Deregulation of microRNA expression in monocytes and CD4⁺ T lymphocytes from patients with axial spondyloarthritis

Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease that mainly affects the spine and sacroiliac joints in young adults. AxSpA includes both radiographic axSpA, also known as ankylosing spondylitis (AS), characterized by radiographic sacroiliitis according to the modified New York criteria [1], and non-radiographic axSpA (nr-axSpA), characterized by the absence of structural damage of the sacroiliac joints on X-rays. The disease is frequently associated with extra-articular manifestations including psoriasis, uveitis, or inflammatory bowel disease [2, 3]. The long-term outcome of AxSpA is determined by structural damages such as the formation of syndesmophytes, excessive new bone formation of the spine [4].

Strong evidence suggests that disease pathogenesis is driven by innate immune cells (monocytes/macrophages, dendritic cells) and CD4⁺ T lymphocytes [3, 5‐8]. Genetic variation associated with AxSpA was found enriched in open chromatin regions in monocytes and CD4⁺ T lymphocytes, suggesting these cell populations might play an important role in the disease [9]. Genetic analyses identified the HLA-B27 and IL23R loci, whose proteins are present at the cell membrane of monocytes and T lymphocytes respectively, as the genetic factors most strongly associated with the disease [10, 11]. Monocytes/macrophages are crucially involved in the disease pathogenesis notably through the HLA-B27-induced unfolded protein response (UPR) stress leading to the release of pro-inflammatory cytokines (IL-1α, TNF, IL-6, and IL-23) [12]. Furthermore, T cells and macrophages have been found in the cellular infiltrates of tissue biopsies from AS patients [13‐18]. Adoptive transfer studies from disease-prone B27 transgenic rats to B27 nude rats demonstrated that CD4⁺ T cells were able to induce the disease [7].

MicroRNAs (miRs) are small non-coding RNAs composed of 18–25 nucleotides that play an important regulatory role at the post-transcriptional level in diverse biological processes including cell differentiation, proliferation, apoptosis, or cellular function [19]. MiRs are involved in immune functions during granulopoiesis, T and B cell ontogenesis, TLR signaling, and cytokine production [20, 21]. We have previously shown that miR expression profiles are highly cell type specific with different expression profiles observed between CD4⁺ T and CD19⁺ B lymphocytes [22], reflecting their specific roles in a broad range of cellular functions. While miR deregulation has been comprehensively investigated in several autoimmune or rheumatic diseases including rheumatoid arthritis [23‐25] and Sjögren’s syndrome [22], similar studies on radiographic or non-radiographic axSpA are scarce.

Given the cell specificity of miR expression profiles and considering the biological importance of CD4⁺ T cells and monocytes in the pathophysiology of the disease, we investigated and validated miR expression profiles in these two cell types from axSpA patients and controls in two independent cohorts, totaling 81 patients and 55 controls.

In this large-scale miR profiling study analysing two independent cohorts, we showed that patients with axSpA present solid evidence for a deregulation of specific miRs in monocytes (n = 13) and CD4⁺ T lymphocytes (n = 11). Only four miRs were consistently differentially expressed between patients and controls in both CD4⁺ and CD14⁺ cells, demonstrating two distinct specific signatures of miR expression profiles in monocytes and CD4⁺ T lymphocytes. The major strength of this study is the large number of miRs screened in sorted cells from a large number of clinically well-characterized subjects.

To date, miR expression profiles have been poorly studied in SpA. Two different approaches have been pursued: (i) the first strategy consisting in analysing miR expression in sorted cells and (ii) the second strategy consisting in analysing circulating miRs isolated from serum or plasma. The first approach is mainly performed to advance our understanding of cell type-specific mechanisms involved in disease pathophysiology; the second aims mainly at identifying biomarkers of the disease in easily accessible biological material for clinical routine. However, miR expression profiles obtained from one strategy cannot be extrapolated to the other, as multiple cell types and mechanisms contribute to the prevalence and abundance of miRs present as cell-free circulating miRs.

We decided to investigate the miR-expression profile of CD4⁺ T lymphocytes and monocytes because of their relevance for the pathophysiology of axSpA [3, 5, 9]. We found that the main factor influencing the miR expression profile is the cell type: almost 90% of the measured miRs were differentially expressed between CD4⁺ and CD14⁺ cells in our study, regardless of the disease status. Thus, the assessment of miR expression profiles in PBMCs or whole blood may be challenging as any observed changes could be related to variable cell subset composition, requiring thus the assessment of miR expression in sorted cells.

