Alternatively activated macrophages (AAMs) contribute to the resolution of inflammation and tissue repair. However, molecular pathways that govern their differentiation have remained incompletely ...understood. Here, we show that uncoupling protein-2-mediated mitochondrial reprogramming and the transcription factor GATA3 specifically controlled the differentiation of pro-resolving AAMs in response to the alarmin IL-33. In macrophages, IL-33 sequentially triggered early expression of pro-inflammatory genes and subsequent differentiation into AAMs. Global analysis of underlying signaling events revealed that IL-33 induced a rapid metabolic rewiring of macrophages that involved uncoupling of the respiratory chain and increased production of the metabolite itaconate, which subsequently triggered a GATA3-mediated AAM polarization. Conditional deletion of GATA3 in mononuclear phagocytes accordingly abrogated IL-33-induced differentiation of AAMs and tissue repair upon muscle injury. Our data thus identify an IL-4-independent and GATA3-dependent pathway in mononuclear phagocytes that results from mitochondrial rewiring and controls macrophage plasticity and the resolution of inflammation.
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•IL-33 induces rapid mitochondrial rewiring in macrophages•Uncoupling of the respiratory chain promotes IL-33-induced AAM differentiation•GATA3 links macrophage metabolism and differentiation in response to IL-33•GATA3 orchestrates the differentiation of pro-resolving macrophages in vivo
Alternatively activated macrophages (AAMs) promote the resolution of inflammation and tissue repair. Faas et al. show that the alarmin IL-33 promotes a mitochondrial rewiring of macrophages and the consecutive activation of the transcription factor GATA3, which orchestrates the IL-4-independent differentiation of AAMs and the resolution of inflammation upon tissue injury.
Microglia cells fulfill key homeostatic functions and essentially contribute to host defense within the CNS. Altered activation of microglia, in turn, has been implicated in neuroinflammatory and ...neurodegenerative diseases. In this study, we identify the nuclear receptor (NR) Nr4a1 as key rheostat controlling the activation threshold and polarization of microglia. In steady-state microglia, ubiquitous neuronal-derived stress signals such as ATP induced expression of this NR, which contributed to the maintenance of a resting and noninflammatory microglia phenotype. Global and microglia-specific deletion of Nr4a1 triggered the spontaneous and overwhelming activation of microglia and resulted in increased cytokine and NO production as well as in an accelerated and exacerbated form of experimental autoimmune encephalomyelitis. Ligand-induced activation of Nr4a1 accordingly ameliorated the course of this disease. Our current data thus identify Nr4a1 as regulator of microglia activation and potentially new target for the treatment of inflammatory CNS diseases such as multiple sclerosis.
Structural remodeling or damage as a result of disease or injury is often not evenly distributed throughout a tissue but strongly depends on localization and extent of damaging stimuli. Skeletal ...muscle as a mechanically active organ can express signs of local or even systemic myopathic damage, necrosis, or repair. Conventionally, muscle biopsies (patients) or whole muscles (animal models) are mechanically sliced and stained to assess structural alterations histologically. Three-dimensional tissue information can be obtained by applying deep imaging modalities,
multiphoton or light-sheet microscopy. Chemical clearing approaches reduce scattering,
through matching refractive tissue indices, to overcome optical penetration depth limits in thick tissues.
Here, we optimized a range of different clearing protocols. We find aqueous solution-based protocols employing (20-80%) 2,2'-thiodiethanol (TDE) to be advantageous over organic solvents (dibenzyl ether, cinnamate) regarding the preservation of muscle morphology, ease-of-use, hazard level, and costs.
Applying TDE clearing to a mouse model of local cardiotoxin (CTX)-induced muscle necrosis, a complete loss of myosin-II signals was observed in necrotic areas with little change in fibrous collagen or autofluorescence (AF) signals. The 3D aspect of myofiber integrity could be assessed, and muscle necrosis in whole muscle was quantified locally
the ratios of detected AF, forward- and backward-scattered Second Harmonic Generation (fSHG, bSHG) signals.
TDE optical clearing is a versatile tool to study muscle architecture in conjunction with label-free multiphoton imaging in 3D in injury/myopathy models and might also be useful in studying larger biofabricated constructs in regenerative medicine.
