The response of resident cells in the synovium is essential to RA pathogenesis and it is conditioned by inflammatory cytokines. Although, the function of the two pivotal cytokines, TNFα and IL6, has been extensively studied in this context, most investigations have focused on the role of TNFα as the main upstream inductor of IL6 expression. Our present work shows that IL6/sIL6R signaling is able to modulate the TNFα inflammatory response elicited in SF, promoting changes of the inflammatory gene expression pattern associated to RA pathogenesis. Furthermore, we provide insight into the molecular mechanisms that regulate the crosstalk between both cytokines.
The mode of action of IL6 is contextual, defined by other factors present within the local milieu. Although IL6 is defined as an inflammatory cytokine, it does not directly induce leukocyte recruitment. Rather, previous studies have shown the ability of IL6, either alone or cooperating with other factors, to modify inflammatory infiltrates[23–27]. Analysis of AIA in IL6+/+ and IL6−/− mice demonstrated that IL6 deficiency is associated with reduced synovial infiltration and is accompanied by a defect in both CCL2 expression and the recruitment of leukocytes bearing the CCL2 receptor CCR2. Our results show that the induction of IL6/sIL6R signaling in SF mainly stimulates the expression of cytokines and chemokines that attract lymphocyte and monocyte cells, although at a lower level than TNFα. However, the cooperative activation of both factors further enhances the expression of common chemokines and cytokines, thus potentially contributing to sustain the leukocyte influx into the synovial tissue. The impact of IL6/sIL6R and TNFα combination in SF may be more relevant in the context of RA where the local increase of sIL6R correlates with the extent of leukocyte infiltration and joint destruction[3, 9]. Because the expression of IL6R within the RA synovial environment is mainly restricted to leukocytes, infiltrating cells may be the source of the synovial sIL6R in response to inflammatory mediators such as TNFα[25, 29–31]. The sIL6R would then be acting as a paracrine mediator on synovial cells, further inducing leukocyte recruitment during inflammation. Several studies have highlighted a role for sIL6R in regulating the expression of chemokines and adhesion molecules[8, 30, 31, 33–36], thus mediating the transition between the early neutrophilic stage of acute inflammation and the more sustained mononuclear cell influx. Interestingly, mouse models of IL6−/− showed reduced neutrophil accumulation at sites of infection or inflammation that seems to be secondary to the effects of IL6 trans-signaling on stromal cells. Our studies demonstrate that IL6/sIL6R shapes the expression program of leukocyte recruiting factors mediated by TNFα in SF. In addition, these results confirm that IL6 trans-signaling directly enhanced TNFα-induced expression of monocyte- and lymphocyte-regulating factors such as il6, ccl2, and ccl8, while inhibiting the expression of the neutrophil-recruiting factor IL8. The functional relevance of these changes in gene expression patterns are highlighted by the increased MNC migration at the expense of PMN recruitment upon IL6/sIL6R trans-signaling. Such scenario may be relevant to arthritis, in which neutrophils may play an essential role in the initiation of RA but mononuclear leukocytes infiltration would explain chronicity[38, 39]. In this context, regulation of the SF inflammatory response by IL6/sIL6R would be playing a major role in the transition to sustained inflammation.
The temporal order of induction and the relative duration of the various inflammatory events induced by TNFα and IL6/sIL6R suggests a gene activation program that ensures a rapid inflammatory response[18–20]. Kinetics and stability analyses of gene expression, as well as the differential decay for each gene induced by TNFα suggest that, similar to what is described in other cells types, SF display a pattern of gene expression controlled by both transcriptional and post-transcriptional mechanisms. In contrast, the fast decay observed in IL6/sIL6R-induced mRNAs suggests that de novo transcriptional mechanisms are mainly, if not exclusively, regulating the expression genes induced by IL6/sIL6R in SF, even for the stable expression of genes such as ccl8 or mmp1. Furthermore, IL6/sIL6R signaling did not modify the stability of TNFα-expressed mRNAs, suggesting an independent transcriptional regulation of commonly induced genes.
Evidences suggest that chronic pathological states are associated with disease-specific stable changes in gene expression, many of them consistent with epigenetic mechanisms. It has been shown that TNFα can display a gene-specific priming effect on RASF, mediated by epigenetic changes, that enhances subsequent inflammatory response to other factors such as IFNs. However, our data do not demonstrate TNFα priming of gene expression induced by IL6/sIL6R. Altogether, our results suggest that the cooperative effect elicited by IL6/sIL6R in TNFα-responses requires the sustained presence of both factors, potentially making SF more responsive to the pharmacological intervention with IL6/sIL6R signaling inhibitors.
IL6/sIL6R activation of SF may not only contribute to synovitis by sustaining leukocyte infiltration within the inflamed joint, but also enhancing their matrix degrading activity. Similar to previous investigations, we observed a consistent up-regulation of MMP1 expression upon IL6/sIL6R stimulation of SF. For most of the MMPs, TNFα induces activation via several transcription factors, including NF-kB and activator protein 1 (AP-1). In contrast, IL6/sIL6R effect on these genes seems to be more specific. For instance, mmp1 and mmp3 genes bear STAT binding sites that would make them susceptible of IL6-induction by promoter-bound STAT3 or STAT1, in contrast to other MMPs gene promoters that do not have STAT motifs. Although differences in the cell sources used in both studies may account for part of the discrepancies between Araki´s results and ours, the fact that mmp3 is not expressed at any dose tested, even under active transcriptional conditions such as those imposed by TNFα, suggests that IL6 does not induce the recruitment of transcription factors to the mmp3 promoter, further supporting that de novo transcription has a major role in the response mediated by IL6/sIL6R signaling.
Of interest is the IL6/sIL6R inhibitory effect on TNFα-mediated expression of mmp10. Mmp10 is linked to the control of the resolution phase of inflammation in models of pulmonary inflammation and experimental colitis, where MMP10 deficiency exacerbates the disease[17, 44, 45]. Therefore, IL6/sIL6R may also potentiate TNFα inflammatory response by restraining this anti-inflammatory loop.