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IL-6 plays a fundamental role in T cell differentiation and is strictly controlled by surface expression and shedding of IL-6R. IL-6 also acts on other cells that might affect T cell maturation. To study the impact of cell-autonomous and uncontrolled IL-6 signaling in T cells, we generated mice with a constitutively active IL-6R gp130 chain (Lgp130) expressed either in all T cells (Lgp130 × CD4Cre mice) or inducible in CD4+ T cells (Lgp130 × CD4CreERT2 mice). Lgp130 × CD4Cre mice accumulated activated T cells, including TH17 cells, in the lung, resulting in severe inflammation. Tamoxifen treatment of Lgp130 × CD4CreERT2 mice caused Lgp130 expression in 40–50% of CD4+ T cells, but mice developed lung disease only after several months. Lgp130+ CD4+ T cells were also enriched for TH17 cells; however, there was concomitant expansion of Lgp130− regulatory T cells, which likely restricted pathologic Lgp130+ T cells. In vitro, constitutive gp130 signaling in T cells enhanced but was not sufficient for TH17 cell differentiation. Augmented TH17 cell development of Lgp130+ T cells was also observed in Lgp130 × CD4CreERT2 mice infected with Staphylococcus aureus, but gp130 activation did not interfere with formation of TH1 cells against Listeria monocytogenes. Lgp130+ CD4+ T cells acquired a memory T cell phenotype and persisted in high numbers as a polyclonal T cell population in lymphoid and peripheral tissues, but we did not observe T cell lymphoma formation. In conclusion, cell-autonomous gp130 signaling alters T cell differentiation. Although gp130 signaling is not sufficient for TH17 cell differentiation, it still promotes accumulation of activated T cells in the lung that cause tissue inflammation.

The identification of tissue-resident memory T cells (TRMcells) has significantly improved our understanding of immunity. In the last decade, studies have demonstrated that TRMcells are induced after an acute T-cell response, remain in peripheral organs for several years, and contribute to both an efficient host defense and autoimmune disease. TRMcells are found in the kidneys of healthy individuals and patients with various kidney diseases. A better understanding of these cells and their therapeutic targeting might provide new treatment options for infections, autoimmune diseases, graft rejection, and cancer. In this review, we address the definition, phenotype, and developmental mechanisms of TRMcells. Then, we further discuss the current understanding of TRMcells in kidney diseases, such as infection, autoimmune disease, cancer, and graft rejection after transplantation.

γδ T cells are involved in the control of

γδ T cells are involved in the control of Staphylococcus aureus infection, but their importance in protection compared to other T cells is unclear. We used a mouse model of systemic S. aureus infection associated with high bacterial load and persistence in the kidney. Infection caused fulminant accumulation of γδ T cells in the kidney. Renal γδ T cells acquired tissue residency and were maintained in high numbers during chronic infection. At day 7, up to 50% of renal γδ T cells produced IL-17A in situ and a large fraction of renal γδ T cells remained IL-17A + during chronic infection. Controlled depletion revealed that γδ T cells restricted renal S. aureus replication in the acute infection and provided protection during chronic renal infection and upon reinfection. Our results demonstrate that kidney-resident γδ T cells are nonredundant in limiting local S. aureus growth during chronic infection and provide enhanced protection against reinfection.

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT “browning” were enhanced in Cd38−/− mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38−/− mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38−/− mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38−/− animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38−/− mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38−/− mice, which might contribute to enhanced BAT activation in Cd38−/− mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis.

In the past, proinflammatory CD11b

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca

Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca2+microdomains. Purinergic signaling is known to be involved in Ca2+influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca2+live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca2+signals but also promote initial Ca2+microdomains tens of milliseconds after T cell stimulation. These Ca2+microdomains were significantly decreased in T cells fromP2rx4−/−andP2rx7−/−mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1–dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca2+microdomains already in the first second of T cell activation.

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+signal that is crucial for T-cell function. The Ca2+channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived fromin vitrostudies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulationin vitro,Trpm2-/-and WT CD4+and CD8+T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation ofTrpm2-/-CD8+T cells and unimpaired differentiation of CD4+T cells into Th1, Th17, and Treg cells under specific polarizing conditions.In vivo,Trpm2-/-and WT CD8+T cells showed equal specific responses toListeria monocytogenesafter infection of WT andTrpm2-/-mice and after transfer of WT andTrpm2-/-CD8+T cells into infected recipients. CD4+T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT andTrpm2-/-CD4+T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

The formation of Ca

During mammalian pregnancy, immune cells are vertically transferred from mother to fetus. The functional role of these maternal microchimeric cells (MMc) in the offspring is mostly unknown. Here we show a mouse model in which MMc numbers are either normal or low, which enables functional assessment of MMc. We report a functional role of MMc in promoting fetal immune development. MMc induces preferential differentiation of hematopoietic stem cells in fetal bone marrow towards monocytes within the myeloid compartment. Neonatal mice with higher numbers of MMc and monocytes show enhanced resilience against cytomegalovirus infection. Similarly, higher numbers of MMc in human cord blood are linked to a lower number of respiratory infections during the first year of life. Our data highlight the importance of MMc in promoting fetal immune development, potentially averting the threats caused by early life exposure to pathogens., Maternal immune cells seed into the foetus during mammalian pregnancy, yet the functional role of these cells is unclear. Here the authors show that maternal immune cells in foetal bone marrow stimulate immune development, subsequently reducing the risk or severity of infections in newborns.

