6-shogaol also strongly reduced the chemotactic migration of endothelial cells in the direction of a serum gradient. Moreover, 30 µM of 6-shogaol blocked the formation of vascular endothelial growth factor (VEGF)-induced endothelial sprouts from HUVEC spheroids and from murine aortic rings. Importantly, this study shows for the first time that 6-shogaol exhibits a vascular-disruptive impact on angiogenic sprouts from murine aortae. Our study demonstrates that the main bioactive ingredient in dried ginger, 6-shogaol, exhibits beneficial characteristics as an inhibitor of inflammation- and angiogenesis-related processes in vascular endothelial cells.
The sphingolipid sphingosine-1-phosphate (S1P) promotes tumor development through a variety of mechanisms including promoting proliferation, survival, and migration of cancer cells. Moreover, S1P emerged as an important regulator of tumor microenvironmental cell function by modulating, among other mechanisms, tumor angiogenesis. Therefore, S1P was proposed as a target for anti-tumor therapy. The clinical success of current cancer immunotherapy suggests that future anti-tumor therapy needs to consider its impact on the tumor-associated immune system. Hereby, S1P may have divergent effects. On the one hand, S1P gradients control leukocyte trafficking throughout the body, which is clinically exploited to suppress auto-immune reactions. On the other hand, S1P promotes pro-tumor activation of a diverse range of immune cells. In this review, we summarize the current literature describing the role of S1P in tumor-associated immunity, and we discuss strategies for how to target S1P for anti-tumor therapy without causing immune paralysis.
Background: Inflammation, particularly cytokine release, contributes to epileptogenesis by influencing the cerebral tissue remodeling and neuronal excitability that occurs after a precipitating epileptogenic insult. While several cytokines have been explored in this process, release kinetics are less well investigated. Determining the time course of cytokine release in the epileptogenic zone is necessary for precisely timed preventive or therapeutic anti-inflammatory interventions. Methods: Hippocampal extracellular levels of six cytokines and chemokines (IL-1β, IL-6, IL-10, CCL2, CCL3, and CCL5) were quantified at various time points during epileptogenesis in a rat model of mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) using microdialysis (MD). Results: The analysis of microdialysates demonstrated consistent elevation at all time points during epileptogenesis for IL-1β and IL-10. IL-10 release was maximal on day 1, IL-1β release peaked at day 8. No correlation between local hippocampal IL-1β concentrations and IL-1β blood levels was found. Conclusion: The release kinetics of IL-1β are consistent with its established pro-epileptogenic properties, while the kinetics of IL-10 suggest a counter-regulatory effect. This proof-of-concept study demonstrates the feasibility of intraindividual longitudinal monitoring of hippocampal molecular inflammatory processes via repetitive MD over several weeks and sheds light on the kinetics of hippocampal cytokine release during epileptogenesis.
LFA-1 (Lymphocyte function-associated antigen-1) is a heterodimeric integrin (CD11a/CD18) present on the surface of all leukocytes; it is essential for leukocyte recruitment to the site of tissue inflammation, but also for other immunological processes such as T cell activation and formation of the immunological synapse. Absent or dysfunctional expression of LFA-1, caused by mutations in the ITGB2 (integrin subunit beta 2) gene, results in a rare immunodeficiency syndrome known as Leukocyte adhesion deficiency type I (LAD I). Patients suffering from severe LAD I present with recurrent infections of the skin and mucosa, as well as inflammatory symptoms complicating the clinical course of the disease before and after allogeneic hematopoietic stem cell transplantation (alloHSCT); alloHSCT is currently the only established curative treatment option. With this review, we aim to provide an overview of the intrinsic role of inflammation in LAD I.
Despite the implementation of consolidative immune checkpoint inhibition after definitive chemoradiotherapy (CRT), the prognosis for locally advanced non-small-cell lung cancer (NSCLC) remains poor. We assessed the impact of the C-reactive protein (CRP) to albumin ratio (CAR) as an inflammation-based prognostic score in patients with locally advanced NSCLC treated with CRT. We retrospectively identified and analyzed 52 patients with primary unresectable NSCLC (UICC Stage III) treated with definitive/neoadjuvant CRT between 2014 and 2019. CAR was calculated by dividing baseline CRP by baseline albumin levels and correlated with clinicopathologic parameters to evaluate prognostic impact. After dichotomizing patients by the median, univariate and multivariate Cox regression analyses were performed. An increased CAR was associated with advanced T-stage (p = 0.018) and poor performance status (p = 0.004). Patients with pre-therapeutic elevated CAR had significantly lower hemoglobin and higher leukocyte levels (hemoglobin p = 0.001, leukocytes p = 0.018). High baseline CAR was shown to be associated with worse local control (LPFS, p = 0.006), shorter progression-free survival (PFS, p = 0.038) and overall survival (OS, p = 0.022), but not distant metastasis-free survival (DMFS). Multivariate analysis confirmed an impaired outcome in patients with high CAR (LPFS: HR 3.562, 95% CI 1.294–9.802, p = 0.011). CAR is an easily available and independent prognostic marker after CRT in locally advanced NSCLC. CAR may be a useful biomarker for patient stratification to individualize treatment concepts.
In recent years, the infrapatellar fat pad (IFP) has gained increasing research interest. The contribution of the IFP to the development and progression of knee osteoarthritis (OA) through extensive interactions with the synovium, articular cartilage, and subchondral bone is being considered. As part of the initiation process of OA, IFP secretes abundant pro-inflammatory mediators among many other factors. Today, the IFP is (partially) resected in most total knee arthroplasties (TKA) allowing better visualization during surgical procedures. Currently, there is no clear guideline providing evidence in favor of or against IFP resection. With increasing numbers of TKAs, there is a focus on preventing adverse postoperative outcomes. Therefore, anatomic features, role in the development of knee OA, and consequences of resecting versus preserving the IFP during TKA are reviewed in the following article.
Interleukin-7 (IL-7) is an important cytokine with pivotal pro-survival functions in the adaptive immune system. However, the role of IL-7 in innate immunity is not fully understood. In the present study, the impact of hepatic IL-7 on innate immune cells was assessed by functional experiments as well as in patients with different stages of liver cirrhosis or acute-on-chronic liver failure (ACLF). Human hepatocytes and liver sinusoidal endothelial cells secreted IL-7 in response to stimulation with interferons (IFNs) of type I and II, yet not type III. De novo translation of interferon-response factor-1 (IRF-1) restricted IL-7 production to stimulation with type I and II IFNs. LPS-primed human macrophages were identified as innate immune target cells responding to IL-7 signaling by inactivation of Glycogen synthase kinase-3 (GSK3). IL-7-mediated GSK3 inactivation augmented LPS-induced secretion of pro-inflammatory cytokines and blunted LPS tolerance of macrophages. The IFN-IRF-1-IL-7 axis was present in liver cirrhosis patients. However, liver cirrhosis patients with or without ACLF had significantly lower concentrations of IL-7 in serum compared to healthy controls, which might contribute to LPS-tolerance in these patients. In conclusion, we propose the presence of an inflammatory cascade where IFNs of type I/II induce hepatocellular IL-7 in an IRF-1-restriced way. Beyond its role in adaptive immune responses, IL-7 appears to augment the response of macrophages to LPS and to ameliorate LPS tolerance, which may improve innate immune responses against invading pathogens.
Background: Polytraumatized patients undergo a strong immunological stress upon insult. Phagocytes (granulocytes and monocytes) play a substantial role in immunological defense against bacteria, fungi and yeast, and in the clearance of cellular debris after tissue injury. We have reported a reduced monocytes phagocytic activity early after porcine polytrauma before. However, it is unknown if both phagocyte types undergo those functional alterations, and if there is a pathogen-specific phagocytic behavior. We characterized the phagocytic activity and capacity of granulocytes and monocytes after polytrauma.
Methods: Eight pigs (Sus scrofa) underwent polytrauma consisting of lung contusion, liver laceration, tibial fracture and hemorrhagic shock with fluid resuscitation and fracture fixation with external fixator. Intensive care treatment including mechanical ventilation for 72 h followed. Phagocytic activity and capacity were investigated using an in vitro ex vivo whole blood stimulation phagocytosis assays before trauma, after surgery, 24, 48, and 72 h after trauma. Blood samples were stimulated with Phorbol-12-myristate-13-acetate and incubated with FITC-labeled E. coli, S. aureus or S. cerevisiae for phagocytosis assessment by flow cytometry.
Results: Early polytrauma-induced significant increase of granulocytes and monocytes declined to baseline values within 24 h. Percentage of E. coli-phagocytizing granulocytes significantly decreased after polytrauma and during further intensive care treatment, while their capacity significantly increased. Interestingly, both granulocytic phagocytic activity and capacity of S. aureus significantly decreased after trauma, although a recovery was observed after 24 h and yet was followed by another decrease. The percentage of S. cerevisiae-phagocytizing granulocytes significantly increased after 24 h, while their impaired capacity after surgery and 72 h later was detected. Monocytic E. coli-phagocytizing percentage did not change, while their capacity increased after 24–72 h. After a significant decrease in S. aureus-phagocytizing monocytes after surgery, a significant increase after 24 and 48 h was observed without capacity alterations. No significant changes in S. cerevisiae-phagocytizing monocytes occurred, but their capacity dropped 48 and 72 h.
Conclusion: Phagocytic activity and capacity of granulocytes and monocytes follow a different pattern and significantly change within 72 h after polytrauma. Both phagocytic activity and capacity show significantly different alterations depending on the pathogen strain, thus potentially indicating at certain and possibly more relevant infection causes after polytrauma.
Multinucleated giant cells (MNGCs) are frequently observed in the implantation areas of different biomaterials. The main aim of the present study was to analyze the long-term polarization pattern of the pro- and anti-inflammatory phenotypes of macrophages and MNGCs for 180 days to better understand their role in the success or failure of biomaterials. For this purpose, silk fibroin (SF) was implanted in a subcutaneous implantation model of Wistar rats as a model for biomaterial-induced MNGCs. A sham operation was used as a control for physiological wound healing. The expression of different inflammatory markers (proinflammatory M1: CCR-7, iNos; anti-inflammatory M2: CD-206, CD-163) and tartrate-resistant acid phosphatase (TRAP) and CD-68 were identified using immunohistochemical staining. The results showed significantly higher numbers of macrophages and MNGCs within the implantation bed of SF-expressed M1 markers, compared to M2 markers. Interestingly, the expression of proinflammatory markers was sustained over the long observation period of 180 days. By contrast, the control group showed a peak of M1 macrophages only on day 3. Thereafter, the inflammatory pattern shifted to M2 macrophages. No MNGCs were observed in the control group. To the best of our knowledge, this is study is the first to outline the persistence of pro-inflammatory MNGCs within the implantation bed of SF and to describe their long-term kinetics over 180 days. Clinically, these results are highly relevant to understand the role of biomaterial-induced MNGCs in the long term. These findings suggest that tailored physicochemical properties may be a key to avoiding extensive inflammatory reactions and achieving clinical success. Therefore, further research is needed to elucidate the correlation between proinflammatory MNGCs and the physicochemical characteristics of the implanted biomaterial.
Influence of antibiotic management on microbial selection and infectious complications after trauma
(2021)
Background: The inflammatory response and post-traumatic complications like infections play an important role in the pathophysiology of severe injuries. This study examines the microbiological aspects in anti-infective treatment of trauma patients and their inflammatory response in post-traumatic infections complications. Patients and Methods: A retrospective analysis of prospectively collected data in trauma patients (ISS ≥ 16) over a 1-year period (01/2018 to 12/2018) is provided. Patient population was stratified into severely injured patients without post-traumatic infection (inf-PT), and severely injured patients who developed an infection (inf+PT).Results: Of 114 trauma patients, 45 suffered from post-traumatic infection during the first 10 days of hospitalization. Severely injured patients with concomitant traumatic brain injury (PT+TBI) showed the highest rate of post-traumatic infection. Pro-inflammatory reaction was tracked by levels of Interleukin (IL-)6 (day 3: inf+T 190.8 ± 359.4 pg/dL > inf-PT 56.2 ± 57.7 pg/mL (mean ± SD); p = 0.008) and C-Reactive-Protein (CRP, day 3: inf+PT 15.3 mg/dL > inf-PT 6.7 mg/dL, p = 0.001) which were significantly higher in trauma patients who develop an infectious complication and showed a significant positive correlation with the occurrence of infection. The leading entity of infection was pneumonia followed by infections of the urinary tract mainly caused by gram-negative Enterobacteriaceae. 67.5% of all trauma patients received single-shot antibiosis during initial care in trauma bay. The development of secondary colonization was not relevant positively correlated with single-shot antibiosis (r = 0.013, p = 0.895) and prophylactically calculated antibiotic administration (r = 0.066, p = 0.500).Conclusion: Severely injured trauma patients have an increased risk for development of infectious complications, which mainly is pneumonia followed by infection of the urinary tract mainly caused by gram-negative Enterobacteriaceae. Based on the data in this study, the one-time antibiotic and prophylactic calculated use of antibiotics, like Cephalosporins must be critically discussed in terms of their role in the development of post-traumatic infections and microbial selection.
Background: Inflammation is essential for the pathogenesis of multiple sclerosis (MS). While the immune system contribution to the development of neurological symptoms has been intensively studied, inflammatory biomarkers for mental symptoms such as depression are poorly understood in the context of MS. Here, we test if depression correlates with peripheral and central inflammation markers in MS patients as soon as the diagnosis is established. Methods: Forty-four patients were newly diagnosed with relapsing-remitting MS, primary progressive MS or clinically isolated syndrome. Age, gender, EDSS, C-reactive protein (CRP), albumin, white blood cells count in cerebrospinal fluid (CSF WBC), presence of gadolinium enhanced lesions (GE) on T1-weighted images and total number of typical MS lesion locations were included in linear regression models to predict Beck Depression Inventory (BDI) score and the depression dimension of the Symptoms Checklist 90-Revised (SCL90RD). Results: CRP elevation and GE predicted significantly BDI (CRP: p = 0.007; GE: p = 0.019) and SCL90RD (CRP: p = 0.004; GE: p = 0.049). The combination of both factors resulted in more pronounced depressive symptoms (p = 0.04). CSF WBC and EDSS as well as the other variables were not correlated with depressive symptoms. Conclusions: CRP elevation and GE are associated with depressive symptoms in newly diagnosed MS patients. These markers can be used to identify MS patients exhibiting a high risk for the development of depressive symptoms in early phases of the disease.
Simple Summary:
Pharmacological activation of tumor suppressor p53 is a promising therapeutic strategy for a range of hematologic and solid cancers. Whether p53 activation augments or suppresses anti-tumor innate immunity is less understood. Here we show that treatment of differentiating human macrophages with a p53 activator idasanutlin suppresses their inflammatory responses to activators of toll-like receptors (TLR) -4 and -7/8. This is accompanied by reduced expression of TLR7, TLR8, as well as TLR4 co-receptor CD14. These data help evaluating the possibilities of combining p53-targeting and immunostimulatory anti-cancer therapies.
Abstract:
The transcription factor p53 has well-recognized roles in regulating cell cycle, DNA damage repair, cell death, and metabolism. It is an important tumor suppressor and pharmacological activation of p53 by interrupting its interaction with the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2) is actively explored for anti-tumor therapies. In immune cells, p53 modulates inflammatory responses, but the impact of p53 on macrophages remains incompletely understood. In this study, we used the MDM2 antagonist idasanutlin (RG7388) to investigate the responses of primary human macrophages to pharmacological p53 activation. Idasanutlin induced a robust p53-dependent transcriptional signature in macrophages, including several pro-apoptotic genes. However, idasanutlin did not generally sensitize macrophages to apoptosis, except for an enhanced response to a Fas-stimulating antibody. In fully differentiated macrophages, idasanutlin did not affect pro-inflammatory gene expression induced by toll-like receptor 4 (TLR4), TLR3, and TLR7/8 agonists, but inhibited interleukin-4-induced macrophage polarization. However, when present during monocyte to macrophage differentiation, idasanutlin attenuated inflammatory responses towards activation of TLR4 and TLR7/8 by low doses of lipopolysaccharide or resiquimod (R848). This was accompanied by a reduced expression of CD14, TLR7, and TLR8 in macrophages differentiated in the presence of idasanutlin. Our data suggest anti-inflammatory effects of pharmacological p53 activation in differentiating human macrophages.
Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.
