Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease

Abstract

Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (T_H1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pT_reg) conversion and promoting T_H1 immunity to dietary antigen. Initiation of T_H1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pT_reg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Fig. 1. T1L blocks the differentiation of pT_regs and promotes T_H1 immunity to dietary antigen at inductive and effector sites of the gut

(A) For each time point, WT mice were inoculated perorally with 10¹⁰ plaque forming units (PFU) of T1L (n = 3 mice; red circles), 10¹⁰ PFU of T3D-RV (n = 3 mice; blue circles), or phosphate-buffered saline (PBS) (sham, n = 3 mice; open circles) and euthanized 6 or 48 hours after inoculation. RNA of mLN, PP, epithelium, and Lp were isolated and analyzed by means of microarray. MST is represented on multidimensional scaling ordination. The MST traces a path of minimum weight through each vertex or node that represents the profile of differentially expressed genes for each sample state shown. The lengths of edges (or connecting paths) indicate the level of dissimilarity between samples. Each sample state and the distances between them are represented in two-dimensional space. The coordinates of each sample along each dimension are indicated by the two axes. (B) Mice were inoculated perorally with 10⁸ PFU of T1L (n = 6 mice), 10⁸ PFU of T3D-RV (n = 6 mice), or PBS (sham, n = 5 mice) for 2 days. The expression of IL-12p40 on gated MHC-II⁺ CD11c⁺ CD103⁺ CD11b⁻ CD8α⁺ mLN DCs was evaluated by means of flow cytometry. Representative dot plots and percentages of IL-12p40 in the mLN are shown in the CD103⁺ CD11b⁻ CD8α⁺ DC subset. (C and D) OT-II⁺ CD45.1⁺ CD4⁺ T cells were transferred into WT CD45.2⁺ mice. One day after transfer, mice were inoculated perorally with 10¹⁰ PFU of T1L (n = 4 to 16 mice), 10¹⁰ PFU of T3D-RV (n = 5 to 14 mice), or PBS (sham, n = 6 to 15 mice) and fed 1.5% OVA in the drinking water (filled circles) or an OVA-containing diet (open circles) for 6 days. The intracellular expression of Foxp3 and T-bet in transferred OT-II⁺ CD45.1⁺ CD4⁺ T cells in the mLN and in the Lp was evaluated by means of flow cytometry. Representative dot plots and percentages of Foxp3⁺ T-bet⁻ and T-bet⁺ Foxp3⁻ cells are shown in the mLN (C) and in Lp (D), respectively. [(B) to (D)] Graphs depict at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way analysis of variance (ANOVA)/Tukey’s multiple comparison.

Fig. 2. Type-1 IFN is required for blockade of pT_reg conversion but not for induction of T_H1 immunity to dietary antigen

(A) WT and IFNAR^−/− mice were inoculated perorally with 10⁸ PFU of T1L (n = 6 mice), 10⁸ PFU of T3D-RV (n = 6 mice), or PBS (sham, n = 6 mice) for 2 days. Mx1 expression in the mLN was analyzed by means of reverse transcription polymerase chain reaction (RT-PCR). (B and C) OT-II⁺ CD45.1⁺ CD4⁺ T cells were transferred into WT CD45.2⁺ or IFNAR^−/− CD45.2⁺ mice. One day after transfer, mice were inoculated perorally with 10⁸ PFU of T1L (n = 6 mice) or PBS (sham, n = 4 or 5 mice) and fed 1.5% OVA in the drinking water for 2 days. The expression of IL-12p40 on gated MHC-II⁺ CD11c⁺ CD103⁺ CD11b⁻ CD8α⁺ mLN DCs (B) and T-bet in OT-II⁺ CD45.1⁺ CD4⁺ T cells (C) in the mLN was evaluated by means of flow cytometry. (D to G) OT-II⁺ CD45.1⁺ CD4⁺ T cells were transferred into WT CD45.2⁺ or IFNAR^−/− CD45.2⁺ mice. One day after transfer, mice were inoculated with PBS (n = 7 mice) or 50 mg of poly(I:C) (n = 7 mice) intraperitoneally or 10¹⁰ PFU of T1L (n = 5 mice) perorally and fed an OVA-containing diet for 6 days. The intracellular expression of Foxp3 and IFN-γ in OT-II⁺ CD45.1⁺ CD4⁺ T cells in the mLN was evaluated by means of flow cytometry. Percentages and absolute numbers of Foxp3 [(D) and (E)] and IFN-γ [(F) and (G)] are shown. [(A) to (G)] Graphs depict at least two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA/Tukey’s multiple comparison.