The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) is required for the maintenance of axonal integrity in neurons and is thought to regulate the intracellular pool of ubiquitin in the brain. In this study, we show that UCH-L1 has an immunological function in dendritic cell (DC) Ag cross-presentation. UCH-L1 is expressed in mouse kidney, spleen, and bone marrow–derived DCs, and its expression and activity are regulated by the immune stimuli LPS and IFN-γ. UCH-L1–deficient mice have significantly reduced ability to cross-prime CD8 T cells in vivo and in vitro because of a reduced ability of DCs to generate MHC class I (MHC I) peptide complexes for cross-presented Ags. Mechanistically, Ag uptake by phagocytosis and receptor-mediated endocytosis as well as phagosome maturation are unaffected by loss of UCH-L1 in DCs. Rather, MHC I recycling is reduced by loss of UCH-L1, which affects the colocalization of intracellular MHC I with late endosomal/lysosomal compartments necessary for cross-presentation of Ag. These results demonstrate a hitherto unrecognized role of the deubiquitinating enzyme UCH-L1 in DC Ag processing.

The transcription factor IRF4 is required for CD8(+) T cell activation, proliferation, and differentiation to effector cells and thus is essential for robust CD8(+) T cell responses. The function of IRF4 in memory CD8(+) T cells yet needs to be explored. To investigate the role of IRF4 for maintaining differentiation state and survival of CD8(+) memory T cells, we used a mouse model with tamoxifen-inducible Irf4 knockout to preclude effects due to inefficient memory cell differentiation in absence of IRF4. We infected mice with ovalbumin-recombinant listeria and induced Irf4 knockout after clearance of the pathogen. Loss of IRF4 resulted in phenotypical changes of CD8(+) memory T cells but did not cause a reduction of the total memory T cell population. However, upon reencounter of the pathogen, CD8(+) memory T cells showed impaired expansion and acquisition of effector functions. When compared to CD8(+) effector memory T cells, CD8(+) tissue-resident memory T cells (T(RM) cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8(+) T(RM)-cell populations, and tamoxifen-induced Irf4 deletion caused a reduction of this cell population. In conclusion, our results demonstrate that IRF4 is required for effective reactivation but not for general survival of CD8(+) memory T cells. Formation and maintenance of CD8(+) T(RM) cells, in contrast, appear to depend on IRF4.

The transcription factor IRF4 is required for CD8+ T cell activation, proliferation, and differentiation to effector cells and thus is essential for robust CD8+ T cell responses. The function of IRF4 in memory CD8+ T cells yet needs to be explored. To investigate the role of IRF4 for maintaining differentiation state and survival of CD8+ memory T cells, we used a mouse model with tamoxifen-inducible Irf4 knockout to preclude effects due to inefficient memory cell differentiation in absence of IRF4. We infected mice with ovalbumin-recombinant listeria and induced Irf4 knockout after clearance of the pathogen. Loss of IRF4 resulted in phenotypical changes of CD8+ memory T cells but did not cause a reduction of the total memory T cell population. However, upon reencounter of the pathogen, CD8+ memory T cells showed impaired expansion and acquisition of effector functions. When compared to CD8+ effector memory T cells, CD8+ tissue-resident memory T cells (TRM cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8+ TRM-cell populations, and tamoxifen-induced Irf4 deletion caused a reduction of this cell population. In conclusion, our results demonstrate that IRF4 is required for effective reactivation but not for general survival of CD8+ memory T cells. Formation and maintenance of CD8+ TRM cells, in contrast, appear to depend on IRF4.