The group of proton-sensing G-protein coupled receptors (GPCRs) consists of the four receptors GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A (GPR132). These receptors are cellular sensors of acidification, a property that has been attributed to the presence of crucial histidine residues. However, the pH detection varies considerably among the group of proton-sensing GPCRs and ranges from pH of 5.5 to 7.8. While the proton-sensing GPCRs were initially considered to detect acidic cellular environments in the context of inflammation, recent observations have expanded our knowledge about their physiological and pathophysiological functions and many additional individual and unique features have been discovered that suggest a more differentiated role of these receptors in health and disease. It is known that all four receptors contribute to different aspects of tumor biology, cardiovascular physiology, and asthma. However, apart from their overlapping functions, they seem to have individual properties, and recent publications identify potential roles of individual GPCRs in mechanosensation, intestinal inflammation, oncoimmunological interactions, hematopoiesis, as well as inflammatory and neuropathic pain. Here, we put together the knowledge about the biological functions and structural features of the four proton-sensing GPCRs and discuss the biological role of each of the four receptors individually. We explore all currently known pharmacological modulators of the four receptors and highlight potential use. Finally, we point out knowledge gaps in the biological and pharmacological context of proton-sensing GPCRs that should be addressed by future studies.
Clonal hematopoiesis of indeterminate potential (CHIP) is caused by recurrent somatic mutations leading to clonal blood cell expansion. However, direct evidence of the fitness of CHIP-mutated human hematopoietic stem cells (HSCs) in blood reconstitution is lacking. Because myeloablative treatment and transplantation enforce stress on HSCs, we followed 81 patients with solid tumors or lymphoid diseases undergoing autologous stem cell transplantation (ASCT) for the development of CHIP. We found a high incidence of CHIP (22%) after ASCT with a high mean variant allele frequency (VAF) of 10.7%. Most mutations were already present in the graft, albeit at lower VAFs, demonstrating a selective reconstitution advantage of mutated HSCs after ASCT. Thus, CHIP-mutated stem and progenitor cells largely gain on clone size upon ASCT-related blood reconstitution, leading to an increased future risk of CHIP-associated complications. CHIP increase with age and is also associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with chronic ischemic heart failure (CHF) in humans is unknown. Therefore, we analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by NGS to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF. Forty-seven mutations with a VAF of at least 2% were found in 18.5% of 200 patients with CHF. The mutations most commonly occurred in the genes DNMT3A and TET2. During a median follow-up of 4.4 years, a significantly worse clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers was notable. Importantly, there was a significant dose-response association between VAF and clinical outcome. Our data suggest that somatic mutations in hematopoietic cells, may be significantly associated with the progression and poor prognosis of CHF.
Mitofusin 2 (MFN2) is a mitochondrial outer membrane GTPase, which modulates mitochondrial fusion and affects the interaction between endoplasmic reticulum and mitochondria. Here, we explored how MFN2 influences mitochondrial functions and inflammatory responses towards zymosan in primary human macrophages. A knockdown of MFN2 by small interfering RNA decreased mitochondrial respiration without attenuating mitochondrial membrane potential and reduced interactions between endoplasmic reticulum and mitochondria. A MFN2 deficiency potentiated zymosan-elicited inflammatory responses of human primary macrophages, such as expression and secretion of pro-inflammatory cytokines interleukin-1β, -6, -8 and tumor necrosis factor α, as well as induction of cyclooxygenase 2 and prostaglandin E2 synthesis. MFN2 silencing also increased zymosan-induced nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases inflammatory signal transduction, without affecting mitochondrial reactive oxygen species production. Mechanistic studies revealed that MFN2 deficiency enhanced the toll-like receptor 2-dependent branch of zymosan-triggered responses upstream of inhibitor of κB kinase. This was associated with elevated, cytosolic expression of interleukin-1 receptor-associated kinase 4 in MFN2-deficient cells. Our data suggest pro-inflammatory effects of MFN2 deficiency in human macrophages.
Circulating P2X7 receptor signaling components as diagnostic biomarkers for temporal lobe epilepsy
(2021)
Circulating molecules have potential as biomarkers to support the diagnosis of epilepsy and to assist with differential diagnosis, for example, in conditions resembling epilepsy, such as in psychogenic non-epileptic seizures (PNES). The P2X7 receptor (P2X7R) is an important regulator of inflammation and mounting evidence supports its activation in the brain during epilepsy. Whether the P2X7R or P2X7R-dependent signaling molecules can be used as biomarkers of epilepsy has not been reported. P2X7R levels were analyzed by quantitative ELISA using plasma samples from controls and patients with temporal lobe epilepsy (TLE) or PNES. Moreover, blood cell P2X7R expression and P2X7R-dependent cytokine signature was measured following status epilepticus in P2X7R-EGFP reporter, wildtype, and P2X7R-knockout mice. P2X7R plasma levels were higher in TLE patients when compared with controls and patients with PNES. Plasma levels of the broad inflammatory marker protein C-Reactive protein (CRP) were similar between the three groups. Using P2X7R-EGFP reporter mice, we identified monocytes as the main blood cell type expressing P2X7R after experimentally evoked seizures. Finally, cytokine array analysis in P2X7R-deficient mice identified KC/GRO as a potential P2X7R-dependent plasma biomarker following status epilepticus and during epilepsy. Our data suggest that P2X7R signaling components may be a promising subclass of circulating biomarkers to support the diagnosis of epilepsy.
Exogenous adenosine and its metabolite inosine exert anti-inflammatory effects in synoviocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients. We analyzed whether these cells are able to synthesize adenosine/inosine and which adenosine receptors (ARs) contribute to anti-inflammatory effects. The functionality of synthesizing enzymes and ARs was tested using agonists/antagonists. Both OA and RA cells expressed CD39 (converts ATP to AMP), CD73 (converts AMP to adenosine), ADA (converts adenosine to inosine), ENT1/2 (adenosine transporters), all AR subtypes (A1, A2A, A2B and A3) and synthesized predominantly adenosine. The CD73 inhibitor AMPCP significantly increased IL-6 and decreased IL-10 in both cell types, while TNF only increased in RA cells. The ADA inhibitor DAA significantly reduced IL-6 and induced IL-10 in both OA and RA cells. The A2AAR agonist CGS 21680 significantly inhibited IL-6 and induced TNF and IL-10 only in RA, while the A2BAR agonist BAY 60-6583 had the same effect in both OA and RA. Taken together, OA and RA synoviocytes express the complete enzymatic machinery to synthesize adenosine/inosine; however, mainly adenosine is responsible for the anti- (IL-6 and IL-10) or pro-inflammatory (TNF) effects mediated by A2A- and A2BAR. Stimulating CD39/CD73 with simultaneous ADA blockage in addition to TNF inhibition might represent a promising therapeutic strategy.
Ginger (Zingiber officinale Roscoe) is widely used as medicinal plant. According to the Committee on Herbal Medicinal Products (HMPC), dried powdered ginger rhizome can be applied for the prevention of nausea and vomiting in motion sickness (well-established use). Beyond this, a plethora of pre-clinical studies demonstrated anti-cancer, anti-oxidative, or anti-inflammatory actions. 6-Shogaol is formed from 6-gingerol by dehydration and represents one of the main bioactive principles in dried ginger rhizomes. 6-Shogaol is characterized by a Michael acceptor moiety being reactive with nucleophiles. This review intends to compile important findings on the actions of 6-shogaol as an anti-inflammatory compound: in vivo, 6-shogaol inhibited leukocyte infiltration into inflamed tissue accompanied with reduction of edema swelling. In vitro and in vivo, 6-shogaol reduced inflammatory mediator systems such as COX-2 or iNOS, affected NFκB and MAPK signaling, and increased levels of cytoprotective HO-1. Interestingly, certain in vitro studies provided deeper mechanistic insights demonstrating the involvement of PPAR-γ, JNK/Nrf2, p38/HO-1, and NFκB in the anti-inflammatory actions of the compound. Although these studies provide promising evidence that 6-shogaol can be classified as an anti-inflammatory substance, the exact mechanism of action remains to be elucidated. Moreover, conclusive clinical data for anti-inflammatory actions of 6-shogaol are largely lacking.
Inflammatory diseases including psoriasis are associated with metabolic and cardiovascular comorbidities, including obesity and metabolic syndrome. Obesity is associated with greater psoriasis disease severity and reduced response to treatment. Therefore, targeting metabolic comorbidities could improve patients’ health status and psoriasis-specific outcomes. METABOLyx is a randomized controlled trial evaluating the combination of a lifestyle intervention program with secukinumab treatment in psoriasis. Here, the rationale, methodology and baseline patient characteristics of METABOLyx are presented. A total of 768 patients with concomitant moderate to severe plaque psoriasis and metabolic syndrome were randomized to secukinumab 300 mg, or secukinumab 300 mg plus a tailored lifestyle intervention program, over 24 weeks. A substudy of immunologic and metabolic biomarkers is ongoing. The primary endpoint of METABOLyx is PASI90 response at week 24. Other endpoints include patient-reported outcomes and safety. METABOLyx represents the first large scale clinical trial of an immunomodulatory biologic in combination with a standardized lifestyle intervention.
Extracts of frankincense, the gum resin of Boswellia species, have been extensively used in traditional folk medicine since ancient times and are still of great interest as promising anti-inflammatory remedies in Western countries. Despite their common therapeutic use and the intensive pharmacological research including studies on active ingredients, modes of action, bioavailability, pharmacokinetics, and clinical efficacy, frankincense preparations are available as nutraceuticals but have not yet approved as a drug on the market. A major issue of commercially available frankincense nutraceuticals is the striking differences in their composition and quality, especially related to the content of boswellic acids (BAs) as active ingredients, mainly due to the use of material from divergent Boswellia species but also because of different work-up and extraction procedures. Here, we assessed three frequently used frankincense-based preparations for their BA content and the interference with prominent pro-inflammatory actions and targets that have been proposed, that is, 5-lipoxygenase and leukotriene formation in human neutrophils, microsomal prostaglandin E2 synthase-1, and inflammatory cytokine secretion in human blood monocytes. Our data reveal striking differences in the pharmacological efficiencies of these preparations in inflammation-related bioassays which obviously correlate with the amounts of BAs they contain. In summary, high-quality frankincense extracts display powerful anti-inflammatory effectiveness against multiple targets which can be traced back to BAs as bioactive ingredients.
The tumor-microenvironment (TME) is an amalgamation of various factors derived from malignant cells and infiltrating host cells, including cells of the immune system. One of the important factors of the TME is microRNAs (miRs) that regulate target gene expression at a post transcriptional level. MiRs have been found to be dysregulated in tumor as well as in stromal cells and they emerged as important regulators of tumorigenesis. In fact, miRs regulate almost all hallmarks of cancer, thus making them attractive tools and targets for novel anti-tumoral treatment strategies. Tumor to stroma cell cross-propagation of miRs to regulate protumoral functions has been a salient feature of the TME. MiRs can either act as tumor suppressors or oncogenes (oncomiRs) and both miR mimics as well as miR inhibitors (antimiRs) have been used in preclinical trials to alter cancer and stromal cell phenotypes. Owing to their cascading ability to regulate upstream target genes and their chemical nature, which allows specific pharmacological targeting, miRs are attractive targets for anti-tumor therapy. In this review, we cover a recent update on our understanding of dysregulated miRs in the TME and provide an overview of how these miRs are involved in current cancer-therapeutic approaches from bench to bedside.
Bioactive lipid mediators play a major role in regulating inflammatory processes. Herein, early pro-inflammatory phases are characterized and regulated by prostanoids and leukotrienes, whereas specialized pro-resolving mediators (SPM), including lipoxins, resolvins, protectins, and maresins, dominate during the resolution phase. While pro-inflammatory properties of prostanoids have been studied extensively, their impact on later phases of the inflammatory process has been attributed mainly to their ability to initiate the lipid-mediator class switch towards SPM. Yet, there is accumulating evidence that prostanoids directly contribute to the resolution of inflammation and return to homeostasis. In this mini review, we summarize the current knowledge of the resolution-regulatory properties of prostanoids and discuss potential implications for anti-inflammatory, prostanoid-targeted therapeutic interventions.
Osteoarthritis: novel molecular mechanisms increase our understanding of the disease pathology
(2021)
Although osteoarthritis (OA) is the most common musculoskeletal condition that causes significant health and social problems worldwide, its exact etiology is still unclear. With an aging and increasingly obese population, OA is becoming even more prevalent than in previous decades. Up to 35% of the world’s population over 60 years of age suffers from symptomatic (painful, disabling) OA. The disease poses a tremendous economic burden on the health-care system and society for diagnosis, treatment, sick leave, rehabilitation, and early retirement. Most patients also experience sleep disturbances, reduced capability for exercising, lifting, and walking and are less capable of working, and maintaining an independent lifestyle. For patients, the major problem is disability, resulting from joint tissue destruction and pain. So far, there is no therapy available that effectively arrests structural deterioration of cartilage and bone or is able to successfully reverse any of the existing structural defects. Here, we elucidate novel concepts and hypotheses regarding disease progression and pathology, which are relevant for understanding underlying the molecular mechanisms as a prerequisite for future therapeutic approaches. Emphasis is placed on topographical modeling of the disease, the role of proteases and cytokines in OA, and the impact of the peripheral nervous system and its neuropeptides.
Bacterial and fungal toll-like receptor activation elicits type I IFN responses in mast cells
(2021)
Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.
Gaining detailed knowledge about sex-related immunoregulation remains a crucial prerequisite for the development of adequate disease models and therapeutic strategies enabling personalized medicine. Here, the key parameter of the production of cytokines mediating disease resolution was investigated. Among these cytokines, STAT3-activating interleukin (IL)-22 is principally associated with recovery from tissue injury. By investigating paradigmatic acetaminophen-induced liver injury, we demonstrated that IL-22 expression is enhanced in female mice. Increased female IL-22 was confirmed at a cellular level using murine splenocytes stimulated by lipopolysaccharide or αCD3/CD28 to model innate or adaptive immunoactivation. Interestingly, testosterone or dihydrotestosterone reduced IL-22 production by female but not by male splenocytes. Mechanistic studies on PMA/PHA-stimulated T-cell-lymphoma EL-4 cells verified the capability of testosterone/dihydrotestosterone to reduce IL-22 production. Moreover, we demonstrated by chromatin immunoprecipitation that testosterone impairs binding of the aryl hydrocarbon receptor to xenobiotic responsive elements within the murine IL-22 promoter. Overall, female mice undergoing acute liver injury and cultured female splenocytes upon inflammatory activation display increased IL-22. This observation is likely related to the immunosuppressive effects of androgens in males. The data presented concur with more pronounced immunological alertness demonstrable in females, which may relate to the sex-specific course of some immunological disorders.
The complex and adaptive nature of malignant neoplasm constitute a major challenge for the development of effective anti-oncogenic therapies. Emerging evidence has uncovered the pivotal functions exerted by the small leucine-rich proteoglycans, decorin and biglycan, in affecting tumor growth and progression. In their soluble forms, decorin and biglycan act as powerful signaling molecules. By receptor-mediated signal transduction, both proteoglycans modulate key processes vital for tumor initiation and progression, such as autophagy, inflammation, cell-cycle, apoptosis, and angiogenesis. Despite of their structural homology, these two proteoglycans interact with distinct cell surface receptors and thus modulate distinct signaling pathways that ultimately affect cancer development. In this review, we summarize growing evidence for the complex roles of decorin and biglycan signaling in tumor biology and address potential novel therapeutic implications.
Anti-inflammatory effects of low-dose irradiation often follow a non-linear dose–effect relationship. These characteristics were also described for the modulation of leukocyte adhesion to endothelial cells. Previous results further revealed a contribution of reactive oxygen species (ROS) and anti-oxidative factors to a reduced leukocyte adhesion. Here, we evaluated the expression of anti-oxidative enzymes and the transcription factor Nrf2 (Nuclear factor-erythroid-2-related factor 2), intracellular ROS content, and leukocyte adhesion in primary human microvascular endothelial cells (HMVEC) upon low-dose irradiation under physiological laminar shear stress or static conditions after irradiation with X-ray or Carbon (C)-ions (0–2 Gy). Laminar conditions contributed to increased mRNA expression of anti-oxidative factors and reduced ROS in HMVEC following a 0.1 Gy X-ray and 0.5 Gy C-ion exposure, corresponding to reduced leukocyte adhesion and expression of adhesion molecules. By contrast, mRNA expression of anti-oxidative markers and adhesion molecules, ROS, and leukocyte adhesion were not altered by irradiation under static conditions. In conclusion, irradiation of endothelial cells with low doses under physiological laminar conditions modulates the mRNA expression of key factors of the anti-oxidative system, the intracellular ROS contents of which contribute at least in part to leucocyte adhesion, dependent on the radiation source.