Abstract Objective Epidemiologic studies suggest that elevated postprandial triglycerides (ppTG) are associated with future cardiovascular events. Monocyte activation plays an important role in ...vascular diseases. Omega-3 fatty acids (n3-FA) lower fasting TG levels. The effects of n3-FA on ppTG and the role of ppTG for monocyte activation are insufficiently understood. Methods and results 23 healthy volunteers and 30 non-diabetic patients with documented coronary artery disease were subjected to an oral TG tolerance test (OTTT) consisting of 80 g cream fat or to water as control (H2 O). Patients were treated with 4 g n3-FA/day or placebo for 3 weeks in a randomized, placebo-controlled, double-blind, crossover study. Relative postprandial TG increase reached its maximum 4 h after fat intake (185.1 ± 10.9% of baseline). n3-FA reduced fasting TG from 137.1 ± 12.9 to 112.2 ± 8.6 mg/dl ( p < 0.05), and maximum ppTG concentrations from 243.6 ± 24.6 to 205.8 ± 17.1 mg/dl ( p < 0.05), while relative TG increase (192.8 ± 12.7%) was comparable to placebo. Relative monocytopenia and neutrophilia were detected following fat intake, which was unaffected by n3-FA and also detectable in the H2 O group. Serum chemotactic cytokine (MCP1 and fractalkine) concentrations and monocyte migration were not affected by fat intake or n3-FA. Monocyte activation markers CD11b and CD14, monocyte subpopulations CD16+ CD14high and CD16+ CD14low , sICAM serum levels and markers of oxidative stress remained unchanged by fat intake or n3-FA. Conclusion The postprandial TG increase does not stimulate monocytes beyond their circadian activation patterns. n3-FA reduce fasting TG and the postprandial TG increase. n3-FA may therefore allow to prospectively study whether selected patients benefit from TG-lowering independent of LDL- and HDL-cholesterol.
Although the control of bone-resorbing osteoclasts through osteocyte-derived RANKL is well defined, little is known about the regulation of osteoclasts by osteocyte death. Indeed, several skeletal ...diseases, such as bone fracture, osteonecrosis, and inflammation are characterized by excessive osteocyte death. Herein we show that osteoclasts sense damage-associated molecular patterns (DAMPs) released by necrotic osteocytes via macrophage-inducible C-type lectin (Mincle), which induced their differentiation and triggered bone loss. Osteoclasts showed robust Mincle expression upon exposure to necrotic osteocytes in vitro and in vivo. RNA sequencing and metabolic analyses demonstrated that Mincle activation triggers osteoclastogenesis via ITAM-based calcium signaling pathways, skewing osteoclast metabolism toward oxidative phosphorylation. Deletion of Mincle in vivo effectively blocked the activation of osteoclasts after induction of osteocyte death, improved fracture repair, and attenuated inflammation-mediated bone loss. Furthermore, in patients with osteonecrosis, Mincle was highly expressed at skeletal sites of osteocyte death and correlated with strong osteoclastic activity. Taken together, these data point to what we believe is a novel DAMP-mediated process that allows osteoclast activation and bone loss in the context of osteocyte death.
Auf Gewebeschäden reagiert das Immunsystem mit der Einleitung von Entzündungsprozessen. Zur Gewährleistung der vollständigen Regeneration nach einer Gewebeschädigung müssen die Vorgänge der ...Entzündung sowie der Entzündungsauflösung fehlerfrei ablaufen. Einen wichtigen Beitrag zu diesen Prozessen leisten alternativ aktivierte Makrophagen, welche im Rahmen der Entzündung differenzieren. Sie nehmen nekrotisches Material auf und fördern durch die Ausschüttung spezifischer Signalmoleküle die Geweberegeneration. Die diesen Phänotyp induzierenden Signalwege konnten bislang jedoch noch nicht ausreichend aufgeklärt werden. Im Rahmen dieser Arbeit wurde Interleukin-33 (IL-33), welches als Alarmin von nekrotischen Zellen in die Umgebung freigesetzt wird, als Auslöser eines alternativ aktivierten Makrophagenphänotyps in sterilen Entzündungsprozessen identifiziert. In Knochenmarksmakrophagen leitet IL-33 zunächst eine frühe Expression proentzündlicher Gene sowie die Entkopplung der Mitochondrien durch das „Uncoupling Protein 2“ (UCP2) ein, woraufhin eine metabolische Umprogrammierung der Zellen zu einem alternativ aktivierten Makrophagenphänotyp mit erhöhter Phagozytosekapazität folgt. Mit Hilfe von in silico Analysen wurde eine Rolle des Transkriptionsfaktors „GATA Binding Protein 3“ (GATA3) im IL-33-Signalweg aufgedeckt, welche sowohl von der initialen IL 33-Signalweiterleitung als auch der darauffolgenden UCP2-Aktivität abhängt. Dementsprechend konnte durch eine konditionelle Gata3-Deletion in mononukleären Phagozyten die Einleitung eines alternativ aktivierten Zellprofils durch IL-33 unterbunden werden. Im Kontext einer sterilen Muskelverletzung führte dies zu einer reduzierten Anzahl entzündungsauflösender, phagozytierender Makrophagen und einer verschlechterten Regeneration des Gewebes. Die Daten enthüllen eine bislang unbekannte, entzündungsmodulierende Wirkung von IL-33 in der Gewebeschädigung, dessen Signalweiterleitung über UCP2 und GATA3 den Makrophagenphänotyp beeinflusst und dadurch die Entzündungsauflösung unterstützt.
