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We assessed the prognostic value of hypoxia (carbonic anhydrase 9; CA9), vessel density (CD31), with macrophages (CD68) and B cells (CD20) that can interact and lead to immune suppression and disease progression using scanning and histological mapping of whole-mount FFPE pancreatectomy tissue sections from 141 primarily resectable pancreatic ductal adenocarcinoma (PDAC) samples treated with surgery and adjuvant chemotherapy. Their expression was correlated with clinicopathological characteristics, and overall survival (OS), progression-free survival (PFS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of stroma density (haematoxylin-eosin) and activity (alpha-smooth muscle actin). The median OS was 21 months after a mean follow-up of 20 months (range, 2–69 months). The median tumor surface area positive for CA9 and CD31 was 7.8% and 8.1%, respectively. Although total expression of these markers lacked prognostic value in the entire cohort, nevertheless, high tumor compartment CD68 expression correlated with worse PFS (p = 0.033) and DMFS (p = 0.047). Also, high CD31 expression predicted for worse OS (p = 0.004), PFS (p = 0.008), LPFS (p = 0.014) and DMFS (p = 0.004) in patients with moderate density stroma. High stromal and peripheral compartment CD68 expression predicted for significantly worse outcome in patients with loose and moderate stroma density, respectively. Altogether, in contrast to the current notion, hypoxia levels in PDAC appear to be comparable to other malignancies. CD31 and CD68 constitute prognostic markers in patient subgroups that vary according to tumor compartment and stromal density. Our study provides important insight on the pathophysiology of PDAC and should be exploited for future treatments.
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.
In vielen Tumorzellen kommt es zu einer Überexpression des Hypoxie-induzierbaren Faktor 1alpha (HIF-1alpha), was zu einer verbesserten Anpassung des Tumors an die intratumorale Hypoxie sowie zu einer Resistenz gegen Strahlen- und Chemotherapie führt. Je nach Tumor kann HIF-1alpha auf verschiedenen Wegen induziert werden. Eine Möglichkeit ist die Hemmung des Abbaus von HIF-1alpha über das 26S-Proteasom, wie z.B. beim van Hippel-Lindau (VHL)-Syndrom aufgrund einer Mutation im VHL Gen. Patienten mit VHL-Syndrom entwickeln häufig renal clearcell carcinomas (RCCs). In diesen Karzinomen kann HIF-1alpha nicht über den klassischen Weg über das 26S-Proteasom abgebaut werden. Um das Verständnis für alternative Regulationsmechanismen von HIF-1alpha zu erweitern, wurde mit RCC4-Zellen gearbeitet. Im ersten Teil der vorliegenden Arbeit konnte gezeigt werden, dass in RCC4-Zellen das HIF-1alpha-Protein unter Hypoxie, in Kombination mit NO, durch die Ca2+-abhängige Protease Calpain abgebaut wird. Unter Hypoxie kam es zu einem Anstieg der Produktion von reaktiven Sauerstoffspezies (ROS) in den Mitochondrien, die mit NO zu Peroxynitrit und weiteren reaktiven Stickstoffintermediaten (RNI) reagierten. Die kombinierte Stimulation der Zellen mit NO und O2- unter Normoxie löste ebenfalls einen Anstieg des intrazellulären Ca2+-Gehaltes und der Calpain-Aktivität aus, was gleichzeitig zu einem reduzierten HIF-1alpha-Proteingehalt führte. Der Calpain-vermittelte HIF-1alpha-Abbau konnte auch in Zellen mit funktionellem VHL-Protein (pVHL) durch NO und O2- ausgelöst werden, wenn der proteasomale Abbau gehemmt war. Diese Ergebnisse beschreiben einen neuen Regulationsmechanismus für das HIF-1alpha-Protein, der unabhängig vom Sauerstoffgehalt und vom 26S-proteasomalen Abbau durch NO/O2- und Calpain erfolgt. Bisher war noch nicht bekannt, dass HIF-1alpha anders als über das 26S Proteasom abgebaut werden kann. In der vorliegenden Arbeit konnte gezeigt werden, dass der Calpain-vermittelte Abbau neben dem proteasomalen Abbau zur Regulierung von HIF 1 beiträgt. In Tumorgeweben stellt nicht nur HIF-1, welches in den Tumorzellen aktiviert ist, einen Selektionsvorteil für die Zellen des Tumorgewebes dar. Ebenso tragen die Zellen des Tumorstromas, darunter die Makrophagen, die in den Tumor einwandern, zur Progression des Tumors durch die Anpassung an die hypoxischen Umgebung bei. Daher wurde im zweiten Teil dieser Arbeit die Regulation von HIF-1alpha in den durch konditioniertes Medium von apoptotischen Zellen (KMAZ) aktivierten Makrophagen und der Bedeutung der daraus resultierenden HIF-1-Aktivierung untersucht. Makrophagen, die durch apoptotische Zellen (AZ) aktiviert werden, stellen einen anti-inflammatorischen, pro-angiogenetischen Phänotyp dar, der vergleichbar mit dem der Tumor-assozierten Makrophagen (TAMs) ist. TAMs infiltrieren in das Tumorgewebe und sind essentiell am Übergang von einem avaskulären zu einem invasiven, vaskularisierten und malignen Tumor beteiligt. Unsere Arbeitsgruppe konnte in Vorarbeiten zeigen, dass Makrophagen zunächst Tumorzellen abtöten, wodurch die apoptotischen Tumorzellen Mediatoren (u.a. Sphingosin-1-Phosphat (S1P)) freisetzen, die eine Polarisierung zu einem alternativen, TAM-ähnlichen-Phänotyp der Makrophagen bewirken. Die Inkubation der Makrophagen mit KMAZ führte zu einer Induktion der HIF-1alpha-mRNA und des -Proteins unter Normoxie, was unabhängig von der Proteinstabilität auf eine gesteigerte Proteinsynthese zurückgeführt werden konnte. Weiterhin führte die Induktion von HIF-1alpha