A type 1 immunity-restricted promoter of the IL-33 receptor gene directs antiviral T-cell responses.

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33-ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33-ST2 axis in infections and inflammatory diseases.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals and that single-nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs) but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33's influence on IL-22-dependent antibacterial defense was dependent on housing conditions of the mice and mediated by IL-33's modulatory effect on the gut microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We conclude that both genetic and environmental factors influence the gut microbiota, thereby impacting the efficacy of antibacterial immune defense and susceptibility to pneumonia.

NOS inhibition reverses TLR2-induced chondrocyte dysfunction and attenuates age-related osteoarthritis.

Osteoarthritis (OA) is a joint disease featuring cartilage breakdown and chronic pain. Although age and joint trauma are prominently associated with OA occurrence, the trigger and signaling pathways propagating their pathogenic aspects are ill defined. Following long-term catabolic activity and traumatic cartilage breakdown, debris accumulates and can trigger Toll-like receptors (TLRs). Here we show that TLR2 stimulation suppressed the expression of matrix proteins and induced an inflammatory phenotype in human chondrocytes. Further, TLR2 stimulation impaired chondrocyte mitochondrial function, resulting in severely reduced adenosine triphosphate (ATP) production. RNA-sequencing analysis revealed that TLR2 stimulation upregulated nitric oxide synthase 2 (NOS2) expression and downregulated mitochondria function-associated genes. NOS inhibition partially restored the expression of these genes, and rescued mitochondrial function and ATP production. Correspondingly, Nos2-/- mice were protected from age-related OA development. Taken together, the TLR2-NOS axis promotes human chondrocyte dysfunction and murine OA development, and targeted interventions may provide therapeutic and preventive approaches in OA.

Individual T helper cells have a quantitative cytokine memory.

The probabilistic expression of cytokine genes in differentiated T helper (Th) cell populations remains ill defined. By single-cell analyses and mathematical modeling, we show that one stimulation featured stable cytokine nonproducers as well as stable producers with wide cell-to-cell variability in the magnitude of expression. Focusing on interferon-γ (IFN-γ) expression by Th1 cells, mathematical modeling predicted that this behavior reflected different cell-intrinsic capacities and not mere gene-expression noise. In vivo, Th1 cells sort purified by secreted IFN-γ amounts preserved a quantitative memory for both probability and magnitude of IFN-γ re-expression for at least 1 month. Mechanistically, this memory resulted from quantitatively distinct transcription of individual alleles and was controlled by stable expression differences of the Th1 cell lineage-specifying transcription factor T-bet. Functionally, Th1 cells with graded IFN-γ production competence differentially activated Salmonella-infected macrophages for bacterial killing. Thus, individual Th cells commit to produce distinct amounts of a given cytokine, thereby generating functional intrapopulation heterogeneity.

Superoxide Dismutase 1 Protects Hepatocytes from Type I Interferon-Driven Oxidative Damage.

Tissue damage caused by viral hepatitis is a major cause of morbidity and mortality worldwide. Using a mouse model of viral hepatitis, we identified virus-induced early transcriptional changes in the redox pathways in the liver, including downregulation of superoxide dismutase 1 (Sod1). Sod1(-/-) mice exhibited increased inflammation and aggravated liver damage upon viral infection, which was independent of T and NK cells and could be ameliorated by antioxidant treatment. Type I interferon (IFN-I) led to a downregulation of Sod1 and caused oxidative liver damage in Sod1(-/-) and wild-type mice. Genetic and pharmacological ablation of the IFN-I signaling pathway protected against virus-induced liver damage. These results delineate IFN-I mediated oxidative stress as a key mediator of virus-induced liver damage and describe a mechanism of innate-immunity-driven pathology, linking IFN-I signaling with antioxidant host defense and infection-associated tissue damage. VIDEO ABSTRACT.

Th2 cells lacking T-bet suppress naive and memory T cell responses via IL-10.