Macrophages facilitate essential homeostatic functions e.g., endocytosis, phagocytosis, and signaling during inflammation, and express a variety of scavenger receptors including CD163 and CD206, which are upregulated in response to inflammation. In healthy individuals, soluble forms of CD163 and CD206 are constitutively shed from macrophages, however, during inflammation pathogen- and damage-associated stimuli induce this shedding. Activation of resident liver macrophages viz. Kupffer cells is part of the inflammatory cascade occurring in acute and chronic liver diseases. We here review the existing literature on sCD163 and sCD206 function and shedding, and potential as biomarkers in acute and chronic liver diseases with a particular focus on Acute-on-Chronic Liver Failure (ACLF). In multiple studies sCD163 and sCD206 are elevated in relation to liver disease severity and established as reliable predictors of morbidity and mortality. However, differences in expression- and shedding-stimuli for CD163 and CD206 may explain dissimilarities in prognostic utility in patients with acute decompensation of cirrhosis and ACLF.
Background: Cytokines are mediators of inflammation that contribute to a low-grade inflammation in different disorders like major depression and obesity. It still remains unclear which psychological and medical factors interact with cytokine regulation. In the current investigation, the association between levels of pro-and anti-inflammatory cytokines and anthropometrics, mood state (depressiveness), physical activity and sleep were investigated in a sample of community-dwelled adults.
Methods: Forty-nine subjects met the inclusion criteria for analyses and were assessed at two time-points (baseline (T1) and follow-up (T2), average T1-T2-interval = 215 days). Serum cytokine measures included the pro-inflammatory cytokines interleukin (IL)-2, IL-12, IFN-γ and TNF-α, the anti-inflammatory cytokines IL-4, IL-5, IL-10 and IL-13 and the granulocyte-macrophage colony-stimulating factor (GM-CSF); anthropometrics were assessed via physical examination, depressiveness was assessed via Beck Depression Inventory (BDI)2, parameters of physical activity (steps, METs) and sleep (night/total sleep duration) were measured via a 1-week actigraphy.
Results: Correlation analyses showed low-to moderate significant relationships between the majority of cytokines and the BDI2 at T1, positive correlation with weight and BMI at T1 and T2, and negative correlations with the number of steps and METs at T2 and T2. Regression analyses for T1 revealed that the BDI2 score was the best positive predictor for the concentrations of all nine cytokines, followed by the number of steps and the nightsleep duration as negative predictors. At T2, the amount of steps was found to be negatively associated with IL-4, IL5, IL-10, GM-CSF, IFN-γ, and TNF-α, whereas the BMI could significantly predict IL-12 and IL-13. The BDI2-score was not significantly associated with any of the cytokines. No associations could be found between dynamics in cytokines from T1 and T2 and changes in any of the variables.
Discussion: The present results indicate an influence of physical activity, subjective well-being and body composition on inflammatory mediators. Since there was no standardized intervention targeting the independent variables between T1 and T2, no assumptions on causality can be drawn from the association results.
Particulate autogenous tooth roots are used for alveolar bone augmentation surgery; however, dental plaque may provoke an inflammatory response that may counteract the desired graft consolidation process. Traditional mechanical cleaning of extracted teeth may be of support to lower a possible inflammatory response of the autograft. To test this assumption, extracted porcine teeth were left either uncleaned or underwent mechanical cleaning with a toothbrush and toothpaste before being fragmented and subjected to acid lysis, termed as unclean acid dentine lysate (ucADL) and clean acid dentine lysate (cADL), respectively. The inflammatory responses of murine macrophage RAW 264.7 cells being exposed to the respective acid dentine lysates were evaluated at the level of inflammatory gene expression and IL6 immunoassays. We report here that acid lysates obtained from uncleaned teeth provoked a robust increase in IL1β, IL6, and COX2 in RAW 264.7 cells. The mechanical removal of dental plaque significantly reduced the inflammatory response. Consistently, Limulus tests revealed that tooth cleaning lowers the presence of endotoxins in dentine lysates. To further prove the involvement of endotoxins, a toll-like receptor 4 (TLR4) inhibitor TAK242 was introduced. TAK242 abolished the inflammatory response provoked by acid lysates obtained from uncleaned teeth in RAW 264.7 cells. Moreover, nuclear translocation and phosphorylation of the TLR4 downstream NFκB-p65 were attenuated at the presence of cleaned versus uncleaned dentine lysates. Taken together, our data support the importance of dental plaque removal of teeth being extracted for alveolar bone augmentation surgery.
Class I and II histone deacetylases (HDAC) are considered important regulators of immunity and inflammation. Modulation of HDAC expression and activity is associated with altered inflammatory responses but reports are controversial and the specific impact of single HDACs is not clear. We examined class I and II HDACs in TLR-4 signaling pathways in murine macrophages with a focus on IκB kinase epsilon (IKKε) which has not been investigated in this context before. Therefore, we applied the pan-HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) as well as HDAC-specific siRNA. Administration of HDACi reduced HDAC activity and decreased expression of IKKε although its acetylation was increased. Other pro-inflammatory genes (IL-1β, iNOS, TNFα) also decreased while COX-2 expression increased. HDAC 2, 3 and 4, respectively, might be involved in IKKε and iNOS downregulation with potential participation of NF-κB transcription factor inhibition. Suppression of HDAC 1–3, activation of NF-κB and RNA stabilization mechanisms might contribute to increased COX-2 expression. In conclusion, our results indicate that TSA and SAHA exert a number of histone- and HDAC-independent functions. Furthermore, the data show that different HDAC enzymes fulfill different functions in macrophages and might lead to both pro- and anti-inflammatory effects which have to be considered in therapeutic approaches.
Background & Aims: Acute‐on‐chronic liver failure (ACLF) is characterized by high short‐term mortality and systemic inflammation (SI). Recently, different cardiodynamic states were shown to independently predict outcomes in cirrhosis. The relationship between cardiodynamic states, SI, and portal hypertension and their impact on ACLF development remains unclear. The aim of this study was therefore to evaluate the interplay of cardiodynamic state and SI on fatal ACLF development in cirrhosis.
Results: At inclusion, hemodynamic measures including cardiac index (CI) and hepatic venous pressure gradient of 208 patients were measured. Patients were followed prospectively for fatal ACLF development (primary endpoint). SI was assessed by proinflammatory markers such as interleukins (ILs) 6 and 8 and soluble IL‐33 receptor (sIL‐33R). Patients were divided according to CI (<3.2; 3.2‐4.2; >4.2 L/min/m2) in hypo‐ (n = 84), normo‐ (n = 69) and hyperdynamic group (n = 55). After a median follow‐up of 3 years, the highest risk of fatal ACLF was seen in hyperdynamic (35%) and hypodynamic patients (25%) compared with normodynamic (14%) (P = .011). Hyperdynamic patients showed the highest rate of SI. The detectable level of IL‐6 was an independent predictor of fatal ACLF development.
Conclusions: Cirrhotic patients with hyperdynamic and hypodynamic circulation have a higher risk of fatal ACLF. Therefore, the cardiodynamic state is strongly associated with SI, which is an independent predictor of development of fatal ACLF.
The aging process is characterized by a chronic, low‐grade inflammatory state, termed “inflammaging.” It has been suggested that macrophage activation plays a key role in the induction and maintenance of this state. In the present study, we aimed to elucidate the mechanisms responsible for aging‐associated changes in the myeloid compartment of mice. The aging phenotype, characterized by elevated cytokine production, was associated with a dysfunction of the hypothalamic–pituitary–adrenal (HPA) axis and diminished serum corticosteroid levels. In particular, the concentration of corticosterone, the major active glucocorticoid in rodents, was decreased. This could be explained by an impaired expression and activity of 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1), an enzyme that determines the extent of cellular glucocorticoid responses by reducing the corticosteroids cortisone/11‐dehydrocorticosterone to their active forms cortisol/corticosterone, in aged macrophages and peripheral leukocytes. These changes were accompanied by a downregulation of the glucocorticoid receptor target gene glucocorticoid‐induced leucine zipper (GILZ) in vitro and in vivo. Since GILZ plays a central role in macrophage activation, we hypothesized that the loss of GILZ contributed to the process of macroph‐aging. The phenotype of macrophages from aged mice was indeed mimicked in young GILZ knockout mice. In summary, the current study provides insight into the role of glucocorticoid metabolism and GILZ regulation during aging.
The coronavirus disease 2019 COVID-19 pandemic is rapidly spreading worldwide and is becoming a major public health crisis. Increasing evidence demonstrates a strong correlation between obesity and the COVID-19 disease. We have summarized recent studies and addressed the impact of obesity on COVID-19 in terms of hospitalization, severity, mortality, and patient outcome. We discuss the potential molecular mechanisms whereby obesity contributes to the pathogenesis of COVID-19. In addition to obesity-related deregulated immune response, chronic inflammation, endothelium imbalance, metabolic dysfunction, and its associated comorbidities, dysfunctional mesenchymal stem cells/adipose-derived mesenchymal stem cells may also play crucial roles in fueling systemic inflammation contributing to the cytokine storm and promoting pulmonary fibrosis causing lung functional failure, characteristic of severe COVID-19. Moreover, obesity may also compromise motile cilia on airway epithelial cells and impair functioning of the mucociliary escalators, reducing the clearance of severe acute respiratory syndrome coronavirus (SARS-CoV-2). Obese diseased adipose tissues overexpress the receptors and proteases for the SARS-CoV-2 entry, implicating its possible roles as virus reservoir and accelerator reinforcing violent systemic inflammation and immune response. Finally, anti-inflammatory cytokines like anti-interleukin 6 and administration of mesenchymal stromal/stem cells may serve as potential immune modulatory therapies for supportively combating COVID-19. Obesity is conversely related to the development of COVID-19 through numerous molecular mechanisms and individuals with obesity belong to the COVID-19-susceptible population requiring more protective measures.
The transcription factor NF-E2 p45-related factor 2 (Nrf2) is an established master regulator of the anti-oxidative and detoxifying cellular response. Thus, a role in inflammatory diseases associated with the generation of large amounts of reactive oxygen species (ROS) seems obvious. In line with this, data obtained in cell culture experiments and preclinical settings have shown that Nrf2 is important in regulating target genes that are necessary to ensure cellular redox balance. Additionally, Nrf2 is involved in the induction of phase II drug metabolizing enzymes, which are important both in degrading and converting drugs into active forms, and into putative carcinogens. Therefore, Nrf2 has also been implicated in tumorigenesis. This must be kept in mind when new therapy approaches are planned for the treatment of sepsis. Therefore, this review highlights the function of Nrf2 in sepsis with a special focus on the translation of rodent-based results into sepsis patients in the intensive care unit (ICU).
A myriad of signaling molecules in a heuristic network of the tumor microenvironment (TME) pose a challenge and an opportunity for novel therapeutic target identification in human cancers. MicroRNAs (miRs), due to their ability to affect signaling pathways at various levels, take a prominent space in the quest of novel cancer therapeutics. The role of miRs in cancer initiation, progression, as well as in chemoresistance, is being increasingly investigated. The canonical function of miRs is to target mRNAs for post-transcriptional gene silencing, which has a great implication in first-order regulation of signaling pathways. However, several reports suggest that miRs also perform non-canonical functions, partly due to their characteristic non-coding small RNA nature. Examples emerge when they act as ligands for toll-like receptors or perform second-order functions, e.g., to regulate protein translation and interactions. This review is a compendium of recent advancements in understanding the role of miRs in cancer signaling and focuses on the role of miRs as novel regulators of the signaling pathway in the TME.
Lichen-forming fungi are symbiotic organisms that synthesize unique natural products with potential for new drug leads. Here, we explored the pharmacological activity of six lichen extracts (Evernia prunastri, Pseudevernia furfuracea, Umbilicaria pustulata, Umbilicaria crustulosa, Flavoparmelia caperata, Platismatia glauca) in the context of cancer and inflammation using a comprehensive set of 11 functional and biochemical in vitro screening assays. We assayed intracellular Ca2+ levels and cell migration. For cancer, we measured tumor cell proliferation, cell cycle distribution and apoptosis, as well as the angiogenesis-associated proliferation of endothelial cells (ECs). Targeting inflammation, we assayed leukocyte adhesion onto ECs, EC adhesion molecule expression, as well as nitric oxide production and prostaglandin (PG)E2 synthesis in leukocytes. Remarkably, none of the lichen extracts showed any detrimental influence on the viability of ECs. We showed for the first time that extracts of F. caperata induce Ca2+ signaling. Furthermore, extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca reduced cell migration. Interestingly, F. caperata extracts strongly decreased tumor cell survival. The proliferation of ECs was significantly reduced by E. prunastri, P. furfuracea, and F. caperata extracts. The extracts did not inhibit the activity of inflammatory processes in ECs. However, the pro-inflammatory activation of leukocytes was inhibited by extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca. After revealing the potential biological activities of lichen extracts by an array of screening tests, a correlation analysis was performed to evaluate particular roles of abundant lichen secondary metabolites, such as atranorin, physodic acid, and protocetraric acid as well as usnic acid in various combinations. Overall, some of the lichen extracts tested in this study exhibit significant pharmacological activity in the context of inflammation and/or cancer, indicating that the group lichen-forming fungi includes promising members for further testing.
Gliflozins are inhibitors of the renal proximal tubular sodium-glucose co-transporter-2 (SGLT-2), that inhibit reabsorption of urinary glucose and they are able to reduce hyperglycemia in patients with type 2 diabetes. A renoprotective function of gliflozins has been proven in diabetic nephropathy, but harmful side effects on the kidney have also been described. In the current project, primary highly purified human renal proximal tubular epithelial cells (PTCs) have been shown to express functional SGLT-2, and were used as an in vitro model to study possible cellular damage induced by two therapeutically used gliflozins: empagliflozin and dapagliflozin. Cell viability, proliferation, and cytotoxicity assays revealed that neither empagliflozin nor dapagliflozin induce effects in PTCs cultured in a hyperglycemic environment, or in co-medication with ramipril or hydro-chloro-thiazide. Oxidative stress was significantly lowered by dapagliflozin but not by empagliflozin. No effect of either inhibitor could be detected on mRNA and protein expression of the pro-inflammatory cytokine interleukin-6 and the renal injury markers KIM-1 and NGAL. In conclusion, empa- and dapagliflozin in therapeutic concentrations were shown to induce no direct cell injury in cultured primary renal PTCs in hyperglycemic conditions.
The macrophage-inducible C-type lectin (mincle) is part of the innate immune system and acts as a pattern recognition receptor for pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs). Ligand binding induces mincle activation which consequently interacts with the signaling adapter Fc receptor, SYK, and NF-kappa-B. There is also evidence that mincle expressed on macrophages promotes intestinal barrier integrity. However, little is known about the role of mincle in hepatic fibrosis, especially in more advanced disease stages. Mincle expression was measured in human liver samples from cirrhotic patients and donors collected at liver transplantation and in patients undergoing bariatric surgery. Human results were confirmed in rodent models of cirrhosis and acute-on-chronic liver failure (ACLF). In these models, the role of mincle was investigated in liver samples as well as in peripheral blood monocytes (PBMC), tissues from the kidney, spleen, small intestine, and heart. Additionally, mincle activation was stimulated in experimental non-alcoholic steatohepatitis (NASH) by treatment with mincle agonist trehalose-6,6-dibehenate (TDB). In human NASH, mincle is upregulated with increased collagen production. In ApoE deficient mice fed high-fat western diet (NASH model), mincle activation significantly increases hepatic collagen production. In human cirrhosis, mincle expression is also significantly upregulated. Furthermore, mincle expression is associated with the stage of chronic liver disease. This could be confirmed in rat models of cirrhosis and ACLF. ACLF was induced by LPS injection in cirrhotic rats. While mincle expression and downstream signaling via FC receptor gamma, SYK, and NF-kappa-B are upregulated in the liver, they are downregulated in PBMCs of these rats. Although mincle expressed on macrophages might be beneficial for intestinal barrier integrity, it seems to contribute to inflammation and fibrosis once the intestinal barrier becomes leaky in advanced stages of chronic liver disease.
