Activation of Myd88-Dependent TLRs Mediates Local and Systemic Inflammation in a Mouse Model of Primary Sjögren's Syndrome

Abstract

Toll-like receptors (TLRs) are important mediators of chronic inflammation in numerous autoimmune diseases, although the role of these receptors in primary Sjögren's syndrome (pSS) remains incompletely understood. Previous studies in our laboratory established Myd88 as a crucial mediator of pSS, although the disease-relevant ligands and the upstream signaling events that culminate in Myd88 activation have yet to be established. The objective of this study was to identify specific Myd88-dependent TLR-related pathways that are dysregulated both locally and systemically in a mouse model of pSS [NOD.B10Sn-H2^b /J (NOD.B10)]. We performed RNA-sequencing on spleens derived from NOD.B10 mice. We then harvested salivary tissue and spleens from Myd88-sufficient and deficient C57BL/10 (BL/10) and NOD.B10 mice and performed flow cytometry to determine expression of Myd88-dependent TLRs. We cultured splenocytes with TLR2 and TLR4 agonists and measured production of inflammatory mediators by ELISA. Next, we evaluated spontaneous and TLR4-mediated inflammatory cytokine secretion in NOD.B10 salivary tissue. Finally, we assessed spontaneous Myd88-dependent cytokine secretion by NOD.B10 salivary cells. We identified dysregulation of numerous TLR-related networks in pSS splenocytes, particularly those employed by TLR2 and TLR4. We found upregulation of TLRs in both the splenic and salivary tissue from pSS mice. In NOD.B10 splenic tissue, robust expression of B cell TLR1 and TLR2 required Myd88. Splenocytes from NOD.B10 mice were hyper-responsive to TLR2 ligation and the endogenous molecule decorin modulated inflammation via TLR4. Finally, we observed spontaneous secretion of numerous inflammatory cytokines and this was enhanced following TLR4 ligation in female NOD.B10 salivary tissue as compared to males. The spontaneous production of salivary IL-6, MCP-1 and TNFα required Myd88 in pSS salivary tissue. Thus, our data demonstrate that Myd88-dependent TLR pathways contribute to the inflammatory landscape in pSS, and inhibition of such will likely have therapeutic utility.

Keywords: B cell; NOD.B10; TLR2; TLR4; decorin; salivary gland.

Figure 1

TLR-related genes are dysregulated in splenic tissue derived from NOD.B10 mice. Spleens were harvested from NOD.B10 females with clinical disease (n = 3) and age and sex-matched BL/10 controls (n = 3) and RNA-sequencing was performed. Bargraph showing the KEGG pathway terms represented in the top 1000 genes enriched in NOD.B10 mice. Dotted line represent the boundary for p = 0.05 (A). Heatmap visualizations showing the relative expression levels of selected DEGs involved in TLR-related signaling pathways (B) and cytokines and chemokines (C) that were enriched in NOD.B10 splenic tissue.

Figure 2

TLR expression is increased in splenic B cells from NOD.B10 mice in a Myd88-dependent manner and differs from that seen in salivary B cells. Spleens were isolated from female BL/10, BL/10^Myd88−/−, NOD.B10, and NOD.B10^Myd88−/− mice (n = 4 each) and SMG tissue was isolated from NOD.B10 females (n = 4 each). Flow cytometry was performed to assess expression of (A) TLR1, (B) TLR2, (C) TLR6, and (D) TLR4 on B220+ B cells. Significance was determined using a one-way ANOVA with the Tukey's multiple comparisons tests. Mean and SEM are shown (^*p ≤ 0.05, ^**p ≤ 0.01, ^****p ≤ 0.0001).

Figure 3

NOD.B10 splenocytes are hyper-responsive to TLR2 ligation. Spleens were isolated from female NOD.B10 mice with clinical disease (n = 8) and age and sex-matched BL/10 controls (n = 10). Splenocytes were cultured as indicated, supernatants were collected, and IL-6 ELISAs performed. All samples were analyzed in duplicate and results of two independent experiments are shown. Significance was determined using the Mann-Whitney test. Mean and SEM are shown (^**p ≤ 0.01).

Figure 4

Dcn and LPS induce distinct inflammatory outcomes in NOD.B10 splenocytes via TLR4. Splenocytes were isolated from NOD.B10 females (n = 6) with clinical disease and age and sex-matched BL/10 controls (n = 3). (A) Cells were cultured in media alone, Dcn and PMB, or S. typhimurium LPS alone or with LPS and PMB. Supernatants were harvested and assayed for IL-6 and TNFα by ELISA. (B) Splenocytes from NOD.B10 females with clinical disease were cultured with the indicated inhibitors and controls and the supernatants harvested. TNFα was measured by ELISA. Combined results of at least 3 independent experiments are shown. All samples were analyzed in duplicate. (C) Splenocytes were isolated from NOD.B10 females (n = 5) with clinical disease and age and sex-matched BL/10 controls (n = 5). Cells were cultured in media alone, Dcn and PMB, or S. typhimurium LPS for 24 h. Supernatants were harvested and MIP-1α, MCP-1, and RANTES levels were quantified by cytokine multiplex array. All samples were evaluated in triplicate. Significance was determined using the Mann–Whitney test. Mean and SEM are shown (^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, N.S., non-significant).

Figure 5

NOD.B10 SMG tissue exhibits spontaneous secretion of pro-inflammatory mediators that are enhanced in the presence of LPS. NOD.B10 female mice with clinical disease (n = 5) were euthanized and SMG tissue harvested and pooled. Cells were cultured in the presence or absence of LPS for 24 h and the supernatant harvested. (A) IL-6, (B) IL-17, (C) TNFα, (D) MCP-1, and (E) RANTES were assessed by cytokine multiplex array. All samples were analyzed in triplicate and results from one of three independent experiments are shown. Significance was determined using Mann–Whitney test. SEM is shown (^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001, N.S., non-significant).

Figure 6

IL-6, MCP-1, and TNFα is decreased in SMG tissue derived from NOD.B10^Myd88−/− females. SMG tissue was harvested from NOD.B10^Myd88+/− and NOD.B10^Myd88−/− mice (n = 6 each). Tissue was dissociated, cultured for 24 h, and the supernatant harvested. Cytokine multiplex arrays were performed for (A) IL-6, (B) MCP-1, (C) TNFα, (D) RANTES, (E) IFNγ, (F) IL-17, (G) IL-4 and (H) IL-1β. Each sample was analyzed in triplicate. Significance was determined using the Mann–Whitney test. Mean and SEM are shown (^*p ≤ 0.05).