Identification of a cytokine network sustaining neutrophil and Th17 activation in untreated early rheumatoid arthritis

Rheumatoid arthritis (RA), the most common chronic autoimmune disease, affects approximately 1% of the population worldwide. This disease comprises a syndrome of pain, stiffness, and symmetrical synovitis which leads to joint destruction, functional disability, and substantial comorbidity due to the involvement of multiple organs and systems. The migration of leukocytes toward the synovium is crucial for the establishment of a chronic inflammatory process in RA [1‐3]. This multi-regulated mechanism involves interactions with endothelial cells through cell adhesion molecules and complex cytokine and chemokine pathways.

Neutrophils specifically play an important role in the onset and perpetuation of RA, not only as interleukin (IL)-producing cells but also as cells responsible for the release of high amounts of reactive oxygen species and destructive enzymes, such as metalloproteases, contributing to joint erosions [4]. Neutrophils are among the first leukocytes to arrive at sites of inflammation. In fact, these cells are the most abundant in the synovial fluid (SF) of patients with active RA, and previous results from our group showed that the synovial tissue is heavily infiltrated by neutrophils in the first weeks of RA onset [5]. Interestingly, in animal models of arthritis, neutrophil depletion prevented joint inflammation if neutrophil-depleting antibodies were given before the induction of arthritis. Moreover, when the depleting antibody was given very early after the induction of arthritis, complete abrogation of the inflammatory symptoms was achieved [6].

T helper 17 (Th17) cells have also been proposed to have a relevant role in the early phase of RA through the production of IL-17 [7, 8]. This cytokine promotes the recruitment and survival of neutrophils, induces the secretion of proinflammatory cytokines and the upregulation of RANKL (receptor activator of nuclear factor-kappa B ligand), and stimulates the activity of matrix metalloproteases, leading to cartilage catabolism and bone resorption [9, 10]. The recruitment, activation, and effector function of Th17 cells and neutrophils are driven by a network of cytokines and chemokines secreted by multiple cellular sources. In established RA, it has been reported that IL-1β, IL-6, IL-8, IL-17, and tumor necrosis factor are elevated in the serum and this correlates with a higher disease activity [11‐13]. Nevertheless, our knowledge of the influence of the cytokine network on RA onset remains limited. The characterization of the cytokine profile at this stage, where the transition from an acute to a chronic inflammatory phase occurs, may lead to the identification of early key players, with potential implications for early treatment strategies.

Thus, the main goal of our work was to determine whether cytokines driving neutrophil and Th17 cell activation and proinflammatory function were already present in very early RA (with less than 6 weeks of disease duration) and how this early cytokine environment differs from established RA. We also evaluated whether the introduction of low-dose corticosteroids and methotrexate (MTX) therapy had any influence on the cytokine profile observed at that early stage of the disease. We found that cytokines related to Th17 polarization and neutrophil recruitment and activation were elevated in early RA and that the conventional therapeutic options, though able to control clinical manifestations of the disease, were ineffective in reversing this underlying proinflammatory drive.

Several studies have previously demonstrated that neutrophils play an important role in the onset of RA [21]. This hypothesis is supported by data from animal models [24]. In fact, neutrophils are the most abundant leukocytes in the SF of patients with active RA, and in early RA, these cells show significantly lower levels of apoptosis when compared with patients with other persistent forms of arthritis or with arthritis that has a self-limited disease course [25]. Additionally, previous results from our group demonstrated that there is a delay in the apoptosis of circulating neutrophils in VERA patients [21] and that these cells heavily infiltrate the synovial tissue during RA onset [5].

In the present study, we demonstrate that a neutrophil- and Th17-driving cytokine pattern is present in untreated VERA patients with less than 6 weeks of disease duration. We consider this observation of interest because the knowledge concerning the immune mechanisms associated with the onset of RA is still elusive. In fact, the majority of early RA studies include patients with 3 to 12 months of disease duration or even more. In accordance with an early participation of neutrophils in RA, our results revealed that VERA patients have increased levels of IL-8 when compared with both VEA and healthy controls, and this could explain the preactivated state of circulating neutrophils [18] and their recruitment toward the SF from the very first weeks of RA onset.

