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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.
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.
Based on increasing evidence suggesting that MS pathology involves alterations in bioactive lipid metabolism, the present analysis was aimed at generating a complex serum lipid-biomarker. Using unsupervised machine-learning, implemented as emergent self-organizing maps of neuronal networks, swarm intelligence and Minimum Curvilinear Embedding, a cluster structure was found in the input data space comprising serum concentrations of d = 43 different lipid-markers of various classes. The structure coincided largely with the clinical diagnosis, indicating that the data provide a basis for the creation of a biomarker (classifier). This was subsequently assessed using supervised machine-learning, implemented as random forests and computed ABC analysis-based feature selection. Bayesian statistics-based biomarker creation was used to map the diagnostic classes of either MS patients (n = 102) or healthy subjects (n = 301). Eight lipid-markers passed the feature selection and comprised GluCerC16, LPA20:4, HETE15S, LacCerC24:1, C16Sphinganine, biopterin and the endocannabinoids PEA and OEA. A complex classifier or biomarker was developed that predicted MS at a sensitivity, specificity and accuracy of approximately 95% in training and test data sets, respectively. The present successful application of serum lipid marker concentrations to MS data is encouraging for further efforts to establish an MS biomarker based on serum lipidomics.
Genetic association studies have shown their usefulness in assessing the role of ion channels in human thermal pain perception. We used machine learning to construct a complex phenotype from pain thresholds to thermal stimuli and associate it with the genetic information derived from the next-generation sequencing (NGS) of 15 ion channel genes which are involved in thermal perception, including ASIC1, ASIC2, ASIC3, ASIC4, TRPA1, TRPC1, TRPM2, TRPM3, TRPM4, TRPM5, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4. Phenotypic information was complete in 82 subjects and NGS genotypes were available in 67 subjects. A network of artificial neurons, implemented as emergent self-organizing maps, discovered two clusters characterized by high or low pain thresholds for heat and cold pain. A total of 1071 variants were discovered in the 15 ion channel genes. After feature selection, 80 genetic variants were retained for an association analysis based on machine learning. The measured performance of machine learning-mediated phenotype assignment based on this genetic information resulted in an area under the receiver operating characteristic curve of 77.2%, justifying a phenotype classification based on the genetic information. A further item categorization finally resulted in 38 genetic variants that contributed most to the phenotype assignment. Most of them (10) belonged to the TRPV3 gene, followed by TRPM3 (6). Therefore, the analysis successfully identified the particular importance of TRPV3 and TRPM3 for an average pain phenotype defined by the sensitivity to moderate thermal stimuli.
Loss of HIF-1α in macrophages attenuates AhR/ARNT-mediated tumorigenesis in a PAH-driven tumor model
(2016)
Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.
BACKGROUND: The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).
RESULTS: 2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.
CONCLUSION: Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.
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.
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.
Epoxyeicotrienoic acids (EETs) are cytochrome P450-dependent anti-hypertensive and anti-inflammatory derivatives of arachidonic acid, which are highly abundant in the kidney and considered reno-protective. EETs are degraded by the enzyme soluble epoxide hydrolase (sEH) and sEH inhibitors are considered treatment for chronic renal failure (CRF). We determined whether sEH inhibition attenuates the progression of CRF in the 5/6-nephrectomy model (5/6-Nx) in mice. 5/6-Nx mice were treated with a placebo, an ACE-inhibitor (Ramipril, 40 mg/kg), the sEH-inhibitor cAUCB or the CYP-inhibitor fenbendazole for 8 weeks. 5/6-Nx induced hypertension, albuminuria, glomerulosclerosis and tubulo-interstitial damage and these effects were attenuated by Ramipril. In contrast, cAUCB failed to lower the blood pressure and albuminuria was more severe as compared to placebo. Plasma EET-levels were doubled in 5/6 Nx-mice as compared to sham mice receiving placebo. Renal sEH expression was attenuated in 5/6-Nx mice but cAUCB in these animals still further increased the EET-level. cAUCB also increased 5-HETE and 15-HETE, which derive from peroxidation or lipoxygenases. Similar to cAUCB, CYP450 inhibition increased HETEs and promoted albuminuria. Thus, sEH-inhibition failed to elicit protective effects in the 5/6-Nx model and showed a tendency to aggravate the disease. These effects might be consequence of a shift of arachidonic acid metabolism into the lipoxygenase pathway.
Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E2 (PGE2), are well-characterized mediators of inflammation. PGE2 is produced in an inducible manner in macrophages (Mϕ) by microsomal PGE2-synthase-1 (mPGES-1), with the notion that it also conveys pro-resolving properties. We aimed to characterize the role of mPGES-1 during resolution of acute, zymosan-induced peritonitis. Experimentally, we applied the mPGES-1 inhibitor compound III (CIII) once the inflammatory response was established and confirmed its potent PGE2-blocking efficacy. mPGES-1 inhibition resulted in an incomplete removal of neutrophils and a concomitant increase in monocytes and Mϕ during the resolution process. The mRNA-seq analysis identified enhanced C-X3-C motif receptor 1 (CX3CR1) expression in resident and infiltrating Mϕ upon mPGES-1 inhibition. Besides elevated Cx3cr1 expression, its ligand CX3CL1 was enriched in the peritoneal lavage of the mice, produced by epithelial cells upon mPGES-1 inhibition. CX3CL1 not only increased adhesion and survival of Mϕ but its neutralization also completely reversed elevated inflammatory cell numbers, thereby normalizing the cellular, peritoneal composition during resolution. Our data suggest that mPGES-1-derived PGE2 contributes to the resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.
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.
Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.
Progranulin deficiency in mice is associated with deregulations of the scavenger receptor signaling of CD36/SCARB3 in immune disease models, and CD36 is a dominant receptor in taste bud cells in the tongue and contributes to the sensation of dietary fats. Progranulin-deficient mice (Grn−/−) are moderately overweight during middle age. We therefore asked if there was a connection between progranulin/CD36 in the tongue and fat taste preferences. By using unbiased behavioral analyses in IntelliCages and Phenomaster cages we showed that progranulin-deficient mice (Grn−/−) developed a strong preference of fat taste in the form of 2% milk over 0.3% milk, and for diluted MCTs versus tap water. The fat preference in the 7d-IntelliCage observation period caused an increase of 10% in the body weight of Grn−/− mice, which did not occur in the wildtype controls. CD36 expression in taste buds was reduced in Grn−/− mice at RNA and histology levels. There were no differences in the plasma or tongue lipids of various classes including sphingolipids, ceramides and endocannabinoids. The data suggest that progranulin deficiency leads to a lower expression of CD36 in the tongue resulting in a stronger urge for fatty taste and fatty nutrition.
The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA4 on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B4 and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA4 (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B4 was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA4. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.
Background: Hyperhomocysteinemia is considered a possible contributor to the complex pathology of Alzheimer’s disease (AD). For years, researchers in this field have discussed the apparent detrimental effects of the endogenous amino acid homocysteine in the brain. In this study, the roles of hyperhomocysteinemia driven by vitamin B deficiency, as well as potentially beneficial dietary interventions, were investigated in the novel AppNL-G-F knock-in mouse model for AD, simulating an early stage of the disease. Methods: Urine and serum samples were analyzed using a validated LC-MS/MS method and the impact of different experimental diets on cognitive performance was studied in a comprehensive behavioral test battery. Finally, we analyzed brain samples immunohistochemically in order to assess amyloid-β (Aβ) plaque deposition. Results: Behavioral testing data indicated subtle cognitive deficits in AppNL-G-F compared to C57BL/6J wild type mice. Elevation of homocysteine and homocysteic acid, as well as counteracting dietary interventions, mostly did not result in significant effects on learning and memory performance, nor in a modified Aβ plaque deposition in 35-week-old AppNL-G-F mice. Conclusion: Despite prominent Aβ plaque deposition, the AppNL-G-F model merely displays a very mild AD-like phenotype at the investigated age. Older AppNL-G-F mice should be tested in order to further investigate potential effects of hyperhomocysteinemia and dietary interventions.
DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50–80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.
