An IFIH1 gene polymorphism associated with risk for autoimmunity regulates canonical antiviral defence pathways in Coxsackievirus infected human pancreatic islets

1 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 www.nature.com/scientificreports An IFIH1 gene polymorphism associated with risk for autoimmunity regulates canonical antiviral defence pathways in Coxsackievirus infected human pancreatic islets Erna Domsgen1,*, Katharina Lind1,*, Lingjia Kong2, Michael H. Hühn1,†, Omid Rasool2, Frank van Kuppeveld3, Olle Korsgren4, Riitta Lahesmaa2 & Malin Flodström-Tullberg1,5 The IFIH1 gene encodes the pattern recognition receptor MDA5. A common polymorphism in IFIH1 (rs1990760, A946T) confers increased risk for autoimmune disease, including type 1-diabetes (T1D). Coxsackievirus infections are linked to T1D and cause beta-cell damage in vitro. Here we demonstrate that the rs1990760 polymorphism regulates the interferon (IFN) signature expressed by human pancreatic islets following Coxsackievirus infection. A strong IFN signature was associated with high expression of IFNλ1 and IFNλ2, linking rs1990760 to the expression of type III IFNs. In the highresponding genotype, IRF-1 expression correlated with that of type III IFN, suggesting a positivefeedback on type III IFN transcription. In summary, our study uncovers an influence of rs1990760 on the canonical effector function of MDA5 in response to an acute infection of primary human parenchymal cells with a clinically relevant virus linked to human T1D. It also highlights a previously unrecognized connection between the rs1990760 polymorphism and the expression level of type III IFNs. The interferon-induced helicase-1 (IFIH1) gene encodes the melanoma differentiation associated protein 5 (MDA5), an intracellular pattern recognition receptor (PRR) of importance for the recognition of certain viruses. The MDA5 protein consists of two N-terminal caspase recruitment domains (CARDs), a central helicase domain, and a C-terminal domain (CTD). MDA5 monomers bind to long double stranded (ds)RNA molecules formed during the viral replication cycle. This initiates the formation of a MDA5 filament along the dsRNA, where multiple CARD domains assemble and recruit the signalling adaptor interferon promoter stimulator 1 (IPS1) (also denoted mitochondrial antiviral-signalling protein, MAVS). Activated IPS1/MAVS initiates the downstream signalling events leading to the activation of the transcription factors NF-κ B and IRF-3, followed by the production of type I and III interferons (IFN)1–4. The rapid production of IFNs is an essential component of the immune response against many viruses. Type I and III IFNs have potent antiviral activities; they act in an autoand paracrine manner to induce the expression of interferon-stimulated genes (ISGs) leading to an antiviral state in uninfected cells. They also promote innate and adaptive immune responses1–5. Activation of type I and III IFN gene transcription is dependent on the 1The Center for Infectious Medicine, Department of Medicine HS, Karolinska Institutet, Karolinska University Hospital, Stockholm, 141 86, Sweden. 2Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, 205 20, Finland. 3Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, 3584, The Netherlands. 4Department of Immunology, Genetics, and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, 751 05, Sweden. 5Institute of Biosciences and Medical Technologies, University of Tampere, Tampere, 33520, Finland. †Present address: Astra Zeneca AB R&D, Pepparedsleden 1, Mölndal, 431 50, Sweden. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.F.-T. (email: [email protected]) Received: 12 August 2016 Accepted: 23 November 2016 Published: 21 December 2016 OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 Figure 1. RNAseq analysis identifies differences in gene expression between CVB3 infected pancreatic islets carrying the rs1990760 TT and TC genotypes. (a and b) Human islets from 23 donors were infected in mock or with CVB3. Islets and supernatants were collected at 48 h p.i. The titers of replicating virus particles in supernatants from infected islets (a) were measured by a standard plaque assay and presented as Log10 (PFU/ml). The expression levels of virus RNA (b) in CVB3 infected islets were measured using quantitative real-time PCR. The expression levels were normalized to that of GAPDH and presented as 2−(ΔCt) (relative www.nature.com/scientificreports/ 3 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 activation of specific transcription factors, NF-κ B and IRF-3. Besides these two, the transcription