Pain is the most frequent cause triggering patients to visit a physician. The worldwide incidence of chronic pain is in the range of 20% of adults, and chronic pain conditions are frequently associated with several comorbidities and a drastic decrease in patients’ quality of life. Although several approved analgesics are available, such therapy is often not satisfying due to insufficient efficacy and/or severe side effects. Therefore, novel strategies for the development of safe and highly efficacious pain killers are urgently needed. To reach this goal, it is necessary to clarify the causes and signal transduction cascades underlying the onset and progression of the different types of chronic pain. The papers in this Special Issue cover a wide variety of mechanisms involved in different pain types such as inflammatory, neuropathic or cancer pain. Therefore, the results summarized here might contribute to a better understanding of the mechanisms in chronic pain and thereby to the development of novel therapeutic strategies for pain patients.
Background: Blunt chest (thoracic) trauma (TxT) and haemorrhagic shock with subsequent resuscitation (H/R) induce strong systemic and local inflammatory response, which is closely associated with apoptotic cell loss and subsequently impaired organ function. The underlying mechanisms are not completely understood, therefore, the treatment of patients suffering from TxT+H/R is challenging. In our recent studies, we have demonstrated local anti-inflammatory effects of ethyl pyruvate (EtP) in lung and liver after TxT+H/R. Here, the therapeutic potential of a reperfusion regime with EtP on the early post-traumatic systemic inflammatory response and apoptotic changes after TxT followed by H/R were investigated.
Methods: Female Lewis rats underwent TxT followed by haemorrhagic shock (60 min). Resuscitation was performed with own blood transfusion and either lactated Ringers solution (LR) or LR supplemented with EtP (50 mg/kg). Sham group underwent the surgical procedures. After 2 h blood as well as lung and liver tissues were obtained for analyses. Systemic activation of neutrophils (expression of CD11b and CD62L), leukocyte phagocytosis, apoptosis (caspase-3/7 activation), pyroptosis (caspase-1 activation) and NF-κB p65 activity were assessed. p < 0.05 was considered significant.
Results: TxT+H/R-induced systemic activation of neutrophils (increased CD11b and reduced CD62L expression) was significantly reduced by EtP. Trauma-induced delayed neutrophil apoptosis was further reduced by EtP reperfusion but remained unaltered in monocytes. Reperfusion with EtP significantly increased the phagocytizing capacity of granulocytes. Trauma-induced inflammasome activation, which was observed in monocytes and not in neutrophils, was significantly reduced by EtP in both cell entities. NF-κB p65 activation, which was increased in neutrophils and monocytes was significantly decreased in monocytes.
Conclusion: TxT+H/R-induced systemic activation of both neutrophils and monocytes concomitant with increased systemic inflammation was reduced by a reperfusion with EtP and was associated with a down-regulation of NF-κB p65 activation.
Background and Aims: Monocyte chemotactic protein-1 (MCP-1) is a potent chemoattractant for monocytes. It is involved in pathogenesis of several inflammatory diseases. Hepatic MCP-1 is a readout of macrophage activation. While inflammation is a major driver of liver disease progression, the origin and role of circulating MCP-1 as a biomarker remains unclear.
Methods: Hepatic CC-chemokine ligand 2 (CCL2) expression and F4/80 staining for Kupffer cells were measured and correlated in a mouse model of chronic liver disease (inhalative CCl4 for 7 weeks). Next, hepatic RNA levels of CCL2 were measured in explanted livers of 39 patients after transplantation and correlated with severity of disease. Changes in MCP-1 were further evaluated in a rat model of experimental cirrhosis and acute-on-chronic liver failure (ACLF). Finally, we analyzed portal and hepatic vein levels of MCP-1 in patients receiving transjugular intrahepatic portosystemic shunt insertion for complications of portal hypertension.
Results: In this mouse model of fibrotic hepatitis, hepatic expression of CCL2 (P = 0.009) and the amount of F4/80 positive cells in the liver (P < 0.001) significantly increased after induction of hepatitis by CCl4 compared to control animals. Moreover, strong correlation of hepatic CCL2 expression and F4/80 positive cells were seen (P = 0.023). Furthermore, in human liver explants, hepatic transcription levels of CCL2 correlated with the MELD score of the patients, and thus disease severity (P = 0.007). The experimental model of ACLF in rats revealed significantly higher levels of MCP-1 plasma (P = 0.028) and correlation of hepatic CCL2 expression (R = 0.69, P = 0.003). Particularly, plasma MCP-1 levels did not correlate with peripheral blood monocyte CCL2 expression. Finally, higher levels of MCP-1 were observed in the hepatic compared to the portal vein (P = 0.01) in patients receiving TIPS. Similarly, a positive correlation of MCP-1 with Child-Pugh score was observed (P = 0.018). Further, in the presence of ACLF, portal and hepatic vein levels of MCP-1 were significantly higher compared to patients without ACLF (both P = 0.039).
Conclusion: Circulating levels of MCP-1 mainly derive from the injured liver and are associated with severity of liver disease. Therefore, liver macrophages contribute significantly to disease progression. Circulating MCP-1 may reflect the extent of hepatic macrophage activation.
Unresolved inflammation maintained by release of danger‐associated molecular patterns, particularly high‐mobility group box‐1 (HMGB1), is crucial for hepatocellular carcinoma (HCC) pathogenesis. To further characterize interactions between leucocytes and necrotic cancerous tissue, a cellular model of necroinflammation was studied in which murine Raw 264.7 macrophages or primary splenocytes were exposed to necrotic lysates (N‐lys) of murine hepatoma cells or primary hepatocytes. In comparison to those derived from primary hepatocytes, N‐lys from hepatoma cells were highly active—inducing in macrophages efficient expression of inflammatory cytokines like C‐X‐C motif ligand‐2 , tumor necrosis factor‐α, interleukin (IL)‐6 and IL‐23‐p19. This activity associated with higher levels of HMGB1 in hepatoma cells and was curbed by pharmacological blockage of the receptor for advanced glycation end product (RAGE)/HMGB1 axis or the mitogen‐activated protein kinases ERK1/2 pathway. Analysis of murine splenocytes furthermore demonstrated that N‐lys did not comprise of functionally relevant amounts of TLR4 agonists. Finally, N‐lys derived from hepatoma cells supported inflammatory splenic Th17 and Th1 polarization as detected by IL‐17, IL‐22 or interferon‐γ production. Altogether, a straightforward applicable model was established which allows for biochemical characterization of immunoregulation by HCC necrosis in cell culture. Data presented indicate a remarkably inflammatory capacity of necrotic hepatoma cells that, at least partly, depends on the RAGE/HMGB1 axis and may shape immunological properties of the HCC microenvironment.
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.
Previous studies reported on the safety and applicability of mesenchymal stem/stromal cells (MSCs) to ameliorate pulmonary inflammation in acute respiratory distress syndrome (ARDS). Thus, multiple clinical trials assessing the potential of MSCs for COVID-19 treatment are underway. Yet, as SARS-inducing coronaviruses infect stem/progenitor cells, it is unclear whether MSCs could be infected by SARS-CoV-2 upon transplantation to COVID-19 patients. We found that MSCs from bone marrow, amniotic fluid, and adipose tissue carry angiotensin-converting enzyme 2 and transmembrane protease serine subtype 2 at low levels on the cell surface under steady-state and inflammatory conditions. We did not observe SARS-CoV-2 infection or replication in MSCs at steady state under inflammatory conditions, or in direct contact with SARS-CoV-2-infected Caco-2 cells. Further, indoleamine 2,3-dioxygenase 1 production in MSCs was not impaired in the presence of SARS-CoV-2. We show that MSCs are resistant to SARS-CoV-2 infection and retain their immunomodulation potential, supporting their potential applicability for COVID-19 treatment.
The sphingolipid sphingosine-1-phosphate (S1P) is produced by sphingosine kinases to either signal through intracellular targets or to activate a family of specific G-protein-coupled receptors (S1PR). S1P levels are usually low in peripheral tissues compared to the vasculature, forming a gradient that mediates lymphocyte trafficking. However, S1P levels rise during inflammation in peripheral tissues, thereby affecting resident or recruited immune cells, including macrophages. As macrophages orchestrate initiation and resolution of inflammation, the sphingosine kinase/S1P/S1P-receptor axis emerges as an important determinant of macrophage function in the pathogenesis of inflammatory diseases such as cancer, atherosclerosis, and infection. In this review, we therefore summarize the current knowledge how S1P affects macrophage biology.
Macrophage S1PR1 signaling alters angiogenesis and lymphangiogenesis during skin inflammation
(2019)
The bioactive lipid sphingosine-1-phosphate (S1P), along with its receptors, modulates lymphocyte trafficking and immune responses to regulate skin inflammation. Macrophages are important in the pathogenesis of psoriasiform skin inflammation and express various S1P receptors. How they respond to S1P in skin inflammation remains unknown. We show that myeloid specific S1P receptor 1 (S1PR1) deletion enhances early inflammation in a mouse model of imiquimod-induced psoriasis, without altering the immune cell infiltrate. Mechanistically, myeloid S1PR1 deletion altered the formation of IL-1β, VEGF-A, and VEGF-C, and their receptors’ expression in psoriatic skin, which subsequently lead to reciprocal regulation of neoangiogenesis and neolymphangiogenesis. Experimental findings were corroborated in human clinical datasets and in knockout macrophages in vitro. Increased blood vessel but reduced lymph vessel density may explain the exacerbated inflammatory phenotype in conditional knockout mice. These findings assign a novel role to macrophage S1PR1 and provide a rationale for therapeutically targeting local S1P during skin inflammation.
Blunt thoracic trauma (TxT) deteriorates clinical post-injury outcomes. Ongoing inflammatory changes promote the development of post-traumatic complications, frequently causing Acute Lung Injury (ALI). Club Cell Protein (CC)16, a pulmonary anti-inflammatory protein, correlates with lung damage following TxT. Whether CC16-neutralization influences the inflammatory course during ALI is elusive. Ninety-six male CL57BL/6N mice underwent a double hit model of TxT and cecal ligation puncture (CLP, 24 h post-TxT). Shams underwent surgical procedures. CC16 was neutralized by the intratracheal application of an anti-CC16-antibody, either after TxT (early) or following CLP (late). Euthanasia was performed at 6 or 24 h post-CLP. Systemic and pulmonary levels of IL-6, IL-1β, and CXCL5 were determined, the neutrophils were quantified in the bronchoalveolar lavage fluid, and histomorphological lung damage was assessed. ALI induced a significant systemic IL-6 increase among all groups, while the local inflammatory response was most prominent after 24 h in the double-hit groups as compared to the shams. Significantly increased neutrophilic infiltration upon double hit was paralleled with the enhanced lung damage in all groups as compared to the sham, after 6 and 24 h. Neutralization of CC16 did not change the systemic inflammation. However, early CC16-neutralization increased the neutrophilic infiltration and lung injury at 6 h post-CLP, while 24 h later, the lung injury was reduced. Late CC16-neutralization increased neutrophilic infiltration, 24 h post-CLP, and was concurrent with an enhanced lung injury. The data confirmed the anti-inflammatory potential of endogenous CC16 in the murine double-hit model of ALI.
Inflammatory activation of astroglia adds to the pathology of various neurological diseases. Astrocytes respond to microglia-derived cytokines such as interleukin-1α (IL-1α) with enhanced inflammatory signaling. This provokes pro-inflammatory gene expression of, among others, the eicosanoid-generating enzyme prostaglandin endoperoxide synthase 2 (Ptgs2). Whereas metabolic regulation of innate immune cell inflammatory responses is intensely studied, pathways related to how metabolism modulates inflammatory signaling in astrocytes are underexplored. Here, we examined how mitochondrial oxidative phosphorylation affects inflammatory responses towards IL-1α and tumor necrosis factor α in neonatal rat astrocytes. Blocking respiratory complex I and III or adenosine triphosphate (ATP) synthase did not affect activation of inflammatory signaling by IL-1α, but did elicit differential effects on inflammatory gene mRNA expression. Remarkably, mRNA and protein expression of Ptgs2 by IL-1α was consistently up-regulated when oxidative phosphorylation was inhibited. The increase of Ptgs2 resulted from mRNA stabilization. Mitochondrial inhibitors also increased IL-1α-triggered secretion of eicosanoids, such as prostaglandin E2, prostaglandin F2α, and 6-keto-prostaglandin F1α, as assessed by liquid chromatography/mass spectrometry. Mechanistically, attenuating oxidative phosphorylation elevated adenosine monophosphate (AMP) and activated AMP-activated protein kinase (AMPK). AMPK silencing prevented Ptgs2 up-regulation by mitochondrial inhibitors, while AMPK activators recapitulated Ptgs2 mRNA stability regulation. Our data indicate modulation of astrocyte inflammatory responses by oxidative metabolism, with relevance towards eicosanoid production.
In cells the interorganelle communication comprises vesicular and non-vesicular mechanisms. Non-vesicular material transfer predominantly takes place at regions of close organelle apposition termed membrane contact sites and is facilitated by a growing number of specialized proteins. Contacts of the endoplasmic reticulum (ER) and mitochondria are now recognized to be essential for diverse biological processes such as calcium homeostasis, phospholipid biosynthesis, apoptosis, and autophagy. In addition to these universal roles, ER-mitochondria communication serves also cell type-specific functions. In this review, we summarize the current knowledge on ER-mitochondria contacts in cells of the innate immune system, especially in macrophages. We discuss ER- mitochondria communication in the context of macrophage fatty acid metabolism linked to inflammatory and ER stress responses, its roles in apoptotic cell engulfment, activation of the inflammasome, and antiviral defense.
Lipoxygenases (LOXs) catalyze the stereo-specific peroxidation of polyunsaturated fatty acids (PUFAs) to their corresponding hydroperoxy derivatives. Human macrophages express two arachidonic acid (AA) 15-lipoxygenating enzymes classified as ALOX15 and ALOX15B. ALOX15, which was first described in 1975, has been extensively characterized and its biological functions have been investigated in a number of cellular systems and animal models. In macrophages, ALOX15 functions to generate specific phospholipid (PL) oxidation products crucial for orchestrating the nonimmunogenic removal of apoptotic cells (ACs) as well as synthesizing precursor lipids required for production of specialized pro-resolving mediators (SPMs) that facilitate inflammation resolution. The discovery of ALOX15B in 1997 was followed by comprehensive analyses of its structural properties and reaction specificities with PUFA substrates. Although its enzymatic properties are well described, the biological functions of ALOX15B are not fully understood. In contrast to ALOX15 whose expression in human monocyte-derived macrophages is strictly dependent on Th2 cytokines IL-4 and IL-13, ALOX15B is constitutively expressed. This review aims to summarize the current knowledge on the regulation and functions of ALOX15 and ALOX15B in human macrophages.
The stimulation of the AMP-activated kinase (AMPK) by 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) has been associated with antihyperalgesia and the inhibition of nociceptive signaling in the spinal cord in models of paw inflammation. The attenuated nociception comes along with a strongly reduced paw edema, indicating that peripheral antiinflammatory mechanisms contribute to antinociception. In this study, we investigated the impact of AICAR on the immune cell composition in inflamed paws, as well as the regulation of inflammatory and resolving markers in macrophages. By using fluorescence-activated cell sorting (FACS) analysis and immunofluorescence, we found a significantly increased fraction of proresolving M2 macrophages and anti-inflammatory interleukin (IL)-10 in inflamed tissue, while M1 macrophages and proinflammatory cytokines such as IL-1 were decreased by AICAR in wild type mice. In AMPKα2 knock-out mice, the M2 polarization of macrophages in the paw was missing. The results were supported by experiments in primary macrophage cultures which also showed a shift to a proresolving phenotype with decreased levels of proinflammatory mediators and increased levels of antiinflammatory mediators. However, in the cell cultures, we did not observe differences between the AMPKα2+/+ and −/− cells, thus indicating that the AICAR-induced effects are at least partially AMPK-independent. In summary, our results indicate that AICAR has potent antiinflammatory and proresolving properties in inflammation which are contributing to a reduction of inflammatory edema and antinociception.