Some studies have addressed the impact of csDMARDs on miR expression in psoriasis or rheumatoid arthritis (RA). However, current data is conflicting. MiR-10a, miR-132-3p, miR-146a-5p, and miR-155-5p measured in whole blood or serum have been shown to be increased after MTX treatment in RA [33, 34] whereas miR-16 in plasma was not altered by the use of MTX in juvenile idiopathic arthritis [35]. In contrast, miR-146a measured in whole blood from patients with psoriasis was decreased after 3 months of methotrexate [36]. Also, in psoriasis, miR-223-3p was found downregulated in PBMC, but not altered in serum after the use of MTX and decreased in serum of patients treated by Etanercept [37, 38]. The authors suggested that the downregulation of miR-223 was not related to the treatment but rather to the immunosuppressive effect of the treatment on cells. We found only a single study reporting the impact of sulfasalazine on miR expression. Mir-21 was found to be negatively associated with PDCD4 expression in whole blood from SpA patients not treated with csDMARDs, while positively associated with the expression of the same gene in patients treated with sulfasalazine [39]. In our study, we did not find miR-21 to be differentially expressed. Considering the presence of multiple cell types in most analyses, it is currently difficult to conclude if the use of csDMARDs has a direct influence on the miR expression profile or leads to a shift in cell populations. In our sorted cell populations, we found little evidence for the modification of the miRNA expression profiles by csDMARDs. However, our absolute number of patients treated with DMARDs were low, and larger studies will be required to fully address this question.

Several miRs found deregulated in CD4⁺ T cells or in monocytes in our study have been previously shown to be deregulated in other cell types in AS. Overexpression of mir-92b-3p, mir-16-5p, and mir-126-3p were also found overexpressed in T cells, fibroblasts, or PBMCs by others [40‐42], reinforcing their likely involvement in the disease. No other study has focused on miR expression in monocytes from AS patients. Contrary to our result, Wang et al. found a lower expression of miR-199a-5p in T cells from 41 AS patients compared to healthy controls [43]. The main difference with our study is that miR expression was assessed on CD3⁺ T cells comprising both CD4⁺ and CD8⁺ T cells.

MiR-16-5p was found upregulated in both monocytes and T lymphocytes in our study and in CD3⁺ T lymphocytes from AS patients in a previous study [41]. It was assigned an anti-inflammatory function as it binds to the 3′-untranslated region (UTR) of Tnfα and Il12b, repressing their expression [44], or to the 3′UTR of Pdcd4 (programmed cell death), resulting in a decreased level of Il-6 or Tnfα in mouse macrophages [45]. Nevertheless, miR-16 has also been shown to be able to create a pro-inflammatory environment [46].

The functional role of miR-126-3p has been well studied in IBD, where it was found upregulated in the colon tissue of patients with active ulcerative colitis [47, 48]. Furthermore, IκB, an inhibitor of NFκB, is a downstream target of miR-126-3p and the transfection of a miR-126-3p mimic decreases the expression of IκB in a dose-dependent manner [48]. MiR-126 also impairs intestinal mucosal barrier function under LPS stimulation by targeting S1PR2 through the PI3k/AKT signaling pathway [49]. Functional experiments are required to further clarify its role in axSpA.

Macrophages play a major role in SpA as demonstrated by their abundant infiltration on biopsies from sacroiliac joints of AS patients, within the synovial tissue in chronic inflammatory arthritis, and in the gut mucosa [13, 50]. The macrophage infiltrate was shown to correlate with disease activity and to decrease after anti-TNF treatment [51, 52]. Macrophages represent a heterogeneous population of classically activated macrophages (M1), the main producers of inflammatory mediators such as TNFα, which are specialized in the clearance of pathogens and alternative M2 macrophages, which are involved in tissue repair and immunosuppression [53]. Whether M1 or M2 are causally implicated in SpA is still a matter of debate. CD163, a marker of M2 macrophages was found increased in the intimal lining layer of the synovial membrane and in gut mucosa from SpA patients [16, 50, 54]. In AS, the IFN-γ signature classically seen in M1 macrophages was impaired and the level of M1-derived cytokines was decreased in synovial fluid compared to patients with rheumatoid arthritis [17, 55]. However, these studies were focused on peripheral SpA and little is known regarding the role of M1 or M2 macrophages in axSpA. In PBMCs, a M2-like predominant polarization of monocytes has been reported, together with a M2/M1 ratio negatively correlated with CRP levels or BASDAI score [56]. A shift toward a M2 macrophage profile could facilitate bacterial survival and growth, especially for intracellular pathogens, such as Chlamydia or Yersinia, that have been involved in reactive arthritis and axial and peripheral arthritis in SKG mice [57, 58].