In addition, Th17 cells are known to be important for the promotion of neutrophil-mediated inflammation by producing IL-17A, a cytokine known to indirectly activate neutrophil chemotaxis and extend their survival [10, 22]. We found a high serum concentration of IL-17A in VERA patients as well as locally within the joints of patients with established RA. This might indicate that an activation of Th17 cells from a very early phase of the disease can promote neutrophil participation in RA pathogenesis [22]. However, we found no evidence for changes in the frequency of T-cell subsets in the peripheral blood of VERA patients [17]. This observation is not unexpected; the relatively small representation of antigen-specific T cells in the circulating pool are the activated T cells that drive the pathology more likely found within the tissues [26]. In a study performed by Kokkonen and colleagues [27], the levels of several cytokines and chemokines were analyzed in blood samples from a group of individuals 3.3 years before RA onset ('pre-patients') and compared with healthy donors and RA patients with 7.7 ± 3.6 months of disease duration. An interesting finding was that IL-17 was present at its highest concentration in pre-patients and the level of this cytokine was lower in patients with RA. This is in accordance with our own results; we observed an increased level of IL-17 in RA patients with less than 6 weeks of disease duration, whereas in patients with established RA, the levels were not significantly different from those of healthy controls. Remarkably, the IL-17 median concentration observed in our established RA cohort (2.6 pg/mL) was even lower than that of RA patients from the work of Kokkonen and colleagues [27] (6.0 pg/mL). Thus, this observation reinforces the role of IL-17 in the initial phase of RA, and as the pathogenesis progresses to a chronic stage, other factors are subsequently brought into action in the peripheral blood. Unlike Kokkonen and colleagues, we have not detected differences in Th1- and Th2-related cytokines between both VERA and patients with established RA in comparison with controls. These discrepancies might be related to the different methodologies used.

Additionally, the elevated levels of IL-1β observed in VERA patients can stimulate endothelial cells, T and B cells, and fibroblasts in the joints to produce IL-6 and IL-8. But importantly, IL-1β and IL-6, both found to be increased in VERA patients, are known to promote the differentiation of Th17 cells, which in turn secrete IL-17A and IL-22 [28, 29], two cytokines that were elevated in VERA patients and have an essential function in the pathogenesis of autoimmune diseases [29].

Currently, the treatment of choice for RA at the time of presentation is MTX. Interestingly, in spite of clinical improvement (DAS28 reduced from 6.1 ± 1.8 to 3.1 ± 1.6), neither therapy with low-dose corticosteroids nor combined therapy with low-dose corticosteroids and MTX corrected the dysregulated cytokine pattern observed in VERA patients. In fact, low-dose corticosteroids and MTX have unclear effects on the RA cytokine network. For instance, corticosteroids fail to reduce serum levels of IL-1β and IL-8 [30] and MTX does not alter serum IL-1β concentration when compared with pre-treatment levels [31, 32]. Our results suggest that the conditions contributing to Th17 cells and neutrophil-mediated inflammation, thus driving early pathogenesis, are not modified with early treatment with low-dose corticosteroids and MTX.

The elevated IL-1β, IL-6, IL-8, and IL-17A levels observed in the SF of patients with RA confirm a local role for these cytokines in the maintenance of synovitis. Moreover, IL-6 can support a continuous recruitment of autoreactive B cells toward the synovium [33, 34], contributing to an exacerbation of the inflammatory process because of the production of autoantibodies and immune complexes.

Taken together, our data reinforce the potential relevance of therapies targeting IL-1β [35, 36] and IL-6 [37, 38] in early RA. In addition, the data establish IL-8 and IL-17A as other potential therapeutic targets at an early stage of the disease. Finally, we found that MTX and corticosteroids, though effective in reducing disease activity in VERA patients, do not appear to correct underlying cytokine dysregulation driving the Th17/neutrophil-mediated inflammation.