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

Abstract

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.

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Figure 1: IFIH1R mediates a modest increase in poly(I:C)-triggered production of IFN-β and leads to a 'basal type I interferon signature'.
Figure 2: Mouse IFIH1T946 displays increased basal and ligand-dependent signaling in vitro.
Figure 3: IFIH1R mice exhibit increased embryonic loss and enhanced basal IFIH1 activity.
Figure 4: Ifih1R mice exhibit protection from EMCV challenge.
Figure 5: IFIH1R mice exhibit enhanced triggering of autoimmune disease.
Figure 6: mIFIH1R mediates enhanced sensitivity to self RNA ligands.

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Acknowledgements

We thank K. Sommer for laboratory management and assistance with manuscript editing; the University of Washington Histology and Imaging Core and Benaroya Clinical Core; M. Kinsman for technical assistance with RNA preparation; and E. Whalen and V. Gersuk for assistance with Fluidigm assays. Supported by the US National Institutes of Health (DP3-DK097672 to J.H.B.; DP3-DK111802 to D.J.R.; R01-AI084914 to D.B.S.; R01-AI104002, R01-AI060389 and R01-AI127463 to M.G.; U19-AI083019 to M.G.; U19-AI100625 to M.G.; R01-DK106718 to P.C.; T32-AR007108 to J.A.G; and T32-GM007270 to K.P.), the Juvenile Diabetes Research Foundation (3-APF-2016-177-A-N to Y.G.), the Children's Guild Association Endowed Chair in Pediatric Immunology and the Benaroya Family Gift Fund (D.J.R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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J.A.G., C.H., A.E.S. and Y.G. designed and performed experiments, analyzed data and wrote and/or edited the manuscript; J.S.E. designed and performed experiments and analyzed data; E.J.A, T.A., C.C., X.D., S.K., K.P., K.C. and M.O. developed required models, strains or reagents and/or performed experiments; D.L., D.B.S., R.G.J., P.C. and M.G. analyzed data and edited the manuscript; J.H.B. designed and interpreted human-subject studies; and D.J.R. conceived of and supervised the study, interpreted data and edited the manuscript.

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Correspondence to David J Rawlings.

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Integrated supplementary information

Supplementary Figure 1 PBMCs from healthy human subjects with IFIH1R manifest increased ISG expression.

(a-b) PBMCs from healthy donors left unstimulated or stimulated with poly(I:C) and quantitative RT-PCR was performed to assess IFIH1 mRNA expression (as described in Fig 1). Each data point represents an individual subject. Bars represent mean. (a) Baseline IFIH1 mRNA levels. (b) IFIH1 mRNA levels following poly(I:C) stimulation for according to IFIH1 genotype or haplotype (as detailed in Fig 1). (c-d) Human PBMCs were thawed and rested for 24 hours and mRNA was analyzed using a custom high-throughput qPCR (Fluidigm) as in Fig 1 to assess ISG expression. Data points show log-transformed relative expression. Welch’s T test was used for statistical analysis. IFIH1R843 was held constant for all individuals. Each data point represents an individual subject. Bars represent median. (c) Additional ISGs with statistically significant increase in IFIH1R subjects. (d) ISGs reaching borderline statistical significance (defined as p=< 0.1). NR=non-risk (encoding IFIH1H843 and IFIH1A946); R=risk (encoding IFIH1R843 and IFIH1T946). *p<0.05.

Supplementary Figure 2 The mIFIH1 protective variant ablates innate signaling, while mIFIH1R promotes protection from West Nile virus.

(a-g) HEK293T cells were transfected with 1 μg of plasmid co-expressing either empty vector (EV) or the indicated mIFIH1 constructs described in Fig. 2. (a-b) Data from four biological replicates showing: (b) Geometric MFI and (c) Percent GFP+ for the histograms shown Fig. 2c. (c-d) IFN B1 mRNA expression in cells transfected with mIFIH1NR or mIFIH1P constructs at time points indicated showing: (c) Representative experiment from one of three biological replicates; (d) Mean ratio of 3 biological replicates. Error bars represent ± SEM. Statistical analysis using a two-tail student T test. (e-g) At 15 hours post-transfection with the indicated mIFIH1 constructs, cells were infected with West Nile Virus (WNV; MOI of 5) or left uninfected (mock) and harvested at the indicated time points. (e) Quantitative RT-PCR for IFNB1 mRNA expression with data normalized as in Fig. 2d. (f) WNV, IFIH1 and β-actin protein expression assessed by Western blotting. Whole cell lysates were resolved by SDS-PAGE and immuno-blotted with indicated antibodies. (+) represents plasmid or virus present, (-) represents plasmid or virus absent. (f) Densitometry analysis of WNV protein expression at the 30 hours normalized to β-actin levels and displayed as relative fold change. Representative data from one of three WNV infection experiments are displayed. **p<0.01, ****p<0.0001.

