The results of the present study show that CTLA4-Ig treatment seems to promote the shift into an M2 phenotype of both cultured HS-M1-MDMs polarised by LPS stimulation and cultured MDMs obtained from RA patients. This shift was detected by the downregulation of the gene expression of M1 phenotype markers (TLR4, CD80, CD86) along with the upregulation of the gene expression of M2 phenotype markers (CD163, CD204, CD206) and their related protein synthesis. Of note, together with the upregulation of specific surface markers of M2 phenotype, the effect of CTLA4-Ig treatment in promoting the M1-M2 shift was observed also at the functional level, through the upregulation of gene expression of MerTK, which seems to play important functions potentially relevant in RA, as recently described [46].

In the pathogenesis of RA and joint damage, monocytes/macrophages are considered to play a central role, including newly differentiated peripheral blood monocytes, compared to fibroblasts, lymphocytes and neutrophils, which cooperate with different functions [7]. In fact, macrophages generate an inflammatory milieu by producing pro-inflammatory cytokines and chemotactic mediators, which further facilitate the synovial tissue invasion of other immune cells [29].

Although the ability of macrophages to assume various polarising profiles including the well-defined pro-inflammatory M1 and alternative M2 polarised phenotypes is frequently observed, an increased frequency of inflammatory M1 macrophages seems to mainly characterise RA patients with high disease activity compared to RA patients under remission, who conversely show an M2 phenotype prevalence [47, 48]. Several studies highlighted the important pathogenic role of M1 monocytes/macrophages in RA. In particular, the PBMCs of RA patients show an aberrant expression of CD14 and a high expression of CD86 (M1 marker) compared to that of HS monocytes, and they are suggested to play an important role in the pathophysiological processes of the disease [48]. Furthermore, Yoon et al. found that monocytes invading the synovial tissue express higher levels of CD80 and TLR4 than peripheral blood monocytes [17]. Moreover, a recent study highlighted that the PBMCs of RA patients express several M1 macrophage molecules, including class II major histocompatibility complex molecules (HLA-DR), CD64, CD86, and CCR5, while in the synovial fluid, they show a high level of HLA-DR, CD40, CD80, CD86, and CD276 [14].

A widely known in vitro model of RA inflammation and M1 phenotype induction is represented by the stimulation of monocytes (both THP1 cell line and peripheral blood monocytes) with LPS [22, 49]. Of note, LPS is able to induce a strong upregulation of CD80 and CD86 costimulatory molecules (among M1 markers), even though it requires other binding partners to induce a robust expression of TLR4 [50, 51]. In accordance with these observations, in our study, the upregulation of the gene expression of TLR4, CD80, and CD86 and the downregulation of that of CD163, CD204, and CD206 following LPS stimulation confirmed its ability to induce a M1 phenotype in cultured human HS-MDMs.

Our data are in line with those described by Degboe et al., although in Degboe’s study, cultured HS-MDMs were stimulated with LPS in combination with IFN-γ instead of LPS alone. However, in these cultured HS-MDMs stimulated with LPS+IFN-γ, the upregulation of CD80 and the downregulation of CD163 and CD206 gene expression were observed [29]. Although the stimulation of HS-MDMs used in our study and in Degboe’s study is different, the resulting induction of the polarisation into an M1 phenotype in these cultured cells seems to be very similar. Nevertheless, it is necessary to highlight that the LPS-induced polarisation of cultured HS-MDMs into a pro-inflammatory M1 phenotype represents an in vitro model, which cannot reflect the pathophysiological activity of RA macrophages. Based on this, to increase the clinical relevance of our study, the MDMs obtained from RA patients have been investigated.

It is noteworthy that monocytes/macrophages may contribute to the T cell activation and expansion during the inflammatory phase, thanks to their expression of HLA-DR and costimulatory molecules (CD80 and CD86), confirming their further functional role as antigen-presenting cells (APCs) [52]. In particular, to fulfil the T cell-naïve activation, the induction of CD80/86-CD28 costimulation is needed in these APCs. This T cell-naïve activation process is inhibited by the binding of CTLA-4 to CD80/86 [52, 53].