factor IRF-7 is required for the transcriptional activation of IFNα and possibly also IFN-λ 2 and -3 genes. IRF-7 is constitutively expressed by some immune cells, but rarely by parenchymal cells. IRF-7 is often induced following exposure to JAK/STAT-activating cytokines, such as the IFNs themselves, thus establishing a positive feedback loop for IFN expression. IRF-1 is another IFN inducible transcription factor. It has binding sites in the type I and III IFN promoters and can also contribute to a positive feedback on IFN gene transcription. Recent studies have suggested that IRF-1 has a role in activating type III and not type I IFNs during infections with RNA viruses3,6,7. The human IFIH1 gene contains numerous single nucleotide polymorphisms (SNPs). The non-synonymous SNP (nsSNP), rs1990760 (A946T), is associated with an increased risk for development of several autoimmune diseases including T1D diabetes, systemic lupus erythematosus and multiple sclerosis (e.g.8–10). Little is known on how this nsSNP regulates risk for development of these inflammatory diseases, and therefore information on the biological effects of this nsSNP may provide useful insights. Previous studies using ectopic overexpression of MDA5 in cell lines have reached contrasting conclusions with regards to the impact of the rs1990760 nsSNP on the activities of MDA5. Some studies reported no influence on function11–13, while another study suggested that the risk allele T (946 T) encodes a constitutively active MDA5 protein that is unable to respond to a natural ligand (encephalomyocarditis virus, EMCV14). These contrasting results illustrate that additional studies are required to reveal if and how the rs1990760 affects the function of MDA5. Recent studies have shown that the functional impact of disease-associated SNPs may vary depending on the cell type studied (e.g.15). This, together with the recent notion that the antiviral effector mechanisms of MDA5 may vary in response to different types of viruses14,16,17, suggest that studies using relevant human primary cells and pertinent natural ligands (i.e. clinically relevant viruses rather than viral mimics such as poly I:C) are needed to gain a better understanding of the function of the disease-associated variants of IFIH1. Enterovirus infections, in particularly those with Coxsackievirus (CVBs), have been associated with the development of T1D in humans. The link between CVB infections and T1D is supported by case reports, epidemiological data and by observations indicating that enteroviruses are present in the remaining beta cells of individuals with T1D diabetes18–25. We have shown that human pancreatic islets express MDA526, and together with others demonstrated that MDA5 is important in the host immune response to CVBs27,28. Human pancreatic islets are easily and reproducibly infected by CVBs in vitro (e.g.25,26,29). This makes the islets a relevant and useful model to study a primary cell response to a CVB infection and to assess whether the rs1990760 nsSNP affect this response. In the present study, we used human pancreatic islets (primary cells) and a virus of clinical relevance (Coxsackievirus) as natural ligand, to provide a better understanding of the functional consequences of this polymorphism. Our study reveals previously not described relationships between the rs1990760 polymorphism and the magnitude of the innate response to Coxsackievirus infection. Results Whole transcriptome analysis identifies an IFN signature in CVB3 infected islets. Infections with CVBs are associated with human diseases, including aseptic meningitis, myocarditis and autoimmune T1D. The serotype B3 (CVB3) replicates in human pancreatic islets, with a peak around 48–72 h post infection (p.i.) (26,29, data not shown). Moreover, robust changes in gene transcription can be measured at 48 h p. i. (29; SupplementaryFig.1). Here, pancreatic islets from a total of 23 human donors were infected with CVB3 for 48 h. The islets were highly permissive to infection as shown by the measurements of high titers of infectious virus particles released into the tissue culture media (Fig.1a) and concomitant expression of viral RNA in the infected islets (Fig.1b). Genotyping for rs1990760 was performed on DNA isolated from each individual islet batch. This analysis demonstrated that the islet cohort consisted of a mixture of donors carrying either the homozygous T1D diabetes risk genotype, TT, or TC, a genotype associated with protection from disease10. None of the donors carried the protective genotype CC (Fig.1c). In parallel, the islet cohort was genotyped for the rs3747517 