High glucosylceramides and low anandamide contribute to sensory loss and pain in Parkinson's disease
(2020)
Background: Parkinson's disease (PD) causes chronic pain in two‐thirds of patients, in part originating from sensory neuropathies. The aim of the present study was to describe the phenotype of PD‐associated sensory neuropathy and to evaluate its associations with lipid allostasis, the latter motivated by recent genetic studies associating mutations of glucocerebrosidase with PD onset and severity. Glucocerebrosidase catalyzes the metabolism of glucosylceramides.
Methods: We used quantitative sensory tests, pain ratings, and questionnaires and analyzed plasma levels of multiple bioactive lipid species using targeted lipidomic analyses. The study comprised 2 sets of patients and healthy controls: the first 128 Israeli PD patients and 224 young German healthy controls for exploration, the second 50/50 German PD patients and matched healthy controls for deeper analyses.
Results: The data showed a 70% prevalence of PD pain and sensory neuropathies with a predominant phenotype of thermal sensory loss plus mechanical hypersensitivity. Multivariate analyses of lipids revealed major differences between PD patients and healthy controls, mainly originating from glucosylceramides and endocannabinoids. Glucosylceramides were increased, whereas anandamide and lysophosphatidic acid 20:4 were reduced, stronger in patients with ongoing pain and with a linear relationship with pain intensity and sensory losses, particularly for glucosylceramide 18:1 and glucosylceramide 24:1.
Conclusions: Our data suggest that PD‐associated sensory neuropathies and PD pain are in part caused by accumulations of glucosylceramides, raising the intriguing possibility of reducing PD pain and sensory loss by glucocerebrosidase substituting or refolding approaches. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.
Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.
Post-exercise hypotension (PEH) is the phenomenon of lowered blood pressure after a single bout of exercise. Only a fraction of people develops PEH but its occurrence correlates well with long-term effects of sports on blood pressure. Therefore, PEH has been suggested as a suitable predictor for the effectivity of exercise as therapy in hypertension. Local vascular bioactive lipids might play a potential role in this context. We performed a cross-over clinical pilot study with 18 healthy volunteers to investigate the occurrence of PEH after a single short-term endurance exercise. Furthermore, we investigated the plasma lipid profile with focus on arachidonic acid (AA)-derived metabolites as potential biomarkers of PEH. A single bout of ergometer cycling induced a significant PEH in healthy volunteers with the expected high inter-individual variability. Targeted lipid spectrum analysis revealed significant upregulation of several lipids in the direct post-exercise phase. Among these changes, only 15- hydroxyeicosatetranoic acid (HETE) correlated significantly with the extent of PEH but in an AA-independent manner, suggesting that 15-HETE might act as specific PEH-marker. Our data indicate that specific lipid modulation might facilitate the identification of patients who will benefit from exercise activity in hypertension therapy. However, larger trials including hypertonic patients are necessary to verify the clinical value of this hypothesis.
Identifying co-expression of lipid species is challenging, but indispensable to identify novel therapeutic targets for breast cancer treatment. Lipid metabolism is often dysregulated in cancer cells, and changes in lipid metabolism affect cellular processes such as proliferation, autophagy, and tumor development. In addition to mRNA analysis of sphingolipid metabolizing enzymes, we performed liquid chromatography time-of-flight mass spectrometry analysis in three breast cancer cell lines. These breast cancer cell lines differ in estrogen receptor and G-protein coupled estrogen receptor 1 status. Our data show that sphingolipids and non-sphingolipids are strongly increased in SKBr3 cells. SKBr3 cells are estrogen receptor negative and G-protein coupled estrogen receptor 1 positive. Treatment with G15, a G-protein coupled estrogen receptor 1 antagonist, abolishes the effect of increased sphingolipid and non-sphingolipid levels in SKBr3 cells. In particular, ether lipids are expressed at much higher levels in cancer compared to normal cells and are strongly increased in SKBr3 cells. Our analysis reveals that this is accompanied by increased sphingolipid levels such as ceramide, sphingadiene-ceramide and sphingomyelin. This shows the importance of focusing on more than one lipid class when investigating molecular mechanisms in breast cancer cells. Our analysis allows unbiased screening for different lipid classes leading to identification of co-expression patterns of lipids in the context of breast cancer. Co-expression of different lipid classes could influence tumorigenic potential of breast cancer cells. Identification of co-regulated lipid species is important to achieve improved breast cancer treatment outcome.
