Stephan Kissler

MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target messenger RNAs. Dysregulation of microRNAs by several mechanisms has been described in various disease states including cardiac disease. Whereas previous studies of cardiac disease have focused on microRNAs that are primarily expressed in cardiomyocytes, the role of microRNAs expressed in other cell types of the heart is unclear. Here we show that microRNA-21 (miR-21, also known as Mirn21) regulates the ERK–MAP kinase signalling pathway in cardiac fibroblasts, which has impacts on global cardiac structure and function. miR-21 levels are increased selectively in fibroblasts of the failing heart, augmenting ERK–MAP kinase activity through inhibition of sprouty homologue 1 (Spry1). This mechanism regulates fibroblast survival and growth factor secretion, apparently controlling the extent of interstitial fibrosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specific antagomir in a mouse pressure-overload-induced disease model reduces cardiac ERK–MAP kinase activity, inhibits interstitial fibrosis and attenuates cardiac dysfunction. These findings reveal that microRNAs can contribute to myocardial disease by an effect in cardiac fibroblasts. Our results validate miR-21 as a disease target in heart failure and establish the therapeutic efficacy of microRNA therapeutic intervention in a cardiovascular disease setting.

In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes

Type 1 diabetes is an autoimmune disease influenced by multiple genetic loci. Although more than 20 insulin-dependent diabetes (Idd) loci have been implicated in the nonobese diabetic (NOD) mouse model, few causal gene variants have been identified. Here we show that RNA interference (RNAi) can be used to probe candidate genes in this disease model. Slc11a1 encodes a phagosomal ion transporter, Nramp1, that affects resistance to intracellular pathogens and influences antigen presentation. This gene is the strongest candidate among the 42 genes in the Idd5.2 region; a naturally occurring mutation in the protective Idd5.2 haplotype results in loss of function of the Nramp1 protein. Using lentiviral transgenesis, we generated NOD mice in which Slc11a1 is silenced by RNAi. Silencing reduced the frequency of type 1 diabetes, mimicking the protective Idd5.2 region. Our results demonstrate a role for Slc11a1 in modifying susceptibility to type 1 diabetes and illustrate that RNAi can be used to study causal genes in a mammalian model organism.

αv Integrin expression by DCs is required for Th17 cell differentiation and development of experimental autoimmune encephalomyelitis in mice

Th17 cells are a distinct lineage of T helper cells that protect the body from bacterial and fungal infection. However, Th17 cells also contribute to inflammatory and autoimmune disorders such as multiple sclerosis. Th17 cell generation requires exposure of naive T cells to the cytokine TGF-β in combination with proinflammatory cytokines. Here we show that differentiation of Th17 cells is also critically dependent on αv integrins. In mice, lack of integrin αv in the immune system resulted in loss of Th17 cells in the intestine and lymphoid tissues. It also led to protection from experimental autoimmune encephalomyelitis (EAE). Further analysis indicated that αv integrins on DCs activated latent TGF-β during T cell stimulation and thereby promoted differentiation of Th17 cells. Furthermore, pharmacologic inhibition of αv integrins using cyclic RGD peptides blocked TGF-β activation and Th17 cell generation in vitro and protected mice from EAE. These data demonstrate that activation of TGF-β by αv-expressing myeloid cells may be a critical step in the generation of Th17 cells and suggest that αv integrins could be therapeutic targets in autoimmune disease.

STIM1-Independent T Cell Development and Effector Function In Vivo

Store-operated Ca2+ entry (SOCE) is believed to be of pivotal importance in T cell physiology. To test this hypothesis, we generated mice constitutively lacking the SOCE-regulating Ca2+ sensor stromal interaction molecule 1 (STIM1). In vitro analyses showed that SOCE and Ag receptor complex-triggered Ca2+ flux into STIM1-deficient T cells is virtually abolished. In vivo, STIM1-deficient mice developed a lymphoproliferative disease despite normal thymic T cell maturation and normal frequencies of CD4+Foxp3+ regulatory T cells. Unexpectedly, STIM1-deficient bone marrow chimeric mice mounted humoral immune responses after vaccination and STIM1-deficient T cells were capable of inducing acute graft-versus-host disease following adoptive transfer into allogeneic hosts. These results demonstrate that STIM1-dependent SOCE is crucial for homeostatic T cell proliferation, but of much lesser importance for thymic T cell differentiation or T cell effector functions.

