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Gene expression profile of the Myd88 gene depletion on mouse salivary glands

ABSTRACT: MyD88 is an important adaptor protein for signal transduction downstream of Toll-like receptors and TACI, receptors for regulation of innate immunity and B cell responses, respectively. The surfaces of oral mucosa are protected from infections by antimicrobial proteins and natural immunoglobulins that are constantly secreted in saliva, serving as principal innate immune defense in the oral cavity. Although MyD88-mediated signaling has a regulatory role in the intestinal mucosal immunity, its specific role in the oral cavity has been remained elusive. To identify the specific roles of MyD88-dependent signaling in gene expression profiles in salivary glands, we performed microarray analysis of the major salivary gland tissues (submandibular gland plus sublingual gland) from control (wild-type C57BL/6) mice and C57BL/6 background Myd88-null mice using Agilent Whole Mouse Genome Oligo Microarrays (8x60K, Design ID 028005).

ORGANISM(S): Mus musculus

PROVIDER: GSE61339 | GEO | 2014/09/19

SECONDARY ACCESSION(S): PRJNA260811

REPOSITORIES: GEO

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Project description:MyD88 is an important adaptor protein for signal transduction downstream of Toll-like receptors and TACI, receptors for regulation of innate immunity and B cell responses, respectively. The surfaces of oral mucosa are protected from infections by antimicrobial proteins and natural immunoglobulins that are constantly secreted in saliva, serving as principal innate immune defense in the oral cavity. Although MyD88-mediated signaling has a regulatory role in the intestinal mucosal immunity, its specific role in the oral cavity has been remained elusive. To identify the specific roles of MyD88-dependent signaling in gene expression profiles in salivary glands, we performed microarray analysis of the major salivary gland tissues (submandibular gland plus sublingual gland) from control (wild-type C57BL/6) mice and C57BL/6 background Myd88-null mice using Agilent Whole Mouse Genome Oligo Microarrays (8x60K, Design ID 028005). Total RNA was extracted from the major salivary gland tissues (submandibular gland plus sublingual gland) of wild-type and Myd88-null mice using using Trizol reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer's instructions. 100 ng of all RNA samples was used as template to produce Cy3-labeled cRNA. The RNA samples were amplified and labeled using the Low Input Quick Amp Labeling Kit (Agilent Technologies, Palo Alto, USA) following the manufacturerM-bM-^@M-^Ys protocol. Cy3 labeled cRNAs were hybridized overnight (17 hours, 65M-BM-0C) to an Agilent Whole Mouse Genome Oligo Microarrays (8x60K, Design ID 028005) using AgilentM-bM-^@M-^Ys recommended hybridization chamber and oven.Finally, microarrays were washed once with wash buffer 1 for 1 min at RT followed by a second wash with wash buffer 2 for 1 min at 37M-BM-0C and a final wash step with acetonitrile for 30 sec at RT. Fluorescence signals of the hybridized Agilent Microarrays were detected using AgilentM-bM-^@M-^Ys Microarray Scanner System (G2505C, Agilent Technologies). The Agilent Feature Extraction Software (FES 10.7.3.1 ) was used to read out and process the microarray image files.

Project description: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 disease, although the upstream signaling events that culminate in Myd88 activation have yet to be identified. To 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-H2b/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 then cultured splenocytes with TLR2 and TLR4 agonists and measured IL-6 secretion 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, B cell TLR1, TLR2, and TLR6 expression was reduced to levels of BL/10 controls in the absence of Myd88. Splenocytes from NOD.B10 mice were hyper-responsive to TLR2 ligation and the endogenous molecule decorin modulated inflammation via TLR4. Finally, we found spontaneous secretion of numerous inflammatory cytokines and this was enhanced following TLR4 ligation in female NOD.B10 salivary tissue as compared to males. Moreover, we found that spontaneous production of salivary IL-6, MCP-1 and TNFa requires 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.

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Gene expression profile of the Myd88 gene depletion on mouse salivary glands

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