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OP0316 Increased expression of microrna-142–3p is associated with the functional defect of regulatory t cells in anti-neutrophil cytoplasmic antibody associated vasculitis
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  1. G. Dekkema1,
  2. T. Bijma,
  3. W. Abdulahad1,2,
  4. P. Jellema3,
  5. A. Van Den Berg1,
  6. B.J. Kroesen1,
  7. C. Stegeman4,
  8. P. Heeringa2,
  9. J.-S. Sanders1,2
  1. 1Pathology and Medical Biology
  2. 2Department of Internal Medicine, division of Nephrology
  3. 3Department of Rheumatology and Clinical Immunology
  4. 4Department of Clinical Immunology, University Medical Center Groningen, Groningen, Netherlands

Abstract

Background Circulating regulatory T cells (Tregs) in anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) are frequently functionally deficient. The mechanism behind their impaired function is however unknown. Small non-coding microRNA (miR) are post-transcriptional regulators of protein synthesis and previous studies have shown that differently expressed miRs in T cells are associated with auto immunity.

Objectives To investigate whether the dysfunctionality of Tregs in AAV is due to altered microRNA (miR) expression.

Methods Tregs (CD4+CD45RO+CD25+CD127-) of healthy controls (HC) and AAV patients in remission without treatment (AAV-REM) were FACS-sorted, and total RNA was isolated. Samples from 8 HCs and 8 AAV-REMs were subjected to miRNA microarray analysis. Based on relative expression and fold change, 5 differentially expressed miRs were validated in an independent cohort using qRT-PCR and a database and literature search was performed to identify potential targets.

Results Nineteen miRs differentially expressed were detected by microarray analysis, of which Let-7g, miR-20a-5p, miR-26a-5p, miR-142–3 p, miR-146a-5p were validated in an independent cohort. Of these, miR-142–3 p was confirmed to be significantly upregulated (2-fold, p=0.03) in Tregs from AAV-REM patients compared to HC Tregs (n=23, n=22). To study the functional impact of miR-142–3 p overexpression, HC Tregs were transfected using either a mimic-miR-142–3 p or a scrambled (SCR)-control. After transfection, live Tregs were co-cultured with T effectors (CD4+CD25-) in a suppression assay to test their suppressive capacity. Transfection with mimic-miR-142–3 p significantly increased the miR-142–3 p levels (2.4 fold, p=0.03) and reduced the suppressive capacity compared to SCR-transduced Tregs (1.9 fold reduction, p=0.02). Moreover, miR-142–3 p levels tended to correlate to the suppressive function of Tregs (p=0.06, rho=−0.591). A database and literature search identified adenylyl cyclase 9 (AC9) as a promising target of miR-142–3 p. mRNA levels of AC9 tended to be lower in AAV-REM patients compared to HC (3.8 fold, p=0.07). In addition, cAMP levels, which are partly produced by AC9, were significantly lower in Tregs from AAV-REM patients after 48 hour of stimulation with anti-CD3 and anti-CD28 (1.7 fold, p=0.003).

Conclusions Increased expression of miR-142–3 p in Tregs of AAV-REM patients is associated with their functional impairment, potentially by targeting the AC9/cAMP mediated suppression.

Disclosure of Interest G. Dekkema: None declared, T. Bijma: None declared, W. Abdulahad Grant/research support from: European Union’s Horizon 2020 research and innovation program under grant No 6 68 036., P. Jellema: None declared, A. Van Den Berg: None declared, B. J. Kroesen: None declared, C. Stegeman: None declared, P. Heeringa Grant/research support from: European Union’s Horizon 2020 research and innovation program under grant No 6 68 036., J.-S. Sanders Grant/research support from: Nierfonds grant no. 13OKJ39

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