Staphylococcus aureus is frequently detected in patients with sepsis and thus represents a major health burden worldwide. CD4+ T helper cells are involved in the immune response to S. aureus by supporting antibody production and phagocytosis. In particular, Th1 and Th17 cells secreting IFN-γ and IL-17A, are involved in the control of systemic S. aureus infections in humans and mice. To investigate the role of T cells in severe S. aureus infections, we established a mouse sepsis model in which the kidney was identified to be the organ with the highest bacterial load and abundance of Th17 cells. In this model, IL-17A but not IFN-γ was required for bacterial control. Using Il17aCre × R26YFP mice we could show that Th17 fate cells produce Th17 and Th1 cytokines, indicating a high degree of Th17 cell plasticity. Single cell RNA-sequencing of renal Th17 fate cells uncovered their heterogeneity and identified a cluster with a Th1 expression profile within the Th17 cell population, which was absent in mice with T-bet/Tbx21-deficiency in Th17 cells (Il17aCre x R26eYFP x Tbx21-flox). Blocking Th17 to Th1 transdifferentiation in Th17 fate cells in these mice resulted in increased S. aureus tissue loads. In summary, we highlight the impact of Th17 cells in controlling systemic S. aureus infections and show that T-bet expression by Th17 cells is required for bacterial clearance. While targeting the Th17 cell immune response is an important therapeutic option in autoimmunity, silencing Th17 cells might have detrimental effects in bacterial infections.

BackgroundHigh serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective.MethodsIL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1.control. Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6rafl/fl×LysMcre+ mice.ResultsOur therapeutic vaccination protocol elicits a strong tumor-specific CD8+ T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8+ T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6rafl/fl×LysMcre+ but not cre-negative control mice, while no differences in the vaccine-induced CD8+ T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages.ConclusionIL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections.

In anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), antibodies and T cells directed against the Goodpasture antigen, the non-collagenous domain of the α3-chain of type IV collagen (α3(IV)NC1), provoke renal inflammation resulting in rapidly progressing crescentic GN. Interleukin 6 (IL-6) is a pleiotropic cytokine with both pro- and anti-inflammatory activities, and IL-6 blockade is successfully used for treatment of diseases associated with acute and chronic inflammation. However, the role of IL-6 in anti-GBM GN is unclear. Here, we use the mouse model of experimental autoimmune glomerulonephritis (EAG) to study the role of IL-6 in anti-GBM GN. DBA/1J mice were immunized with α3(IV)NC1 and developed fatal crescentic GN. Treatment of mice with neutralizing anti-IL-6 antibodies impaired the generation of α3(VI)NC1-specific TH1 and TH17 cells. However, despite lasting reduction of the TH17 cell response, antibody treatment did not prevent crescentic GN. Antibody treatment was also ineffective in a therapeutic setting with pre-existing autoantibodies and T cells. In conclusion, our results indicate that although the blockade of IL-6 impairs the development of autoimmunity against α3(VI)NC1, this treatment does not ameliorate crescentic GN both in a preemptive and a therapeutic approach.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.

Different strategies to boost NAD

Background & Aims Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. Methods We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. Results Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. Conclusions Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. Lay summary Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (T

Different strategies to boost NAD+ levels are considered promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme involved in the regulation of different cell functions. In the context of systemic energy metabolism, it has been demonstrated that brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis. In this study we aim to understand the functional relevance of CD38 for NAD+ and energy metabolism in BAT and WAT, also using a CD38−/− mouse model. During cold exposure, an increase in NAD+ levels occurred in BAT of wild type mice, together with a marked downregulation of CD38, as detected at the mRNA and protein level. CD38 downregulation was observed also in WAT of cold-exposed mice, where it was accompanied by a strong increase in NADP(H) levels. Accordingly, NAD kinase and glucose-6-phosphate dehydrogenase activities were enhanced in WAT (but not in BAT). Increased NAD+ levels were observed in BAT/WAT from CD38−/− compared with wild type mice, in line with CD38 being a major NAD+-consumer in AT. CD38−/− mice kept at 6 °C had higher levels of Ucp1 and Pgc-1α in BAT and WAT, and increased levels of phosphorylated hormone-sensitive lipase in BAT, compared with wild type mice. These results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT.

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide with rising incidence. The inflammatory cytokine, interleukin-6 (IL-6), is a critical mediator of HCC development. It can signal through two distinct pathways: the IL-6 classic and the IL-6 trans-signaling pathway. Whereas IL-6 classic signaling is important for innate and acquired immunity, IL-6 trans-signaling has been linked to accelerated liver regeneration and several chronic inflammatory pathologies. However, its implication in liver tumorigenesis has not been addressed yet. Here, we show that IL-6 trans-signaling, but not IL-6 classic signaling, is essential to promote hepatocellular carcinogenesis by two mechanisms: First, it prevents DNA-damage-induced hepatocyte apoptosis through suppression of p53 and enhances β-catenin activation and tumor proliferation. Second, IL-6 trans-signaling directly induces endothelial cell proliferation to promote tumor angiogenesis. Consequently, soluble gp130 fused to Fc transgenic mice lacking IL-6 trans-signaling are largely protected from tumor formation in a diethylnitrosamine/3,3',5,5'-tetrachloro-1,4-bis(pyridyloxy)benzene model of HCC.IL-6 trans-signaling, and not IL-6 classic signaling, is mandatory for development of hepatocellular carcinogenesis. Therefore, specific inhibition of IL-6 trans-signaling, rather than total inhibition of IL-6 signaling, is sufficient to blunt tumor initiation and impair tumor progression without compromising IL-6 classic signaling-driven protective immune responses. (Hepatology 2017;65:89-103).