SNP (SupplementaryTableS1). The results from this genotyping were in line with the previously published strong linkage disequilibrium with rs19907609,16,30,31. expression). Data is shown as mean ± SEM, ****p < 0.0001, Wilcoxons matched-pairs signed rank test. n.d., not detected. (c) Human islet donors included in the study were genotyped for the polymorphisms rs1990760 (A946T). Allele distribution is shown in numbers and as percentage of the whole cohort (n = 23). (d) Pie chart showing the enrichment of process networks based on the 166 DEGs identified by RNAseq. Data was generated using the MetaCoreTM enrichment analysis tool with a cut off p-value of 0.05. The content of the process networks is defined and annotated by Thomson Reuters scientists. Each process represents a pre-set network of interactions characteristic for the process. (e) List of 10 genes displaying the highest log2 fold change in expression in infected human islets versus uninfected, as identified by RNAseq. (f) Pie chart showing the number and percentage of the 166 DEGs identified as ISGs or non-ISGs based on the Interferome database. (g) Venn diagrams showing the number of ISGs (upper diagram, yellow) and non-ISGs (including IFNs, lower diagram, red) commonly or differentially expressed in the two rs1990760 genotype populations (TT and TCs). (h) Heat map of differentially expressed ISGs (n = 134). Log2 (CPM) values were mean-centred and scaled for each gene (white, low expression difference; blue high expression difference). The Pearson correlation was used to compute distances between genes and samples, and the clustering was performed using average linkage. Each column corresponds to one islet donor with indicated rs1990760 genotype and each row corresponds to a specific gene. www.nature.com/scientificreports/ 4 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 To address whether the rs1990760 nsSNP has an influence on the human islet response to CVB3 infection, islets from eight of the 23 donors (TT, n = 4; TC, n = 4) were subjected to whole-transcriptome analysis (RNAseq). Using a stringent statistical method (see Methods) this analysis identified a total of 166 mRNA transcripts that were significantly altered in infected compared to uninfected islets (FDR < 0.05, Fig.1d, SupplementaryTableS2). Functional annotation of the differentially expressed coding genes (DEGs) using MetaCoreTM (Thomson Reuters) suggested significant enrichment of biological pathways related to inflammation, innate immunity and response to viral infection (Fig.1d). With the same software, we identified several transcription factors critical for robust antiviral responses (e.g. IRF-1, STAT-1 and -2), as well as both type I (IFNβ ) and III IFN (IFNλ 1–3) genes (Fig.1e, and SupplementaryTableS2). We also observed an increased expression of genes encoding negative regulators Figure 2. CVB3 infection of human pancreatic islets results in the upregulated expression of type I and III IFNs and ISGs. (a–c,e and f) mRNA expression levels of IFNβ (a), IFNλ 1 and IFNλ 2 (b), PRRs MDA5, RIG-I and TLR3 (c), MxA (e) and CXCL10 (f) were measured at 48 h p.i. in the mockor CVB3 infected islets described in Fig.1 (n = 23) using quantitative real-time PCR. The expression levels were normalized to that of GAPDH and presented as 2−(ΔCt) (relative expression) for all islets. (d) Protein was isolated from a total of 6 islet donors. At 48 h p.i. the protein expression levels of MDA5 (3 donors) and RIG-I (3 donors) were measured in mock and CVB3 infected islets using western blot. Actin was used as loading control. Data from one donor is shown for each protein. The displayed figures are cropped images. Images of uncropped blots can be viewed in SupplementaryFig.S2. (g) The amount of secreted CXCL10 protein was measured in supernatant harvested from mock-infected and CVB3 infected islets (n = 23) using ELISA and expressed as ng/ng RNA. Data is shown as mean ± SEM, ****p < 0.0001, Wilcoxons matched-pairs signed rank test. n.d., not detected. www.nature.com/scientificreports/ 5 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 of IFN expression and signalling (e.g. USBP18, TRIM21, GBP4, SupplementaryTableS2)5. Because we noted an upregulated expression of type I and III IFNs, which are known to induce a wide range of genes (so-called ISGs), we used the Interferome v2.0 database32 to identify how many of the DEGs were ISGs. This analysis revealed that the majority (80.7%) of the 166 DEGs were ISGs (Fig.1f). The magnitude of the human islet immune response to CVB3 infection is linked to the rs1990760 genotype. Most of the DEGs