The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 (IFIH1), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1A946T) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1A946T risk variant, (IFIH1R) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1R compared to non-risk Ifih1 (Ifih1NR) mice and a significant acceleration of diabetes onset in Ifih1R females. Ifih1R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1NR, indicative of increased IFN I signaling. Ifih1R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8+ T cells. Our results indicate that IFIH1R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

The IFIH1-A946T risk variant promotes diabetes in a sex-dependent manner.

Type 1 diabetes (T1D) is an autoimmune disease in which pancreatic islet ß-cells are attacked by the immune system, resulting in insulin deficiency and hyperglycemia. One of the top non-synonymous single-nucleotide polymorphisms (SNP) associated with T1D is in the interferon-induced helicase C domain-containing protein 1 ( IFIH1 ), which encodes an anti-viral cytosolic RNA sensor. This SNP results in an alanine to threonine substitution at amino acid 946 (IFIH1 A946T ) and confers an increased risk for several autoimmune diseases, including T1D. We hypothesized that the IFIH1 A946T risk variant, ( IFIH1 R ) would promote T1D pathogenesis by stimulating type I interferon (IFN I) signaling leading to immune cell alterations. To test this, we developed Ifih1 R knock-in mice on the non-obese diabetic (NOD) mouse background, a spontaneous T1D model. Our results revealed a modest increase in diabetes incidence and insulitis in Ifih1 R compared to non-risk Ifih1 ( Ifih1 NR ) mice and a significant acceleration of diabetes onset in Ifih1 R females. Ifih1 R mice exhibited a significantly enhanced interferon stimulated gene (ISG) signature compared to Ifih1 NR , indicative of increased IFN I signaling. Ifih1 R mice exhibited an increased frequency of plasma cells as well as tissue-dependent changes in the frequency and activation of CD8 + T cells. Our results indicate that IFIH1 R may contribute to T1D pathogenesis by altering the frequency and activation of immune cells. These findings advance our knowledge on the connection between the rs1990760 variant and T1D. Further, these data are the first to demonstrate effects of Ifih1 R in NOD mice, which will be important to consider for the development of therapeutics for T1D.

The Autoimmune Risk R262W Variant of the Adaptor SH2B3 Improves Survival in Sepsis.

The single-nucleotide polymorphism (SNP) rs3184504 is broadly associated with increased risk for multiple autoimmune and cardiovascular diseases. Although the allele is uniquely enriched in European descent, the mechanism for the widespread selective sweep is not clear. In this study, we find the rs3184504*T allele had a strong association with reduced mortality in a human sepsis cohort. The rs3184504*T allele associates with a loss-of-function amino acid change (p.R262W) in the adaptor protein SH2B3, a likely causal variant. To better understand the role of SH2B3 in sepsis, we used mouse modeling and challenged SH2B3-deficient mice with a polymicrobial cecal-ligation puncture (CLP) procedure. We found SH2B3 deficiency improved survival and morbidity with less organ damage and earlier bacterial clearance compared with control mice. The peritoneal infiltrating cells exhibited augmented phagocytosis in Sh2b3 -/- mice with enriched recruitment of Ly6Chi inflammatory monocytes despite equivalent or reduced chemokine expression. Rapid cycling of monocytes and progenitors occurred uniquely in the Sh2b3 -/- mice following CLP, suggesting augmented myelopoiesis. To model the hypomorphic autoimmune risk allele, we created a novel knockin mouse harboring a similar point mutation in the murine pleckstrin homology domain of SH2B3. At baseline, phenotypic changes suggested a hypomorphic allele. In the CLP model, homozygous knockin mice displayed improved mortality and morbidity compared with wild-type or heterozygous mice. Collectively, these data suggest that hypomorphic SH2B3 improves the sepsis response and that balancing selection likely contributed to the relative frequency of the autoimmune risk variant.

Germline SAMD9L truncation variants trigger global translational repression.

SAMD9L is an interferon-induced tumor suppressor implicated in a spectrum of multisystem disorders, including risk for myeloid malignancies and immune deficiency. We identified a heterozygous de novo frameshift variant in SAMD9L in an infant with B cell aplasia and clinical autoinflammatory features who died from respiratory failure with chronic rhinovirus infection. Autopsy demonstrated absent bone marrow and peripheral B cells as well as selective loss of Langerhans and Purkinje cells. The frameshift variant led to expression of a truncated protein with interferon treatment. This protein exhibited a gain-of-function phenotype, resulting in interference in global protein synthesis via inhibition of translational elongation. Using a mutational scan, we identified a region within SAMD9L where stop-gain variants trigger a similar translational arrest. SAMD9L variants that globally suppress translation had no effect or increased mRNA transcription. The complex-reported phenotype likely reflects lineage-dominant sensitivities to this translation block. Taken together, our findings indicate that interferon-triggered SAMD9L gain-of-function variants globally suppress translation.

The TYK2-P1104A Autoimmune Protective Variant Limits Coordinate Signals Required to Generate Specialized T Cell Subsets.

TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within TYK2 encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant TYK2A1104 (TYK2P ). To study this variant in vivo, we developed a knock-in murine model of this allele. Murine Tyk2P expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous Tyk2P T cells exhibited diminished in vitro Th1 skewing. Surprisingly, despite these signaling changes, in vivo formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that Tyk2P expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 in vitro. Consistent with these findings, homozygous, but not heterozygous, Tyk2P mice were fully protected in a murine model of MS. Homozygous Tyk2P mice had fewer infiltrating CD4+ T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17+/IFNγ+, double positive, pathogenic CD4+ T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the Tyk2P allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2P diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades.

The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity.

The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1T946) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1T946 exhibited heightened basal and ligand-triggered production of type I interferons. Consistent with those findings, mice with a knock-in mutation encoding IFIH1T946 displayed enhanced basal expression of type I interferons, survived a lethal viral challenge and exhibited increased penetrance in autoimmune models, including a combinatorial effect with other risk variants. Furthermore, IFIH1T946 mice manifested an embryonic survival defect consistent with enhanced responsiveness to RNA self ligands. Together our data support a model wherein the production of type I interferons driven by an autoimmune risk variant and triggered by ligand functions to protect against viral challenge, which probably accounts for its selection within human populations but provides this advantage at the cost of modestly promoting the risk of autoimmunity.

Epigenetic regulation of autophagy by the methyltransferase G9a.

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy.

Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks.

The Arp2/3-activator Wiskott-Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1(+) endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex-associated proteins or EEA1. In addition, we observed global collapse of LAMP1(+) lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.

The cytoskeletal adaptor protein IQGAP1 regulates TCR-mediated signaling and filamentous actin dynamics.

The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.