Pregnant women are at the highest risk to develop severe and even fatal influenza. The high vulnerability of women against influenza A virus infections during pregnancy was repeatedly highlighted during influenza pandemics including the pandemic of this century. In 2009, mortality rates were particularly high among otherwise healthy pregnant women. However, our current understanding of the molecular mechanisms involved in severe disease development during pregnancy is still very limited. In this review, we summarize the knowledge on the clinical observations in influenza A virus-infected pregnant women. In addition, knowledge obtained from few existing experimental infections in pregnant animal models is discussed. Since clinical data do not provide in-depth information on the pathogenesis of severe influenza during pregnancy, adequate animal models are urgently required that mimic clinical findings. Studies in pregnant animal models will allow the dissection of involved molecular disease pathways that are key to improve patient management and care.

UNLABELLED Death receptor (DR) ligands such as tumor necrosis factor (TNF) have been identified as fundamental mediators of liver damage both in mouse models and in humans. While the essential site of function of DR signaling is conceivably the hepatocyte, a systematic analysis is missing. Using mice with conditional gene ablation, we analyzed the tissue-specific function of DR signaling in T cell-dependent (concanavalin A) and independent (lipopolysaccharide/galactosamine) hepatitis and in models of bacterial infection (Listeria monocytogenes, lipopolysaccharide). We report that lipopolysaccharide/galactosamine-induced liver injury depends on hepatocyte-intrinsic TNF receptor 1 (p55, TNFR1). In contrast, we show that T cell-induced hepatitis was independent of TNFR1 signaling in hepatocytes, T cells, or endothelial cells. Moreover, T cell-induced hepatitis was independent of hepatocyte-intrinsic Fas-associated protein with death domain, TNF-related apoptosis-inducing ligand receptor, or Fas signaling. Instead, concanavalin A-induced hepatitis was completely prevented in mice with myeloid-derived cell (MDC)-specific deletion of TNFR1. Significantly, however, mice lacking TNFR1 in MDCs succumbed to listeria infection, although they displayed similar sensitivity toward endotoxin-induced septic shock when compared to control mice. These results suggest that TNFR1 signaling in MDCs is a critical mediator of both the detrimental and the protective functions of TNF in T cell-induced hepatitis and bacterial infection, respectively. CONCLUSION The critical site of action of DRs is completely dependent on the nature of hepatitis; the data specify MDCs as the essential cell type of TNFR1 function in T cell-mediated hepatitis and in the response to listeria, thereby identifying the opposing role of MDC TNFR1 in autoimmunity and bacterial infection. (Hepatology 2016;64:508-521).

IL-6 is required for the response of mice against Listeria monocytogenes. Control of infection depends on classical IL-6 signaling via membrane IL-6Rα, but IL-6 target cells and protective mechanisms remain unclear. We used mice with IL-6Rα-deficiency in T cells (Il6rafl/fl×CD4cre) or myeloid cells (Il6rafl/fl×LysMcre) to define the role of these cells in IL-6-mediated protection. Abrogation of IL-6Rα in T cells did not interfere with bacteria control and induction of TH1 and CD8+ T-cell responses. IL-6Rα-deficiency in myeloid cells caused significant defects in listeria control. This defect was not associated with reduced recruitment of granulocytes and inflammatory monocytes, and both cell populations were activated and not impaired in cytokine production. However, IL-6Rα-deficient inflammatory monocytes displayed diminished expression of IL-4Rα and of CD38, a protein required for phagocytosis and innate control of listeria. In vitro studies revealed that IL-4 and IL-6 cooperated in induction of CD38. In listeria-infected mice, phagocytic activity of inflammatory monocytes correlated with CD38 expression levels on cells and inflammatory monocytes of Il6rafl/fl×LysMcre mice were significantly impaired in phagocytosis. In conclusion, we demonstrate that inhibition of classical IL-6 signaling in myeloid cells causes alterations in differentiation and function of these cells, which subsequently prevent effective control of L. monocytogenes.

Chemokines and chemokine receptors are implicated in regulatory T cell (Treg) trafficking to sites of inflammation and suppression of excessive immune responses in inflammatory and autoimmune diseases; however, the specific requirements for Treg migration into the inflamed organs and the positioning of these cells within the tissue are incompletely understood. Here, we report that Tregs expressing the TH1-associated chemokine receptor CXCR3 are enriched in the kidneys of patients with ANCA-associated crescentic GN and colocalize with CXCR3(+) effector T cells. To investigate the functional role of CXCR3(+) Tregs, we generated mice that lack CXCR3 in Tregs specifically (Foxp3(eGFP-Cre) × Cxcr3(fl/fl)) and induced experimental crescentic GN. Treg-specific deletion of CXCR3 resulted in reduced Treg recruitment to the kidney and an overwhelming TH1 immune response, with an aggravated course of the nephritis that was reversible on anti-IFNγ treatment. Together, these findings show that a subset of Tregs expresses CXCR3 and thereby, acquires trafficking properties of pathogenic CXCR3(+) TH1 cells, allowing Treg localization and control of excessive TH1 responses at sites of inflammation.