Short Summary: Extracellular vesicles (EVs), released during tissue/cell injury, contain a "barcode" indicating specific microRNAs (miRs) that can uncover their origin. We examined whether systemic EVs possessing hepatic miR-signatures would indicate ongoing liver injury and clinical complications in trauma patients (TP). We grouped the patients of alcoholic drinkers into "alcohol-drinkers with liver injury (LI)" (EtOH with LI) or "alcohol-drinkers without LI" (EtOH w/o LI) and we compared these groups to "non-drinkers" (no EtOH). When we examined patient blood from the EtOH with LI group we found the total number of EVs to be increased, along with an increase in miR-122 and let7f—two EV-associated miRNAs—and several inflammation-associating cytokines, such as interleukin (IL)-6 and IL-33. In contrast, all of the aforementioned readouts were found to be decreased in the EtOH w/o LI group. These novel data demonstrate that hepatocyte damage in alcohol-intoxicated trauma patients presenting with liver injury can be reflected by an increase in circulating serum EVs, their specific miR-"barcode" and the concomitant increase of systemic inflammatory markers IL-6 and IL-33. Anti-inflammatory effect of alcohol-drinking in EtOH w/o LI can be presented by a reduced number of hepato-derived EVs, no upregulation of IL-6 and IL-33, and a miR "barcode" different from patients presenting with liver injury.
Background: Alcohol abuse is associated with (neuro)protective effects related to (head) injuries, and with negative effects regarding infection rates and survival in severely injured trauma patients (TP). Extracellular vesicles (EVs), which are released during tissue and/or cell injury, can contain a "barcode" including specific microRNAs (miRs) that uncover their origin. We examined whether EVs with a hepatic miR signature can be systemically measured, and whether they can indicate ongoing liver injury in alcohol-intoxicated TP and foretell clinical complications.
Patients/Methods: We enrolled 35 TP and measured blood EVs, IL-6, TNF-alpha, IL-1beta, IL-10 and IL-33, alcohol (ethanol, EtOH) concentration (BAC), GLDH, GGT, AST, ALT, leukocytes, platelets, and bilirubin. Within circulating EVs we measured the expression levels of miR-122, let7f, miR21, miR29a, miR-155, and miR-146a. Patients of alcohol-drinkers were grouped into "alcohol drinkers with liver injury (LI)" (EtOH with LI) or "alcohol drinkers without LI" (EtOH w/o LI) and compared to "non-drinkers" (no EtOH). We assessed systemic injury characteristics and the outcome of hospitalization with regard to sepsis, septic shock, pneumonia, or mortality.
Results: EtOH with LI patients had significantly increased rates of pneumonia vs. the EtOH w/o LI group. EVs, IL-6, and IL-33 levels were significantly increased in EtOH with LI vs. EtOH w/o LI group (p < 0.05). EV number correlated positively with ALT and IL-6 (p < 0.0001). Two miRs, miR-122 and let7f, were increased only in the blood EVs from the EtOH with LI group (p < 0.05). Five miRs, miR-122, let7f, miR-21, miR-29a, and miR-146a, were reduced in the blood EVs from the EtOH w/o LI group, vs. no EtOH (p < 0.05). Notably miR-122 correlated significantly with increased bilirubin levels in the EtOH with LI group (p < 0.05).
Conclusions: Liver injury in alcohol-intoxicated TP is reflected by increased EV numbers, their specific miR barcode, and the correlated increase of systemic inflammatory markers IL-6 and IL-33. Interestingly, severely injured TP without liver injury were found to have a reduced number of liver-derived EVs, no observed inflammatory infiltration and reduced specific miR "barcode."
MicroRNAs (miRs) significantly contribute to the regulation of gene expression, by virtue of their ability to interact with a broad, yet specific set of target genes. MiRs are produced and released by almost every cell type and play an important role in horizontal gene regulation in the tumor microenvironment (TME). In the TME, both tumor and stroma cells cross-communicate via diverse factors including miRs, which are taking central stage as a therapeutic target of anti-tumor therapy. One of the immune escape strategies adopted by tumor cells is to release miRs as a Trojan horse to hijack circulating or tumor-localized monocytes/macrophages to tune them for pro-tumoral functions. On the other hand, macrophage-derived miRs exert anti-tumor functions. The transfer of miRs from host to recipient cells depends on the supramolecular structure and composition of miR carriers, which determine the distinct uptake mechanism by recipient cells. In this review, we provide a recent update on the miR-mediated crosstalk between tumor cells and macrophages and their mode of uptake in the TME.
Over the last years, many microRNAs (miRNAs) have been identified that regulate the formation of bioactive lipid mediators such as prostanoids and leukotrienes. Many of these miRNAs are involved in complex regulatory circuits necessary for the fine-tuning of biological functions including inflammatory processes or cell growth. A better understanding of these networks will contribute to the development of novel therapeutic strategies for the treatment of inflammatory diseases and cancer. In this review, we provide an overview of the current knowledge of miRNA regulation in eicosanoid pathways with special focus on novel miRNA functions and regulatory circuits of leukotriene and prostaglandin biosynthesis.
Pretubulysin (PT), a biosynthetic precursor of the myxobacterial compound tubulysin D, was recently identified as a novel microtubule-targeting agent (MTA) causing microtubule destabilization. MTAs are the most frequently used chemotherapeutic drugs. They are well studied regarding their direct cytotoxic effects against various tumors as well as for their anti-angiogenic and vascular-disrupting action addressing endothelial cells of the tumor vasculature. However, the impact of MTAs on endothelial cells of the non-tumor vasculature has been largely neglected, although tumor cell interactions with the healthy endothelium play a crucial role in the process of cancer metastasis. Besides their use as potent anti-cancer drugs, some MTAs such as colchicine are traditionally used or recommended for the therapy of inflammatory diseases. Here, too, the role of endothelial cells has been largely neglected, although the endothelium is crucially involved in regulating the process of inflammation.
In the present study, the impact of PT on tumor-endothelial cell interactions was therefore analyzed in vitro to gain insights into the mechanism underlying its anti-metastatic effect that was recently confirmed in vivo. In the second part of this work, the influence of PT and other MTAs, namely the microtubule-destabilizing compounds vincristine (VIN) and colchicine (COL) and the microtubule-stabilizing drug paclitaxel (PAC), on leukocyte-endothelial cell interactions was investigated in vitro and in vivo (only PT). It is important to mention that in all in vitro experiments solely endothelial cells and not tumor cells or leukocytes were treated with the MTAs to strictly focus on the role of the endothelium in the action of these compounds.
The impact of PT on tumor-endothelial cell interactions was analyzed in vitro by cell adhesion and transendothelial migration assays as well as immunocytochemistry using the breast cancer cell line MDA-MB-231 and primary human umbilical vein endothelial cells (HUVECs). The treatment of HUVECs with PT increased the adhesion of MDA cells onto the endothelial monolayer, whereas their transendothelial migration was reduced by the compound. Thereafter, the influence of PT on the endothelial cell adhesion molecules (CAMs) E-selectin, N-cadherin, ICAM-1, VCAM-1 and galectin-3 and on the CXCL12/CXCR4 chemokine system was examined, since they might be involved in the PT-triggered tumor cell adhesion. Interestingly, although PT induced the upregulation of ICAM-1, VCAM-1, N-cadherin and CXCL12, cell adhesion assays using neutralizing antibodies or the CXCL12 inhibitor AMD3100 revealed that all these molecules were dispensable for the PT-evoked tumor cell adhesion. As PT induces the formation of interendothelial gaps and MDA cells might adhere onto components of the underlying extracellular matrix (ECM), the precise location of MDA cells attached to the PT-treated endothelial monolayer was investigated. Instead of a direct interaction between tumor and endothelial cells, this work showed that MDA cells preferred to adhere to the ECM component collagen that was exposed within PT-triggered endothelial gaps. Both the PT-evoked increase in tumor cell adhesion onto and the decrease in trans-endothelial migration were completely abolished when β1-integrins were blocked on MDA cells. Similar results were obtained when endothelial cells were treated with VIN and COL but not PAC, indicating that the observed effects of PT depend on its microtubule-destabilizing activity.
The impact of PT, VIN, COL and PAC on leukocyte-endothelial cell interactions was analyzed in vivo (only PT) by intravital microscopy of the mouse cremaster muscle and in vitro by cell adhesion assays using the monocyte-like cell line THP-1 and TNFα-activated human dermal microvascular endothelial cells (HMEC-1). While PT did not affect the rolling of leukocytes on the endothelium, their firm adhesion onto and transmigration through the activated endothelium was reduced by PT in vivo. In accordance, the treatment of HMEC-1 with PT, VIN and COL decreased the TNFα-induced adhesion of THP-1 cells onto the endothelial monolayer, whereas PAC had no influence on this process. Thereafter, the influence of PT, VIN, COL and PAC on endothelial ICAM-1 and VCAM-1 was examined, since these molecules are substantially involved in the firm adhesion of leukocytes onto the endothelium. The cell surface protein expression of ICAM-1 and VCAM-1 was reduced by PT, VIN and COL in activated endothelial cells, whereas PAC did only slightly affect the TNFα-induced upregulation of VCAM-1. As the pro-inflammatory transcription factor NFκB plays a crucial role in the TNFα-induced expression of these CAMs, the impact of the MTAs on the NFκB promotor activity was investigated. While PT, VIN and COL decreased the activation of NFκB in activated endothelial cells, PAC did not affect this process. However, in contrast to the strong effects regarding the cell surface protein expression of ICAM-1 and VCAM-1, the effects of PT, VIN and COL on the NFκB activity was rather low. Thus, the used MTAs might also affect other relevant signaling pathways and/or the intracellular transport of CAMs might be influenced by the impact of the MTAs on the microtubule network.
Taken together, the current study provides – at least in part – an explanation for the anti-metastatic potential of PT and gives first insights into the use of PT and VIN as anti-inflammatory drugs. Moreover, this work highlights the endothelium as an attractive target for the development of new anti-cancer and anti-inflammatory drugs.
Atherosclerosis and its sequelae, such as myocardial infarction and stroke, are the leading cause of death worldwide. Vascular endothelial cells (EC) play a critical role in vascular homeostasis and disease. Atherosclerosis as well as its independent risk factors including diabetes, obesity, and aging, are hallmarked by endothelial activation and dysfunction. Metabolic pathways have emerged as key regulators of many EC functions, including angiogenesis, inflammation, and barrier function, processes which are deregulated during atherogenesis. In this review, we highlight the role of glucose, fatty acid, and amino acid metabolism in EC functions during physiological and pathological states, specifically atherosclerosis, diabetes, obesity and aging.
Natural Killer T cells (NKT cells) are emerging as critical regulators of pro- and anti-tumor immunity, both at baseline and in therapeutic settings. While type I NKT cells can promote anti-tumor immunity, their activity in the tumor microenvironment may be limited by negative regulators such as inhibitory immune checkpoints. We observed dominant expression of B- and T-lymphocyte attenuator (BTLA) on type I NKT cells in polyoma middle T oncogene-driven (PyMT) murine autochthonous mammary tumors. Other immune checkpoint receptors, such as programmed cell death 1 (PD-1) were equally distributed among T cell populations. Interference with BTLA using neutralizing antibodies limited tumor growth and pulmonary metastasis in the PyMT model in a therapeutic setting, correlating with an increase in type I NKT cells and expression of cytotoxic marker genes. While therapeutic application of an anti-PD-1 antibody increased the number of CD8+ cytotoxic T cells and elevated IL-12 expression, tumor control was not established. Expression of ZBTB16, the lineage-determining transcription factor of type I NKT cells, was correlated with a favorable patient prognosis in the METABRIC dataset, and BTLA levels were instrumental to further distinguish prognosis in patents with high ZBTB16 expression. Taken together, these data support a role of BTLA on type I NKT cells in limiting anti-tumor immunity.
Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate (S1P), which acts as a key regulator of inflammatory and fibrotic reactions, mainly via S1P receptor activation. Here, we show that in the human renal proximal tubular epithelial cell line HK2, the profibrotic mediator transforming growth factor β (TGFβ) induces SK-1 mRNA and protein expression, and in parallel, it also upregulates the expression of the fibrotic markers connective tissue growth factor (CTGF) and fibronectin. Stable downregulation of SK-1 by RNAi resulted in the increased expression of CTGF, suggesting a suppressive effect of SK-1-derived intracellular S1P in the fibrotic process, which is lost when SK-1 is downregulated. In a further approach, the S1P transporter Spns2, which is known to export S1P and thereby reduces intracellular S1P levels, was stably downregulated in HK2 cells by RNAi. This treatment decreased TGFβ-induced CTGF and fibronectin expression, and it abolished the strong induction of the monocyte chemotactic protein 1 (MCP-1) by the pro-inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1β. Moreover, it enhanced the expression of aquaporin 1, which is an important water channel that is expressed in the proximal tubules, and reverted aquaporin 1 downregulation induced by IL-1β/TNFα. On the other hand, overexpression of a Spns2-GFP construct increased S1P secretion and it resulted in enhanced TGFβ-induced CTGF expression. In summary, our data demonstrate that in human renal proximal tubular epithelial cells, SK-1 downregulation accelerates an inflammatory and fibrotic reaction, whereas Spns2 downregulation has an opposite effect. We conclude that Spns2 represents a promising new target for the treatment of tubulointerstitial inflammation and fibrosis.
Chronic kidney disease and diabetes mellitus are associated with extensive media calcification, which leads to increased cardiovascular morbidity and mortality. Here, we investigated the role of autophagy in the pathogenesis of uremic vascular media calcification. DBA/2 mice were fed with high-phosphate diet (HPD) in order to cause vascular calcification. DBA/2 mice on standard chow diet were used as control. In parallel, autophagy and its response to rapamycin, 3-methyladenine (3-MA), and bafilomycin were studied in an in vitro model using mouse vascular smooth muscle cells (MOVAS). DBA/2 mice on HPD developed severe vascular media calcification, which is mirrored in vitro by culturing MOVAS under calcifying conditions. Both, in vitro and in vivo, autophagy significantly increased in MOVAS under calcifying conditions and in aortas of HPD mice, respectively. Histologically, autophagy was located to the aortic Tunica media, but also vascular endothelial cells, and was found to continuously increase during HPD treatment. 3-MA as well as bafilomycin blocked autophagy in MOVAS and increased calcification. Vice versa, rapamycin treatment further increased autophagy and resulted in a significant decrease of vascular calcification in vitro and in vivo. Rapamycin reduced Runx2 transcription levels in aortas and MOVAS to control levels, whereas it increased α-smooth muscle actin and Sm22α transcription in MOVAS to control levels. Furthermore, rapamycin-treated HPD mice survived significantly longer compared to HPD controls. These findings indicate that autophagy is an endogenous response of vascular smooth muscle cells (VSMC) to protect from calcification in uremia. Induction of autophagy by rapamycin protects cells and mice from uremic media calcification possibly by inhibiting osteogenic transdifferentiation of VSMC.
Aim: Reactive oxygen species (ROS) produced by enzymes of the NADPH oxidase family serve as second messengers for cellular signaling. Processes such as differentiation and proliferation are regulated by NADPH oxidases. In the intestine, due to the exceedingly fast and constant renewal of the epithelium both processes have to be highly controlled and balanced. Nox1 is the major NADPH oxidase expressed in the gut, and its function is regulated by cytosolic subunits such as NoxO1. We hypothesize that the NoxO1-controlled activity of Nox1 contributes to a proper epithelial homeostasis and renewal in the gut.
Results: NoxO1 is highly expressed in the colon. Knockout of NoxO1 reduces the production of superoxide in colon crypts and is not subsidized by an elevated expression of its homolog p47phox. Knockout of NoxO1 increases the proliferative capacity and prevents apoptosis of colon epithelial cells. In mouse models of dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS induced colon cancer, NoxO1 has a protective role and may influence the population of natural killer cells.
Conclusion: NoxO1 affects colon epithelium homeostasis and prevents inflammation.