Supplementary Figure 3 Generation of Ifih1R knock-in mice.

(a) Strategy for generating the Ifih1R knock-in mice with introduction of point mutation within exon 15 of Ifih1 designed to introduce the risk variant allele. Also shown are location of neomycin cassette with flanking FRT sites and the location of LoxP sites introduced in order to permit future generation of lineage-specific Ifih1deletion via intercrossing with Cre-expressing strains. (b) DNA sequencing reaction showing the Ifih1 coding change from GCA (Ala) to ACA (Thr) in homologous knock-in (Ifih1R/R) mice. (c) Quantitative RT-PCR to assess Ifih1 mRNA expression. Ct values were normalized to Hprt. (d) Splenocytes were isolated from 2-12 month old animals of the indicated genotypes. Fold increase relative to the average WT values for each experiment is displayed. Each dot represents an individual animal. Error bars represent ± SEM and significance was assessed using one-way ANOVA (c) or Kruskal-Wallis test (d).

Supplementary Figure 4 Ifih1R mice display enhanced triggering of autoimmune disease.

(a-b) Mice were injected with STZ for 4 days (55 mg/kg) and monitored for development of diabetes as described in Figure 5c. Tissues were harvested at week 15 post-STZ treatment for genotypes that developed disease [Ifih1NR/NRPtpn22NR/R (n=2) or Ifih1NR/RPtpn22NR/R (n=2)] and analyzed in a blinded fashion for histological changes. Scale bars equal 100 μm. (a) STZ treated heterozygous Ifih1NR/NRPtpn22NR/R mice. Both diabetic (A1, 20X) and nondiabetic (A2, 20X) animals had mild cellular changes evident in islets in H&E stained sections. Immuno-histochemical stains revealed reduced numbers of dark brown insulin positive cells in diabetics (B1, 20X) compared to nondiabetics (B2, 20X). Inflammation was rare and scant. (b) Compound heterozygous Ifih1NR/RPtpn22NR/R diabetic mice exhibited inflammatory changes in the pancreas with lesion severity ranging from scattered mild lymphocytic insulitis (arrow, C1, 40X) to atrophy and sclerosis with replacement by fibrous tissue containing pancreatic ductal remnants (arrows, C2, 10X). Immuno-histochemical staining for insulin and CD3 demonstrated atrophic and fibrosing pancreatitis containing whole or fragments of islets (arrows showing brown foci, D1, 20X) or CD3+ inflammatory cells (scattered brown foci, D2, 20X) dispersed among an isolated islet (arrow) and remnants of pancreatic ducts (stars). (b-c) BM12 CD4+ T cell were adoptively transferred into mice of indicated genotypes as described in Figure 5. Each dot represents results from an individual animal. (b) ELISAs for IgG anti-smRNP (upper panel) and IgG2c anti-smRNP (lower panel) autoantibodies at time points indicated. (c) Splenic cell numbers at week 15 post injection. Error bars represent ± SEM.

Supplementary Figure 5 Full-length immunoblots.

(a) Full western blot from Fig. 2b immuno-blotted with anti-IFIH1 (left) and anti-β-actin (right) antibodies. (b) Full western blots from Sup. Fig. 2f immuno-blotted with anti-IFIH1 (top), anti-β-actin (middle), and anti-WNV (bottom) antibodies. The rightmost three lanes are from an unrelated experiment. Positions of loading controls used to assess relative protein molecular weight are indicated.

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Gorman, J., Hundhausen, C., Errett, J. et al. The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity. Nat Immunol 18, 744–752 (2017). https://doi.org/10.1038/ni.3766

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