The Soluble CTLA-4 Splice Variant Protects From Type 1 Diabetes and Potentiates Regulatory T-Cell Function

OBJECTIVE CTLA4 gene variation associates with multiple autoimmune disorders, including type 1 diabetes. The CTLA4 susceptibility allele was found to generate decreased levels of mRNA encoding soluble CTLA-4 (sCTLA-4) relative to the full-length isoform, the functional consequence of which is as yet unknown. In this study, we investigated the contribution of sCTLA-4 to immune regulation with the aim to elucidate the functional basis of the disease association of CTLA4. RESEARCH DESIGN AND METHODS To model the disease-associated splicing variation of CTLA4, we generated NOD mice in which sCTLA-4 mRNA is silenced by RNA interference. RESULTS We found that loss of sCTLA-4 impairs the function of regulatory T (Treg) cells. This functional defect could be attributed, at least in part, to the failure of sCTLA-4 knockdown (KD) Treg cells to downregulate dendritic cell costimulation. sCTLA-4 KD Treg cells, in contrast with wild-type Treg cells, failed to inhibit colitis induced by transfer of CD4+CD45RBhi cells into NOD.SCID animals. Furthermore, diminished sCTLA-4 expression accelerated the onset of autoimmune diabetes in transgenic mice. CONCLUSIONS Our results demonstrate that sCTLA-4 participates in immune regulation by potentiating the function of Treg cells. The functional outcome of silencing this splice variant in the NOD model provides an explanation for the association of CTLA4 variation with autoimmunity. Lower sCTLA-4 expression from the susceptibility allele may directly affect the suppressive capacity of Treg cells and thereby modulate disease risk. Our unprecedented approach establishes the feasibility of modeling splicing variations relevant to autoimmunity.

IL-17 Silencing Does Not Protect Nonobese Diabetic Mice from Autoimmune Diabetes

The long-held view that many autoimmune disorders are primarily driven by a Th1 response has been challenged by the discovery of Th17 cells. Since the identification of this distinct T cell subset, Th17 cells have been implicated in the pathogenesis of several autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. Type 1 diabetes has also long been considered a Th1-dependent disease. In light of the emerging role for Th17 cells in autoimmunity, several recent studies investigated the potential of this subset to initiate autoimmune diabetes. However, direct evidence supporting the involvement of Th17 cells in actual pathogenesis, particularly during spontaneous onset, is lacking. In this study, we sought to directly address the role of IL-17, the cytokine by which Th17 cells are primarily characterized, in the pathogenesis of autoimmune diabetes. We used lentiviral transgenesis to generate NOD mice in which IL-17 is silenced by RNA interference. The loss of IL-17 had no effect on the frequency of spontaneous or cyclophosphamide-induced diabetes. In contrast, IL-17 silencing in transgenic NOD mice was sufficient to reduce the severity of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, consistent with reports that IL-17 deficiency is protective in this experimental model of multiple sclerosis. We concluded that IL-17 is dispensable, at least in large part, in the pathogenesis of autoimmune diabetes.

PTPN22 Silencing in the NOD Model Indicates the Type 1 Diabetes-Associated Allele Is Not a Loss-of-Function Variant

PTPN22 encodes the lymphoid tyrosine phosphatase (LYP) and is the second strongest non-HLA genetic risk factor for type 1 diabetes. The PTPN22 susceptibility allele generates an LYP variant with an arginine-to-tryptophan substitution at position 620 (R620W) that has been reported by several studies to impart a gain of function. However, a recent report investigating both human cells and a knockin mouse model containing the R620W homolog suggested that this variation causes faster protein degradation. Whether LYP R620W is a gain- or loss-of-function variant, therefore, remains controversial. To address this issue, we generated transgenic NOD mice (nonobese diabetic) in which Ptpn22 can be inducibly silenced by RNA interference. We found that Ptpn22 silencing in the NOD model replicated many of the phenotypes observed in C57BL/6 Ptpn22 knockout mice, including an increase in regulatory T cells. Notably, loss of Ptpn22 led to phenotypic changes in B cells opposite to those reported for the human susceptibility allele. Furthermore, Ptpn22 knockdown did not increase the risk of autoimmune diabetes but, rather, conferred protection from disease. Overall, to our knowledge, this is the first functional study of Ptpn22 within a model of type 1 diabetes, and the data do not support a loss of function for the PTPN22 disease variant.

