Yilang Tang

The deubiquitinating enzyme CYLD regulates the differentiation and maturation of thymic medullary epithelial cells

The cross talk between thymocytes and the thymic epithelium is critical for T‐cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) are located in the thymic medulla and mediate the elimination of self‐reactive thymocytes, thereby preventing the onset of autoimmunity. Previous studies identified the deubiquitinating enzyme CYLD as a critical regulator of T‐cell development by activating proximal T‐cell receptor signaling during the transition of double‐positive to single‐positive thymocytes. Here we evaluated the impact of the naturally occurring short‐splice variant of the cyld gene (sCYLD) on the development and maturation of mTECs. We found that thymi of CYLDex7/8 mice, solely expressing sCYLD, displayed a reduced number of mature mTECs caused by a developmental block during the transition of immature to mature mTECs. Further, we could demonstrate an impaired negative selection of thymocytes in these mice. Our data demonstrate that inefficient negative selection in the thymus of CYLDex7/8 mice result from a defect in mTEC maturation.

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.

IL-4 Receptor Alpha Signaling through Macrophages Differentially Regulates Liver Fibrosis Progression and Reversal

Chronic hepatitis leads to liver fibrosis and cirrhosis. Cirrhosis is a major cause of worldwide morbidity and mortality. Macrophages play a key role in fibrosis progression and reversal. However, the signals that determine fibrogenic vs fibrolytic macrophage function remain ill defined. We studied the role of interleukin-4 receptor α (IL-4Rα), a potential central switch of macrophage polarization, in liver fibrosis progression and reversal. We demonstrate that inflammatory monocyte infiltration and liver fibrogenesis were suppressed in general IL-4Rα−/− as well as in macrophage-specific IL-4Rα−/− (IL-4RαΔLysM) mice. However, with deletion of IL-4RαΔLysM spontaneous fibrosis reversal was retarded. Results were replicated by pharmacological intervention using IL-4Rα-specific antisense oligonucleotides. Retarded resolution was linked to the loss of M2-type resident macrophages, which secreted MMP-12 through IL-4 and IL-13-mediated phospho-STAT6 activation. We conclude that IL-4Rα signaling regulates macrophage functional polarization in a context-dependent manner. Pharmacological targeting of macrophage polarization therefore requires disease stage-specific treatment strategies. Research in Context Alternative (M2-type) macrophage activation through IL-4Rα promotes liver inflammation and fibrosis progression but speeds up fibrosis reversal. This demonstrates context dependent, opposing roles of M2-type macrophages. During reversal IL-4Rα induces fibrolytic MMPs, especially MMP-12, through STAT6. Liver-specific antisense oligonucleotides efficiently block IL-4Rα expression and attenuate fibrosis progression.

NG2 plays a role in neuroinflammation but is not expressed by immune cells

latter group used a different immunization protocol that included a single administration of 200 μg of MOG and two injections of 400 ng of pertussis toxin (48 h apart). In the present report, we confirm that NG2-deficient mice develop milder EAE clinical score compared to control mice (Fig. 1a, b), similar to what was observed by Ferrara et al. We utilized an NG2-EYFP knock-in reporter mouse line we have previously generated, where homozygous EYFP integration into the NG2 locus yields NG2-deficient mice (NG2) [7]. Twenty-five days after immunization, the extent of demyelination in spinal cords of NG2deficient mice was negligible in comparison to control animals (Fig. 1c, e). In addition, infiltrating immune cells were scarcely present in the CNS of NG2-deficient mice at this time point of disease progression (Fig. 1d, f). It should be noted that our immunization protocol differed from those applied in aforementioned studies, consisting of a single injection of MOG35–55 (50 μg)/CFA and two injections of 200 ng of pertussis toxin administered on the day and 2 days after immunization. Importantly, application of another lot of pertussis toxin, with different activity values, resulted in the development of disease in NG2 mice, although the severity was still milder in comparison to controls (Supplemental Fig. 1). The mechanism of pertussis toxin action in EAE is not completely understood, but some of the proposed functions include modulation of the blood–brain barrier permeability and promotion of pathogenic T helper 17 (Th17) cell development and expansion [3, 10]. Therefore, it is likely that after a stronger boost of the immune system, such as varying concentrations of MOG, CFA and/or pertussis toxin, the threshold for the disease onset can be reached also in NG2-deficient mice. However, the proportion of Th1 and Th17 cell subsets was similar between knockout and control mice, based on the expression of signature cytokines (Supplemental Fig. 2). Nerve/glial antigen 2 (NG2) is a large transmembrane chondroitin sulphate proteoglycan expressed mainly by oligodendrocyte precursor cells in the developing and adult central nervous system (CNS) and pericytes in various organs throughout the body [14]. Numerous studies reported increased levels of this molecule after CNS injury, but its relevance remained questionable [5, 6, 9, 11]. In recent years, contradicting studies have been published concerning the role of NG2 molecule in experimental autoimmune encephalomyelitis (EAE). Using an immunization protocol consisting of two 100 μg doses of MOG35–55, emulsified in CFA, on days 0 and 7 and two injections of 300 ng of pertussis toxin on days 0 and 2 postimmunization, Moransard et al. demonstrated that NG2 is dispensable for EAE development [11]. In contrast, Ferrara et al. reported recently that mice of the same NG2-deficient line displayed milder EAE clinical score [4]. Of note, the

NF-κB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.