Background/Purpose:Some reports on DNA microarray analysis in labial salivary glands (LSGs) of Sjögren’s syndrome (SS) and healthy controls (HCs) showed that the genes associated with mononuclear infiltration were up-regulated in SS. However, since the controls were healthy individuals, it is possible that the results reflected nonspecific gene expression due to inflammatory cell infiltration. The purpose of this study was to examine the genes expressed specifically in LSGs of patients with SS by comparing them with those of patients with IgG4-related disease (IgG4-RD), which also show mononuclear inflammation and to identify the genes involved in the pathogenesis of SS.

Methods:

1) Gene expression in LSGs of SS patients (n=5), IgG4-RD patients (n=5) and HCs (n=3) was analyzed by DNA microarray. All patients with SS and IgG4-RD fulfilled the Japanese Ministry of Health criteria for the diagnosis of SS (1999) and the diagnostic criteria for IgG4-RD proposed in 2011 by the All Japan IgG4 team, respectively. After the obtained microarray data were normalized by FARMS algorithm, differentially expressed genes (DEGs) up-regulated in SS were identified in pairwise comparisons with IgG4-RD (false discovery rate<0.05) by rank products method. Approval for this study was obtained from the local ethics committee and a signed informed consent was obtained from each subject.

2) Validation of the results was performed by quantitative PCR (qPCR) using LSGs obtained from other patients with SS (n=15), IgG4-RD (n=12), and HC (n=6) than those examined by DNA microarray.

3) Protein production of the validated gene in LSGs was examined by immunofluorescence (IF) assay.

4) The molecular functions of the validated gene under the pathological condition of SS were examined using peripheral blood CD4⁺ T cells in vitro.

Results:

1) In patients with SS, 1785 up-regulated probe sets (corresponding to 1320 up-regulated genes) were identified as DEGs in comparison with those with IgG4-RD.

2) CXCL9, NR4A2, CD26, SGK1, IRF4 and PDK1 were selected as candidate genes for validation, according to rank<150, high expression levels, small variance and relation to T cell functions. qPCR confirmed up-regulation of NR4A2 in LSGs of SS compared with IgG4-RD.

3) IF staining showed higher production of NR4A2 in nuclei of CD4⁺ T cells and IL-17-producing cells in LSGs of SS, compared with IgG4-RD.

4) Overexpression of NR4A2 mRNA was observed in peripheral CD4⁺ T cells of SS patients, compared with HCs. The percentage of IL-17-producing peripheral CD4⁺ T cells under Th17-polarizing conditions correlated significantly with NR4A2 mRNA expression at baseline. Nuclear NR4A2 expression in Th17-polarized CD4⁺ T cells determined by cellular IF was significantly higher in SS than in HC. Importazole, an inhibitor of importin-β, inhibited nuclear transport of NR4A2 and Th17 polarization via suppression of IL-21 expression in naïve CD4⁺ T cells under Th17-polarizing conditions, but did not alter RORC expression.

Conclusion: NR4A2 seems to promote Th17 polarization via increased expression and intranuclear localization in CD4⁺ T cells of SS patients, which could play a critical role in the pathogenesis of SS.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/dna-microarray-analysis-identifies-nuclear-receptor-subfamily-4-group-a-member-2-nr4a2-as-a-novel-molecule-involved-in-the-pathogenesis-of-sjogrens-syndrome/