Integrin Alpha E (CD103) Limits Virus-Induced IFN-I Production in Conventional Dendritic Cells

Abstract

Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E (Itgae, CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection.

Keywords: AKT; CD103; GWAS; IFN-I; Itgae; genome wide association screen; mTOR; vesicular stomatitis virus.

Figure 1

GWAS screen reveals CD103 as regulator of viral replication. (A) Virus titers in spleens of different inbred mouse strains which were intravenously (i.v) infected with 2x10⁸ PFU of VSV and analyzed after 8 h (n = 3). (B) Manhattan plot showing the distribution of SNPs present in each chromosome in exon regions on x-axis with its associated p-values on y-axis of the EMMA analysis based on the virus titers measured in (A). Five most significantly regulated genes are highlighted with red arrows. (C) Plot representing distribution of reference SNPs based on classification accuracy (x-axis), Bayesian Cohen’s d (y-axis) and Cohen’s kappa (colour code) of the genphen analysis of the data in (A). The list of all SNPs present in the top refSNP which is represented by dotted circle is within Table S2 . The table depicted at top of (C), shows the top SNPs located in the exon regions. (D) Viral titers from spleen and liver of WT or Sirpa^−/− or Itgae^−/− mice after i.v. infection with 2x10⁸ PFU of VSV, measured after 8h (n = 4 for WT and Sirpa^−/−; n = 10 for WT and Itgae^−/−, pooled from two independent experiments). Horizontal dotted lines represent the detection threshold (D). Data are shown as mean ± SEM. ***P < 0.001; ****P < 0.0001 (Student’s t-test).

Figure 2

CD103 limits early Interferon production and enhances susceptibility to virus infection. (A) Representative FACS plots for CD103 expression on different splenic immune cells of naïve WT and Itgae^−/− mice (n = 4). Values in the plots represents the frequency of cells within CD103 positive bar. (B) Total IFN-α in serum of WT and Itgae^−/− mice, which were infected with 2x10⁸ PFU of VSV and at various time points as indicated (n = 3-10). (C) Gene expression data determined by RT-PCR in various FACS sorted DC subtypes from spleens of WT and Itgae^−/− mice which were left untreated or were infected i.v with 2x10⁹ PFU of VSV for 2h or 4h (n = 3). (D) Representative FACS plots showing staining for VSV glycoprotein (GP) on splenic DC subtypes of naïve WT, and WT, Itgae^−/− and IFNAR^−/− mice which were infected i.v with 2x10⁹ PFU of VSV for 4h (n = 3). (E) Survival of WT and Itgae^−/− mice, which were infected with 2x10⁷ PFU of VSV (n = 14-16). Data are shown as mean ± SEM or Kaplan Meier graph. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; (Student’s t-test for B and C, and Mentel-Cox survival test for C).

Figure 3

CD103 limits IFN-I response in bone marrow derived dendritic cells. (A) Representative FACS plot showing CD103 expression on MHCII⁺CD11c⁺ cells from naïve BMDC culture from WT or Itgae^−/− mice (n = 3). Values in the plots represents the frequency of cells within CD103 positive bar. (B) Expression of Ifna4 and Ifnb1 determined by RT-PCR in WT and Itgae^−/− BMDC which were left untreated or were treated with VSV (MOI = 1 for 3 h, n = 3). (C) Representative FACS plot showing VSV-GP staining from naïve or VSV treated (MOI-0.01 for 24h) BMDC generated from WT or Itgae^−/− mice (n = 3). (D, E) BMDC generated from WT and Itgae^−/− mice (n = 6) incubated with VSV-WT (MOI = 1, D) or VSV- EBOV (MOI = 1, E) and after 1h media was replaced with virus free fresh complete media and virus titers measured in supernatant at indicated time points. (F) Virus titers in supernatants of WT and Itgae^−/− BMDC, which were treated with anti-IFNAR antibody (40µg/ml) or isotype control antibody (40µg/ml), measured after infection with VSV (MOI = 0.001, n = 6). (G) Virus titers in supernatants of WT and Itgae^−/− BMDC, which were treated with IFNa4 (100U/ml), measured after infection with VSV (MOI = 0.01, n = 6). Data are shown as mean ± SEM. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Student’s t-test).

Figure 4

CD103 modulates AKT activation and mTOR signaling. (A) Representative western blots for naïve WT and Itgae^−/− BMDC detected with antibodies to phospho-AKT (Thr308), phospho-AKT (Ser473), AKT (pan), phospho-GSK-3β (Ser9), GSK-3β (pan) and GAPDH. Graphs show the quantifications for p-AKT (Thr308), p-AKT (Ser473), and phospho-GSK-3β (Ser9) from naïve WT and Itgae^−/− BMDCs (n = 3). (B) Representative western blots for naïve WT and Itgae^−/− BMDC detected with antibodies to p-mTOR (Ser2481) and mTOR (pan). Graph shows the quantifications for p-mTOR (Ser2481) from naïve WT and Itgae^−/− BMDCs (n = 3). (C) Virus titers at different time intervals after virus infection in supernatants of WT and Itgae^−/− BMDC, which were treated with or without the AKT inhibitor GSK2141795 (4500 nM) for 24h and infection with VSV (MOI = 0.01, n = 6). Data shown is pooled from two independent experiments. (D) Virus titers at different time intervals after virus infection in supernatants of WT and Itgae^−/− BMDCs, which were treated with or without the mTOR inhibitor Rapamycin (200 nM) for 24h and infection with VSV (MOI = 0.01, n = 3). (E) Expression of Ifnb1 determined by RT-PCR in WT and Itgae^−/− BMDC which were treated with or without rapamycin (200nM) for 24h and infected with VSV or left without infection for 3h (MOI = 1). Data are shown as mean ± SEM. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Student’s t-test).