Elsevier

Human Immunology

Volume 71, Issue 2, February 2010, Pages 206-211
Human Immunology

Research article
miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis

https://doi.org/10.1016/j.humimm.2009.11.008Get rights and content

Abstract

miRNAs have recently emerged as key regulators of the immune system, being involved in lymphocyte selection and proliferation, in Treg cells differentiation, and in hematopoiesis in general. Rheumatoid arthritis (RA) is an autoimmune pathology the etiology of which is still obscure. Although a multifactorial pathogenesis has been hypothesized, the precise mechanisms leading to the disease are still poorly understood at the molecular level. miRNA expression profile analysis highlighted that miR-223 is the only miRNA that is strikingly deregulated in peripheral T-lymphocytes from RA patients compared with healthy donors. Further analysis by quantitative reverse transcription–polymerase chain analysis confirmed that miR-223 is overexpressed in T-lymphocytes from RA patients (n = 28) compared with healthy donors (n = 10). Moreover, purification of different T-lymphocyte populations from RA patients highlights that miR-223 is expressed at higher levels in naive CD4+ lymphocytes, whereas its expression is barely detectable in Th-17 cells. In summary, our data provide a first characterization of the miRNA expression profiles of peripheral T-lymphocytes of RA patients, identifying miR-223 as overexpressed in CD4+ naive T-lymphocytes from these individuals. A deeper analysis of the biologic functions and effects of the expression of miR-223 in T-lymphocytes is needed to clarify the exact link between our observation and the disease.

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of joint synovial tissue and progressive damage to the cartilage and bone tissue, ultimately leading to disability [1]. The mechanisms underlying pathology initiation and progression are still poorly understood. However, it is largely accepted that an important role is played by the immune system. In fact, synovia are infiltrated with macrophages and CD4+ T-lymphocytes [2], [3]. Moreover, a decrease of TCR repertoire diversity has been observed in RA patients. This decrease is paralleled by an age inappropriate telomere erosion in peripheral T-lymphocytes, suggesting a defect in T-cell homeostasis [4].

Recently, several studies highlighted that microRNAs (miRNAs) are important regulators of the immune response [5]. These tiny noncoding RNAs, 19–23 nt long, act as negative regulators of gene expression at post-transcriptional level, inhibiting target mRNA translation and/or inducing specific degradation of their target mRNAs [6]. miRNAs playing a role in the immune system include miR-181a, which has been shown to control TCR sensitivity [7], thus possibly affecting thymic selection; miR-150, which plays a key role in lymphocyte differentiation [8], [9]; miR-155, acting as a regulator of B-lymphocyte proliferation [10]; and the miRNA cluster 17-92, the expression of which leads to uncontrolled lymphocyte proliferation, lymphomas, and impairment of peripheral tolerance [11], [12]. miRNAs have also been shown to play a role in innate immunity: for example, miR-146a is a negative regulator of TLR-4 signaling involved in innate immune response [13] and miR-223 is specifically expressed upon retinoic acid induced differentiation of the promielocytic leukemia cell line HL-60 and has been shown to play a role in granulocytic differentiation [14], [15], [16].

The role of miRNAs in inflammatory diseases has been recently explored. Stanczyk et al.[17] revealed a prominent upregulation of miR-155 and miR-146a in RA synovial fibroblasts (RASFs) compared with those in patients with osteoarthritis (OA). These authors also demonstrated that the expression of miR-155 in synovial fibroblasts could be further induced by tumor necrosis factor (TNF)–α, interleukin-1β, lipopolysaccharide, poly(I-C), and bacterial lipoprotein. Nakasa et al.[18] confirmed the increased expression of miRNA-146 in synovial tissue from patients with RA compared with OA and normal controls also by in situ hybridization and immunohistochemistry of tissue sections. The authors also concluded that miR-146 expressed in RA synovial tissue may be induced by stimulation with TNF-α and IL-1β. More recently, a significant increase of miR-146a, miR-155, miR-132, and miR-16 in peripheral blood mononuclear cells from RA patients compared with healthy and diseased control individuals has been highlighted, suggesting that miRNAs can be involved at different levels in the pathogenesis of RA [19].

The aim of the present study was to investigate the role of miRNAs in RA; in particular, as T-lymphocytes have been reported to play a role in the pathogenesis of RA, we focused on the role of miRNAs in this cell lineage. Our analyses highlight miR-223 as the only miRNA dramatically upregulated in peripheral blood CD3+ lymphocytes from RA patients compared with healthy controls. Moreover, our data suggest that miR-223 overexpression occurs mainly in CD4+ naïve T-lymphocytes. We suggest that this aberrant overexpression of miR-223 in RA patients lymphocytes could contribute to pathogenesis of the disease. Identification of miR-223 targets in T-lymphocytes could therefore contribute to elucidate at least in part some of the molecular mechanisms that lead to RA.

Section snippets

Patients and controls

We studied 37 RA patients, 27 female and 10 male, 22–82 years of age (median 58 years), with a disease duration between 2 and 336 months (median 132 months). All patients fulfilled the 1987 ACR criteria for the classification of RA [20], all had active disease as defined according to DAS28 score. In all, 75% were IgM rheumatoid factor (RF) positive, and 67.5% had anticyclic citrullinated peptide antibodies (anti-CCP) in serum. Nine of them were taking no drug at all, whereas 28 were under

miR-223 is upregulated in CD3+ T-lymphocytes from peripheral blood of RA patients

In the first step, three patients with active RA, who were taking no drugs, and a healthy control subject were tested for 470 miRNAs by microarray technique (Fig. 1A). The test was performed on CD3+ sorted blood cells. The miRNA expression profile in the four samples was strikingly similar for all miRNAs but miR-223, which appeared to be dramatically upregulated in the three RA patients only (Fig. 1B). A few other miRNAs showed a less marked differential expression between RA patients and

Discussion

MicroRNAs (miRNA) are a new class of modulators of gene expression, and a large body of evidence can be found in the literature demonstrating a role for miRNAs in adaptive immunity [26]. Because T-lymphocytes have been suggested to play a role in RA [3], we have studied miRNAs expression in T-lymphocytes from peripheral blood of RA patients and healthy donors. Notably, to avoid measuring artifactual effects on miRNAs expression caused by therapy, we focused our analysis on RA patients without

Acknowledgments

Research was supported by grants to GM by Associazione Italiana per la Ricerca sul Cancro (AIRC) and by the EU integrated project Silencing RNAs: Organisers and Coordinators of Complexity in eukaryotic Organisms (SIROCCO).

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