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
(2017)
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.
Hyperhomocysteinemia has been suggested potentially to contribute to a variety of pathologies, such as Alzheimer’s disease (AD). While the impact of hyperhomocysteinemia on AD has been investigated extensively, there are scarce data on the effect of AD on hyperhomocysteinemia. The aim of this in vivo study was to investigate the kinetics of homocysteine (HCys) and homocysteic acid (HCA) and effects of AD-like pathology on the endogenous levels. The mice received a B-vitamin deficient diet for eight weeks, followed by the return to a balanced control diet for another eight weeks. Serum, urine, and brain tissues of AppNL-G-F knock-in and C57BL/6J wild type mice were analyzed for HCys and HCA using LC-MS/MS methods. Hyperhomocysteinemic levels were found in wild type and knock-in mice due to the consumption of the deficient diet for eight weeks, followed by a rapid normalization of the levels after the return to control chow. Hyperhomocysteinemic AppNL-G-F mice had significantly higher HCys in all matrices, but not HCA, compared to wild type control. Higher serum concentrations were associated with elevated levels in both the brain and in urine. Our findings confirm a significant impact of AD-like pathology on hyperhomocysteinemia in the AppNL-G-F mouse model. The immediate normalization of HCys and HCA after the supply of B-vitamins strengthens the idea of a B-vitamin intervention as a potentially preventive treatment option for HCys-related disorders such as AD.
Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis
(2017)
Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the ‘symptom-free’ intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach.
Around 20% of the American population have chronic pain and estimates in other Western countries report similar numbers. This represents a major challenge for global health care systems. Additional problems for the treatment of chronic and persistent pain are the comparably low efficacy of existing therapies, the failure to translate effects observed in preclinical pain models to human patients and related setbacks in clinical trials from previous attempts to develop novel analgesics. Drug repurposing offers an alternative approach to identify novel analgesics as it can bypass various steps of classical drug development. In recent years, several approved drugs were attributed analgesic properties. Here, we review available data and discuss recent findings suggesting that the approved drugs minocycline, fingolimod, pioglitazone, nilotinib, telmisartan, and others, which were originally developed for the treatment of different pathologies, can have analgesic, antihyperalgesic, or neuroprotective effects in preclinical and clinical models of inflammatory or neuropathic pain. For our analysis, we subdivide the drugs into substances that can target neuroinflammation or substances that can act on peripheral sensory neurons, and highlight the proposed mechanisms. Finally, we discuss the merits and challenges of drug repurposing for the development of novel analgesics.
Background: The quantification of global DNA methylation has been established in epigenetic screening. As more practicable alternatives to the HPLC-based gold standard, the methylation analysis of CpG islands in repeatable elements (LINE-1) and the luminometric methylation assay (LUMA) of overall 5-methylcytosine content in “CCGG” recognition sites are most widely used. Both methods are applied as virtually equivalent, despite the hints that their results only partly agree. This triggered the present agreement assessments.
Results: Three different human cell types (cultured MCF7 and SHSY5Y cell lines treated with different chemical modulators of DNA methylation and whole blood drawn from pain patients and healthy volunteers) were submitted to the global DNA methylation assays employing LINE-1 or LUMA-based pyrosequencing measurements. The agreement between the two bioassays was assessed using generally accepted approaches to the statistics for laboratory method comparison studies. Although global DNA methylation levels measured by the two methods correlated, five different lines of statistical evidence consistently rejected the assumption of complete agreement. Specifically, a bias was observed between the two methods. In addition, both the magnitude and direction of bias were tissue-dependent. Interassay differences could be grouped based on Bayesian statistics, and these groups allowed in turn to re-identify the originating tissue.
Conclusions: Although providing partly correlated measurements of DNA methylation, interchangeability of the quantitative results obtained with LINE-1 and LUMA was jeopardized by a consistent bias between the results. Moreover, the present analyses strongly indicate a tissue specificity of the differences between the two methods.