MiRs play a critical role in controlling macrophage differentiation [59]. MiR-16 was able to induce differentiation of mouse peritoneal macrophages into M1 macrophages from either the basal M0 or M2 polarized state and to activate purified CD4⁺ T lymphocytes, thus creating a pro-inflammatory environment [46]. In contrast, miR-124 and miR-223-3p were reported to contribute to M2 polarization or to limit M1 activation [60, 61]. The increased expression of miR-223-3p in our analysis suggests that circulating monocytes should be more prone to evolve into M2 macrophages in peripheral tissue. The function of miR-223-3p in SpA has never been addressed before, but it was found to be up-regulated in psoriasis and in inflammatory bowel disease [37, 62‐64]. A dual role has been attributed to miR-223-3p in IBD: a protective role through the decreased in the release of inflammatory mediators and a facilitating role by increasing intestinal permeability. The overexpression of miR-223-3p in RAW 264.7 macrophages inhibited LPS-stimulated secretion of Il-6 or Il-1 by targeting STAT3 [60]. In H. pylori-infected THP-1 monocytes, increased expression of miR-223-3p was correlated with decreased expression of NLRP3 [65]. MiR-223^−/− mice exhibit exacerbated DSS-induced colitis and an increased level of IL-1β through the loss of repressive function on NLRP3 targeted by miR-223-3p [64]. MiR-223-3p may also increase the intestinal permeability by targeting CLDN8 in response to IL-23 [62]. Therapeutic approaches in mice model have been elaborated targeting this dual role of miR-223. A systemic treatment with an antagomiR-223 was able to attenuate the phenotype in TNBS-induced colitis in mice [62]. By contrast, an intravenous nanoparticle delivery of a miR-223 mimic attenuated DSS-colitis in mice by targeting Nlrp3 [64]. The upregulation of miR-223-3p among SpA patients in our study could both reflect the activated state of the monocyte/macrophages cells and an induced anti-inflammatory regulatory loop.

MiR-146a-5p is the most studied miR in autoimmune or inflammatory diseases. Also called anti-inflammoMir, miR-146a can bind to many targets involved in inflammatory signaling such as IRAK1, TLR2/4 or NFκB. It acts as an effective regulator to prevent an overstimulated inflammatory state [31, 66]. IRAK1^−/− CD4⁺ T cells are less prone to produce IL17 under TGF-B and IL-6 stimuli and expressed a higher level of Foxp3 under TGF-B stimulation compared to WT CD4⁺ T cells [67]. Thus, IRAK1 can control the Th17/regulatory T-cells balance under specific stimuli, promoting Th17 differentiation. Keratinocytes from miR-146a^−/− mice were more sensitive to IL-17A stimulation that keratinocytes from WT mice, suggesting that mir-146a is a potent suppressor of IL17-driven disease [68]. MiR-146a-5p expression was found to be increased in skin biopsies and PBMCs from patients with psoriasis [69]. In SpA, discordant results have been published with an overexpression in fibroblasts and a decreased expression in PBMCs [70, 71]. We found a downregulation of miR-146a-5p in monocytes and its level was negatively correlated with ASDAS and CRP. Given its target genes, miR-146a-5p deregulation in monocytes might facilitate the polarization of Th17 cells from naïve CD4⁺ T cells through the release of pro-inflammatory cytokines.

We have demonstrated a cell type-specific miRNA expression profile, whose deregulation could be related to the pathophysiology of the disease. More specifically, this study has highlighted some pathways that could be crucial in the onset or the maintenance of the disease such as the role of macrophage polarization, the impact of intestinal permeability or the modulation of the expression of pro-inflammatory cytokines.