The CTLA4-Ig fusion protein (abatacept) is a bDMARD known to modulate RA inflammation, blocking T cell costimulation, and its effects on different cell types, involved in the immune-inflammatory reaction, have been widely analysed and demonstrated [31, 54]. Our previous studies highlighted the efficacy of CTLA4-Ig treatment to reduce the in vitro pro-inflammatory cytokine production (IL-6, TNFα, IL-1β) as well as the CD86 expression in cultured synovial macrophages, and this effect seems to be mediated by the upregulation of the NF-kB inhibitor IkB-a [36,37,38]. Moreover, Rochman et al. demonstrated the effect of abatacept in reducing the gene expression of NF-kB and AP-1 transcription factors along with the remarkable proliferation decrease of T regulatory cells [55]. Additionally, Lorenzetti et al. reported a dose-dependent decrease of CD80 and CD86 induced by the treatment with abatacept in B cells [56]. Lastly, abatacept is involved in the synovial cell infiltration regression [35, 57].

Since all these observations highlight the role of abatacept in reducing the inflammatory process, the present study investigated if this anti-inflammatory effect might be linked to its ability to promote an in vitro induction of the shift from an M1 to an M2 phenotype in cultured HS-M1-MDMs and RA-MDMs. However, current data regarding the in vitro activity of bDMARD to induce the shift from M1 to M2 macrophages are limited. Recently, Degboe et al. demonstrated the ability of the anti-TNFα bDMARDs to decrease CD80 and to increase CD163 and MerTK protein synthesis in cultured polarised M1-MDMs, whereas anti-IL-6 receptor or anti-CD20 agents (such as tocilizumab and rituximab) did not induce any upregulation of M2 phenotype markers [29]. The fact that anti-IL-6 receptor agents may not affect macrophages as well as their shift into an M2 polarised status is also confirmed by Chatzidionysiou’s study, in which it was demonstrated that no reduction in the number of macrophages was detectable in the synovial tissue samples of RA patients treated with tocilizumab [58].

The results of our study show that CTLA4-Ig treatment increased surface M2 phenotype markers (CD163, CD206, and CD204) in both cultured HS-M1-MDMs and RA-MDMs, at both gene and protein level (12 and 24 h, respectively), with variable statistically significant results. Interestingly, CTLA4-Ig treatment seems to induce a remarkable reduction in the gene expression of M1 markers (CD80, CD86, and TLR4) already at early stage (3 h), in distinct statistically significant manner.

Therefore, our observations may suggest a rapid onset of CTLA4-Ig action on the downregulation of M1 gene expression, remaining significantly stable over time (12 h), and followed by a clear induction of M2 phenotype that might confirm the M1–M2 shift. Noteworthy, CTLA4-Ig treatment already at low dosage (100 μM) seems to fulfil its maximum pharmacological action on M1 and M2 gene expression.

It should be considered that there are few “strong” monotone associations between CTLA4-Ig dosages and their modulatory effect on gene expression and protein synthesis of M1 and M2 markers in both cultured HS-M1-MDMs and RA-MDMs. It means that the gene expression or the protein synthesis (as dependent variable) always decrease (M1) or increase (M2) with the rising CTLA4-Ig dosages (as independent variable). The presence of moderate or weak associations might support the hypothesis that abatacept at early dosage (100 μM) already induce the gene modulation observed in this in vitro study.

Several studies confirmed that M2 macrophages are necessary for the correct resolution of inflammation, suggesting that the induction of shift from M1 to M2 phenotype can block the progression of many rheumatic diseases, including RA [19, 47]. The upregulation of CD163 on monocytes/macrophages surface expression induced by CTLA4-Ig in our experiments may suggest that the treatment could accelerate the maturation process towards the M2 phenotype reverting the M1/M2 disequilibrium which characterises the chronic inflammation of RA.