IL-6 can mediate proinflammatory effects, and IL-6 receptor (IL-6R) blockade as a treatment for inflammatory diseases has entered clinical practice. However, opposing effects of IL-6 have been observed in models of GN. Although IL-6 is proinflammatory in murine lupus nephritis, protective effects have been observed for IL-6 in the nephrotoxic nephritis (NTN) model of acute crescentic GN. In light of the potential dangers of IL-6-directed treatment, we studied the mechanisms underlying the contradictory findings in GN. IL-6 can signal through the membrane-bound IL-6R, which is expressed only on hepatocytes and certain leukocytes (classic), or through the soluble IL-6R, which binds the ubiquitously expressed gp130 (alternative). Preemptive treatment of mice with anti-IL-6R or anti-IL-6 worsened NTN, whereas selective blockade of alternative IL-6 signaling by the fusion protein sgp130Fc did not. FACS analysis of mouse spleen cells revealed proinflammatory macrophages express the highest levels of IL-6Rα, and in vitro treatment with IL-6 blocked macrophage proliferation. Furthermore, proinflammatory macrophages were expanded during inflammation in IL-6(-/-) mice. Late application of anti-IL-6 after establishment of adaptive nephritogenic immunity was sufficient to aggravate NTN within 2.5 days, a period when macrophages are active. Finally, NTN was aggravated in mice with macrophage-specific impairment of IL-6 classic signaling, coincident with enhanced macrophage proliferation and accumulation in the kidney. Our data thus reveal a novel mechanism in which IL-6-mediated dampening of macrophage activation protects tissues from overshooting immune responses. This finding has important implications for potential IL-6-directed therapies and supports the careful choice of recipient patients and timing.

Autoimmunity against the Goodpasture antigen α3IV-NC1 results in crescentic glomerulonephritis (GN). Both antibodies and T cells directed against α3IV-NC1 have been implicated in disease development and progression. Using the model of experimental autoimmune glomerulonephritis (EAG) in DBA/1 mice, we aimed to characterize the frequency and function of α3IV-NC1-specific CD4(+) T cells in the kidneys. DBA/1 mice repeatedly immunized with human α3IV-NC1 developed necrotizing/crescentic GN. Kidneys with crescentic GN contained CD4(+) cells responding to α3IV-NC1 with the production of IFN-γ or IL-17A, demonstrating the accumulation of both α3IV-NC1-specific TH1 and TH17 cells. To test the functional relevance of TH1 and TH17 cells, EAG was induced in DBA/1 mice deficient in IFN-γR, IL-17A or IL-23p19. Mice of all knockout groups mounted α3IV-NC1 IgG, developed nephrotic range proteinuria, and IgG deposition to the glomerular basement membranes at levels similar to immunized wild-type mice. However, all knockout groups showed significantly fewer glomerular crescents and attenuated tubulointerstitial damage. Our results suggest that both α3IV-NC1-specific TH1 and TH17 cells accumulate in the kidneys and are crucial for the development of necrotizing/crescentic GN.

Regulatory T cells (Tregs) use different mechanisms to exert their suppressive function, among them the conversion of ATP to adenosine initiated by the ectonucleotidase CD39. In humans, the expression of CD39 on Tregs shows a high interindividual variation, and is especially high at sites of inflammation, like the synovia of patients with arthritis. How CD39 expression is regulated, and the functional consequences of different levels of CD39 expression is not known. We show here that stimulation of CD39 − Tregs results in a modest upregulation of CD39, which cannot explain the high levels observed in many donors. Moreover, CD39 + Tregs are present in naive compartments such as cord blood and thymus, and the individual frequency of CD39 + Tregs remains stable over time, suggesting inherent regulation of CD39 expression. Indeed, we show that a single nucleotide polymorphism in the CD39 gene determines expression levels in Tregs. CD39 + Tregs suppress T cell proliferation and inflammatory cytokine production more efficiently than CD39 − Tregs. Accordingly, Tregs from donors with the GG (high CD39) genotype have a higher capacity to suppress IFN-γ and IL-17 production by effector cells than Tregs from AA (low CD39) donors. Our study demonstrates that the expression of CD39 in Tregs is primarily genetically driven, and this may determine interindividual differences in the control of inflammatory responses.

