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The submandibular and sublingual glands maintain oral microbial homeostasis through multiple antimicrobial proteins

ABSTRACT: Oral microbial homeostasis is a key factor affecting oral health, and saliva plays a significant role in maintaining oral microbial homeostasis. The submandibular gland (SMG) and sublingual gland (SLG) together produce the most saliva at rest. Organic ingredients, including antimicrobial proteins, are rich and distinctive and depend on the type of acinar cells in the SMG and SLG. However, the functions of the SMG and SLG in maintaining oral microbial homeostasis have been difficult to identify and distinguish, given their unique anatomical structures. Therefore, we analyzed each gland using single-cell RNA sequencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE216476 | GEO | 2023/01/03

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:Application of allergens onto the sublingual epithelium is used to desensitize allergic individuals, a treatment known as sublingual immunotherapy. However, the response of sublingual epithelial cells to house dust mite allergen and potential tolerance-promoting adjuvants such as Toll-like receptor ligands and calcitriol has not been investigated. In order to study this, primary sublingual epithelial cells were isolated from dogs and cultured in vitro. After 24h incubation with Dermatophagoides farinae extract, TLR2 ligands (FSL-1, heat-killed Listeria monocytogenes, Pam3CSK4), a TLR3 ligand (poly I:C), a TLR4 ligand (LPS) and calcitriol (1,25-dihydroxyvitamin D3), viability of the cells was analyzed using an MTT test and their secretion of IL-6, IL-10, CXCL8 and TGF-β1 was measured by ELISA. Additionally, to evaluate its potential effect as an adjuvant, sublingual epithelial cells were incubated with calcitriol in combination with D. farinae extract followed by measurement of CXCL8 secretion. Furthermore, the effect of D. farinae and calcitriol on the transcriptome was assessed by RNA-sequencing. The viability of the sublingual epithelial cells was significantly decreased by poly I:C, but not by the other stimuli. CXCL8 secretion was significantly increased by D. farinae extract and all TLR ligands apart from LPS. Calcitriol significantly decreased CXCL8 secretion and co-administration with D. farinae extract reduced CXCL8 concentrations to levels seen in unstimulated sublingual epithelial cells. Although detectable, TGF-β1 secretion could not be modulated by any of the stimuli. IL-6 and IL-10 could not be detected at the protein nor at the mRNA level. It can be concluded that D. farinae extract and TLR ligands augment the secretion of the pro-inflammatory chemokine CXCL8, which might interfere with sublingual desensitization. On the other hand, CXCL8 secretion was reduced by co-application of calcitriol and D. farinae extract. Calcitriol therefore seems to be a suitable candidate to be used as adjuvant during sublingual immunotherapy.

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The submandibular and sublingual glands maintain oral microbial homeostasis through multiple antimicrobial proteins

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