Furthermore, the concomitant increase in gene expression and protein synthesis of CD204 and CD206 might suggest that CTLA4-Ig treatment is able to induce the shift from M1 to all well-characterised subsets of M2 macrophages compared to other bDMARDs like anti-TNF-α bDMARDs [29]. They in fact seem to primarily promote the shift to an M2c macrophage subset, by inducing CD163 and MerTK expression through the increased production of IL-10, but not CD204 and CD206 expression, which is mediated by IL-4 instead [29]. In our in vitro study, the capability of CTLA4-Ig treatment to induce the upregulation of CD163 and MerTK gene expression, together with that of CD204 and CD206, in cultured RA-MDMs seems to promote the shift into an M2 macrophages phenotype which might have a role in the reduction of inflammation in RA patients. In support of this statement, an impressive rise in the presence of CD163 and CD206 markers on synovial tissue macrophages, together with MerTK, has been reported in RA patients in clinical remission [46]. As already mentioned, between M1 and M2, several other “M2-like” subtypes are observed; in fact, in other autoimmune connective tissue diseases, such as systemic sclerosis, circulating cells expressing both M1 and M2 phenotype markers were observed [59, 60]. In particular, CD163 is a scavenger receptor involved in haemoglobin-haptoglobin (HbHp) complex clearance leading to the release of IL-10 and carbon oxide (CO), which in turn exhibit strong anti-inflammatory activities [61, 62].

Besides a role in the resolution of inflammation, CD163 may also affect the initial steps of an adaptive immune response [7]. In fact, the M2 marker CD163 has been demonstrated to be involved in the adherence of human monocytes to endothelium, as well as in the inhibition of T lymphocyte proliferation in vitro [63, 64]. Moreover, in an ex vivo study by Ambarus et al., the presence of a cluster of CD163+ macrophages was identified in the intima layer of synovial tissue compared to the vascular/fibrous synovial layer [65].

Therefore, the shift towards CD163-expressing macrophages induced with abatacept might represent an additional aspect of its anti-inflammatory activity to attenuate inflammation in RA.

Some limitations characterise the present study. For example, even if the modulation of specific surface markers induced by CTLA4-Ig is important for the assessment of the shift from M1 towards M2 macrophages in RA, the evaluation of cytokines/chemokines produced by the M2 macrophages was not tested, to complete the evaluation, and must deserve further analysis. In addition, the less evident in vitro reactivity to the CTLA-4-Ig treatment for the RA-MDMs compared to the HS-M1-MDMs, might be partially due to the concomitant treatment of the RA patients with low-dose prednisone and/or csDMARDs [66]. However, ethical reasons justified the inclusion criteria and the concomitant minimal treatment. Finally, the limited number of RA patients analysed was due to the need to select patients with similar clinical and therapeutical characteristics.

Although this study demonstrated the in vitro capability of CTLA4-Ig to promote the shift from an M1 to an M2 phenotype in RA-MDMs, future experiments on cultured synovial macrophages need to be considered in order to improve the relevance of these results and the potential therapeutical importance of CTLA4-Ig treatment in RA patients.

Nevertheless, based on a higher effect of the high tested CTLA4-Ig concentration (500 μg/mL) in increasing the protein synthesis of tested M2 markers observed in cultured HS-M1-MDMs at longer lasting period, further experiments are needed to confirm a possible dose-dependent effect also in cultured RA-MDMs. The results of the present in vitro study, together with the knowledges reported in previous studies by our group as well as other groups, suggest a contribution of CTLA4-Ig treatment in ameliorating the clinical aspect in RA patients through the downregulation of the pro-inflammatory status that is determined by the presence at peripheral and tissue level of M1 polarised monocytes/macrophages which cooperate in the high disease activity. The reduction of this inflammatory status exerted by CTLA4-Ig in RA macrophages seems to occur through a rapid downregulation of specific markers of M1 phenotype (CD80, CD86, and TLR4) and the reduction of specific pro-inflammatory cytokines (IL-6, IL-1β, and TNFα), followed by the upregulation of specific markers of anti-inflammatory M2 phenotype (CD163, CD204, CD206, and MerTK). Moreover, starting from the results observed in this study, a future perspective which certainly might improve the impact of CTLA4-Ig treatment should be to investigate the capability of CTLA4-Ig to induce the shift from M1 to M2 phenotype in macrophages isolated from RA patients resistant to a previous therapy with anti-TNF drugs as well as in macrophages isolated from patients characterised by genetic variability in CTLA4 (such as those with a single nucleotide polymorphism CTLA-4 rs231775) that was recently demonstrated to decrease the risk of RA [67]. Based on the results described in this in vitro study, the release of specific anti-inflammatory cytokines and chemokines in these cultured shifted RA-MDMs along with their capability to activate a T-helper 2 response will be another future perspective of this study, primarily focusing on MDMs and synovial macrophages of naïve RA patients.

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