Therapeutic potential of TCR antagonists is determined by their ability to modulate a diverse repertoire of autoreactive T cells

The use of altered peptide ligands (APL) with TCR antagonist properties holds promise as an antigen‐specific therapy for autoimmune disorders. We are investigating the therapeutic potential of APL in experimental autoimmune encephalomyelitis (EAE) using the Ac1 – 9 peptide of myelin basic protein in H‐2u mice. Encephalitogenic T cells recognize Ac1 – 9 using residues 3Gln and 6Pro as the major TCR contact sites. Use of position 6 APL is compromised by the heterogeneous nature of the Ac1 – 9‐specific repertoire. Here we identify two position 3 APL that act as TCR antagonists on transgenic T cells expressing Ac1 – 9‐specific TCR and that inhibit EAE in H‐2u mice. However, the therapeutic capacity of these two APL correlated directly with the ability to maximally inhibit activation of a heterogeneous T cell pool. The implications of these findings for the requirements for EAE induction, the relative contribution of a given T cell subpopulation to pathology and the mechanism underlying EAE inhibition in this model are discussed.

The Autoimmunity-Associated Gene CLEC16A Modulates Thymic Epithelial Cell Autophagy and Alters T Cell Selection

CLEC16A variation has been associated with multiple immune-mediated diseases, including type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, celiac disease, Crohns disease, Addisons disease, primary biliary cirrhosis, rheumatoid arthritis, juvenile idiopathic arthritis, and alopecia areata. Despite strong genetic evidence implicating CLEC16A in autoimmunity, this genes broad association with disease remains unexplained. We generated Clec16a knock-down (KD) mice in the nonobese diabetic (NOD) model for type 1 diabetes and found that Clec16a silencing protected against autoimmunity. Disease protection was attributable to T cell hyporeactivity, which was secondary to changes in thymic epithelial cell (TEC) stimuli that drive thymocyte selection. Our data indicate that T cell selection and reactivity were impacted by Clec16a variation in thymic epithelium owing to Clec16as role in TEC autophagy. These findings provide a functional link between human CLEC16A variation and the immune dysregulation that underlies the risk of autoimmunity.

Deficiency of HIF1α in Antigen-Presenting Cells Aggravates Atherosclerosis and Type 1 T-Helper Cell Responses in Mice

Objective—Although immune responses drive the pathogenesis of atherosclerosis, mechanisms that control antigen-presenting cell (APC)–mediated immune activation in atherosclerosis remain elusive. We here investigated the function of hypoxia-inducible factor (HIF)-1&agr; in APCs in atherosclerosis. Approach and Results—We found upregulated HIF1&agr; expression in CD11c+ APCs within atherosclerotic plaques of low-density lipoprotein receptor–deficient (Ldlr−/−) mice. Conditional deletion of Hif1a in CD11c+ APCs in high-fat diet–fed Ldlr−/− mice accelerated atherosclerotic plaque formation and increased lesional T-cell infiltrates, revealing a protective role of this transcription factor. HIF1&agr; directly controls Signal Transducers and Activators of Transcription 3 (Stat3), and a reduced STAT3 expression was found in HIF1&agr;-deficient APCs and aortic tissue, together with an upregulated interleukin-12 expression and expansion of type 1 T-helper (Th1) cells. Overexpression of STAT3 in Hif1a-deficient APCs in bone marrow reversed enhanced atherosclerotic lesion formation and reduced Th1 cell expansion in chimeric Ldlr−/− mice. Notably, deletion of Hif1a in LysM+ bone marrow cells in Ldlr−/− mice did not affect lesion formation or T-cell activation. In human atherosclerotic lesions, HIF1&agr;, STAT3, and interleukin-12 protein were found to colocalize with APCs. Conclusions—Our findings identify HIF1&agr; to antagonize APC activation and Th1 T cell polarization during atherogenesis in Ldlr−/− mice and to attenuate the progression of atherosclerosis. These data substantiate the critical role of APCs in controlling immune mechanisms that drive atherosclerotic lesion development.