Fig. 3. A central role for IRF1 in reovirus-mediated T_H1 immunity to dietary antigen

(A) WT and IFNAR^−/− mice were inoculated perorally with 10⁸ PFU of T1L (n = 6 mice), 10⁸ PFU of T3D-RV (n = 6 mice), or PBS (sham, n = 6 mice) for 2 days. Irf1 expression in the mLN was analyzed by means of RT-PCR. (B) OT-II⁺ CD45.1⁺ CD4⁺ T cells were transferred into WT CD45.2⁺ or IRF1^−/− CD45.2⁺ mice. One day after transfer, mice were inoculated perorally with 10⁸ PFU of T1L (n = 4 to 6 mice) or PBS (sham, n = 4 to 6 mice) and fed 1.5% OVA in the drinking water for 2 days. The expression of IL-12p40 on gated MHC-II⁺ CD11c⁺ CD103⁺ CD11b⁻ CD8α⁺ mLN DCs was evaluated by means of flow cytometry. (C to F) OT-II⁺ CD45.1⁺ CD4⁺ T cells were transferred into WT CD45.2⁺ or IRF1^−/− CD45.2⁺ mice. One day after transfer, mice were inoculated perorally with 10¹⁰ PFU of T1L (n = 5 or 6 mice) or PBS (sham, n = 4 to 6 mice) and fed 1.5% OVA in the drinking water for 6 days. Intracellular expression of Foxp3 and IFN-γ was evaluated by means of flow cytometry. Representative dot plots (C), percentages of Foxp3 (D), representative dot plots (E), and percentages of IFN-γ (F) are shown in transferred OT-II⁺ CD4⁺ T cells in the mLN. [(A) to (F)] Graphs depict two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way ANOVA/Tukey’s multiple comparison.

Fig. 4. Role of reovirus infection in loss of oral tolerance to gluten and TG2 activation

(A and B) DQ8tg mice were inoculated perorally with 10⁸ PFU of T1L (n = 5 mice) or PBS (sham, n = 5 mice) for 2 days. (A) Levels of Irf1 expression in the mLN were analyzed by means of RT-PCR. (B) The expression of IL-12p40 on gated CD11c⁺ CD103⁺ CD11b⁻ CD8α⁺ mLN DCs was evaluated by means of flow cytometry. (C and D) DQ8tg mice were inoculated perorally with 10¹⁰ PFU of T1L at the initiation of an oral tolerance/delayed type hypersensitivity protocol. Mice were fed orally with gliadin (Glia) for 2 days and then immunized subcutaneously with a CFA-Glia emulsion. (C) Levels of Glia-specific IgG2c antibodies in the serum were quantified at day 18 by means of enzyme-linked immunosorbent assay (ELISA). (D) On day 28, mice were challenged subcutaneously with Glia, and the degree of ear swelling was determined 24 hours after challenge. Sham, n = 4 or 5 mice; Glia, n = 6 mice; and Glia + T1L, n = 5 mice. (E) DQ8tg mice were inoculated perorally with 10¹⁰ PFU of T1L (n = 8 mice) or PBS (sham, n = 4 mice). TG2^−/− mice were inoculated perorally with 10¹⁰ PFU of T1L (n = 2 mice) and used as a negative control. Mice were euthanized at 18 hours after infection, and small intestines were collected and frozen in optimal cutting temperature compound. Representative images from stained frozen sections of the proximal small intestines are shown. Scale bars, 100 mm. Staining with 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue, TG2 protein is shown in green, and TG2 enzymatic activity as assessed by means of 5BP cross-linking is shown in red.TG2 enzymatic activity normalized to TG2 protein levels was quantified for each villus. The mean enzymatic activity in the proximal small intestine per mouse is shown. [(A) to (E)] Graph depicts two independent experiments. (A), (B), and (E), **P < 0.01, ***P < 0.001; unpaired t test. (C) and (D), **P < 0.01; one-way ANOVA/Tukey’s multiple comparison.

Fig. 5. Role of human reovirus infections in celiac disease pathogenesis

(A and D) Boxplots showing levels of reovirus antibody titers (A) and rotavirus antibody titers (D) in control (n = 73) and CeD patients (n = 160). (B and E) Percentage of control patients (gray), active CeD patients (blue), CeD patients on a gluten-free diet (GFD) (orange), and active CeD and GFD patients combined (red) that have reovirus (B) or rotavirus (E) antibody titers above an increasingly higher cutoff (left to right). Cutoffs were determined by defining the deciles of the distribution of reovirus (B) or rotavirus (E) antibody titers observed in the patient samples analyzed. GFD and CeD (Active + GFD) patient groups are significantly overrepresented among individuals with reovirus titers above a PRNT60 = 156 (6th decile) and PRNT60 = 1597 (9th decile), respectively. (C and F) IRF1 expression in small intestinal biopsies of GFD patients (n = 38) was analyzed by means of RT-PCR. To avoid any confounding factors associated with increased inflammation, analysis of IRF1 expression was performed in CeD patients on a GFD who, unlike active CeD patients, display normal levels of IFN-γ. Relative expression level of IRF1 in GFD patients with low (left) and high (right) levels of reovirus (C) or rotavirus (F) antibody titers is shown. (C) Reovirus low and reovirus high were respectively defined as individuals with antibody titers below and above the median PRNT60 = 47 [(B), 5th decile]. (F) Rotavirus low and rotavirus high were respectively defined as individuals with antibody titers below and above the median PRNT60 = 53 [(E), 5th decile]. (G and H) Reovirus and rotavirus antibody titers in serum of patients were determined by means of plaque-reduction neutralization assay. Levels of HSV-1 antibody titers were determined by means of ELISA. Correlations between the levels of reovirus antibody titers and rotavirus antibody titers (G) or HSV-1 antibody titers (H) in GFD patients are shown. r, Pearson correlation. (A) to (F), *P < 0.05; Mann-Whitney U test.