GTP cyclohydrolase (GCH1) governs de novo synthesis of the enzyme cofactor, tetrahydrobiopterin (BH4), which is essential for biogenic amine production, bioactive lipid metabolism and redox coupling of nitric oxide synthases. Overproduction of BH4 via upregulation of GCH1 in sensory neurons is associated with nociceptive hypersensitivity in rodents, and neuron‐specific GCH1 deletion normalizes nociception. The translational relevance is revealed by protective polymorphisms of GCH1 in humans, which are associated with a reduced chronic pain. Because myeloid cells constitute a major non‐neuronal source of BH4 that may contribute to BH4‐dependent phenotypes, we studied here the contribution of myeloid‐derived BH4 to pain and itch in lysozyme M Cre‐mediated GCH1 knockout (LysM‐GCH1−/−) and overexpressing mice (LysM‐GCH1‐HA). Unexpectedly, knockout or overexpression in myeloid cells had no effect on nociceptive behaviour, but LysM‐driven GCH1 knockout reduced, and its overexpression increased the scratching response in Compound 48/80 and hydroxychloroquine‐evoked itch models, which involve histamine and non‐histamine dependent signalling pathways. Mechanistically, GCH1 overexpression increased BH4, nitric oxide and hydrogen peroxide, and these changes were associated with increased release of histamine and serotonin and degranulation of mast cells. LysM‐driven GCH1 knockout had opposite effects, and pharmacologic inhibition of GCH1 provided even stronger itch suppression. Inversely, intradermal BH4 provoked scratching behaviour in vivo and BH4 evoked an influx of calcium in sensory neurons. Together, these loss‐ and gain‐of‐function experiments suggest that itch in mice is contributed by BH4 release plus BH4‐driven mediator release from myeloid immune cells, which leads to activation of itch‐responsive sensory neurons.
Acetaminophen [paracetamol, N-acetyl-p-aminophenol (APAP)]-induced acute liver injury (ALI) not only remains a persistent clinical challenge but likewise stands out as well-characterized paradigmatic model of drug-induced liver damage. APAP intoxication associates with robust hepatic necroinflammation the role of which remains elusive with pathogenic but also pro-regenerative/-resolving functions being ascribed to leukocyte activation. Here, we shine a light on and put forward a unique role of the interleukin (IL)-1 family member IL-18 in experimental APAP-induced ALI. Indeed, amelioration of disease as previously observed in IL-18-deficient mice was further substantiated herein by application of the IL-18 opponent IL-18-binding protein (IL-18BPd:Fc) to wild-type mice. Data altogether emphasize crucial pathological action of this cytokine in APAP toxicity. Adding recombinant IL-22 to IL-18BPd:Fc further enhanced protection from liver injury. In contrast to IL-18, the role of prototypic pro-inflammatory IL-1 and tumor necrosis factor-α is controversially discussed with lack of effects or even protective action being repeatedly reported. A prominent detrimental function for IL-18 in APAP-induced ALI as proposed herein should relate to its pivotal role for hepatic expression of interferon-γ and Fas ligand, both of which aggravate APAP toxicity. As IL-18 serum levels increase in patients after APAP overdosing, targeting IL-18 may evolve as novel therapeutic option in those hard-to-treat patients where standard therapy with N-acetylcysteine is unsuccessful. Being a paradigmatic experimental model of ALI, current knowledge on ill-fated properties of IL-18 in APAP intoxication likewise emphasizes the potential of this cytokine to serve as therapeutic target in other entities of inflammatory liver diseases.
Background/Aims: Alcohol (ethanol, EtOH) as significant contributor to traumatic injury is linked to suppressed inflammatory response, thereby influencing clinical outcomes. Alcohol-induced immune-suppression during acute inflammation (trauma) was linked to nuclear factor-kappaB (NF-ĸB). Here, we analyzed alcohol`s effects and mechanisms underlying its influence on NF-ĸB-signaling during acute inflammation in human lung epithelial cells. Methods: A549-cells were stimulated with interleukin (IL)-1β, or sera from trauma patients (TP) or healthy volunteers, with positive/negative blood alcohol concentrations (BAC), and subsequently exposed to EtOH (170 Mm, 1h). IL-6-release and neutrophil adhesion to A549 were analyzed. Specific siRNA-NIK mediated downregulation of non-canonical, and IKK-NBD-inhibition of canonical NF-ĸB signaling were performed. Nuclear levels of activated p50 and p52 NF-ĸB-subunits were detected using TransAm ELISA. Results: Both stimuli significantly induced IL-6-release (39.79±4.70 vs. 0.58±0.8 pg/ml) and neutrophil adhesion (132.30±8.80 vs. 100% control, p<0.05) to A549-cells. EtOH significantly decreased IL-6-release (22.90±5.40, p<0.05) and neutrophil adherence vs. controls (105.40±14.5%, p<0.05). IL-1β-induced significant activation of canonical/p50 and non-canonical/p52 pathways. EtOH significantly reduced p50 (34.90±23.70 vs. 197.70±36.43, p<0.05) not p52 activation. Inhibition of canonical pathway was further increased by EtOH (less p50-activation), while p52 remained unaltered. Inhibition of non-canonical pathway was unchanged by EtOH. Conclusion: Here, alcohol`s anti-inflammatory effects are mediated via decreasing nuclear levels of activated p50-subunit and canonical NF-ĸB signaling pathway.
Systemic sclerosis (SSc) is a rare multi-organ autoimmune disease characterized by progressive skin fibrosis. Inflammation, type 2 immunity, and fibrogenic processes are involved in disease development and may be affected by sphingolipids. However, details about early-stage pathophysiological mechanisms and implicated mediators remain elusive. The sphingolipid sphingosine-1-phosphate (S1P) is elevated in the sera of SSc patients, and its receptor S1P5 is expressed in skin tissue. Nevertheless, almost nothing is known about the dermatological contribution of S1P5 to inflammatory and pro-fibrotic processes leading to the pathological changes seen in SSc. In this study, we observed a novel effect of S1P5 on the inflammatory processes during low-dose bleomycin (BLM)-induced fibrogenesis in murine skin. By comparing 2-week-treated skin areas of wild-type (WT) and S1P5-deficient mice, we found that S1P5 is important for the transcriptional upregulation of the Th2 characteristic transcription factor GATA-3 under treatment-induced inflammatory conditions, while T-bet (Th1) and FoxP3 (Treg) mRNA expression was regulated independently of S1P5. Additionally, treatment caused a regulation of S1P receptor 1 and S1P receptor 3 mRNA as well as a regulation of long-chain ceramide profiles, which both differ significantly between the genotypes. Despite S1P5-dependent differences regarding inflammatory processes, similar macroscopic evidence of fibrosis was detected in the skin histology of WT and S1P5-deficient mice after 4 weeks of subcutaneous BLM treatment. However, at the earlier 2-week point in time, the mRNA data of pro-collagen type 1 and SMAD7 indicate a pro-fibrotic S1P5 contribution in the applied SSc mouse model. In conclusion, we propose that S1P5 plays a role as a novel modulator during the early phase of BLM-caused fibrogenesis in murine skin. An immediate relationship between dermal S1P5 expression and fibrotic processes leading to skin alterations, such as formative for SSc pathogenesis, is indicated but should be studied more profound in further investigations. Therefore, this study is an initial step in understanding the role of S1P5-mediated effects during early stages of fibrogenesis, which may encourage the ongoing search for new therapeutic options for SSc patients.
Background: Caloric restriction is associated with broad therapeutic potential in various diseases and an increase in health and life span. In this study, we assessed the impact of caloric restriction on acute and inflammatory nociception in mice, which were either fed ad libitum or subjected to caloric restriction with 80% of the daily average for two weeks.
Results: The behavioral tests revealed that inflammatory nociception in the formalin test and in zymosan-induced mechanical hypersensitivity were significantly decreased when mice underwent caloric restriction. As potential mediators of the diet-induced antinociception, we assessed genes typically induced by inflammatory stimuli, AMP-activated kinase, and the endocannabinoid system which have all already been associated with nociceptive responses. Zymosan-induced inflammatory markers such as COX-2, TNFα, IL-1β, and c-fos in the spinal cord were not altered by caloric restriction. In contrast, AMPKα2 knock-out mice showed significant differences in comparison to C57BL/6 mice and their respective wild type littermates by missing the antinociceptive effects after caloric restriction. Endocannabinoid levels of anandamide and 2-arachidonyl glyceroldetermined in serum by LC-MS/MS were not affected by either caloric restriction alone or in combination with zymosan treatment. However, cannabinoid receptor type 1 expression in the spinal cord, which was not altered by caloric restriction in control mice, was significantly increased after caloric restriction in zymosan-induced paw inflammation. Since increased cannabinoid receptor type 1 signaling might influence AMP-activated kinase activity, we analyzed effects of anandamide on AMP-activated kinase in cell culture and observed a significant activation of AMP-activated kinase. Thus, endocannabionoid-induced AMP-activated kinase activation might be involved in antinociceptive effects after caloric restriction.
Conclusion: Our data suggest that caloric restriction has an impact on inflammatory nociception which might involve AMP-activated kinase activation and an increased activity of the endogenous endocannabinoid system by caloric restriction-induced cannabinoid receptor type 1 upregulation.
The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.
Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.
Chronic inflammation as an important epigenetic and environmental factor for putative tumorigenesis and tumor progression may be associated with specific activation of Toll-like receptors (TLR). Recently, carcinogenesis has been suggested to be dependent on TLR7 signaling. In the present study, we determined the role of both TLR7 and TLR8 expression and signaling in tumor cell proliferation and chemoresistance in pancreatic cancer. Expression of TLR7/TLR8 in UICC stage I-IV pancreatic cancer, chronic pancreatitis, normal pancreatic tissue and human pancreatic (PANC1) cancer cell line was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of (un-)stimulated TLR expression on tumor cell proliferation and chemoresistance. TLR expression was increased in pancreatic cancer, with stage-dependent upregulation in advanced tumors, compared to earlier stages and chronic pancreatitis. Stimulation of TLR7/TLR8 overexpressing PANC1 cells resulted in elevated NF-κB and COX-2 expression, increased cancer cell proliferation and reduced chemosensitivity. More importantly, TLR7/TLR8 expression increased tumor growth in vivo. Our data demonstrate a stage-dependent upregulation of both TLR7 and TLR8 expression in pancreatic cancer. Functional analysis in human pancreatic cancer cells point to a significant role of both TLRs in chronic inflammation-mediated TLR7/TLR8 signaling leading to tumor cell proliferation and chemoresistance.
The processing of pain undergoes several changes in aging that affect sensory nociceptive fibers and the endogenous neuronal inhibitory systems. So far, it is not completely clear whether age-induced modifications are associated with an increase or decrease in pain perception. In this study, we assessed the impact of age on inflammatory nociception in mice and the role of the hormonal inhibitory systems in this context. We investigated the nociceptive behavior of 12-month-old versus 6–8-week-old mice in two behavioral models of inflammatory nociception. Levels of TRP channels, and cortisol as well as cortisol targets, were measured by qPCR, ELISA, and Western blot in the differently aged mice. We observed an age-related reduction in nociceptive behavior during inflammation as well as a higher level of cortisol in the spinal cord of aged mice compared to young mice, while TRP channels were not reduced. Among potential cortisol targets, the NF-κB inhibitor protein alpha (IκBα) was increased, which might contribute to inhibition of NF-κB and a decreased expression and activity of the inducible nitric oxide synthase (iNOS). In conclusion, our results reveal a reduced nociceptive response in aged mice, which might be at least partially mediated by an augmented inflammation-induced increase in the hormonal inhibitory system involving cortisol.
Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.
Hypoxia potentiates palmitate-induced pro-inflammatory activation of primary human macrophages
(2015)
Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.
Altered mucosal immune response after acute lung injury in a murine model of Ataxia Telangiectasia
(2014)
Background: Ataxia telangiectasia (A-T) is a rare but devastating and progressive disorder characterized by cerebellar dysfunction, lymphoreticular malignancies and recurrent sinopulmonary infections. In A-T, disease of the respiratory system causes significant morbidity and is a frequent cause of death.
Methods: We used a self-limited murine model of hydrochloric acid-induced acute lung injury (ALI) to determine the inflammatory answer due to mucosal injury in Atm (A-T mutated)- deficient mice (Atm−/−).
Results: ATM deficiency increased peak lung inflammation as demonstrated by bronchoalveolar lavage fluid (BALF) neutrophils and lymphocytes and increased levels of BALF pro-inflammatory cytokines (e.g. IL-6, TNF). Furthermore, bronchial epithelial damage after ALI was increased in Atm−/− mice. ATM deficiency increased airway resistance and tissue compliance before ALI was performed.
Conclusions: Together, these findings indicate that ATM plays a key role in inflammatory response after airway mucosal injury.
5-Lipoxygenase (5LO) is a key enzyme in biosynthesis of leukotrienes (LTs), lipid mediators of inflammation. To study the roles of the 5LO accessory proteins coactosin-like protein (CLP) and 5LO-activating protein (FLAP), we knocked down these proteins in human monocytic cells. Our results show that expression of CLP was required for full cellular 5LO activity when cells were activated with Ca2+ ionophore, as well as with a physiological stimulus (lipopolysaccharide followed by N-formylmethionyl-leucyl-phenylalanine). During LT biosynthesis in stimulated cells, 5LO typically translocates to the nuclear membrane. This redistribution, from cytosolic to perinuclear, was clearly compromised in both CLP- and FLAP-deficient cells. Our results suggest that the CLP–5LO interaction may be a target for reduced LT production.
Interleukin (IL)-10 and IL-22 are key members of the IL-10 cytokine family that share characteristic properties such as defined structural features, usage of IL-10R2 as one receptor chain, and activation of signal transducer and activator of transcription (STAT)-3 as dominant signaling mode. IL-10, formerly known as cytokine synthesis inhibitory factor, is key to deactivation of monocytes/macrophages and dendritic cells. Accordingly, pre-clinical studies document its anti-inflammatory capacity. However, the outcome of clinical trials assessing the therapeutic potential of IL-10 in prototypic inflammatory disorders has been disappointing. In contrast to IL-10, IL-22 acts primarily on non-leukocytic cells, in particular epithelial cells of intestine, skin, liver, and lung. STAT3-driven proliferation, anti-apoptosis, and anti-microbial tissue protection is regarded a principal function of IL-22 at host/environment interfaces. In this hypothesis article, hidden/underappreciated pro-inflammatory characteristics of IL-10 and IL-22 are outlined and related to cellular priming by type I interferon. It is tempting to speculate that an inherent inflammatory potential of IL-10 and IL-22 confines their usage in tissue protective therapy and beyond that determines in some patients efficacy of type I interferon treatment.
Immunomodulatory properties and molecular effects in inflammatory diseases of low-dose X-irradiation
(2012)
Inflammatory diseases are the result of complex and pathologically unbalanced multicellular interactions. For decades, low-dose X-irradiation therapy (LD-RT) has been clinically documented to exert an anti-inflammatory effect on benign diseases and chronic degenerative disorders. By contrast, experimental studies to confirm the effectiveness and to reveal underlying cellular and molecular mechanisms are still at their early stages. During the last decade, however, the modulation of a multitude of immunological processes by LD-RT has been explored in vitro and in vivo. These include leukocyte/endothelial cell adhesion, adhesion molecule and cytokine/chemokine expression, apoptosis induction, and mononuclear/polymorphonuclear cell metabolism and activity. Interestingly, these mechanisms display comparable dose dependences and dose-effect relationships with a maximum effect in the range between 0.3 and 0.7 Gy, already empirically identified to be most effective in the clinical routine. This review summarizes data and models exploring the mechanisms underlying the immunomodulatory properties of LD-RT that may serve as a prerequisite for further systematic analyses to optimize low-dose irradiation procedures in future clinical practice.
Ubiquitylation in immune disorders and cancer: from molecular mechanisms to therapeutic implications
(2012)
Conjugation of ubiquitin to proteins (ubiquitylation) has emerged to be one of the most crucial post-translational modifications controlling virtually all cellular processes. What was once regarded as a mere signal for protein degradation has turned out to be a major regulator of molecular signalling networks. Deregulation of ubiquitin signalling is closely associated with various human pathologies. Here, we summarize the current knowledge of ubiquitin signalling in immune deficiencies and cancer as well as the available therapeutic strategies targeting the ubiquitin system in combating these pathogenic conditions.
Research over the past few years has provided fascinating results indicating that biglycan, besides being a ubiquitous structural component of the extracellular matrix (ECM), may act as a signaling molecule. Proteolytically released from the ECM, biglycan acts as a danger signal signifying tissue stress or injury. As a ligand of innate immunity receptors and activator of the inflammasome, biglycan stimulates multifunctional proinflammatory signaling linking the innate to the adaptive immune response. By clustering several types of receptors on the cell surface and orchestrating their downstream signaling events, biglycan is capable to autonomously trigger sterile inflammation and to potentiate the inflammatory response to microbial invasion. Besides operating in a broad biological context, biglycan also displays tissue-specific affinities to certain receptors and structural components, thereby playing a crucial role in bone formation, muscle integrity, and synapse stability at the neuromuscular junction. This review attempts to provide a concise summary of recent data regarding the involvement of biglycan in the regulation of inflammation and the musculoskeletal system, pointing out both a signaling and a structural role for this proteoglycan. The potential of biglycan as a novel therapeutic target or agent for the treatment of inflammatory diseases and skeletal muscular dystrophies is also addressed.
