Florian Wanke

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.

Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins

Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.

Subclinical CNS Inflammation as Response to a Myelin Antigen in Humanized Mice

Multiple sclerosis is a demyelinating autoimmune disease of the CNS. Its animal model experimental autoimmune encephalomyelitis is commonly induced by active immunization with myelin antigens. To investigate human immune responses against myelin antigens in vivo we established a new subclinical experimental autoimmune encephalomyelitis model in humanized mice. NOD/Scidγc−/− animals were transferred with peripheral blood mononuclear cells from healthy human donors and immunized with myelin antigens in complete Freund’s adjuvant and antigen-pulsed autologous dendritic cells. Human T cells recovered from these animals reacted specifically to the soluble domain of myelin oligodendrocyte glycoprotein and secreted proinflammatory cytokines. Furthermore, immunized animals developed subclinical CNS inflammation with infiltrating CD4+ and CD8+ T cells and production of encephalitogenic cytokines. Thus, this model of myelin-induced CNS inflammation by human T cells may allow testing of new human-specific therapeuticals for multiple sclerosis.

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.

Single-cell profiling reveals GPCR heterogeneity and functional patterning during neuroinflammation

GPCR expression was intensively studied in bulk cDNA of leukocyte populations, but limited data are available with respect to expression in individual cells. Here, we show a microfluidic-based single-cell GPCR expression analysis in primary T cells, myeloid cells, and endothelial cells under naive conditions and during experimental autoimmune encephalomyelitis, the mouse model of multiple sclerosis. We found that neuroinflammation induces characteristic changes in GPCR heterogeneity and patterning, and we identify various functionally relevant subgroups with specific GPCR profiles among spinal cord-infiltrating CD4 T cells, macrophages, microglia, or endothelial cells. Using GPCRs CXCR4, S1P1, and LPHN2 as examples, we show how this information can be used to develop new strategies for the functional modulation of Th17 cells and activated endothelial cells. Taken together, single-cell GPCR expression analysis identifies functionally relevant subpopulations with specific GPCR repertoires and provides a basis for the development of new therapeutic strategies in immune disorders.

Regulation of IL-22BP in psoriasis

IL-22 is a potent pro-inflammatory cytokine upregulated in psoriasis and in other inflammatory diseases. The function of IL-22 is regulated by the soluble scavenging receptor, IL-22 binding protein (IL-22BP or IL-22RA2). However, the role and regulation of IL-22BP itself in the pathogenesis of inflammatory disease remain unclear. We used the TLR7 agonist Imiquimod (IMQ) to induce a psoriasis-like skin disease in mice and found a strong downregulation of IL-22BP in the affected skin as well as in the lymph nodes of animals treated with IMQ. We also analysed psoriatic skin of patients and compared this to skin of healthy donors. Interestingly, IL-22BP expression was similarly downregulated in skin biopsies of psoriasis patients compared to the skin of healthy donors. Since IL-22BP is expressed foremost in dendritic cells, we characterized its expression in monocyte-derived dendritic cells (MoDC) during maturation. In this way, we found Prostaglandin E2 (PGE2) to be a potent suppressor of IL-22BP expression in vitro. We conclude that regulation of IL-22BP by inflammatory mediators is an important step for the progression of inflammation in the skin and possibly also in other autoimmune diseases.

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.

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

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

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.