Salivary gland hypofunction induced by activation of innate immunity is dependent on type I interferon signaling

Abstract

Background: Activation of innate immunity through polyinosinic:polycytidylic acid [poly(I:C)] causes acute salivary gland hypofunction. As a major consequence of poly(I:C) treatment is type I interferon (IFN) production, this study was undertaken to investigate their role in salivary gland dysfunction.

Methods: Different strains of mice deficient in either interferon alpha receptor (IFNAR1(-/-)) or IL-6(-/-), or IL-10(-/-), or EBI3(-/-) were treated with poly(I:C). Salivary gland function was determined by measuring pilocarpine-induced saliva volume. Gene expression levels were measured by real-time PCR. Ca(2+) mobilization studies were performed using ex-vivo acinar cells.

Results: A single injection of poly(I:C) rapidly induced salivary gland hypofunction in wild-type B6 mice (41% drop in saliva volumes compared to PBS-treated mice). In contrast, the loss of function in poly(I:C)-treated IFNAR(-/-) mice was only 9.6%. Gene expression analysis showed reduced levels of Il-6, Il-10, and Il-27 in submandibular glands of poly(I:C)-treated IFNAR(-/-) mice. While salivary gland dysfunction in poly(I:C)-treated IL-10(-/-) and EBI3(-/-) mice was comparable to wild-type mice, the IL-6(-/-) mice were more resistant, with only a 21% drop in function. Pilocarpine-induced Ca(2+) flux was significantly suppressed in acinar cells obtained from poly(I:C)-treated wild-type mice.

Conclusions: Our data demonstrate that a combined action of type I IFNs and IL-6 contributes toward salivary gland hypofunction. This happens through interference with Ca(2+) mobilization within acinar cells. Thus, in acute viral infections and diseases like Sjögren's syndrome, elevated levels of type I IFNs and IL-6 can directly affect glandular function.

Figure 1. Poly(I:C) induced loss of salivary gland function is much lower in the absence of type I IFN signaling

IFNAR^−/− and IFNAR^+/+ (wild type mice) mice were injected either with PBS or poly(I:C) and pilocarpine induced saliva was measured. The results are expressed as ratio of saliva volume (μl) to gm body weight. In the IFNAR^−/−mice, the mean saliva volume ± SEM in poly(I:C) group is 5.88 ± 0.07 and in PBS group it is 6.52 ± 0.13. In wild type mice, the mean saliva volume ± SEM in poly(I:C) group is 3.73 ± 0.15 and in PBS treated group it is 6.33 ± 0.18. Thus, the loss of function in IFNAR^−/− mice is 9.6% in comparison to 41% loss in IFNAR^+/+ mice. Similar results were obtained in an independent cohort of mice.

Figure 2. In presence of type I IFN signaling, gene expression levels of Il-6, Il-10 and Il-27p28 are amplified within the salivary glands

IFNAR^+/+ and IFNAR^−/− mice were injected with poly(I:C) and submandibular glands harvested after 3.5 hr. Expression levels of different genes were determined by real time PCR using Taqman gene expression assays. Gapdh was used as house keeping gene and results are expressed as relative change in gene expression. The ratio of mean gene expression level in IFNAR^+/+ mice to that in IFNAR^−/− mice is shown as fold (X) increase or decrease. Mx1 is an IFN responsive gene and was used as positive control. The relative gene expression levels of Il-6, Il-10 and Il-27p28 were 34, 16 and 11 folds higher in IFNAR^+/+ mice. Similar results were obtained in an additional experiment performed in an independent cohort of mice.

Figure 3. IL-6 contributes towards the loss of salivary gland function

Mice deficient in IL-6, IL-10 and IL27B, were either injected with poly(I:C) or PBS and pilocarpine induced saliva measured. The mean saliva volume in all groups of poly(I:C) treated mice are significantly lower than the mean saliva volumes in respective PBS treated controls. Saliva collections in IL-6^−/− mice were done at 2 time points (12wks and 30wks of age) and the loss in function was 26% and 21% respectively. The loss of function in IL-27^−/− (35%) IL-10^−/− (38%) mice was comparable to that observed in wild type mice (41%). Similar results were obtained in an additional experiment.

Figure 4. Ca²⁺ mobilization is suppressed in acini from poly(I:C) treated mice

C57BL/6 mice were either treated with poly(I:C) or PBS and submandibular glands were removed for acinar cell preparation. Cells were loaded with fura-2 AM and one set of cells were loaded with Cell Tracker Red. Following 30 min incubation, cells were washed, placed in recording chamber and calcium changes recorded following exposure to 5 μM of pilocarpine-hydrochloride. The data shown is mean ratio (340/380nm) ± SEM from 5 independent experiments with 70 acini from PBS treated mice and 64 acini from poly(I:C) treated mice. The panel on left shows the whole trace. The differences in baseline between poly(I:C) and PBS treated mice were statistically not significant. The right panel shows baseline subtracted mean ratio ± SEM seen at maximum stimulation. The mean Ca²⁺ 340/380nm ratio in acini from poly(I:C) treated mice is significantly lower (p=0.0003) than that observed in acini from PBS treated mice.