Non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the leading causes of liver disease worldwide. To identify disease-specific pathomechanisms, we analyzed the lipidome, metabolome and immune cell recruitment in livers in both diseases. Mice harboring ASH or NASH had comparable disease severities regarding mortality rate, neurological behavior, expression of fibrosis marker and albumin levels. Lipid droplet size was higher in NASH than ASH and qualitative differences in the lipidome were mainly based on incorporation of diet-specific fatty acids into triglycerides, phosphatidylcholines and lysophosphatidylcholines. Metabolomic analysis showed downregulated nucleoside levels in both models. Here, the corresponding uremic metabolites were only upregulated in NASH suggesting stronger cellular senescence, which was supported by lower antioxidant levels in NASH as compared to ASH. While altered urea cycle metabolites suggest increased nitric oxide synthesis in both models, in ASH, this depended on increased L-homoarginine levels indicating a cardiovascular response mechanism. Interestingly, only in NASH were the levels of tryptophan and its anti-inflammatory metabolite kynurenine upregulated. Fittingly, high-content immunohistochemistry showed a decreased macrophage recruitment and an increased polarization towards M2-like macrophages in NASH. In conclusion, with comparable disease severity in both models, higher lipid storage, oxidative stress and tryptophan/kynurenine levels were seen in NASH, leading to distinct immune responses.
Background: SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. Methods: CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. Results: Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. Conclusion: Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.
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.
The experience of pain is mediated by a specialized sensory system, the nociceptive system. There is considerable evidence that the cGMP/cGMP kinase I (cGKI) signaling pathway modulates the nociceptive processing within the spinal cord. However, downstream targets of cGKI in this context have not been identified to date. In this study we investigated whether cysteine-rich protein 2 (CRP2) is a downstream effector of cGKI in the spinal cord and is involved in nociceptive processing. Immunohistochemistry of the mouse spinal cord revealed that CRP2 is expressed in superficial laminae of the dorsal horn. CRP2 is colocalized with cGKI and with markers of primary afferent C fibers. Importantly, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and CRP2 is phosphorylated in a cGMP-dependent manner. To elucidate the functional role of CRP2 in nociception, we investigated the nociceptive behavior of CRP2-deficient (CRP2-/-) mice. Touch perception and acute thermal nociception were unaltered in CRP2-/- mice. However, CRP2-/- mice showed an increased nociceptive behavior in models of persistent pain as compared to wild type mice. Intrathecal administration of cGKI activating cGMP analogs increased the nociceptive behavior in wild type but not in CRP2-/- mice, indicating that the presence of CRP2 was essential for cGMP/cGKI-mediated nociception. These data indicate that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.
Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice
(2007)
Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E2 synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE2 synthase-1 (mPGES-1) isomerizes COX-2-derived PGH2 to PGE2. Here, we evaluated the effect of mPGES-1-deficiency on the noci-ceptive behavior in various models of nociception that depend on PGE2 synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE2 synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE2 synthesis to PGD2, PGF2α and 6-keto-PGF1α (stable metabolite of PGI2). Since the latter prostaglandins serve also as mediators of noci-ception they may compensate the loss of PGE2 synthesis in mPGES-1-deficient mice.
Consequences of a human TRPA1 genetic variant on the perception of nociceptive and olfactory stimuli
(2014)
Background: TRPA1 ion channels are involved in nociception and are also excited by pungent odorous substances. Based on reported associations of TRPA1 genetics with increased sensitivity to thermal pain stimuli, we therefore hypothesized that this association also exists for increased olfactory sensitivity.
Methods: Olfactory function and nociception was compared between carriers (n = 38) and non-carriers (n = 43) of TRPA1 variant rs11988795 G.A, a variant known to enhance cold pain perception. Olfactory function was quantified by assessing the odor threshold, odor discrimination and odor identification, and by applying 200-ms pulses of H2S intranasal. Nociception was assessed by measuring pain thresholds to experimental nociceptive stimuli (blunt pressure, electrical stimuli, cold and heat stimuli, and 200-ms intranasal pulses of CO2).
