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. 2011 Jul;41(7):2029-39.
doi: 10.1002/eji.201040757. Epub 2011 May 27.

Altered miR-146a expression in Sjögren's syndrome and its functional role in innate immunity

Kaleb M Pauley  1 , Carol M Stewart, Adrienne E Gauna, Lauren C Dupre, Riya Kuklani, Annie L Chan, Brad A Pauley, Westley H Reeves, Edward K L Chan, Seunghee Cha
Affiliations

Altered miR-146a expression in Sjögren's syndrome and its functional role in innate immunity

Kaleb M Pauley et al. Eur J Immunol. 2011 Jul.

Abstract

MicroRNAs (miRNAs), small non-coding RNA molecules that post-transcriptionally regulate gene expression, are known to play key roles in regulating immune responses and autoimmunity. We investigated miR-146a expression in Sjögren's syndrome (SjS) patients as well as in the SjS-prone C57BL/6.NOD-Aec1Aec2 mouse model, to elucidate its involvement in SjS pathogenesis. Expression of miR-146a was examined in the PBMCs of 25 SjS patients and ten healthy donors, as well as in PBMCs, salivary and lacrimal glands of SjS-prone mice and WT C57BL/6J mice. Functional assays using THP-1 human monocytes were conducted to determine the biological roles of miR-146a in innate immunity. Expression of miR-146a was significantly increased in SjS patients compared with healthy controls, and was upregulated in the salivary glands and PBMCs of the SjS-prone mouse at both 8 wk (prior to disease onset) and 20 wk (full-blown disease) of age. More importantly, functional analysis revealed roles for miR-146a in increasing phagocytic activity and suppressing inflammatory cytokine production while migration, nitric oxide production and expression of antigen-presenting/costimulatory molecules are not affected in human monocytic THP-1 cells. Taken together, our data suggest that abnormal expression/regulation of microRNAs in innate immunity may contribute to, or be indicative of, the initiation and progression of SjS.

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Conflict of interest statement

Conflict of Interest: The authors declare they have no conflicts of interest.

Figures

Figure 1
Figure 1. SjS patients exhibit increased miR-146a and miR-155 expression in PBMCs compared to healthy controls
PBMC total RNA was isolated from healthy control individuals (n=10) and SjS-patients (n=25) and relative expression levels of A) miR-146a, B) miR-155 and C) miR-132 were analyzed by qRT-PCR using U44 RNA as an internal control. D) MX-1 gene expression was analyzed by qRT-PCR as a measure of the IFN signature in SjS patients compared to healthy controls. Bars indicate mean, asterisks (**) indicate p<0.0001, (*) p<0.05 as determined by Mann Whitney test for panels A-D. E) Relative expression of miR-146a versus MX-1 was plotted for healthy controls and SjS patients (F) to determine the presence of any correlations. No significant correlation was determined by linear regression with p=0.377 for healthy controls and p=0.930 for SjS patients.
Figure 2
Figure 2. miR-146a expression is increased in SjS-prone mouse SMX and PBMCs
qRT-PCR was used to examine miR-146a expression in SjS-prone (B6DC) and wild-type (C57BL/6) mice at 8 and 20 weeks old. A) PBMC (n=2 replicates of pooled sample), B) Submandibular gland (SMX, n=6), C) lacrimal gland (LAC, n=10), and D) kidney (KID, n=6) were analyzed. Data are presented as relative expression after normalization to U44 RNA and C57BL/6 8 week samples. Asterisks (*) indicate p<0.05 compared to age-matched wild-type control as determined by Mann Whitney, bars represent mean.
Figure 3
Figure 3. miR-146a has no effect on migration or expression of antigen presenting molecules
THP-1 cells were transfected with miR-146a mimic, miR-146a inhibitor, or negative control as described in Methods. Two days after transfection, migration and expression of antigen presenting and costimulatory moleucles were monitored and compared to mock transfected cells. A) miR-146a, TRAF6, and IRAK-1 expression after transfection of mimic/inhibitor/negative control was determined by qRT-PCR. B) Migration assay was conducted using 8 μm transwell membranes as described in Methods (n=5 independent experiments, bars represent mean with standard error). C) The surface expression of MHC class I, MHC class II, CD54, and CD86 was detected by flow cytometry after 48 hours of stimulation with 50ng/ml of IFNγ. The THP-1 cells were transfected with either miR-146a mimic (filled peak), negative control miRNA (solid line), or mock transfected (bold solid line). The mouse K isotype moAb controls for FITC, PE, and APC (dotted lines) are also shown. Data are representative of 3 independent experiments.
Figure 4
Figure 4. miR-146a upregulates phagocytic activity and suppresses cytokine production in human monocytic THP-1 cells
THP-1 cells were transfected with miR-146a mimic or miR-146a inhibitor as described in Methods, and 48 hours after transfection, phagocytic activity and LPS-induced cytokine production were monitored and compared to mock transfected cells. A) Phagocytosis was quantitatively measured as described in Methods. Cells were transfected with miR-146a mimic (n=23), miR-146a inhibitor (n=18), or mock transfected (n=27). Asterisk (**) indicates p<0.0001 or (*) p<0.05 compared to mock transfected cells as determined by t test. Bars represent mean with standard error. Data shown from at least 5 independent experiments. B) Phagocytic activity was also monitored by flow cytometry as described in Methods. Cells were transfected with miR-146a mimic (n=6 independent experiments), miR-146a inhibitor (n=6 independent experiments), negative control miRNA mimic (n=6 independent experiments), or mock transfected (n=6 independent experiments). Mean fluorescence intensity (MFI) shown, asterisk (*) indicates p<0.05 compared to mock as determined by one-way ANOVA. Bars represent mean with standard error. C) After transfection, cells were stimulated with 1 μg/ml LPS for 4, 8, or 24 hours or left untreated. Culture supernatants were collected and cytokine production was quantitatively measured using multiplex analysis as described in Methods (n=3, asterisk indicates p<0.05 as determined by t test). Bars represent mean with standard error. D) LPS-induced IL-1β production was detected by ELISA after transfection of miR-146a mimic/inhibitor or negative control mimic (n=3 independent experiments, asterisk indicates p<0.05 as determined by one-way ANOVA). Bars represent mean with standard error.
Figure 5
Figure 5. Summary of potential role of miRNA regulation in SjS
Based on our data showing increased miR-146a expression in SjS and miR-146a's role in upregulating phagocytosis and downregulating pro-inflammatory cytokine production, we propose this model. In normal individuals, an initial stimuli triggers and inflammatory response and increased miR-146a expression which then acts to negatively regulate cytokine production and enhance phagocytic activity. This suppressed inflammation and efficient phagocytic clearance results in the resolution of inflammation. However, in SjS, it is unclear whether miR-146a can properly function which could potentially lead to chronic inflammation, autoimmunity, and prolonged miR-146a elevation. It is also speculated that enhanced phagocytic clearance of apoptotic debri in SjS target tissues could contribute to loss of tolerance and activation of the adaptive immune response. Question marks indicate speculated points.
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