Ilgiz A. Mufazalov

IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells

Interleukin‐1 (IL‐1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL‐1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL‐1 receptor type 1 (IL‐1R1)‐dependent IL‐1β expression by myeloid cells in the draining lymph nodes. This myeloid‐derived IL‐1β did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM‐CSF+ Th17 cell subset, thereby enhancing its encephalitogenic potential. Lack of expansion of GM‐CSF‐producing Th17 cells led to ameliorated disease in mice deficient for IL‐1R1 specifically in T cells. Importantly, pathogenicity of IL‐1R1‐deficient T cells was fully restored by IL‐23 polarization and expansion in vitro. Therefore, our data demonstrate that IL‐1 functions as a mitogenic mediator of encephalitogenic Th17 cells rather than qualitative inducer of their generation.

Oral epithelial cells orchestrate innate type 17 responses to Candida albicans through the virulence factor candidalysin

Candidalysin, a hypha-associated fungal peptide, drives interleukin-17 responses to Candida albicans. Calibrating antifungal responses Immune responses to fungal infections are complicated by the fact that fungi can exist in multiple forms depending on environmental cues. Here, Verma et al. have evaluated innate immune responses to Candida albicans, a fungus that transitions from yeast to filamentous hyphae as infection progresses. They find that candidalysin, a hypha-associated protein and virulence factor, serves as a danger signal that potentiates the immune response to C. albicans. Candidalysin-deficient strains of C. albicans caused minimal epithelial damage and elicited a blunted type 17 immune response. The authors propose that the innate antifungal responses to C. albicans are driven by a synergy between cellular damage triggered by candidalysin that is further amplified by interleukin-17 driven inflammation. Candida albicans is a dimorphic commensal fungus that causes severe oral infections in immunodeficient patients. Invasion of C. albicans hyphae into oral epithelium is an essential virulence trait. Interleukin-17 (IL-17) signaling is required for both innate and adaptive immunity to C. albicans. During the innate response, IL-17 is produced by γδ T cells and a poorly understood population of innate-acting CD4+ αβ T cell receptor (TCRαβ)+ cells, but only the TCRαβ+ cells expand during acute infection. Confirming the innate nature of these cells, the TCR was not detectably activated during the primary response, as evidenced by Nur77eGFP mice that report antigen-specific signaling through the TCR. Rather, the expansion of innate TCRαβ+ cells was driven by both intrinsic and extrinsic IL-1R signaling. Unexpectedly, there was no requirement for CCR6/CCL20-dependent recruitment or prototypical fungal pattern recognition receptors. However, C. albicans mutants that cannot switch from yeast to hyphae showed impaired TCRαβ+ cell proliferation and Il17a expression. This prompted us to assess the role of candidalysin, a hyphal-associated peptide that damages oral epithelial cells and triggers production of inflammatory cytokines including IL-1. Candidalysin-deficient strains failed to up-regulate Il17a or drive the proliferation of innate TCRαβ+ cells. Moreover, candidalysin signaled synergistically with IL-17, which further augmented the expression of IL-1α/β and other cytokines. Thus, IL-17 and C. albicans, via secreted candidalysin, amplify inflammation in a self-reinforcing feed-forward loop. These findings challenge the paradigm that hyphal formation per se is required for the oral innate response and demonstrate that establishment of IL-1– and IL-17–dependent innate immunity is induced by tissue-damaging hyphae.

Characterization of a conditional interleukin‐1 receptor 1 mouse mutant using the Cre/LoxP system

IL‐1 is a key cytokine known to drive chronic inflammation and to regulate many physiological, immunological, and neuroimmunological responses via actions on diverse cell types of the body. To determine the mechanisms of IL‐1 actions as part of the inflammatory response in vivo, we generated a conditional IL‐1 receptor 1 (IL‐1R1) mouse mutant using the Cre/LoxP system (IL‐1R1fl/fl). In the mutant generated, exon 5, which encodes part of the extracellular‐binding region of the receptor, is flanked by LoxP sites, thereby inactivating the two previously described functional IL‐1R1 gene transcripts after Cre‐mediated recombination. Using keratin 14‐Cre driver mice, new IL‐1R1 deficient (−/−) mice were subsequently generated, in which all signaling IL‐1 receptor isoforms are deleted ubiquitously. Furthermore, using vav‐iCre driver mice, we deleted IL‐1 receptor isoforms in the hematopoietic system. In these mice, we show that both the IL‐17 and IL‐22 cytokine response is reduced, when mice are challenged by the helminth Trichuris muris. We are currently crossing IL‐1R1fl/fl mice with different Cre‐expressing mice in order to study mechanisms of acute and chronic inflammatory diseases.

Generation of a Novel T Cell Specific Interleukin-1 Receptor Type 1 Conditional Knock Out Mouse Reveals Intrinsic Defects in Survival, Expansion and Cytokine Production of CD4 T Cells.

Interleukin-1 (IL-1) plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1). To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN)-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment.

Isolation of Central Nervous System (CNS) Infiltrating Cells.

Leukocyte infiltration of the central nervous system (CNS) occurs under certain pathogenic conditions and most often results in severe disorders. Therefore, the isolation and analysis of such infiltrating cell populations is necessary for elucidating the underlying pathogenic mechanisms. Here we describe a simple and straightforward protocol for cell isolation from the inflamed CNS, which combines mechanical dissociation and enzymatic degradation of the tissue. Additionally, purification by Percoll gradient centrifugation provides a great yield of the infiltrating material. The isolated cells can be further used for downstream applications such as cell sorting, cellular or molecular analysis.