Exacerbated immune responses and loss of self-tolerance lead to the development of autoimmunity and immunopathology. Novel therapies to target autoreactive T cells are still needed. Here, we report that Th2-polarized T cells lacking the transcription factor T-bet harbor strong immunomodulatory potential and suppress antigen-specific CD8+ T cells via IL-10. Tbx21-/- Th2 cells protected mice against virus-induced type 1 diabetes development and suppressed not only naive but also memory CD8+ T cell responses. IL-10-producing, but not IL-10-deficient Tbx21-/- Th2 cells down-regulated costimulatory molecules on dendritic cells and reduced their IL-12 production after lymphocytic choriomeningitis virus infection. Impaired dendritic cell activation hindered effector and cytotoxic CD8+ T cell development after infection. These findings indicate that Tbx21-/- Th2 cells strongly suppress proinflammatory responses of naive and memory T cells via IL-10. Thus, in vivo IL-10-secreting Th2 cells could harbor a therapeutic potential for the treatment of T cell-mediated inflammatory disorders.

Vaccine-elicited CD4 T cells prevent the deletion of antiviral B cells in chronic infection.

Chronic viral infections subvert protective B cell immunity. An early type I interferon (IFN-I)-driven bias to short-lived plasmablast differentiation leads to clonal deletion, so-called "decimation," of antiviral memory B cells. Therefore, prophylactic countermeasures against decimation remain an unmet need. We show that vaccination-induced CD4 T cells prevented the decimation of naïve and memory B cells in chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. Although these B cell responses were largely T independent when IFN-I was blocked, preexisting T help assured their sustainability under conditions of IFN-I-driven inflammation by instructing a germinal center B cell transcriptional program. Prevention of decimation depended on T cell-intrinsic Bcl6 and Tfh progeny formation. Antigen presentation by B cells, interactions with antigen-specific T helper cells, and costimulation by CD40 and ICOS were also required. Importantly, B cell-mediated virus control averted Th1-driven immunopathology in LCMV-challenged animals with preexisting CD4 T cell immunity. Our findings show that vaccination-induced Tfh cells represent a cornerstone of effective B cell immunity to chronic virus challenge, pointing the way toward more effective B cell-based vaccination against persistent viral diseases.

Eosinophils are required for the maintenance of plasma cells in the bone marrow.

Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils localized together with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported the survival of plasma cells by secreting the proliferation-inducing ligand APRIL and interleukin-6 (IL-6). In eosinophil-deficient mice, plasma cell numbers were much lower in the bone marrow both at steady state and after immunization. Reconstitution experiments showed that eosinophils were crucial for the retention of plasma cells in the bone marrow. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. Our findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils.

Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus.

The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.

Type I interferon protects antiviral CD8+ T cells from NK cell cytotoxicity.

Despite development of new antiviral drugs, viral infections are still a major health problem. The most potent antiviral defense mechanism is the innate production of type I interferon (IFN-I), which not only limits virus replication but also promotes antiviral T cell immunity through mechanisms, which remain insufficiently studied. Using the murine lymphocytic choriomeningitis virus model system, we show here that IFN-I signaling on T cells prevented their rapid elimination in vivo. Microarray analyses uncovered that IFN-I triggered the expression of selected inhibitory NK-cell-receptor ligands. Consequently, T cell immunity of IFN-I receptor (IFNAR)-deficient T cells could be restored by NK cell depletion or in NK-cell-deficient hosts (Nfil3(-/-)). The elimination of Ifnar1(-/-) T cells was dependent on NK-cell-mediated perforin expression. In summary, we identified IFN-I as a key player regulating the protection of T cells against regulatory NK cell function.

The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes.

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor-mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell-mediated immune responses and open new therapeutic possibilities.

[The DRFZ-a pioneer in research on the interaction between immune and stromal cells during de- and regeneration of the musculoskeletal system]. / Das DRFZ ­ ein Vorreiter bei der Erforschung des Zusammenspiels von Immun- und Stromazellen bei der De- und Regeneration des Bewegungsapparats.