Results: Among the 11 subjects with moderate hyposmia, carriers of the minor A allele (n = 2) were underrepresented (34 carriers among the 70 normosmic subjects; p = 0.049). Moreover, carriers of the A allele discriminated odors significantly better than non-carriers (13.161.5 versus 12.361.6 correct discriminations) and indicated a higher intensity of the H2S stimuli (29.2613.2 versus 21612.8 mm VAS, p = 0.006), which, however, could not be excluded to have involved a trigeminal component during stimulation. Finally, the increased sensitivity to thermal pain could be reproduced.
Conclusions: The findings are in line with a previous association of a human TRPA1 variant with nociceptive parameters and extend the association to the perception of odorants. However, this addresses mainly those stimulants that involve a trigeminal component whereas a pure olfactory effect may remain disputable. Nevertheless, findings suggest that future TRPA1 modulating drugs may modify the perception of odorants.
Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin–renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. Conclusion: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.
Oral presentation from 4th International Conference of cGMP Generators, Effectors and Therapeutic Implications ; Regensburg, Germany. 19–21 June 2009 Background: An exaggerated pain sensitivity is the dominant feature of inflammatory and neuropathic pain both in the clinical setting and in experimental animal models. It manifests as pain in response to normally innocuous stimuli (allodynia), increased response to noxious stimuli (hyperalgesia) or spontaneous pain, and can persist long after the initial injury is resolved. Research over the last decades has revealed that several signaling pathways in the spinal cord essentially contribute to the pain sensitization. To test the contribution of cGMP produced by NO-sensitive guanylyl cyclase (NO-GC) to pain sensitization, we investigated the localization of NO-GC in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). Results: We show that NO-GC (β1 subunit) is distinctly expressed in neurons of the mouse spinal cord, while its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by spinal administration of drugs releasing NO. Surprisingly, during spinal nociceptive processing cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), while cGKI can be activated by natriuretic peptide receptor-B (NPR-B) dependent cGMP production. Conclusion: Taken together, our results provide evidence that NO-GC has a dominant role in the development of exaggerated pain sensitivity during inflammatory and neuropathic pain. Furthermore, beside the NO-mediated cGMP synthesis, cGMP produced by NPR-B contributes to pain sensitization by activation of cGKI.
FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.
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.
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.
Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15−/− mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15−/− mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15−/− mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.
Alkylglycerol monooxygenase (AGMO) is a tetrahydrobiopterin (BH4)-dependent enzyme with major expression in the liver and white adipose tissue that cleaves alkyl ether glycerolipids. The present study describes the disclosure and biological characterization of a candidate compound (Cp6), which inhibits AGMO with an IC50 of 30–100 µM and 5–20-fold preference of AGMO relative to other BH4-dependent enzymes, i.e., phenylalanine-hydroxylase and nitric oxide synthase. The viability and metabolic activity of mouse 3T3-L1 fibroblasts, HepG2 human hepatocytes and mouse RAW264.7 macrophages were not affected up to 10-fold of the IC50. However, Cp6 reversibly inhibited the differentiation of 3T3-L1 cells towards adipocytes, in which AGMO expression was upregulated upon differentiation. Cp6 reduced the accumulation of lipid droplets in adipocytes upon differentiation and in HepG2 cells exposed to free fatty acids. Cp6 also inhibited IL-4-driven differentiation of RAW264.7 macrophages towards M2-like macrophages, which serve as adipocyte progenitors in adipose tissue. Collectively, the data suggest that pharmacologic AGMO inhibition may affect lipid storage.
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.
Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund's Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states.
Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.
