Background/Purpose:

Sjögren’s syndrome (SS) causes severe dry mouth and eyes. The presence of immune cell infiltration in the salivary (SG) and lacrimal glands suggests a response to local antigens, presumably from resident antigen presenting cells (APCs) and exocrine epithelium stimulation of autoreactive CD4+ T cells. Recent studies in our lab identified co-stimulatory molecule CD80 as a target of microRNA (miR)-146a and showed reduced CD80 in the SG cells of SS-prone mice (C57BL/6.NOD-Aec1Aec2, B6DC). Co-stimulatory molecule CD80 is presumed to promote T cell regulatory phenotypes by binding to CTLA-4 or PD-L1. Therefore, we sought to identify which cell subsets were contributing to the reduced CD80 in SGs and whether there were alterations in regulatory (Treg) and conventional (Tconv) T cell functional markers in B6DC mice.

 Methods:

Single cell suspension of submandibular SGs and draining lymph nodes (dLNs) of 20-24-week-old B6DC and C57BL/6 (B6) mice (n = 5-9 per group) were evaluated by flow cytometry. To identify potential APC subsets, cells were assessed for CD19, CD11c, CD11b, and F4/80. SG epithelial cells (SGECs) were negatively selected using anti-CD45 microbeads and identified using CD326. APCs and SGECs were evaluated for co-stimulatory molecules CD80 and CD86. In addition, Treg (CD4+Foxp3+) and Tconv (CD4+Foxp3-) cells were assessed for cell surface proteins CD25, CTLA-4, PD-L1, and intracellular IL-10 and TGF-β1. Quantitative real-time-PCR was used to evaluate the expression of miR-146a in SGECs relative to RNU6b. Data were analyzed by two-tailed-unpaired t-test or linear regression where P < 0.05 was considered significant.

Results:  

SG APCs contributed to less than 5% of total SG cells. The CD19+CD11c- B cells, CD11c+CD11b+F4/80+CD19+ macrophages, CD11c+CD11b+F4/80-CD19- dendritic cells and CD11c+CD11b+F4/80+CD19- macrophages showed reduced percentages of CD86+CD80+ cells. Analyses of B6DC SGECs revealed a 29% reduction in CD80 mean fluorescence intensity (MFI), but no alteration in CD86 MFI in comparison to B6 mice. The ratio of CD86:CD80 MFI was increased and the relative expression level of miR-146a was elevated by 2.09-fold in B6DC SGECs. Furthermore, CD80 MFI showed a negative relationship to the relative expression level of miR-146a (slope = -0.376, r² = 0.511). Also, B6DC SG Tregs were increased (+56%) and had increased CTLA-4 (+81%) and PD-L1 (+37%) positive cells; while Tconv had increased PD-L1 (+43%) and decreased IL-10 (-60%) positive cells. The dLN Tregs had increased  CTLA-4 (45+%) or PD-L1 (+8.7%) positive cells along with 28% decrease in IL-10 positive cells; whereas Tconv cells showed 8.8% increase in PD-L1 positive cells.

Conclusion:

SGECs are major contributors to reduced total CD80 expression in the SG since contributions of resident APCs were minimal. Therefore, it is presumed that the imbalance of CD86:CD80 in SGECs, due to aberrantly expressed miR-146a, could contribute to the abnormal activation of T cells in the affected tissues of SS. This is supported by the observed reduction in regulatory cytokine IL-10 in SG T cells despite elevated expression of regulatory CTLA-4, PD-L1, and the number of Foxp3+ Tregs.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microrna-146a-in-salivary-gland-epithelial-cells-inhibits-co-stimulatory-molecule-cd80-expression-and-increases-autoreactive-t-cell-activation-in-sjogrens-syndrome/