Leukotrienes (LTs) are pro-inflammatory lipid mediators that belong to the group of eicosanoids, which are oxygenated metabolites of one common precursor, the aracidonic acid (AA). This polyunsaturated fatty acid is esterified at the sn-2 position of cellular membrane phospholipids and can be released by cytosolic phospholipase A2 alpha (cPLA2alpha) enzymatic deacylation. AA can be converted into LTs by the catalytic reaction of 5-lipoxygenase (5-LO). Enzymatic activation of cPLA2alpha as well as of 5-LO is regulated by similar determinants. In response to cellular stimuli that elevate the intracellular Ca2+ level and/or activate MAP kinase pathways, cPLA2alpha and 5-LO comigrate from a soluble cell compartment (mainly the cytosol) to the nuclear membrane, where AA is released und converted into LTs. LTs play a significant role in promoting inflammatory reactions and immune processes. They have been shown to be released from leukocytes in response to bacterial and viral infections and substantially contribute to an effective immune reaction for host defense. Innate immune pathogen recognition is mediated to a substantial part by the Toll-like receptor (TLR) family. So far, 10 human TLR subtypes have been identified, all of which detect distinct highly conserved microbial structures and trigger the induction of signaling pathways that lead to the expression of numerous immune and inflammatory genes. TLR signaling culminates in the activation NF-kappaB and/or MAP kinases, which as well are known to be involved in the regulation of cellular LT biosynthesis. In this regard, it seemed conceivable that the release of LTs might be regulated by TLR activation. Present studies were undertaken in order to verify and characterize a possible influence of TLR activation on the LT biosynthesis, and furthermore to identify the involved signaling pathways and underlying mechanisms. First experiments revealed that pre-incubation of differentiated Mono Mac 6 (MM6) cells with a TLR4 ligand, a TLR5 ligand, as well as with different TLR2 ligands led to an about 2-fold enhancement of Ca2+ ionophore induced LT biosynthesis. Ligands of other TLR subtypes did not show any influence. These observations could also be confirmed in primary human monocytes stimulated with ionophore or fMLP. With focus on TLR2 ligands, further studies were carried out to characterize the observed enhancement of LT biosynthesis in MM6 cells. It was demonstrated that the extent of LT formation was dependent on the ligand concentration used, but was also dependent on the duration of pre-incubation. Ligand pre-incubation of 15 minutes was optimal to maximally enhance LT formation and further prolongation of pre-incubation decreased LT formation again. Moreover, simultaneous addition of TLR2 ligands with ionophore did also not enhance LT formation. These results indicated that TLR2 ligands seemed to prime human monocytes for an enhanced response upon ionophore stimulation, but did not act as costimuli, which per se were not capable of directly stimulating the biosynthesis of LTs. To analyze the underlying mechanism, the impact of TLR2 ligands on the two key enzymes of the LT biosynthesis pathway, cPLA2alpha and 5-LO, was investigated. In this regard, 5-LO could not been shown to be positively regulated by TLR ligand priming. Neither a direct stimulation, nor an enhancement of 5-LO activity by TLR ligands was detectable in MM6 cells. Similarly, TLR2 ligands did also not enhance ionophore induced 5-LO translocation to the nuclear membrane. However, it was shown that TLR2 ligands enhanced ionophore induced release of AA in MM6 cells, which occurred with a similar time course as LT formation, displaying a maximum at 10 minutes of pre-incubation. A direct stimulation of AA release, however, could not been detected. Inhibitor studies revealed cPLA2alpha to be essential for AA release in TLR2 ligand primed, ionophore stimulated MM6 cells, but also sPLA2 was found to be involved. However, the priming effect of TLR2 ligands was mediated exclusively by cPLA2alpha. Western Blot analyses revealed that p38 MAP kinase, as well as ERK1/2, are activated in MM6 cells in response to TLR2 ligands, and also Ser-505 phosphorylation of cPLA2alpha was detected, which is known to be mediated by MAP kinases and to increase cPLA2alpha activity in vitro. Maximal cPLA2alpha phosphorylation occurred after 5-10 minutes of TLR2 ligand incubation, slightly preceding maximal AA release at 10 minutes and maximal LT formation at 15 minutes of priming. The combined use of a specific p38 MAPK inhibitor with an inhibitor of the ERK1/2 signaling pathway resulted in a complete prevention of cPLA2alpha phosphorylation and TLR2 ligand mediated enhancement of AA release. Thus, both MAPK pathways seem to play a role for TLR2 ligand mediated priming effects on the release of AA. An impact of other kinases such as Mnk-1 and CamKII, which can also regulate cPLA2alpha by phosphorylation, was excluded. Finally, an anti-hTLR2 antibody significantly reduced enhanced AA release, confirming the priming effects to be dependent on TLR2 activation. In summary, it was concluded that the increase of LT biosynthesis by TLR2 ligand priming is considerably due to an enhanced cellular AA supply, which arises from a MAPK mediated phosphorylation and up-regulation of cPLA2alpha. TLR dependent enhancement of LT biosynthesis represents an interesting link between activation of innate immune receptors and the rapid formation of pro-inflammatory lipid mediators. On the one hand, this support the role of LTs in host defence and infectious diseases, but may also be relevant in pathophysiological processes, which involve TLRs as well as LTs, as it has been shown for the pathogenesis of atherosclerosis or allergic diseases.
Wassergefiltertes Infrarot A (wIRA) ist eine spezielle Form der Wärmestrahlung mit hohem Penetrationsvermögen ins Gewebe bei geringer thermischer Oberflächenbelastung. wIRA entspricht dem Großteil der Sonnenwärmestrahlung, die in gemäßigten Klimazonen die Erdoberfläche wasserdampfgefiltert erreicht. wIRA steigert die drei energetisch für die Wundheilung wichtigen Faktoren Temperatur, Sauerstoffpartialdruck und Durchblutung im Gewebe. wIRA mindert Schmerzen, Entzündung und Wundsekretion. Entsprechend kann wIRA sehr gut zur Verbesserung der Wundheilung bei akuten und chronischen Wunden eingesetzt werden.
Water-filtered infrared-A (wIRA), as a special form of heat radiation with a high tissue penetration and a low thermal load to the skin surface, can improve the healing of acute and chronic wounds both by thermal and thermic as well as by non-thermal and non-thermic effects. wIRA increases tissue temperature (+2.7°C at a tissue depth of 2 cm), tissue oxygen partial pressure (+32% at a tissue depth of 2 cm) and tissue perfusion. These three factors are decisive for a sufficient supply of tissue with energy and oxygen and consequently also for wound healing and infection defense.
wIRA can considerably alleviate pain (without any exception during 230 irradiations) with substantially less need for analgesics (52–69% less in the groups with wIRA compared to the control groups). It also diminishes exudation and inflammation and can show positive immunomodulatory effects. The overall evaluation of the effect of irradiation as well as the wound healing and the cosmetic result (assessed on visual analogue scales) were markedly better in the group with wIRA compared to the control group. wIRA can advance wound healing (median reduction of wound size of 90% in severely burned children already after 9 days in the group with wIRA compared to 13 days in the control group; on average 18 versus 42 days until complete wound closure in chronic venous stasis ulcers) or improve an impaired wound healing (reaching wound closure and normalization of the thermographic image in otherwise recalcitrant chronic venous stasis ulcers) both in acute and in chronic wounds including infected wounds. After major abdominal surgery there was a trend in favor of the wIRA group to a lower rate of total wound infections (7% versus 15%) including late infections following discharge from hospital (0% versus 8%) and a trend towards a shorter postoperative hospital stay (9 versus 11 days).
Even the normal wound healing process can be improved.
The mentioned effects have been proven in six prospective studies, with most of the effects having an evidence level of Ia/Ib.
wIRA represents a valuable therapy option and can generally be recommended for use in the treatment of acute as well as of chronic wounds.
End-stage renal disease has been denominated a vasculopathic state, owing to the accelerated arterial stiffening, which occurs in addition to and independent of atherosclerosis and bears an increased cardiovascular risk. The altered metabolic milieu in uraemia leads to an increased oxidative stress, heightened inflammatory burden, and an abnormal calcium-phosphate metabolism, which are thought to be responsible for the vascular changes. The pulse wave velocity (PWV) is a widely employed surrogate parameter of arteriosclerosis. The purpose of this study was to gain more insight into the pathogenesis of arterial stiffness, by investigating the influence of markers of oxidative stress, procoagulation, and inflammation, and of the calcium-phosphate product on the PWV. We conducted a cross-sectional study in 53 stable patients aged 59 ± 16 years, who had been on haemodialysis for at least 4 months (68 ± 48). Carotid-radial PWV was measured using a semi-automated device, Complior SP (Artech Medical, France). Advanced glycosylation end-products (AGE) and advanced oxidation protein products (AOPP), were quantified according to previously described methods. High sensitive CRP was measured using ELISA, whereas the other biochemical parameters, i.e. fibrinogen, albumin, calcium, phosphate, cholesterol, and triglycerides, were determined using routine methods. For statistical calculations we employed SPSS (Statistical Package of Social Science, 12.0, 2003). The correlations between PWV, as the dependent variable, and many dependent variables were assessed by means of multiple regression analysis, in which we controlled for the influence of the traditional cardiovascular risk factors and some of the patients’ medication (calcium-channel blockers and statins). PWV was found to be significantly correlated to serum CRP (p=0.003), LDLcholesterol (p<0.001), triglycerides (p<0.001), AGE (p=0.002), calcium (p<0.001), phosphate (p=0.001), and fibrinogen (p=0.020). Between PWV and dialysis duration (months) an interesting quadratic relationship (p=0.058) was noted. Against expectation, regression analysis showed a negative correlation between AOPP and PWV (p=0.001). We failed to confirm the correlation between PWV and age, systolic blood pressure, or heart rate. Among traditional cardiovascular risk factors only LDL-cholesterol was positively correlated to PWV. In this cross-sectional analysis we could put forward that PWV correlates positively and significantly with fibrinogen, CRP, AGEs, calcium, phosphate, and LDL-cholesterol in haemodialysis patients. It seems procoagulatory and proinflammatory pathways, oxidative stress, and the calcium-phosphate product exert a synergistic effect on disturbances of vascular architecture in ESRD patients.
The central portion of chronic wounds is often hypoxic and relatively hypothermic, representing a deficient energy supply of the tissue, which impedes wound healing or even makes it impossible. Water-filtered infrared-A (wIRA) is a special form of heat radiation with a high tissue penetration and a low thermal load to the skin surface. wIRA produces a therapeutically usable field of heat and increases temperature, oxygen partial pressure and perfusion of the tissue. These three factors are decisive for a sufficient tissue supply with energy and oxygen and consequently as well for wound healing, especially in chronic wounds, and infection defense. wIRA acts both by thermal and thermic as well as by non-thermal and non-thermic effects. wIRA can advance wound healing or improve an impaired wound healing process and can especially enable wound healing in non-healing chronic wounds. wIRA can considerably alleviate the pain and diminish wound exudation and inflammation and can show positive immunomodulatory effects.
In a prospective, randomized, controlled study of 40 patients with chronic venous stasis ulcers of the lower legs irradiation with wIRA and visible light (VIS) accelerated the wound healing process (on average 18 vs. 42 days until complete wound closure, residual ulcer area after 42 days 0.4 cm² vs. 2.8 cm²) and led to a reduction of the required dose of pain medication in comparison to the control group of patients treated with the same standard care (wound cleansing, wound dressing with antibacterial gauze, and compression garment therapy) without the concomitant irradiation.
Another prospective study of 10 patients with non-healing chronic venous stasis ulcers of the lower legs included extensive thermographic investigation. Therapy with wIRA(+VIS) resulted in a complete or almost complete wound healing in 7 patients and a marked reduction of the ulcer size in another 2 of the 10 patients, a clear reduction of pain and required dose of pain medication, and a normalization of the thermographic image.
In a current prospective, randomized, controlled, blinded study patients with non-healing chronic venous stasis ulcers of the lower legs are treated with compression garment therapy, wound cleansing, wound dressings and 30 minutes irradiation five times per week over 9 weeks. A preliminary analysis of the first 23 patients of this study has shown in the group with wIRA(+VIS) compared to a control group with VIS an advanced wound healing, an improved granulation and in the later phase of treatment a decrease of the bacterial burden.
Some case reports have demonstrated that wIRA can also be used for mixed arterial-venous ulcers or arterial ulcers, if irradiation intensity is chosen appropriately low and if irradiation is monitored carefully. wIRA can be used concerning decubital ulcers both in a preventive and in a therapeutic indication. wIRA can improve the resorption of topically applied substances also on wounds.
An irradiation with VIS and wIRA presumably acts with endogenous protoporphyrin IX (or protoporphyrin IX of bacteria) virtually similar as a mild photodynamic therapy (endogenous PDT-like effect). This could lead to improved cell regeneration and wound healing and to antibacterial effects.
In conclusion, these results indicate that wIRA generally should be considered for the treatment of chronic wounds.
Water-filtered infrared-A (wIRA) as a special form of heat radiation with a high tissue penetration and with a low thermal load to the skin surface acts both by thermal and thermic as well as by non-thermal and non-thermic effects. wIRA produces a therapeutically usable field of heat in the tissue and increases tissue temperature, tissue oxygen partial pressure, and tissue perfusion. These three factors are decisive for a sufficient tissue supply with energy and oxygen and consequently as well for wound healing and infection defense.
wIRA can considerably alleviate the pain (with remarkably less need for analgesics) and diminish an elevated wound exudation and inflammation and can show positive immunomodulatory effects. wIRA can advance wound healing or improve an impaired wound healing both in acute and in chronic wounds including infected wounds. Even the normal wound healing process can be improved.
A prospective, randomized, controlled, double-blind study with 111 patients after major abdominal surgery at the University Hospital Heidelberg, Germany, showed with 20 minutes irradiation twice a day (starting on the second postoperative day) in the group with wIRA and visible light VIS (wIRA(+VIS), approximately 75% wIRA, 25% VIS) compared to a control group with only VIS a significant and relevant pain reduction combined with a markedly decreased required dose of analgesics: during 230 single irradiations with wIRA(+VIS) the pain decreased without any exception (median of decrease of pain on postoperative days 2-6 was 13.4 on a 100 mm visual analog scale VAS 0-100), while pain remained unchanged in the control group (p<0.001). The required dose of analgesics was 57-70% lower in the subgroups with wIRA(+VIS) compared to the control subgroups with only VIS (median 598 versus 1398 ml ropivacaine, p<0.001, for peridural catheter analgesia; 31 versus 102 mg piritramide, p=0.001, for patient-controlled analgesia; 3.4 versus 10.2 g metamizole, p=0.005, for intravenous and oral analgesia). During irradiation with wIRA(+VIS) the subcutaneous oxygen partial pressure rose markedly by approximately 30% and the subcutaneous temperature by approximately 2.7°C (both in a tissue depth of 2 cm), whereas both remained unchanged in the control group: after irradiation the median of the subcutaneous oxygen partial pressure was 41.6 (with wIRA) versus 30.2 mm Hg in the control group (p<0.001), the median of the subcutaneous temperature was 38.9 versus 36.4°C (p<0.001). The overall evaluation of the effect of irradiation, including wound healing, pain and cosmesis, assessed on a VAS (0-100 with 50 as indifferent point of no effect) by the surgeon (median 79.0 versus 46.8, p<0.001) or the patient (79.0 versus 50.2, p<0.001) was markedly better in the group with wIRA compared to the control group. This was also true for single aspects: Wound healing assessed on a VAS by the surgeon (median 88.6 versus 78.5, p<0.001) or the patient (median 85.8 versus 81.0, p=0.040, trend) and cosmetic result assessed on a VAS by the surgeon (median 84.5 versus 76.5, p<0.001) or the patient (median 86.7 versus 73.6, p=0.001). In addition there was a trend in favor of the wIRA group to a lower rate of total wound infections (3 of 46, approximately 7%, versus 7 of 48, approximately 15%, p=0.208) including late infections after discharge, caused by the different rate of late infections after discharge: 0 of 46 in the wIRA group and 4 of 48 in the control group. And there was a trend towards a shorter postoperative hospital stay: 9 days in the wIRA group versus 11 days in the control group (p=0.037). The principal finding of this study was that postoperative irradiation with wIRA can improve even a normal wound healing process.