Based on accumulating evidence of a role of lipid signaling in many physiological and pathophysiological processes including psychiatric diseases, the present data driven analysis was designed to gather information needed to develop a prospective biomarker, using a targeted lipidomics approach covering different lipid mediators. Using unsupervised methods of data structure detection, implemented as hierarchal clustering, emergent self-organizing maps of neuronal networks, and principal component analysis, a cluster structure was found in the input data space comprising plasma concentrations of d = 35 different lipid-markers of various classes acquired in n = 94 subjects with the clinical diagnoses depression, bipolar disorder, ADHD, dementia, or in healthy controls. The structure separated patients with dementia from the other clinical groups, indicating that dementia is associated with a distinct lipid mediator plasma concentrations pattern possibly providing a basis for a future biomarker. This hypothesis was subsequently assessed using supervised machine-learning methods, implemented as random forests or principal component analysis followed by computed ABC analysis used for feature selection, and as random forests, k-nearest neighbors, support vector machines, multilayer perceptron, and naïve Bayesian classifiers to estimate whether the selected lipid mediators provide sufficient information that the diagnosis of dementia can be established at a higher accuracy than by guessing. This succeeded using a set of d = 7 markers comprising GluCerC16:0, Cer24:0, Cer20:0, Cer16:0, Cer24:1, C16 sphinganine, and LacCerC16:0, at an accuracy of 77%. By contrast, using random lipid markers reduced the diagnostic accuracy to values of 65% or less, whereas training the algorithms with randomly permuted data was followed by complete failure to diagnose dementia, emphasizing that the selected lipid mediators were display a particular pattern in this disease possibly qualifying as biomarkers.
Persistent and, in particular, neuropathic pain is a major healthcare problem with still insufficient pharmacological treatment options. This triggered research activities aimed at finding analgesics with a novel mechanism of action. Results of these efforts will need to pass through the phases of drug development, in which experimental human pain models are established components e.g. implemented as chemical hyperalgesia induced by capsaicin. We aimed at ranking the various readouts of a human capsaicin–based pain model with respect to the most relevant information about the effects of a potential reference analgesic. In a placebo‐controlled, randomized cross‐over study, seven different pain‐related readouts were acquired in 16 healthy individuals before and after oral administration of 300 mg pregabalin. The sizes of the effect on pain induced by intradermal injection of capsaicin were quantified by calculating Cohen's d. While in four of the seven pain‐related parameters, pregabalin provided a small effect judged by values of Cohen's d exceeding 0.2, an item categorization technique implemented as computed ABC analysis identified the pain intensities in the area of secondary hyperalgesia and of allodynia as the most suitable parameters to quantify the analgesic effects of pregabalin. Results of this study provide further support for the ability of the intradermal capsaicin pain model to show analgesic effects of pregabalin. Results can serve as a basis for the designs of studies where the inclusion of this particular pain model and pregabalin is planned.
The single nucleotide polymorphism 118A>G of the human micro-opioid receptor gene OPRM1, which leads to an exchange of the amino acid asparagine (N) to aspartic acid (D) at position 40 of the extracellular receptor region, alters the in vivo effects of opioids to different degrees in pain-processing brain regions. The most pronounced N40D effects were found in brain regions involved in the sensory processing of pain intensity. Using the mu-opioid receptor-specific agonist DAMGO, we analyzed the micro-opioid receptor signaling, expression, and binding affinity in human brain tissue sampled postmortem from the secondary somatosensory area (SII) and from the ventral posterior part of the lateral thalamus, two regions involved in the sensory processing and transmission of nociceptive information. We show that the main effect of the N40D micro-opioid receptor variant is a reduction of the agonist-induced receptor signaling efficacy. In the SII region of homo- and heterozygous carriers of the variant 118G allele (n=18), DAMGO was only 62% as efficient (p=0.002) as in homozygous carriers of the wild-type 118A allele (n=15). In contrast, the number of [3H]DAMGO binding sites was unaffected. Hence, the micro-opioid receptor G-protein coupling efficacy in SII of carriers of the 118G variant was only 58% as efficient as in homozygous carriers of the 118A allele (p<0.001). The thalamus was unaffected by the OPRM1 118A>G SNP. In conclusion, we provide a molecular basis for the reduced clinical effects of opioid analgesics in carriers of mu-opioid receptor variant N40D.
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.
Patient therapy is based mainly on a combination of diagnosis, suitable monitoring or support devices and drug treatment and is usually employed for a pre-existing disease condition. Therapy remains predominantly symptom-based, although it is increasingly clear that individual treatment is possible and beneficial. However, reasonable precision medicine can only be realized with the coordinated use of diagnostics, devices and drugs in combination with extensive databases (4Ds), an approach that has not yet found sufficient implementation. The practical combination of 4Ds in health care is progressing, but several obstacles still hamper their extended use in precision medicine.