identified in the RNAseq analysis (97%) were shared between donors carrying the TT and TC genotypes, with only 4 DEGs unique to donors with the TC genotype (Fig.1g). Hierarchical clustering based on the 134 genes identified as ISGs distinguished two major clusters, in which the majority (3/4) of donors carrying the TT genotype were placed in a cluster with low relative gene expression levels. The majority of islets carrying the TC genotype (3/4) were clustered together based on high relative gene expression (Fig.1h). Taken together, the transcriptome analysis demonstrated that CVB3 infection induces a robust immune response in human pancreatic islets. These studies also indicated that the strength of this response might be regulated by the rs1990760 genotype. The rs1990760 TC genotype is associated with a high type III IFN response and a strong IFN signature. We next validated some of the results from the transcriptome analysis using conventional qPCR. By analysing cDNA from the whole islet cohort (n = 23) we verified that type I (IFNβ ) and III IFNs (IFNλ 1 and IFNλ) showed increased expression following CVB3 infection (Fig.2a and b). Similarly, the expression levels of a number of the identified DEGs including PRRs (e.g. the IFIH1/MDA5 gene itself and DDX58/RIG-I), and genes encoding proteins involved in cellular antiviral defence (e.g. Mx1/MXA) or recruitment of lymphocytes and with relevance for T1D (CXCL1033) (Fig.2c,e,f) were elevated. Western blot or ELISA measurements revealed that the induced gene expression translated into increased protein expression (Fig.2d) and secretion (Fig.2g). We attempted to measure secreted type I and III IFNs, but our pilot studies using an ELISA and a sensitive bioassay failed to detect IFNs in the culture media of both control and CVB3 infected islets (data not shown). This is likely due to that enteroviruses, including CVB3, have developed mechanisms to impair type I and III IFN production and release34,35, and that the secreted IFNs are locally consumed due to binding to receptors expressed by the cultured cells. When stratifying our donors according to the rs1990760 genotype no statistically significant difference was detected in the expression of IFNβ mRNA between the islets carrying the TT and TC genotypes (Fig.3a). In contrast, islets from donors carrying the TC genotype expressed significantly higher levels of type III IFNs when compared to islets carrying the TT genotype (p < 0.05; Fig.3b). Similarly, TC islets expressed higher levels of the studied genes (ISGs), and the difference was observed also when comparing the fold induction (i.e. Δ Δ Ct-values) (p < 0.05 and p < 0.01; Fig.3c–g). In line with the gene expression data, the secretion of the chemokine CXCL10 was also significantly higher in islets from donors with the TC genotype compared to those with the TT genotype (Fig.3h). In contrast, the expression levels of the studied genes did not differ between the mock-infected islets of the TT and TC genotypes Fig.3c–g). The titers of virus accumulating in the media during the whole 48 h culture period, as well as the expression of viral RNA at 48 h p.i., were not statistically different between the donors, although the latter was close to reaching significance (p = 0.09, Fig.3i). Collectively, this shows that the human islets carrying the rs1990760 TC genotype have a higher type III IFN and ISG response to CVB3 than islets carrying the rs1990760 TT (risk) genotype. A high type III IFN response to CVB3 infection in TC donors is associated with a strong expression of IRF-1. IFN expression is regulated by the activities of transcription factors as well as negative regulators3,36,37. Our transcriptome analyses identified several transcription factors as being inducible by CVB3 infection (Fig.4a). A few of these showed significantly different expression levels between the TT and TC genotypes (BATF2 and STAT1) (Fig.4a). In addition to this, the expression of IRF-1 was significantly increased only in infected islets with the TC genotype (p < 0.01; Fig.4a). Four negative regulators of type 1 IFN signalling, IFI35, IFIT1, OASL, USP185, showed a different level of expression in the two genotypes (Fig.4b). All four were expressed at a higher level in the TC genotype (high responders) as compared to the TT genotype (low responders). Thus, it is unlikely that these genes contributed to a higher expression of type III IFNs in islets from donors with the TC genotype per se, and these four genes were therefore not further studied. The transcription factor IRF-1 has been shown to specifically regulate type III and not type I IFN expression