Differential cell type-specific function of the aryl hydrocarbon receptor and its repressor in diet-induced obesity and fibrosis.

OBJECTIVE: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor regulating xenobiotic responses as well as physiological metabolism. Dietary AhR ligands activate the AhR signaling axis, whereas AhR activation is negatively regulated by the AhR repressor (AhRR). While AhR-deficient mice are known to be resistant to diet-induced obesity (DIO), the influence of the AhRR on DIO has not been assessed so far. METHODS: In this study, we analyzed AhRR-/- mice and mice with a conditional deletion of either AhRR or AhR in myeloid cells under conditions of DIO and after supplementation of dietary AhR ligands. Moreover, macrophage metabolism was assessed using Seahorse Mito Stress Test and ROS assays as well as transcriptomic analysis. RESULTS: We demonstrate that global AhRR deficiency leads to a robust, but not as profound protection from DIO and hepatosteatosis as AhR deficiency. Under conditions of DIO, AhRR-/- mice did not accumulate TCA cycle intermediates in the circulation in contrast to wild-type (WT) mice, indicating protection from metabolic dysfunction. This effect could be mimicked by dietary supplementation of AhR ligands in WT mice. Because of the predominant expression of the AhRR in myeloid cells, AhRR-deficient macrophages were analyzed for changes in metabolism and showed major metabolic alterations regarding oxidative phosphorylation and mitochondrial activity. Unbiased transcriptomic analysis revealed increased expression of genes involved in de novo lipogenesis and mitochondrial biogenesis. Mice with a genetic deficiency of the AhRR in myeloid cells did not show alterations in weight gain after high fat diet (HFD) but demonstrated ameliorated liver damage compared to control mice. Further, deficiency of the AhR in myeloid cells also did not affect weight gain but led to enhanced liver damage and adipose tissue fibrosis compared to controls. CONCLUSIONS: AhRR-deficient mice are resistant to diet-induced metabolic syndrome. Although conditional ablation of either the AhR or AhRR in myeloid cells did not recapitulate the phenotype of the global knockout, our findings suggest that enhanced AhR signaling in myeloid cells deficient for AhRR protects from diet-induced liver damage and fibrosis, whereas myeloid cell-specific AhR deficiency is detrimental.

TGFß depletion does neither modulate acute E. coli-induced inflammatory immune responses nor impair the protective effect by chronic filarial infection.

TGFß is an anti-inflammatory molecule that suppresses pro-inflammatory immune responses. Previously, we demonstrated that chronic filarial infection has a beneficial impact on Escherichia coli-induced sepsis. In the present study, we investigated whether this protective effect is dependent on TGFß signaling and whether depletion of TGFß before E. coli challenge alters the early course of sepsis per se. In vivo depletion of TGFß before E. coli challenge did not alter levels of pro-inflammatory cytokines/chemokines and did neither increase the bacterial burden nor worsen E. coli-induced hypothermia six hours post E. coli challenge. Similarly, in the co-infection model, despite TGFß depletion, mice infected with the filarial nematode Litomosoides sigmodontis exhibited milder E. coli-induced hypothermia, reduced bacterial load and pro-inflammatory immune responses. Thus, we conclude that TGFß is not essentially modulating the initial pro-inflammatory phase during sepsis and that the protective effect of a chronic filarial infection against sepsis is independent of TGFß signaling.

CCL17 exerts a neuroimmune modulatory function and is expressed in hippocampal neurons.

Chemokines are important signaling molecules in the immune and nervous system. Using a fluorescence reporter mouse model, we demonstrate that the chemokine CCL17, a ligand of the chemokine receptor CCR4, is produced in the murine brain, particularly in a subset of hippocampal CA1 neurons. We found that basal expression of Ccl17 in hippocampal neurons was strongly enhanced by peripheral challenge with lipopolysaccharide (LPS). LPS-mediated induction of Ccl17 in the hippocampus was dependent on local tumor necrosis factor (TNF) signaling, whereas upregulation of Ccl22 required granulocyte-macrophage colony-stimulating factor (GM-CSF). CCL17 deficiency resulted in a diminished microglia density under homeostatic and inflammatory conditions. Further, microglia from naïve Ccl17-deficient mice possessed a reduced cellular volume and a more polarized process tree as assessed by computer-assisted imaging analysis. Regarding the overall branching, cell surface area, and total tree length, the morphology of microglia from naïve Ccl17-deficient mice resembled that of microglia from wild-type mice after LPS stimulation. In line, electrophysiological recordings indicated that CCL17 downmodulates basal synaptic transmission at CA3-CA1 Schaffer collaterals in acute slices from naïve but not LPS-treated animals. Taken together, our data identify CCL17 as a homeostatic and inducible neuromodulatory chemokine affecting the presence and morphology of microglia and synaptic transmission in the hippocampus.