Rheumatoid arthritis and osteoarthritis are two related chronic diseases of the musculoskeletal system which are particularly pronounced in the region of joints and bones. Their pathogeneses are associated with chronic inflammation, which can disrupt homeostasis in bones and articular cartilage. Degradation products deriving from articular cartilage can contribute to the exacerbation of inflammation in the joint region. Mechanical stimuli and blood vessels also play a central role in both the regulation of bone growth as well as in the regeneration of bone tissue. Not only chronic inflammatory processes but also hormonal changes after menopause or undesired effects of glucocorticoid therapy have an influence on the balance between bone resorption and deposition, by promoting the former and reducing the latter. This results in decreased bone quality and, in some cases, considerable loss of bone or osteoporosis. An in-depth understanding of these processes at the molecular, cellular, and tissue level, as well as of the changes present in chronic inflammatory diseases, has been the focus of research at the German Rheumatism Research Center (Deutsches Rheuma-Forschungszentrum, DRFZ) since its foundation. Based on an improved understanding of these mechanisms, the DRFZ aims to develop improved prevention and treatment strategies with effects even in early disease stages.

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non-overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long-term maintenance of immune cells.

The alarmin IL-33 promotes regulatory T-cell function in the intestine.

FOXP3(+) regulatory T cells (Treg cells) are abundant in the intestine, where they prevent dysregulated inflammatory responses to self and environmental stimuli. It is now appreciated that Treg cells acquire tissue-specific adaptations that facilitate their survival and function; however, key host factors controlling the Treg response in the intestine are poorly understood. The interleukin (IL)-1 family member IL-33 is constitutively expressed in epithelial cells at barrier sites, where it functions as an endogenous danger signal, or alarmin, in response to tissue damage. Recent studies in humans have described high levels of IL-33 in inflamed lesions of inflammatory bowel disease patients, suggesting a role for this cytokine in disease pathogenesis. In the intestine, both protective and pathological roles for IL-33 have been described in murine models of acute colitis, but its contribution to chronic inflammation remains ill defined. Here we show in mice that the IL-33 receptor ST2 is preferentially expressed on colonic Treg cells, where it promotes Treg function and adaptation to the inflammatory environment. IL-33 signalling in T cells stimulates Treg responses in several ways. First, it enhances transforming growth factor (TGF)-ß1-mediated differentiation of Treg cells and, second, it provides a necessary signal for Treg-cell accumulation and maintenance in inflamed tissues. Strikingly, IL-23, a key pro-inflammatory cytokine in the pathogenesis of inflammatory bowel disease, restrained Treg responses through inhibition of IL-33 responsiveness. These results demonstrate a hitherto unrecognized link between an endogenous mediator of tissue damage and a major anti-inflammatory pathway, and suggest that the balance between IL-33 and IL-23 may be a key controller of intestinal immune responses.

Manipulation of the balance between Th2 and Th2/1 hybrid cells affects parasite nematode fitness in mice.

T-helper type 2 (Th2) responses are central to the control of helminth infections, but sensitive to opposing cytokine signals favoring Th1 priming. We previously reported on GATA-3+ T-bet+ Th2/1 hybrid cell differentiation in helminth mono-infections, resulting in a substantial proportion of cells coproducing IFN-γ next to Th2 cytokines. Here, we demonstrate Th2/1 cells as the major source of parasite-specific IFN-γ production in acute and chronic infections with the enteric nematode Heligmosomoides polygyrus. Th2/1 cells differentiated from naive precursors and accumulated in spleen and intestine of infected mice, resulting in increased systemic and mucosal IFN-γ production. IFN-γ supplementation early during infection supported Th2/1 differentiation, associated with elevated parasite fecundity and the maintenance of high worm burdens in the chronic stage of infection, whereas mice lacking IFN-γ signals generated poor Th2/1 responses and restricted parasite fecundity more efficiently. These findings suggest that Th2/1 hybrid responses take part in immune regulation during helminth infection and restrain effective anti-helminth immunity.

IL-33 in T Cell Differentiation, Function, and Immune Homeostasis.

Recent studies have highlighted a role for the alarmin interleukin (IL)-33 in CD4(+) and CD8(+) T cell activation and function, and have also revealed important distinctions. The IL-33 receptor ST2 is constitutively and abundantly expressed on T-helper-2 (Th2) and GATA-3(+) regulatory T cells in a GATA-3- and STAT5-dependent manner. Upon activation, Th1 and cytotoxic T cells express ST2 transiently, driven by T-bet and/or STAT4. We review these findings here, and critically examine evidence indicating that IL-33 enhances the differentiation and functionality of various T cell subsets through positive feedback loops involving lineage-specifying transcription factors. In this context, we discuss how quantitative and qualitative differences in ST2 expression between effector and GATA-3(+) regulatory T cells may contribute to immune homeostasis, and outline important areas of future inquiry.

