Activation of innate immune responses through Toll-like receptor 3 causes a rapid loss of salivary gland function

Abstract

Background: Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögren's syndrome (SS) patients. As viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function.

Methods: Female New Zealand Black/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid [poly(I:C)]. TLR3 expression within submandibular glands was studied using immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time polymerase chain reaction. Pilocarpine induced saliva volume was used as an index of glandular function.

Results: Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules, and ducts. Poly(I:C) treatment rapidly up-regulated the mRNA levels of type I interferon (IFN) and inflammatory cytokines in the submandibular glands. One week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the phosphate-buffered saline (PBS) treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped.

Conclusions: Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.

Figure 1. Epithelial cells of murine salivary glands express TLR3

Representative photomicrographs of immunohistochemical staining of salivary glands from NZB/WF1 (A, B) and C57BL/6 (C) female mouse for TLR3 expression. (A) TLR3 is detected on the base of secretory cells of mucus (m) and serous (s) acini in the salivary glands and is indicated with arrows. (B) In addition, TLR3 staining was observed in the cytoplasm of the ductal (d) and granular convoluted tubular (gct) epithelium. (C) A similar distribution of TLR3 expression was seen in a non-autoimmune C57BL/6 female. (D) Sections incubated with rabbit polyclonal serum antibody served as negative controls (magnification 40X).

Figure 2. Expression of type I IFN, IFN responsive genes and proinflammatory cytokine genes is upregulated in the submandibular glands of poly(I:C) treated mice

Expression levels of IFN-β1, Mx1, PRKR, IFI44, IL-6, TNF-α, IL-1β, and CCL5 in submandibular glands of poly(I:C) (n=5) and PBS (n=5) treated mice were determined by real time PCR using Taqman gene expression assays. Each data point represents RNA expression in one mouse and data are represented as mean duplicate relative expression calculated by the 2(−Delta Delta C(T)) method. Numbers denoted with ‘x’ represent fold increase in RNA expression in poly(I:C) treated mice in comparison with PBS treated mice. Also shown are p values determined using non parametric student’s t-test. NS; not significant.

Figure 3. Poly(I:C) treatment causes rapid loss of salivary gland function

NZB/WF1 mice were treated either with poly(I:C) (n=10) or PBS (n=9) for one week (days 0, 2, 4 and 6). Pilocarpine induced saliva volume was determined on day 7. Data are represented as saliva volume/body weight (μl/gm). Note poly(I:C) treatment causes a significant (p<0.0001) drop in the saliva produced compared to untreated and PBS treated mice.

Figure 4. Glandular function recovers after stopping poly(I:C) treatment

Mice (n=5 per group) were either treated with poly(I:C) or PBS every other day for 2 weeks. Pilocarpine induced saliva was determined on days 16, and 40 (2 and 26 days after stopping treatment). Data are represented as mean±SEM saliva volume/body weight (μl/gm). Note that mean saliva volume in poly(I:C) treated mice was significantly lower than the PBS treated mice 2 days after stopping the treatment. However, on day 26 after stopping the treatment, the difference in mean saliva volume between the 2 groups of mice was not significant.