TGF-β inhibitor Smad7 regulates dendritic cell-induced autoimmunity

Significance Smad7 is a negative regulator of TGF-β signaling, a cytokine with anti-inflammatory properties. Although TGF-β was implicated in the development and function of dendritic cells (DCs), the in vivo role of Smad7 in DCs remains elusive. Here, we demonstrate that DC-specific Smad7 deletion affects the development of splenic CD8+CD103+ DCs by regulating expression of the transcription factors Batf3 and IRF8. In addition, Smad7 directs DC function by regulating the expression of indoleamine 2,3-dioxygenase in response to IFN-γ signaling. Hence, absent Smad7 in DCs mediates resistance of mice to the development of autoimmunity via protective regulatory T-cell induction. These findings demonstrate that Smad7 expression governs splenic DC subset differentiation and affects tolerogenic DC function in vivo. TGF-β is an anti-inflammatory cytokine whose signaling is negatively controlled by Smad7. Previously, we established a role for Smad7 in the generation of autoreactive T cells; however, the function of Smad7 in dendritic cells (DCs) remains elusive. Here, we demonstrate that DC-specific Smad7 deficiency resulted in elevated expression of the transcription factors Batf3 and IRF8, leading to increased frequencies of CD8+CD103+ DCs in the spleen. Furthermore, Smad7-deficient DCs expressed higher levels of indoleamine 2,3-dioxygenase (IDO), an enzyme associated with tolerance induction. Mice devoid of Smad7 specifically in DCs are resistant to the development of experimental autoimmune encephalomyelitis (EAE) as a result of an increase of protective regulatory T cells (Tregs) and reduction of encephalitogenic effector T cells in the central nervous system. In agreement, inhibition of IDO activity or depletion of Tregs restored disease susceptibility. Intriguingly, when Smad7-deficient DCs also lacked the IFN-γ receptor, the mice regained susceptibility to EAE, demonstrating that IFN-γ signaling in DCs mediates their tolerogenic function. Our data indicate that Smad7 expression governs splenic DC subset differentiation and is critical for the promotion of their efficient function in immunity.

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, thereby regulating NF-κB-mediated gene expression. This study thus reveals intrinsic functions of Bcl-3 in Treg cells, identifies Bcl-3 as a potential prognostic marker for colitis and illustrates the mechanism by which Bcl-3 regulates NF-κB activity in Tregs to prevent colitis.

Expression of IL-17F is associated with non-pathogenic Th17 cells

IL-17A and IL-17F share the highest sequence homology of the IL-17 family and signal via the same IL-17RA/RC receptor heterodimer. To better explore the expression of these two cytokines, we used a double reporter mouse strain (IL-17DR mice), where IL-17A expressing cells are marked by enhanced green fluorescent protein (eGFP) while red fluorescence protein (RFP) reports the expression of IL-17F. In steady state, we found that Th17 and γδ T cells only expressed IL-17A, while IL-17F expression was restricted to CD8 T cells (Tc17) and innate lymphoid cells (ILC type 3) of the gut. In experimental autoimmune encephalomyelitis, the vast majority of CNS-infiltrating Th17 cells expressed IL-17A but not IL-17F. In contrast, anti-CD3-induced, TGF-β-driven Th17 cells in the gut expressed both of these IL-17 cytokines. In line with this, in vitro differentiation of Th17 cells in the presence of IL-1β led primarily to IL-17A expressing T cells, while TGF-β induced IL-17F co-expressing Th17 cells. Our results suggest that expression of IL-17F is associated with non-pathogenic T cells, pointing to a differential function of IL-17A versus IL-17F.Key messagesNaïve mice: CD4+ T cells and γδ T cells express IL-17A, and Tc17 cells express IL-17F. Gut ILC3 show differential expression of IL17A and F.Th17 differentiation with TGF-β1 induces IL-17A and F, whereas IL-1β induced cells expressing IL-17A.Th17 cells in EAE in CNS express IL-17A only.Gut Th17 cells induced by anti-CD3 express IL-17A and F together as skin γδ T cells of IMQ-treated mice.

Expression of the G-protein coupled receptor EBI2 in T cells is highly regulated and confers pathogenicity to myelin specific Th17 cells

Institute for Molecular Medicine, Johannes Gutenberg-University, Mainz, Germany; Institute of Experimental Immunology, Neuroand Tumorimmunology, University of Zurich, Zurich, Switzerland; Department of Pharmacology, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany; Institute for Neuropathology, University of Münster, Münster, Germany; Developmental & Molecular Pathways, Novartis Institutes for BioMedical Research, Basel, Switzerland; Max-DelbrückCenter for Molecular Medicine, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; IFOM-IEO Campus, IFOM-IEO Campus, Milan, Italy

Balanced Bcl‐3 expression in murine CD4+ T cells is required for generation of encephalitogenic Th17 cells

The function of NF‐κB family members is controlled by multiple mechanisms including the transcriptional regulator Bcl‐3, an atypical member of the IκB family. By using a murine model of conditional Bcl‐3 overexpression specifically in T cells, we observed impairment in the development of Th2, Th1, and Th17 cells. High expression of Bcl‐3 promoted CD4+ T‐cell survival, but at the same time suppressed proliferation in response to TCR stimulation, resulting in reduced CD4+ T‐cell expansion. As a consequence, T‐cell‐specific overexpression of Bcl‐3 led to reduced inflammation in the small intestine of mice applied with anti‐CD3 in a model of gut inflammation. Moreover, impaired Th17‐cell development resulted in the resistance of Bcl‐3 overexpressing mice to EAE, a mouse model of multiple sclerosis. Thus, we concluded that fine‐tuning expression of Bcl‐3 is needed for proper CD4+ T‐cell development and is required to sustain Th17‐cell mediated pathology.