Objective The CD4+ T cell immune response plays a pivotal role in the immunopathogenesis of human and experimental lupus nephritis, but the contribution of the Th17/interleukin-17 (IL-17) immune pathway to renal tissue injury in systemic lupus erythematosus (SLE) remains to be elucidated. The aim of this study was to characterize the function of the Th17/IL-17A immune response in 2 murine models of lupus nephritis. Methods IL-17A–deficient MRL/MPJ-Faslpr/2J (MRL/lpr) mice were generated, and the clinical course of nephritis was monitored by assessing the levels of albuminuria, extent of renal tissue injury, and functional parameters. In addition, lupus-prone (NZB × NZW)F1 (NZB/NZW) mice were treated with anti–IL-17A and anti–interferon-γ (anti-IFNγ) antibodies, and their effects on the clinical course of lupus nephritis were assessed. Results Characterization of renal IL-17A–producing and IFNγ-producing T cells in MRL/lpr and NZB/NZW mice revealed low numbers of infiltrating CD3+IL-17A+ cells. Renal IL-17A was mainly produced by CD4/CD8 double-negative CD3+ T cells and CD4+ Th17 cells. In contrast, the number of renal CD3+IFNγ+ cells continuously increased over time and largely consisted of typical CD4+ Th1 cells. IL-17A deficiency did not affect the morphologic or functional parameters in MRL/lpr mice with lupus nephritis, nor did IL-17A neutralization affect the clinical course of nephritis in NZB/NZW mice, but anti-IFNγ treatment attenuated the severity of the disease. Conclusion The Th17/IL-17A immune response plays no major role in the immunopathogenesis of lupus nephritis in MRL/lpr and NZB/NZW mice. Thus, the results of this study do not support the hypothesis that IL-17A targeting could be an intriguing new therapeutic approach for the management of proliferative lupus nephritis in SLE patients.

M6P-dependent transport routes are essential for B cell functions in vivo and humoral immunity in mouse and human., Antigen processing and presentation and cytotoxic targeting depend on the activities of several lysosomal enzymes that require mannose 6-phosphate (M6P) sorting signals for efficient intracellular transport and localization. In this paper, we show that mice deficient in the formation of M6P residues exhibit significant loss of cathepsin proteases in B cells, leading to lysosomal dysfunction with accumulation of storage material, impaired antigen processing and presentation, and subsequent defects in B cell maturation and antibody production. The targeting of lysosomal and granular enzymes lacking M6P residues is less affected in dendritic cells and T cells and sufficient for maintenance of degradative and lytic functions. M6P deficiency also impairs serum immunoglobulin levels and antibody responses to vaccination in patients. Our data demonstrate the critical role of M6P-dependent transport routes for B cell functions in vivo and humoral immunity in mice and human.

It has become evident that nonlymphoid tissues are populated by distinct subsets of innate and adaptive lymphocytes that are characterized by minimal exchange with recirculating counterparts. Especially at barrier sites, such as the skin, gut, and lung, these tissue-resident lymphocyte populations are ideally positioned to quickly respond to pathogens and other environmental stimuli. The kidney harbors several classes of innate and innate-like lymphocytes that have been described to contribute to this tissue-resident population in other organs, including innate lymphoid cells, natural killer cells, natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Additionally, a substantial proportion of the adaptive lymphocytes that are found in the kidney displays a surface phenotype suggestive of tissue residency, such as CD69+CD4+ T cells. In this review, we summarize recent advances in the understanding of tissue-resident lymphocyte populations, review the available evidence for the existence of these populations in the kidney, and discuss the potential physiologic and pathophysiologic roles thereof in kidney.

Prenatal glucocorticoids are routinely administered to pregnant women at risk of preterm delivery in order to improve survival of the newborn. However, in half of the cases, birth occurs outside the beneficial period for lung development. Glucocorticoids are potent immune modulators and cause apoptotic death of immature T cells, and we have previously shown that prenatal betamethasone treatment at doses eliciting lung maturation induce profound thymocyte apoptosis in the offspring. Here, we asked if there are long-term consequences on the offspring's immunity after this treatment. In the non-obese diabetic mouse model, prenatal betamethasone clearly decreased the frequency of pathogenic T cells and the incidence of type 1 diabetes (T1D). In contrast, in the lupus-prone MRL/lpr strain, prenatal glucocorticoids induced changes in the T cell repertoire that resulted in more autoreactive cells. Even though glucocorticoids transiently enhanced regulatory T cell (Treg) development, these cells did not have a protective effect in a model for multiple sclerosis which relies on a limited repertoire of pathogenic T cells for disease induction that were not affected by prenatal betamethasone. We conclude that prenatal steroid treatment, by inducing changes in the T cell receptor repertoire, has unforeseeable consequences on development of autoimmune disease. Our data should encourage further research to fully understand the consequences of this widely used treatment.

