Innate immune signaling induces interleukin-7 production from salivary gland cells and accelerates the development of primary Sjögren's syndrome in a mouse model

Abstract

Elevated IL-7 in the target tissues is closely associated with multiple autoimmune disorders, including Sjögren's syndrome (SS). We recently found that IL-7 plays an essential role in the development and onset of primary SS (pSS) in C57BL/6.NOD-Aec1Aec2 mice, a well-defined mouse model of primary SS. However, environmental signals that cause excessive IL-7 production are not well-characterized. Innate immune signaling plays a critical role in shaping the adaptive immune responses including autoimmune responses. We and others have previously shown that innate immune signaling can induce IL-7 expression in lungs and intestines of C57BL/6 mice. In this study, we characterized the effects of poly I:C, a double-stranded RNA analog and toll-like receptor 3 agonist, on the induction of IL-7 expression in salivary glands and on pSS development. We showed that poly I:C administration to C57BL/6 mice rapidly induced IL-7 expression in the salivary glands in a type 1 IFN- and IFN-γ-dependent manner. Moreover, poly I:C-induced IL-7 contributed to the optimal up-regulation of CXCL9 in the salivary glands, which may subsequently promote recruitment of more IFN-γ-producing T cells. Repeated administration of poly I:C to C57BL/6.NOD-Aec1Aec2 mice accelerated the development of SS-like exocrinopathy, and this effect was abolished by the blockade of IL-7 receptor signaling with a neutralizing antibody. Finally, poly I:C or a combination of IFN-α and IFN-γ induced IL-7 gene expression and protein production in a human salivary gland epithelial cell line. Hence, we demonstrate that IL-7 expression in the salivary gland cells can be induced by poly I:C and delineate a crucial mechanism by which innate immune signals facilitate the development of pSS, which is through induction of IL-7 in the target tissues.

Figure 1. Poly I:C induces IL-7 expression in the submandibular glands.

(A) Real-time PCR analysis of gene expression in submandibular glands from C57BL/6 mice 6 hours post poly I:C injection, presented relative to that of β-actin. Data are the average of analyses of 6 individual mice (3 mice per experiment, total 2 independent experiments). (B) Immunofluorescence staining of IL-7 in submandibular gland sections from C57BL/6 mice 24 hourse post poly I:C injection. Differential interference contrast (DIC) image of the same sample is also shown. Data are representative of analyses of 6 individual mice (3 mice per experiment, total 2 independent experiments). (C) C57BL/6 mice were pretreated with anti-IFNAR1 or anti-IFN-γ 2 hours prior to poly I:C injection. After 6 hours, relative IL-7 mRNA levels in lung tissue were measured by real-time RT-PCR. Data are from analyses of 6 individual mice (2 mice per experiment, total 3 independent experiments).

Figure 2. Poly I:C upregulates expression of CXCR3 ligands in submandibular glands in an IL-7-dependent fashion

C57BL/6 mice were pre-treated with anti-IL-7Rα 2 hours prior to poly I:C injection. (A) Levels of CXCL9, 10, 11 and CXCR3 mRNA were measured from submandibular glands 24 hours after poly I:C administration, presented relative to that of β-actin. (B) Submandibular gland sections stained with CXCL9 and DRAQ5. All data are the average of or representative of 6 individual mice (2 mice per experiment, total 3 independent experiments).

Figure 3. NK cells contribute to the early up-regulation of CXCR3 ligands induced by poly I:C.

(A) C57BL/6 mice were treated with poly I:C by for the indicated amount of time. Left panels, surface staining for immune cell populations in the submandibular glands, with the gating indicated above the plots. Right panels, Percentage of NK1.1⁺ TCR-β⁺, TCR-γδ⁺, CD4⁺ and CD8⁺ T cells among total submandibular gland cells. All data are the average of or representative of 5 individual mice (2-3 mice per experiment, total 2 independent experiments). (B) RAG-1^-/- mice were pre-treated with anti-NK1.1 antibody 2 days prior to poly I:C injection. Submandibular glands were harvested 6 hours after poly I:C injection and measured for gene expression by real time PCR. Data are from analyses of 4 individual mice (2 mice per experiment, total 2 independent experiments).

Figure 4. Poly I:C accelerates the development of SS in an IL-7-dependent manner.

Poly I:C, together with either IgG or anti-IL-7Rα antibody, was administered to 12-week old B6.NOD-Aec mice 3 times weekly for 8 weeks. All the following parameters were measured after 8 weeks of consecutive injections. (A) H&E staining of submandibular gland sections (left panels) and focus score of leukocyte infiltration in submandibular glands (right panel). (B) Detection of serum ANA, left panels, and percentages of mice that are serum ANA positive, right panel. All data are representative or the average of analyses of 7 individual mice (1-3 mice per experiment, total 4 independent experiments).

Figure 5. Poly I:C enhances Th1 and Tc1 responses in an IL-7-dependent manner.

B6.NOD-Aec mice were injected with poly I:C plus IgG or anti-IL-7Rα antibody as described in Figure 4. (A) IL-7 mRNA levels in the submandibular glands of non-poly I:C- or poly I:C-treated B6.NOD-Aec mice. (B) Flow cytometric analyses of leukocyte subpopulations among mononuclear cells in submandibular glands. (C) Percentage of IFN-γ⁺ T cells in total submandibular gland cells, splenocytes and dr LN cells based on flow cytometric analysis. Data are representative of or the average of the analyses of 7 individual mice (1-3 mice per experiment, total 4 independent experiments).

Figure 6. Poly I:C induces production of IL-7 in human salivary gland epithelial cells.

(A) HSG cells were cultured in vitro with or without poly I:C for 1 and 3 days. IL-7 mRNA levels, left panel, and protein concentrations in culture supernatants, right panel, assessed by real time PCR and ELISA, respectively. (B) HSG cells were treated with IFN-α, IFN-γ or both for 1 and 3 days, and then measured for IL-7 mRNA levels. (C) HSG cells were treated with poly I:C in the presence of control IgG or anti-human IL-7 for 1 or 3 days, and then measured for BAFF mRNA expression. All PCR results are presented relative to that of GAPDH. All data are the average of analyses of 6 independent samples for each group (3 samples per experiment, total 2 independent experiments).