RNA Aptamers Recognizing Murine CCL17 Inhibit T Cell Chemotaxis and Reduce Contact Hypersensitivity In Vivo.

The chemokine CCL17, mainly produced by dendritic cells (DCs) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers, which were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 toward CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevented ear swelling and T cell infiltration into the ears of sensitized mice after challenge with the contact sensitizer. The results of this proof-of-principle study establish aptamers as potent inhibitors of CCL17-mediated chemotaxis. Potentially, CCL17-specific aptamers may be used therapeutically in humans to treat or prevent allergic and inflammatory diseases.

Gut microbial translocation corrupts myeloid cell function to control bacterial infection during liver cirrhosis.

OBJECTIVE: Patients with liver cirrhosis suffer from increased susceptibility to life-threatening bacterial infections that cause substantial morbidity. METHODS: Experimental liver fibrosis in mice induced by bile duct ligation or CCl4 application was used to characterise the mechanisms determining failure of innate immunity to control bacterial infections. RESULTS: In murine liver fibrosis, translocation of gut microbiota induced tonic type I interferon (IFN) expression in the liver. Such tonic IFN expression conditioned liver myeloid cells to produce high concentrations of IFN upon intracellular infection with Listeria that activate cytosolic pattern recognition receptors. Such IFN-receptor signalling caused myeloid cell interleukin (IL)-10 production that corrupted antibacterial immunity, leading to loss of infection-control and to infection-associated mortality. In patients with liver cirrhosis, we also found a prominent liver IFN signature and myeloid cells showed increased IL-10 production after bacterial infection. Thus, myeloid cells are both source and target of IFN-induced and IL-10-mediated immune dysfunction. Antibody-mediated blockade of IFN-receptor or IL-10-receptor signalling reconstituted antibacterial immunity and prevented infection-associated mortality in mice with liver fibrosis. CONCLUSIONS: In severe liver fibrosis and cirrhosis, failure to control bacterial infection is caused by augmented IFN and IL-10 expression that incapacitates antibacterial immunity of myeloid cells. Targeted interference with the immune regulatory host factors IL-10 and IFN reconstitutes antibacterial immunity and may be used as therapeutic strategy to control bacterial infections in patients with liver cirrhosis.

Filarial Infection or Antigen Administration Improves Glucose Tolerance in Diet-Induced Obese Mice.

Helminths induce type 2 immune responses and establish an anti-inflammatory milieu in their hosts. This immunomodulation was previously shown to improve diet-induced insulin resistance which is linked to chronic inflammation. In the current study, we demonstrate that infection with the filarial nematode Litomosoides sigmodontis increased the eosinophil number and alternatively activated macrophage abundance within epididymal adipose tissue (EAT) and improved glucose tolerance in diet-induced obese mice in an eosinophil-dependent manner. L. sigmodontis antigen (LsAg) administration neither altered the body weight of animals nor adipose tissue mass or adipocyte size, but it triggered type 2 immune responses, eosinophils, alternatively activated macrophages, and type 2 innate lymphoid cells in EAT. Improvement in glucose tolerance by LsAg treatment remained even in the absence of Foxp3+ regulatory T cells. Furthermore, PCR array results revealed that LsAg treatment reduced inflammatory immune responses and increased the expression of genes related to insulin signaling (Glut4, Pde3b, Pik3r1, and Hk2) and fatty acid uptake (Fabp4 and Lpl). Our investigation demonstrates that L. sigmodontis infection and LsAg administration reduce diet-induced EAT inflammation and improve glucose tolerance. Helminth-derived products may, therefore, offer new options to improve insulin sensitivity, while loss of helminth infections in developing and developed countries may contribute to the recent increase in the prevalence of type 2 diabetes.

Chronic filarial infection provides protection against bacterial sepsis by functionally reprogramming macrophages.