A prospective, randomized, controlled, double-blind study with 45 severely burned children at the Children’s Hospital Park Schönfeld, Kassel, Germany, showed with 30 minutes irradiation once a day (starting on the first day, day of burn as day 1) in the group with wIRA and visible light VIS (wIRA(+VIS), approximately 75% wIRA, 25% VIS) compared to a control group with only VIS a markedly faster reduction of wound size. On the fifth day (after 4 days with irradiation) decision was taken, whether surgical debridement of necrotic tissue was necessary because of deeper (second degree, type b) burns (11 of 21 in the group with wIRA, 14 of 24 in the control group) or non-surgical treatment was possible (second degree, type a, burns). The patients treated conservatively were kept within the study and irradiated till complete reepithelialization. The patients in the group with wIRA showed a markedly faster reduction of wound area: a median reduction of wound size of 50% was reached already after 7 days compared to 9 days in the control group, a median reduction of wound size of 90% was already achieved after 9 days compared to 13 days in the control group. In addition the group with wIRA showed superior results till 3 months after the burn in terms of the overall surgical assessment of the wound, cosmesis, and assessment of effects of irradiation compared to the control group.
In a prospective, randomized, controlled study with 12 volunteers at the University Medical Center Charité, Berlin, Germany, within each volunteer 4 experimental superficial wounds (5 mm diameter) as an acute wound model were generated by suction cup technique, removing the roof of the blister with a scalpel and a sterile forceps (day 1). 4 different treatments were used and investigated during 10 days: no therapy, only wIRA(+VIS) (approximately 75% wIRA, 25% VIS; 30 minutes irradiation once a day), only dexpanthenol (= D-panthenol) cream once a day, wIRA(+VIS) and dexpanthenol cream once a day. Healing of the small experimental wounds was from a clinical point of view excellent with all 4 treatments. Therefore there were only small differences between the treatments with slight advantages of the combination wIRA(+VIS) and dexpanthenol cream and of dexpanthenol cream alone concerning relative change of wound size and assessment of feeling of the wound area. However laser scanning microscopy with a scoring system revealed differences between the 4 treatments concerning the formation of the stratum corneum (from first layer of corneocytes to full formation) especially on the days 5-7: fastest formation of the stratum corneum was seen in wounds treated with wIRA(+VIS) and dexpanthenol cream, second was wIRA(+VIS) alone, third dexpanthenol cream alone and last were untreated wounds. Bacterial counts of the wounds (taken every 2 days) showed, that wIRA(+VIS) and the combination of wIRA(+VIS) with dexpanthenol cream were able to inhibit the colonisation with physiological skin flora up to day 5 when compared with the two other groups (untreated group and group with dexpanthenol cream alone). At any investigated time, the amount of colonisation under therapy with wIRA(+VIS) alone was lower (interpreted as more suppressed) compared with the group with wIRA(+VIS) and dexpanthenol cream.
During rehabilitation after hip and knee endoprosthetic operations the resorption of wound seromas and wound hematomas was both clinically and sonographically faster and pain was reduced by irradiation with wIRA(+VIS).
wIRA can be used successfully for persistent postoperative pain e.g. after thoracotomy.
As perspectives for wIRA it seems clinically prudent to use wIRA both pre- and postoperatively, e.g. in abdominal and thoracic operations. wIRA can be used preoperatively (e.g. during 1-2 weeks) to precondition donor and recipient sites of skin flaps, transplants or partial-thickness skin grafts, and postoperatively to improve wound healing and to decrease pain, inflammation and infections at all mentioned sites. wIRA can be used to support routine pre- or intraoperative antibiotic administration or it might even be discussed to replace this under certain conditions by wIRA.
Principles and working mechanisms of water-filtered infrared-A (wIRA) in relation to wound healing
(2007)
The experience of the pleasant heat of the sun in moderate climatic zones arises from the filtering of the heat radiation of the sun by water vapor in the atmosphere of the earth. The filter effect of water decreases those parts of infrared radiation (most parts of infrared-B and -C and the absorption bands of water within infrared-A), which would cause – by reacting with water molecules in the skin – only an undesired thermal load to the surface of the skin. Technically water-filtered infrared-A (wIRA) is produced in special radiators, whose full spectrum of radiation of a halogen bulb is passed through a cuvette, containing water, which absorbs or decreases the described undesired wavelengths of the infrared radiation. Within infrared the remaining wIRA (within 780-1400 nm) mainly consists of radiation with good penetration properties into tissue and therefore allows – compared to unfiltered heat radiation – a multiple energy transfer into tissue without irritating the skin, similar to the sun’s heat radiation in moderate climatic zones. Typical wIRA radiators emit no ultraviolet (UV) radiation and nearly no infrared-B and -C radiation and the amount of infrared-A radiation in relation to the amount of visible light (380-780 nm) is emphasized.
Water-filtered infrared-A as a special form of heat radiation with a high tissue penetration and with a low thermal load to the skin surface acts both by thermal (related to heat energy transfer) and thermic (temperature depending, with a relevant change of temperature) as well as by non-thermal (without a relevant transfer of heat energy) and non-thermic (not depending on temperature, without a relevant change of temperature) effects. wIRA produces a therapeutically usable field of heat in the tissue and increases tissue temperature, tissue oxygen partial pressure, and tissue perfusion. These three factors are vital for a sufficient tissue supply with energy and oxygen. As wound healing and infection defense (e.g. granulocyte function including their antibacterial oxygen radical formation) depend decisively on a sufficient supply with energy and oxygen, one explanation for the good clinical effect of wIRA on wounds and wound infections can be the improvement of both the energy supply per time (increase of metabolic rate) and the oxygen supply. In addition wIRA has non-thermal and non-thermic effects, which are based on putting direct stimuli on cells and cellular structures.
wIRA can considerably alleviate the pain (with remarkably less need for analgesics) and diminish an elevated wound exudation and inflammation and can show positive immunomodulatory effects. wIRA can advance wound healing or improve an impaired wound healing both in acute and in chronic wounds including infected wounds. Even the normal wound healing process can be improved.
Keywords: water-filtered infrared-A (wIRA), infrared-A radiation, wound healing, thermal and non-thermal effects, thermic and non-thermic effects, energy supply, oxygen supply, tissue oxygen partial pressure, tissue temperature, tissue blood flow, reduction of pain, wound exudation, inflammation, immunomodulatory effects, acute wounds, chronic venous stasis ulcers of the lower legs, problem wounds, wound infections, infection defense, contact-free method, absent expenditure of material, quality of life, prospective, randomized, controlled, double-blind studies
Water-filtered infrared-A (wIRA) as a special form of heat radiation with a high tissue penetration and with a low thermal load to the skin surface acts both by thermal and thermic as well as by non-thermal and non-thermic effects. wIRA produces a therapeutically usable field of heat in the tissue and increases tissue temperature, tissue oxygen partial pressure, and tissue perfusion. These three factors are decisive for a sufficient tissue supply with energy and oxygen and consequently as well for wound healing and infection defense.
wIRA can considerably alleviate the pain (with remarkably less need for analgesics) and diminish an elevated wound exudation and inflammation and can show positive immunomodulatory effects. wIRA can advance wound healing or improve an impaired wound healing both in acute and in chronic wounds including infected wounds. Even the normal wound healing process can be improved.
A prospective, randomized, controlled, double-blind study with 111 patients after major abdominal surgery at the University Hospital Heidelberg, Germany, showed with 20 minutes irradiation twice a day (starting on the second postoperative day) in the group with wIRA and visible light VIS (wIRA(+VIS), approximately 75% wIRA, 25% VIS) compared to a control group with only VIS a significant and relevant pain reduction combined with a markedly decreased required dose of analgesics: during 230 single irradiations with wIRA(+VIS) the pain decreased without any exception (median of decrease of pain on postoperative days 2-6 was 13.4 on a 100 mm visual analog scale VAS 0-100), while pain remained unchanged in the control group (p<0.001). The required dose of analgesics was 57-70% lower in the subgroups with wIRA(+VIS) compared to the control subgroups with only VIS (median 598 versus 1398 ml ropivacaine, p<0.001, for peridural catheter analgesia; 31 versus 102 mg piritramide, p=0.001, for patient-controlled analgesia; 3.4 versus 10.2 g metamizole, p=0.005, for intravenous and oral analgesia). During irradiation with wIRA(+VIS) the subcutaneous oxygen partial pressure rose markedly by approximately 30% and the subcutaneous temperature by approximately 2.7°C (both in a tissue depth of 2 cm), whereas both remained unchanged in the control group: after irradiation the median of the subcutaneous oxygen partial pressure was 41.6 (with wIRA) versus 30.2 mm Hg in the control group (p<0.001), the median of the subcutaneous temperature was 38.9 versus 36.4°C (p<0.001). The overall evaluation of the effect of irradiation, including wound healing, pain and cosmesis, assessed on a VAS (0-100 with 50 as indifferent point of no effect) by the surgeon (median 79.0 versus 46.8, p<0.001) or the patient (79.0 versus 50.2, p<0.001) was markedly better in the group with wIRA compared to the control group. This was also true for single aspects: Wound healing assessed on a VAS by the surgeon (median 88.6 versus 78.5, p<0.001) or the patient (median 85.8 versus 81.0, p=0.040, trend) and cosmetic result assessed on a VAS by the surgeon (median 84.5 versus 76.5, p<0.001) or the patient (median 86.7 versus 73.6, p=0.001). In addition there was a trend in favor of the wIRA group to a lower rate of total wound infections (3 of 46, approximately 7%, versus 7 of 48, approximately 15%, p=0.208) including late infections after discharge, caused by the different rate of late infections after discharge: 0 of 46 in the wIRA group and 4 of 48 in the control group. And there was a trend towards a shorter postoperative hospital stay: 9 days in the wIRA group versus 11 days in the control group (p=0.037). The principal finding of this study was that postoperative irradiation with wIRA can improve even a normal wound healing process.
A prospective, randomized, controlled, double-blind study with 45 severely burned children at the Children’s Hospital Park Schönfeld, Kassel, Germany, showed with 30 minutes irradiation once a day (starting on the first day, day of burn as day 1) in the group with wIRA and visible light VIS (wIRA(+VIS), approximately 75% wIRA, 25% VIS) compared to a control group with only VIS a markedly faster reduction of wound size. On the fifth day (after 4 days with irradiation) decision was taken, whether surgical debridement of necrotic tissue was necessary because of deeper (second degree, type b) burns (11 of 21 in the group with wIRA, 14 of 24 in the control group) or non-surgical treatment was possible (second degree, type a, burns). The patients treated conservatively were kept within the study and irradiated till complete reepithelialization. The patients in the group with wIRA showed a markedly faster reduction of wound area: a median reduction of wound size of 50% was reached already after 7 days compared to 9 days in the control group, a median reduction of wound size of 90% was already achieved after 9 days compared to 13 days in the control group. In addition the group with wIRA showed superior results till 3 months after the burn in terms of the overall surgical assessment of the wound, cosmesis, and assessment of effects of irradiation compared to the control group.
In a prospective, randomized, controlled study with 12 volunteers at the University Medical Center Charité, Berlin, Germany, within each volunteer 4 experimental superficial wounds (5 mm diameter) as an acute wound model were generated by suction cup technique, removing the roof of the blister with a scalpel and a sterile forceps (day 1). 4 different treatments were used and investigated during 10 days: no therapy, only wIRA(+VIS) (approximately 75% wIRA, 25% VIS; 30 minutes irradiation once a day), only dexpanthenol (= D-panthenol) cream once a day, wIRA(+VIS) and dexpanthenol cream once a day. Healing of the small experimental wounds was from a clinical point of view excellent with all 4 treatments. Therefore there were only small differences between the treatments with slight advantages of the combination wIRA(+VIS) and dexpanthenol cream and of dexpanthenol cream alone concerning relative change of wound size and assessment of feeling of the wound area. However laser scanning microscopy with a scoring system revealed differences between the 4 treatments concerning the formation of the stratum corneum (from first layer of corneocytes to full formation) especially on the days 5-7: fastest formation of the stratum corneum was seen in wounds treated with wIRA(+VIS) and dexpanthenol cream, second was wIRA(+VIS) alone, third dexpanthenol cream alone and last were untreated wounds. Bacterial counts of the wounds (taken every 2 days) showed, that wIRA(+VIS) and the combination of wIRA(+VIS) with dexpanthenol cream were able to inhibit the colonisation with physiological skin flora up to day 5 when compared with the two other groups (untreated group and group with dexpanthenol cream alone). At any investigated time, the amount of colonisation under therapy with wIRA(+VIS) alone was lower (interpreted as more suppressed) compared with the group with wIRA(+VIS) and dexpanthenol cream.
During rehabilitation after hip and knee endoprosthetic operations the resorption of wound seromas and wound hematomas was both clinically and sonographically faster and pain was reduced by irradiation with wIRA(+VIS).
wIRA can be used successfully for persistent postoperative pain e.g. after thoracotomy.
As perspectives for wIRA it seems clinically prudent to use wIRA both pre- and postoperatively, e.g. in abdominal and thoracic operations. wIRA can be used preoperatively (e.g. during 1-2 weeks) to precondition donor and recipient sites of skin flaps, transplants or partial-thickness skin grafts, and postoperatively to improve wound healing and to decrease pain, inflammation and infections at all mentioned sites. wIRA can be used to support routine pre- or intraoperative antibiotic administration or it might even be discussed to replace this under certain conditions by wIRA.
Principles and working mechanisms of water-filtered infrared-A (wIRA) in relation to wound healing
(2007)
The experience of the pleasant heat of the sun in moderate climatic zones arises from the filtering of the heat radiation of the sun by water vapor in the atmosphere of the earth. The filter effect of water decreases those parts of infrared radiation (most parts of infrared-B and -C and the absorption bands of water within infrared-A), which would cause – by reacting with water molecules in the skin – only an undesired thermal load to the surface of the skin. Technically water-filtered infrared-A (wIRA) is produced in special radiators, whose full spectrum of radiation of a halogen bulb is passed through a cuvette, containing water, which absorbs or decreases the described undesired wavelengths of the infrared radiation. Within infrared the remaining wIRA (within 780-1400 nm) mainly consists of radiation with good penetration properties into tissue and therefore allows – compared to unfiltered heat radiation – a multiple energy transfer into tissue without irritating the skin, similar to the sun’s heat radiation in moderate climatic zones. Typical wIRA radiators emit no ultraviolet (UV) radiation and nearly no infrared-B and -C radiation and the amount of infrared-A radiation in relation to the amount of visible light (380-780 nm) is emphasized. Water-filtered infrared-A as a special form of heat radiation with a high tissue penetration and with a low thermal load to the skin surface acts both by thermal (related to heat energy transfer) and thermic (temperature depending, with a relevant change of temperature) as well as by non-thermal (without a relevant transfer of heat energy) and non-thermic (not depending on temperature, without a relevant change of temperature) effects. wIRA produces a therapeutically usable field of heat in the tissue and increases tissue temperature, tissue oxygen partial pressure, and tissue perfusion. These three factors are vital for a sufficient tissue supply with energy and oxygen. As wound healing and infection defense (e.g. granulocyte function including their antibacterial oxygen radical formation) depend decisively on a sufficient supply with energy and oxygen, one explanation for the good clinical effect of wIRA on wounds and wound infections can be the improvement of both the energy supply per time (increase of metabolic rate) and the oxygen supply. In addition wIRA has non-thermal and non-thermic effects, which are based on putting direct stimuli on cells and cellular structures. wIRA can considerably alleviate the pain (with remarkably less need for analgesics) and diminish an elevated wound exudation and inflammation and can show positive immunomodulatory effects. wIRA can advance wound healing or improve an impaired wound healing both in acute and in chronic wounds including infected wounds. Even the normal wound healing process can be improved. wIRA is contact-free, easily applied, without discomfort to the patient, with absent consumption of material and with a good effect in the depth. The irradiation of the typically uncovered wound is carried out with a wIRA radiator.