during RNA virus infections3,37. Thus, we next analysed the expression of IRF-1 in the whole islet cohort by real-time PCR and confirmed that CVB3 infection induces the expression of IRF-1 (Fig.5a). We found no difference in the basal expression levels of IRF-1 when control islets from TT and TC donors were compared (Fig.5a). After CVB3 infection, islets with the TC genotype expressed higher levels of IRF-1 mRNA compared to islets with the TT genotype (Fig.5a). Infected islets with the TC genotype also had a stronger increase in their IRF-1 mRNA expression relative to uninfected control islets (i.e. “fold induction”) as compared to islets with the TT genotype (Fig.5a). We next investigated whether there was a correlation between the levels of IRF-1 and type I and III IFN mRNA expression. When analysed for the entire human islet cohort the levels of expressed IRF-1 mRNA correlated with the expressed levels of IFNβ , IFNλ 1 and IFNλ 2 (Fig.5b). However, when analysing the TT and TC donors separately it was found that the levels of IRF-1 mRNA correlated with the levels of the type III IFNs (IFNλ 1 and IFNλ 2) only in TC and not TT donors (Fig.5c,d). This suggests that higher IRF-1 expression in the TC donors contributes to the stronger expression of type III IFNs via a feedback mechanism (Fig.6). www.nature.com/scientificreports/ 6 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 Figure 3. Homozygosity for the T (Thr946) allele of the rs1990760 polymorphism is associated with low human islet type III IFN and ISG mRNA expression levels, as well as amounts of secreted CXCL10, following CVB3 infection. (a and b) IFNβ (a), IFNλ 1 and IFNλ 2 (b) mRNA expression levels measured at 48 h p.i. in the CVB3 infected islets described in Fig.2 (n = 23) were re-analysed and the donors stratified according to the rs1990760 genotype (TT or TC). The expression levels were normalized to that of GAPDH and presented as 2−(ΔCt) (relative expression) for all islets. (c–g) mRNA expression levels of PRRs (MDA5, RIG-I, TLR3; (c–e), genes involved in antiviral defence (MxA; (f) or chemoattraction (CXCL10; (g), measured at 48 h p.i. in the mock-infected (left column, C) and CVB3 infected (two right columns, CVB3) islets described in Fig.2 (n = 23) were re-analysed by dividing the donors according to the rs1990760 genotype (TT or TC). The expression levels were normalized to that of GAPDH and presented as 2−(ΔCt) (relative expression; the two left columns), or normalized to GAPDH and compared to the expression levels in mock-infected islets from the same donor and presented as 2−(ΔΔCt) (fold induction: the right column). Fold induction calculation was not applicable to IFNβ , IFNλ 1–2 and CXCL10 mRNA, which were undetectable in mock-infected islets). (h) The amount of secreted CXCL10 protein (see Fig.2g) was measured in supernatant harvested from mock-infected (left column, C) and CVB3 infected (right column, CVB3) islets using ELISA and expressed as ng/ng isolated RNA. The islet donors were stratified according to rs1990760 genotype TT or TC. (i) The data described in Fig.2 was re-analysed and the islets were divided according to the rs1990760 genotype (TT or TC). Titers of replicating virus in supernatants harvested at 48 h p.i. are presented as Log10(PFU/ml). The expression levels of virus RNA in CVB3 infected islets were measured using quantitative real-time PCR. The expression levels were normalized to that of GAPDH and presented as 2−(ΔCt) (relative expression). Data is shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, Mann-Whitney test. www.nature.com/scientificreports/ 7 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 The expression of T1D-related genes does not differ between donors with the rs1990760 TT and TC genotypes. Risk for T1D development has been linked to polymorphisms in numerous genes besides IFIH1. Many of these genes are expressed by pancreatic beta cells and/or islets38. Using our gene transcription data (SupplementaryTableS1) and the T1Dbase.org we identified some of these genes as having an altered expression level following CVB3 infection, including the upregulated expression of TAGAP, CD69 and Figure 4. The gene expression of several transcription factors and genes involved in negative regulation of type 1 IFN signalling is increased in human islets upon CVB3 infection. RNA sequencing was performed on RNA isolated from non-infected and CVB3 infected primary human islets harvested at 48 h p.i. (8 donors in total; 4 TT and 4 TC) Among the genes demonstrating a differential expression between control and infected islets, 10 were identified as