Helminths immunomodulate their hosts and induce a regulatory, anti-inflammatory milieu that prevents allergies and autoimmune diseases. Helminth immunomodulation may benefit sepsis outcome by preventing exacerbated inflammation and severe pathology, but the influence on bacterial clearance remains unclear. To address this, mice were chronically infected with the filarial nematode Litomosoides sigmodontis (L.s.) and the outcome of acute systemic inflammation caused by i.p. Escherichia coli injection was determined. L.s. infection significantly improved E. coli-induced hypothermia, bacterial clearance and sepsis survival and correlated with reduced concentrations of associated pro-inflammatory cytokines/chemokines and a less pronounced pro-inflammatory macrophage gene expression profile. Improved sepsis outcome in L.s.-infected animals was mediated by macrophages, but independent of the alternatively activated macrophage subset. Endosymbiotic Wolbachia bacteria that are present in most human pathogenic filariae, as well as L.s., signal via TLR2 and modulate macrophage function. Here, gene expression profiles of peritoneal macrophages from L.s.-infected mice revealed a downregulation of genes involved in TLR signaling, and pulsing of macrophages in vitro with L.s. extract reduced LPS-triggered activation. Subsequent transfer improved sepsis outcome in naïve mice in a Wolbachia- and TLR2-dependent manner. In vivo, phagocytosis was increased in macrophages from L.s.-infected wild type, but not TLR2-deficient animals. In association, L.s. infection neither improved bacterial clearance in TLR2-deficient animals nor ameliorated E. coli-induced hypothermia and sepsis survival. These results indicate that chronic L.s. infection has a dual beneficial effect on bacterial sepsis, reducing pro-inflammatory immune responses and improving bacterial control. Thus, helminths and their antigens may not only improve the outcome of autoimmune and allergic diseases, but may also present new therapeutic approaches for acute inflammatory diseases that do not impair bacterial control.

Escherichia coli-induced immune paralysis is not exacerbated during chronic filarial infection.

Sepsis initially starts with a systemic inflammatory response (SIRS phase) and is followed by a compensatory anti-inflammatory response syndrome (CARS) that causes impaired adaptive T-cell immunity, immune paralysis and an increased susceptibility to secondary infections. In contrast, parasitic filariae release thousands of microfilariae into the peripheral blood without triggering inflammation, as they induce regulatory, anti-inflammatory host responses. Hence, we investigated the impact of chronic filarial infection on adaptive T-cell responses during the SIRS and CARS phases of a systemic bacterial infection and analysed the development of T-cell paralysis following a subsequent adenovirus challenge in BALB/c mice. Chronic filarial infection impaired adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ responses in the absence of a bacterial challenge and led to higher numbers of splenic CTLA-4(+)  CD4(+) T cells, whereas splenic T-cell expression of CD69 and CD62 ligand, serum cytokine levels and regulatory T-cell frequencies were comparable to naive controls. Irrespective of filarial infection, the SIRS phase dominated 6-24 hr after intravenous Escherichia coli challenge with increased T-cell activation and pro-inflammatory cytokine production, whereas the CARS phase occurred 6 days post E. coli challenge and correlated with high levels of transforming growth factor-ß and increased CD62 ligand T-cell expression. Escherichia coli-induced impairment of adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ production was not additionally impaired by chronic filarial infection. This suggests that filarial immunoregulation does not exacerbate E. coli-induced T-cell paralysis.

ST2 deficiency does not impair type 2 immune responses during chronic filarial infection but leads to an increased microfilaremia due to an impaired splenic microfilarial clearance.

BACKGROUND: Interactions of the Th2 cytokine IL-33 with its receptor ST2 lead to amplified Type 2 immune responses. As Type 2 immune responses are known to mediate protection against helminth infections we hypothesized that the lack of ST2 would lead to an increased susceptibility to filarial infections. METHODOLOGY/PRINCIPAL FINDING: ST2 deficient and immunocompetent BALB/c mice were infected with the filarial nematode Litomosoides sigmodontis. At different time points after infection mice were analyzed for worm burden and their immune responses were examined within the thoracic cavity, the site of infection, and systemically using spleen cells and plasma. Absence of ST2 led to significantly increased levels of peripheral blood microfilariae, the filarial progeny, whereas L. sigmodontis adult worm burden was not affected. Development of local and systemic Type 2 immune responses were not impaired in ST2 deficient mice after the onset of microfilaremia, but L. sigmodontis infected ST2-ko mice had significantly reduced total numbers of cells within the thoracic cavity and spleen compared to infected immunocompetent controls. Pronounced microfilaremia in ST2-ko mice did not result from an increased microfilariae release by adult female worms, but an impaired splenic clearance of microfilariae. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the absence of ST2 does not impair the establishment of adult L. sigmodontis worms, but is important for the splenic clearance of microfilariae from peripheral blood. Thus, ST2 interactions may be important for therapies that intend to block the transmission of filarial disease.

Helminth protection against autoimmune diabetes in nonobese diabetic mice is independent of a type 2 immune shift and requires TGF-ß.

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-ß alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-ß, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-ß.