Osteoclasts are specialised bone resorbing cells that control both physiological and pathological bone turnover. Functional changes in the differentiation and activity of osteoclasts are accompanied ...by active metabolic reprogramming. However, the biological significance and the in vivo relevance of these events has remained unclear. Here we show that bone resorption of differentiated osteoclasts heavily relies on increased aerobic glycolysis and glycolysis-derived lactate production. While pharmacological inhibition of glycolysis did not affect osteoclast differentiation or viability, it efficiently blocked bone resorption in vitro and in vivo and consequently ameliorated ovariectomy-induced bone loss. Our experiments thus highlight the therapeutic potential of interfering with osteoclast-intrinsic metabolic pathways as possible strategy for the treatment of diseases characterized by accelerated bone loss.
An animal model for preeclampsia was developed by means of an ultra-low-dose endotoxin infusion protocol in conscious pregnant rats.
Rats received a permanent jugular vein cannula on day 0 of ...pregnancy, through which endotoxin (1.0 micrograms/kg body weight) (n = 10) or saline solution (n = 6) was infused during 1 hour on day 14 of pregnancy. Blood pressure, albuminuria, and platelet counts were measured, and histopathologic studies was performed in these rats.
A significant increase of blood pressure (p < 0.05) and of urinary albumin excretion (p < 0.05) was observed in endotoxin-treated pregnant animals, in contrast to control pregnant rats receiving saline solution. Platelet coagulopathy was found and glomerular fibrinogen deposits could be detected only in the endotoxin-treated pregnant rats. In addition, the activity of the glomerular antithrombotic enzyme adenosine diphosphatase was decreased in endotoxin-treated pregnant rats compared with saline solution-treated pregnant rats.
Because histopathologic and clinical events in this model mimic predominant features of human preeclampsia, this model may enable further study into the pathophysiologic mechanisms of this complication of pregnancy.
Objective: The aim of this study was to search for activation markers of peripheral leukocytes in experimental preeclampsia in the rat.
Study Design: Experimental preeclampsia was induced in ...14-day-pregnant rats by infusion of endotoxin (1.0 μg/kg body weight). For comparison, rats with normal pregnancies that were infused with sodium chloride solution and cyclic rats that were infused with either endotoxin or sodium chloride solution were used. At various points before and after the infusion, blood samples were withdrawn and analyzed by means of whole-blood flow cytometry to evaluate expression of inflammation-associated adhesion molecules (CD11b, CD11a, CD49d, and CD62L) and CD14 on the leukocytes.
Results: Normal pregnancy was associated with increased CD11b (granulocytes and monocytes), CD11a (monocytes and lymphocytes), and CD49d (granulocytes, monocytes, and lymphocytes) expression. In addition to these changes found in normal pregnancy, reduced CD62L and increased CD11a and CD49d expression was found on granulocytes after endotoxin treatment of pregnant rats. No effect of endotoxin was observed in cyclic rats.
Conclusion: Leukocytes of rats with experimental preeclampsia and, to a lesser extent, those of rats with normal pregnancies had an activated phenotype. These results are consistent with our previous findings in human subjects and suggest that (experimental) preeclampsia results from a generalized inflammatory response. (Am J Obstet Gynecol 2000;182:351-7.)
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