transcription factors and 8 have been described as negative regulators of type I IFN signalling5. The mRNA abundance is shown as log2(CPM) of identified transcription factors (a) and regulators of IFN signalling (b) from control and CVB3 infected islets carrying the rs1990760 TT (TT control, TT infected) or TC genotype (TC control, TC infected). Data is shown as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Bonferroni correction. www.nature.com/scientificreports/ 8 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 Figure 5. Higher expression levels of type III IFNs in TC donors correlate with the expression levels of the transcription factor IRF-1. (a) mRNA expression levels of IRF-1 were measured at 48 h p.i. in the mockand CVB3 infected islets described in Fig.1 (n = 23) using quantitative real-time PCR. The expression levels were normalized to that of GAPDH and presented as 2−(ΔCt) (relative expression) for the whole islet cohort (first column). The results were re-analysed in that the donors were divided according to their rs1990760 genotype (TT or TC). The expression levels in the mock infected (C, second column) and CVB3 infected human islets (CVB3, third column) were normalized to that of GAPDH and presented as 2−(ΔCt) (relative expression), or normalized to GAPDH and compared to the expression levels in mock-infected islets from the same donor and presented as 2−(ΔΔCt) (fold induction: fourth column). (b) Correlation analysis was performed by comparing the mRNA levels for selected genes expressed by CVB3 infected islets at 48 h p.i. (n = 23). The 2−(ΔCt) values of IRF-1 were correlated with that of IFNλ 1, IFNλ 2 and IFNβ in CVB3 infected islets. (c,d) The correlations described in (b) were separately performed for donors with the rs1990760 TT (c) and TC (d) genotypes. Linear regression and the correlation coefficient r, R2 and p values were calculated using Pearson correlation test (b–d). p < 0.05 was considered statistically significant. www.nature.com/scientificreports/ 9 Scientific RepoRts | 6:39378 | DOI: 10.1038/srep39378 NF-κB IKKβ IKKα MAVS MDA5 IFNAR IFNλR ISGF3 ISGs IκB Degradation Type I interferons CVB3 Type III interferons P Peroxisome 946T Cell surface Nucleus 946T TAK1 TBK IKKε MAVS MDA5 TRAF6 P IRF3 P IRF1 IRF3 JAK TYK2 JAK TYK2 STAT1 STAT2 IRF9 TT (rs1990760) 946T autocrine, paracrine TC (rs1990760) 946A/T NF-κB IKKβ IKKα MAVS MDA5 IFNAR IFNλR ISGF3 IκB Degradation CVB3 P Peroxisome 946A/T Cell surface Nucleus 946A/T TAK1 TBK IKKε MAVS MDA5 TRAF6 P IRF3 P IRF1 IRF3 JAK TYK2 JAK TYK2 STAT1 STAT2 IRF9 Feedback autocrine, paracrine Type I interferons Type III interferons ISGs e.g. IRF1 Figure 6. Proposed model of type I and type III IFN induction and action in islets from donors carrying the TT and TC rs1990760 genotypes. CVB3 infection of islets from donors carrying the rs1990760 TT genotype (MDA5, 946 T) (upper model) results in replication of viral RNA and the generation of a viral intermediate (dsRNA). MDA5 binds dsRNA and activates mitochondrial and peroxisomal associated MAVS. This initiates a signalling cascade leading to the phosphorylation and dimerization of IRF-3 through TBK-IKKε , and the degradation of Iκ B from the NF-κ B complex through TRAF6 and TAK1. The activated transcription factors IRF-3 and NF-κ B translocate to the nucleus where they bind to the promoter regions of type I and type III IFNs and initiate their expression. In addition, peroxisome associated MAVS leads through unknown mechanisms to the activation of the transcription factor IRF-1, which binds solely to the promoter regions of type III IFNs and selectively initiate their expression. Expressed and secreted type I and type III IFNs bind in an autoand paracrine manner to their receptors IFNAR and IFNλ R, respectively. This in turn activates JAK-TYK2 kinases leading to the formation of the ISGF3 complex and the expression of a large number of ISGs. Infection of islets from donors carrying the rs1990760 TC genotype (MDA5, 946 T/A) (lower model) results in stronger type III IFN gene expression compared to that induced in islets from TT donors, possibly through the peroxisome associated MAVS mediated activation of IRF-1. The stronger type III IFN expression is accompanied by a more robust ISG expression in TC donors compared to TT donors. This includes the expression of IRF-1, which may further amplify the type III IFN expression through a positive feedback mechanism. Why MDA5 activation in donors carrying the rs1990760 TC genotype leads to a stronger activation of peroxisome associated MAVS compared to mitochondrial associated MAVS is still to be discovered.