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Abstract Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a life-threatening autoimmune disease that often results in kidney failure caused by crescentic glomerulonephritis (GN). To date, treatment of most patients with ANCA-GN relies on non-specific immunosuppressive agents, which may have serious adverse effects and be only partially effective. Here, using spatial and single-cell transcriptome analysis, we characterize inflammatory niches in kidney samples from 34 patients with ANCA-GN and identify proinflammatory, cytokine-producing CD4+ and CD8+ T cells as a pathogenic signature. We then utilize these transcriptomic profiles for digital pharmacology and identify ustekinumab, a monoclonal antibody targeting IL-12 and IL-23, as the strongest therapeutic drug to use. Moreover, four patients with relapsing ANCA-GN are treated with ustekinumab in combination with low-dose cyclophosphamide and steroids, with ustekinumab given subcutaneously (90 mg) at weeks 0, 4, 12, and 24. Patients are followed up for 26 weeks to find this treatment well-tolerated and inducing clinical responses, including improved kidney function and Birmingham Vasculitis Activity Score, in all ANCA-GN patients. Our findings thus suggest that targeting of pathogenic T cells in ANCA-GN patients with ustekinumab might represent a potential approach and warrants further investigation in clinical trials.

CD8+ T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca2+ microdomains in CD4+ T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8+ T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca2+ microdomains, global Ca2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8+ T cells lacking P2X4 and P2X7 channels, global Ca2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca2+ concentration ([Ca2+]i). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8+ T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8+ T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca2+ events during CD8+ T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions.

IntroductionDuring thermogenesis, adipose tissue (AT) becomes more active and enhances oxidative metabolism. The promotion of this process in white AT (WAT) is called “browning” and, together with the brown AT (BAT) activation, is considered as a promising approach to counteract obesity and metabolic diseases. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), is an ion channel that allows extracellular Ca2+ influx into the cytosol, and is gated by adenosine diphosphate ribose (ADPR), produced from NAD+ degradation. The aim of this study was to investigate the relevance of TRPM2 in the regulation of energy metabolism in BAT, WAT, and liver during thermogenesis.MethodsWild type (WT) and Trpm2-/- mice were exposed to 6°C and BAT, WAT and liver were collected to evaluate mRNA, protein levels and ADPR content. Furthermore, O2 consumption, CO2 production and energy expenditure were measured in these mice upon thermogenic stimulation. Finally, the effect of the pharmacological inhibition of TRPM2 was assessed in primary adipocytes, evaluating the response upon stimulation with the β-adrenergic receptor agonist CL316,243.ResultsTrpm2-/- mice displayed lower expression of browning markers in AT and lower energy expenditure in response to thermogenic stimulus, compared to WT animals. Trpm2 gene overexpression was observed in WAT, BAT and liver upon cold exposure. In addition, ADPR levels and mono/poly-ADPR hydrolases expression were higher in mice exposed to cold, compared to control mice, likely mediating ADPR generation.DiscussionOur data indicate TRPM2 as a fundamental player in BAT activation and WAT browning. TRPM2 agonists may represent new pharmacological strategies to fight obesity.

The transcription factor Interferon Regulatory Factor 4 (IRF4) is central in control of T cell activation and differentiation. Deficiency of IRF4 results in severe immune deficiency and affects maturation and function of most if not all T cell subsets. Here we use mouse infection models for Citrobacter rodentium and Strongyloides ratti to analyze the function of IRF4 in T helper (Th) 17 and Th2 cell responses, respectively. IRF4 deficient mice were impaired in the control of both pathogens, failed to mount Th17 and Th2 cell responses and showed impaired recruitment of T helper cells to the intestine, the infection site of both pathogens. Compromised intestinal migration was associated with reduced expression of the intestinal homing receptors α4β7 integrin, CCR9 and GPR15. Identification of IRF4 binding sites in the gene loci of these receptors suggests a direct control of their expression by IRF4. Competitive T cell transfer assays further demonstrated that loss of one functional Irf4 allele already affected intestinal accumulation and Th2 and Th17 cell generation, indicating that lower IRF4 levels are of disadvantage for Th2 and Th17 cell differentiation as well as their migration to the intestine. Conversion of peripheral CD4+ T cells from an Irf4 wildtype to an Irf4 heterozygous or from an Irf4 heterozygous to a homozygous mutant genotype after C. rodentium or S. ratti infection did not reduce their capacity to produce Th17 or Th2 cytokines and only partially affected their persistence in the intestine, revealing that IRF4 is not essential for maintenance of the Th2 and Th17 phenotype and for survival of these T helper cells in the intestine. In conclusion, we demonstrate that the expression levels of IRF4 determine Th2 and Th17 cell differentiation and their intestinal accumulation but that IRF4 expression is not crucial for Th2 and Th17 cell survival.

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.

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 from in vitro studies 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 stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- 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 to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- 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 and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

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+Ly6Chi monocytes were predominantly considered as a uniform population. However, recent investigations suggests that this population is far more diverse than previously thought. For example, in mouse models of Entamoeba (E.) histolytica and Listeria (L.) monocytogenes liver infections, it was shown that their absence had opposite effects. In the former model, it ameliorated parasite-dependent liver injury, whereas in the listeria model it exacerbated liver pathology. Here, we analyzed Ly6Chi monocytes from the liver of both infection models at transcriptome, protein, and functional levels. Paralleled by E. histolytica- and L. monocytogenes-specific differences in recruitment-relevant chemokines, both infections induced accumulation of Ly6C+ monocytes at infection sites. Transcriptomic analysis revealed a high similarity between monocytes from naïve and parasite-infected mice and a clear proinflammatory phenotype of listeria-induced monocytes. This was further reflected by the upregulation of M2-related transcription factors (e.g., Mafb, Nr4a1, Fos) and higher CD14 expression by Ly6Chi monocytes in the E. histolytica infection model. In contrast, monocytes from the listeria infection model expressed M1-related transcription factors (e.g., Irf2, Mndal, Ifi204) and showed higher expression of CD38, CD74, and CD86, as well as higher ROS production. Taken together, proinflammatory Ly6Chi monocytes vary considerably depending on the causative pathogen. By using markers identified in the study, Ly6Chi monocytes can be further subdivided into different populations.