Interferons direct Th2 cell reprogramming to generate a stable GATA-3(+)T-bet(+) cell subset with combined Th2 and Th1 cell functions.

Current T cell differentiation models invoke separate T helper 2 (Th2) and Th1 cell lineages governed by the lineage-specifying transcription factors GATA-3 and T-bet. However, knowledge on the plasticity of Th2 cell lineage commitment is limited. Here we show that infection with Th1 cell-promoting lymphocytic choriomeningitis virus (LCMV) reprogrammed otherwise stably committed GATA-3(+) Th2 cells to adopt a GATA-3(+)T-bet(+) and interleukin-4(+)interferon-gamma(+) "Th2+1" phenotype that was maintained in vivo for months. Th2 cell reprogramming required T cell receptor stimulation, concerted type I and type II interferon and interleukin-12 signals, and T-bet. LCMV-triggered T-bet induction in adoptively transferred virus-specific Th2 cells was crucial to prevent viral persistence and fatal immunopathology. Thus, functional reprogramming of unfavorably differentiated Th2 cells may facilitate the establishment of protective immune responses. Stable coexpression of GATA-3 and T-bet provides a molecular concept for the long-term coexistence of Th2 and Th1 cell lineage characteristics in single memory T cells.

Synovial Fibroblasts Selectively Suppress Th1 Cell Responses through IDO1-Mediated Tryptophan Catabolism.

The development of rheumatoid arthritis (RA) is linked to functional changes in synovial fibroblasts (SF) and local infiltration of T lymphocytes. Fibroblasts possess the capacity to suppress T cell responses, although the molecular mechanisms of this suppression remain incompletely understood. In this study, we aimed to define the mechanisms by which noninflammatory SF modulate Th cell responses and to determine the immunosuppressive efficacy of RASF. Hence, the influence of SF from osteoarthritis or RA patients on total Th cells or different Th cell subsets of healthy donors was analyzed in vitro. We show that SF strongly suppressed the proliferation of Th cells and the secretion of IFN-γ in a cell contact-independent manner. In cocultures of SF and Th cells, tryptophan was completely depleted within a few days, resulting in eukaryotic initiation factor 2α phosphorylation, TCRζ-chain downregulation, and proliferation arrest. Blocking IDO1 activity completely restored Th cell proliferation, but not IFN-γ production. Interestingly, only the proliferation of Th1 cells, but not of Th2 or Th17 cells, was affected. Finally, RASF had a significantly lower IDO1 expression and a weaker Th cell suppressive capacity compared with osteoarthritis SF. We postulate that the suppression of Th cell growth by SF through tryptophan catabolism may play an important role in preventing inappropriate Th cell responses under normal conditions. However, expansion of Th17 cells that do not induce IDO1-mediated suppression and the reduced capacity of RASF to restrict Th cell proliferation through tryptophan metabolism may support the initiation and propagation of synovitis in RA patients.

Professional memory CD4+ T lymphocytes preferentially reside and rest in the bone marrow.

CD4(+) T lymphocytes are key to immunological memory. Here we show that in the memory phase of specific immune responses, most of the memory CD4(+) T lymphocytes had relocated into the bone marrow (BM) within 3-8 weeks after their generation-a process involving integrin alpha2. Antigen-specific memory CD4(+) T lymphocytes highly expressed Ly-6C, unlike most splenic CD44(hi)CD62L(-) CD4(+) T lymphocytes. In adult mice, more than 80% of Ly-6C(hi)CD44(hi)CD62L(-) memory CD4(+) T lymphocytes were in the BM. In the BM, they associated to IL-7-expressing VCAM-1(+) stroma cells. Gene expression and proliferation were downregulated, indicating a resting state. Upon challenge with antigen, they rapidly expressed cytokines and CD154 and efficiently induced the production of high-affinity antibodies by B lymphocytes. Thus, in the memory phase of immunity, memory helper T cells are maintained in BM as resting but highly reactive cells in survival niches defined by IL-7-expressing stroma cells.