A pathogenic role for Th17 cells in inflammatory renal disease is well established. The mechanisms underlying their counter-regulation are, however, largely unknown. Recently, Th17 lineage-specific regulatory T cells (Treg17) that depend on activation of the transcription factor Stat3 were identified. We studied the function of Treg17 in the nephrotoxic nephritis (NTN) model of crescentic GN. The absence of Treg17 cells in Foxp3(Cre)×Stat3(fl/fl) mice resulted in the aggravation of NTN and skewing of renal and systemic immune responses toward Th17. Detailed analysis of Stat3-deficient Tregs revealed that the survival, activation, proliferation, and suppressive function of these cells remained intact. However, Tregs from Foxp3(Cre)×Stat3(fl/fl) mice lacked surface expression of the chemokine receptor CCR6, which resulted in impaired renal trafficking. Furthermore, aggravation of NTN was reversible in the absence of Th17 responses, as shown in CD4(Cre)×Stat3(fl/fl) mice lacking both Treg17 and Th17 cells, suggesting that Th17 cells are indeed the major target of Treg17 cells. Notably, immunohistochemistry revealed CCR6-bearing Treg17 cells in kidney biopsy specimens of patients with GN. CCR6 expression on human Treg17 cells also appears dependent on STAT3, as shown by analysis of Tregs from patients with dominant-negative STAT3 mutations. Our data indicate the presence and involvement of Stat3/STAT3-dependent Treg17 cells that specifically target Th17 cells in murine and human crescentic GN, and suggest the kidney-specific action of these Treg17 cells is regulated by CCR6-directed migration into areas of Th17 inflammation.

It is well established that CD8(+) T cells constitute an important branch of adaptive immunity contributing to clearance of intracellular pathogens and providing long-term protection. These functions are mostly fulfilled by the best characterized subpopulation of CD8(+) T cells, the cytotoxic T lymphocytes (also called Tc1 cells), owing to their ability to kill infected cells and to secrete cytokines such as interferon-γ and tumor necrosis factor-α. However, there is growing evidence for alternative CD8(+) T cell fates influencing CD4(+) T-cell-mediated responses in the context of allergy, autoimmunity and infections. Thus, like subpopulations of CD4(+) T cells, also CD8(+) T cells under particular conditions acquire the expression of interleukin (IL)-4, IL-5, IL-9, IL-13, IL-17 or suppressive activity and thereby influence immune responses. The process of CD8(+) T-cell differentiation is dictated by antigen strength, co-stimulatory molecules and cytokines. These environmental cues induce transcription factors further specifying CD8(+) T-cell decision into Tc1, Tc2, Tc9, Tc17 or CD8(+) T regulatory fate. Here, we discuss our current understanding about functional diversity of effector CD8(+) T cells and contribution of transcription factors to this process.

The intestinal microbiota influences not only metabolic processes, but also the mucosal and systemic immune systems. Here, we compare innate and adaptive immune responses against the intracellular pathogen Listeria monocytogenes in germfree (GF) and conventional mice. We show that animals without endogenous microbiota are highly susceptible to primary infection with impaired activation and accumulation of phagocytes to the site of infection. Unexpectedly, secondary infection with otherwise lethal dose resulted in survival of all GF animals which cleared bacteria more rapidly and developed a stronger antilisterial CD8+ memory T-cell response compared to conventional mice. In summary, lack of the intestinal microbiota impairs early innate immunity, but enhances activation and expansion of memory T cells.

Successful host defense against numerous pulmonary infections depends on bacterial clearance by polymorphonuclear leukocytes (PMNs); however, excessive PMN accumulation can result in life-threatening lung injury. Local expression of CXC chemokines is critical for PMN recruitment. The impact of chemokine-dependent PMN recruitment during pulmonary Mycobacterium tuberculosis infection is not fully understood. Here, we analyzed expression of genes encoding CXC chemokines in M. tuberculosis-infected murine lung tissue and found that M. tuberculosis infection promotes upregulation of Cxcr2 and its ligand Cxcl5. To determine the contribution of CXCL5 in pulmonary PMN recruitment, we generated Cxcl5(-/-) mice and analyzed their immune response against M. tuberculosis. Both Cxcr2(-/-) mice and Cxcl5(-/-) mice, which are deficient for only one of numerous CXCR2 ligands, exhibited enhanced survival compared with that of WT mice following high-dose M. tuberculosis infection. The resistance of Cxcl5(-/-) mice to M. tuberculosis infection was not due to heightened M. tuberculosis clearance but was the result of impaired PMN recruitment, which reduced pulmonary inflammation. Lung epithelial cells were the main source of CXCL5 upon M. tuberculosis infection, and secretion of CXCL5 was reduced by blocking TLR2 signaling. Together, our data indicate that TLR2-induced epithelial-derived CXCL5 is critical for PMN-driven destructive inflammation in pulmonary tuberculosis.