zu einer gesteigerten HIF-1-Aktivität. Die Differenzierung von Stammzellen zu CD31+-Endothelzellen wurde durch die Überstände von den durch KMAZ polarisierten Makrophagen HIF-1-abhängig hervorgerufen und ist ein Indiz für die Ausbildung des HIF 1-vermittelten pro-angiogenetischen Phänotyps der Makrophagen. Als Mediatoren, die von den AZ freigesetzt wurden und an der HIF-1alpha-mRNA-Induktion beteiligt sind, konnten S1P und transforming growth factor-beta (TGF-beta) identifiziert werden. Des Weiteren kommt es zu einer Aktivierung des nuclear factor of activated T-cells (NFAT), der an den HIF-1alpha-Promotor bindet und die Transkription induziert. Aufgrund der verstärkten Synthese kommt es zur Akkumulation von HIF-1alpha und zur Aktivierung von HIF-1. Bisher ist die Aktivierung von HIF-1 in TAMs durch die Lokalisation in hypoxischen Arealen erklärt und nicht weiter untersucht worden. Die Erkenntnisse über die Regulierung von HIF-1 durch AZ beschreiben einen neuen Mechanismus, der zur HIF-1-Aktivierung auch unter Normoxie führt. Dabei vermitteln AZ statt der beschriebenen hypoxischen Stabilisierung des HIF-1α-Proteins eine Induktion der HIF-1alpha-mRNA. Weiterhin zeigen die Ergebnisse eine Möglichkeit auf, wie TAMs bereits unter Normoxie zur Angiogenese Induktion in Tumoren beitragen können und erweitern damit das Verständnis, wie die Tumor-unterstützende Wirkung der TAMs vermittelt wird.
Sepsis is a serious clinical condition which can cause life-threatening organ dysfunction, and has limited therapeutic options. The paradigm of limiting excessive inflammation and promoting anti-inflammatory responses is a simplified concept. Yet, the absence of intrinsic anti-inflammatory signaling at the early stage of an infection can lead to an exaggerated activation of immune cells, including monocytes and macrophages. There is emerging evidence that endogenous molecules control those mechanisms. Here we aimed to identify and describe the dynamic changes in monocyte and macrophage subsets and lung damage in CL57BL/6N mice undergoing blunt chest trauma with subsequent cecal ligation and puncture. We showed that early an increase in systemic and activated Ly6C+CD11b+CD45+Ly6G− monocytes was paralleled by their increased emigration into lungs. The ratio of pro-inflammatory Ly6ChighCD11b+CD45+Ly6G− to patrolling Ly6ClowCD11b+CD45+Ly6G− monocytes significantly increased in blood, lungs and bronchoalveolar lavage fluid (BALF) suggesting an early transition to inflammatory phenotypes during early sepsis development. Similar to monocytes, the level of pro-inflammatory Ly6ChighCD45+F4/80+ macrophages increased in lungs and BALF, while tissue repairing Ly6ClowCD45+F4/80+ macrophages declined in BALF. Levels of inflammatory mediators TNF-α and MCP-1 in blood and RAGE in lungs and BALF were elevated, and besides their boosting of inflammation via the recruitment of cells, they may promote monocyte and macrophage polarization, respectively, toward the pro-inflammatory phenotype. Neutralization of uteroglobin increased pro-inflammatory cytokine levels, activation of inflammatory phenotypes and their recruitment to lungs; concurrent with increased pulmonary damage in septic mice. In in vitro experiments, the influence of uteroglobin on monocyte functions including migratory behavior, TGF-β1 expression, cytotoxicity and viability were proven. These results highlight an important role of endogenous uteroglobin as intrinsic anti-inflammatory signal upon sepsis-induced early lung injury, which modules the early monocyte/macrophages driven inflammation.
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.
During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls’ staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.
Efferocytosis is critical for tissue homeostasis, as its deregulation is associated with several autoimmune pathologies. While engulfing apoptotic cells, phagocytes activate transcription factors, such as peroxisome proliferator-activated receptors (PPAR) or liver X receptors (LXR) that orchestrate metabolic, phagocytic, and inflammatory responses towards the ingested material. Coordination of these transcription factors in efferocytotic human macrophages is not fully understood. In this study, we evaluated the transcriptional profile of macrophages following the uptake of apoptotic Jurkat T cells using RNA-seq analysis. Results indicated upregulation of PPAR and LXR pathways but downregulation of sterol regulatory element-binding proteins (SREBP) target genes. Pharmacological inhibition and RNA interference pointed to LXR and PPARδ as relevant transcriptional regulators, while PPARγ did not substantially contribute to gene regulation. Mechanistically, lysosomal digestion and lysosomal acid lipase (LIPA) were required for PPAR and LXR activation, while PPARδ activation also demanded an active lysosomal phospholipase A2 (PLA2G15). Pharmacological interference with LXR signaling attenuated ABCA1-dependent cholesterol efflux from efferocytotic macrophages, but suppression of inflammatory responses following efferocytosis occurred independently of LXR and PPARδ. These data provide mechanistic details on LXR and PPARδ activation in efferocytotic human macrophages.
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.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
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.