PROBLEM: Steroid hormones such as progesterone and glucocorticoids rise during pregnancy and are accountable for the adaptation of the maternal immune system to pregnancy. How steroid hormones induce fetal tolerance is not fully understood. We hypothesized that steroid hormones selectively regulate the T cell response by promoting T cell death. METHOD OF STUDY: We incubated murine spleen cells isolated from non-pregnant and pregnant mice with physiological concentrations of steroid hormones in vitro and analyzed T cell subsets after 48 h of incubation. RESULTS: We found that progesterone and the synthetic glucocorticoid dexamethasone induce T cell death. CD4(+) regulatory T cells (T(reg)) were refractory towards progesterone-induced cell death, in contrast to conventional CD4(+) T cells, which resulted in a preferential enrichment of CD4(+) T(reg) cells in culture. T cells isolated from pregnant mice at early and late gestation showed comparable sensitivity to steroid-induced cell death. The target receptor for progesterone in immune cells is controversially discussed. We provide here support of progesterone binding to the glucocorticoid receptor as only T cells lacking the glucocorticoid but not the progesterone receptor showed resistance against progesterone-induced death. CONCLUSIONS: Our results indicate that high levels of progesterone during pregnancy can induce selective T cell death by binding the glucocorticoid receptor. Although physiological hormone concentrations were used, due to different bioavailability of steroid hormones in vivo these results have to be validated in an in vivo model. This mechanism might ensure immunological tolerance at the feto-maternal interface at gestation.

Robust cytotoxic CD8 + T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8 + T-cell response to the intracellular bacterium Listeria monocytogenes . IRF4-deficient ( Irf4 −/− ) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenes -specific CD8 + T cells with impaired effector phenotype and function. Transfer of wild-type CD8 + T cells into Irf4 −/− mice improved bacterial clearance, suggesting an intrinsic defect of CD8 + T cells in Irf4 −/− mice. Following transfer into wild-type recipients, Irf4 −/− CD8 + T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4 −/− CD8 + T cells rescued the defect. During acute infection, Irf4 −/− CD8 + T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4 −/− CD8 + T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4 −/− CD8 + T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4 + T-cell differentiation, the identification of its decisive role in peripheral CD8 + T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.

The CD8αβ coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8(+) T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD(+) to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD(+) , ART2.2 caused ADP-ribosylation of CD8-β on murine CD8(+) T cells in vitro and in vivo. Treatment with NAD(+) prevented binding of anti-CD8-β mAb YTS156.7.7 but not of mAb H35-17.2, indicating that NAD(+) caused modification of certain epitopes and not a general loss of CD8-β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-β occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8(+) T cells with NAD(+) interfered with binding of OVA257-264 :MHC-I tetramers. In line with this result, treatment of WT mice with NAD(+) resulted in reduced CD8(+) T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD(+) .

Pregnant women are at high risk for severe influenzadisease outcomes, yet insights into the underlyingmechanisms are limited. Here, we present models ofH1N1 infection in syngenic and allogenic pregnantmice; infection in the latter mirrors the severe courseof 2009 pandemic influenza in pregnant women. Wefound that the anti-viral immune response in the pregnanthost was significantly restricted as compared tothe non-pregnant host. This included a reduced type Iinterferon response as well as impaired migration ofCD8+ T cells into the lung. The multi-faceted failureto mount an anti-viral response in allogenic pregnantmice resulted in a less stringent selective environmentthat promoted the emergence of 2009 H1N1 virusvariants that specifically counteract type I interferonresponse and mediate increased viral pathogenicity.These insights underscore the importance of influenzavaccination compliance in pregnant womenand may open novel therapeutic avenues. Fil: Engels, Géraldine. University Medical Center Hamburg-Eppendorf; Alemania. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Hierweger, Alexandra Maximiliane. University Medical Center Hamburg-Eppendorf; Alemania Fil: Hoffmann, Julia. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Thieme, René. University Medical Center Hamburg-Eppendorf; Alemania Fil: Thiele, Swantje. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Bertram, Stephanie. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Dreier, Carola. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Resa-Infante, Patricia. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Jacobsen, Henning. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Thiele, Kristin. University Medical Center Hamburg-Eppendorf; Alemania Fil: Alawi, Malik. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Indenbirken, Daniela. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Grundhoff, Adam. Leibniz Institut Für Experimentelle Virologie; Alemania Fil: Siebels, Svenja. University Medical Center Hamburg-Eppendorf; Alemania Fil: Fischer, Nicole. University Medical Center Hamburg-Eppendorf; Alemania Fil: Stojanovska, Violeta. University of Groningen; Países Bajos Fil: Muzzio, Damián Oscar. University of Greifswald; Alemania Fil: Jensen, Cristian Federico. University of Greifswald; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Karimi, Khalil. University Medical Center Hamburg-Eppendorf; Alemania Fil: Mittrücker, Hans-Willi. University Medical Center Hamburg-Eppendorf; Alemania Fil: Arck, Petra Clara. University Medical Center Hamburg-Eppendorf; Alemania Fil: Gabriel, Gülsah. Leibniz Institut Für Experimentelle Virologie; Alemania