Increase in circulating Th17 cells during anti-TNF therapy is associated with ultrasonographic improvement of synovitis in rheumatoid arthritis

We conducted a longitudinal investigation of patients with RA during the initial 12 weeks of anti-TNF treatment using clinical, ultrasonographic and T cell assessments to gain an understanding of immune correlates of treatment response. This longitudinal evaluation allowed us to identify a link between changes in circulating Th17 cells, evaluated by cellular assays, and resolving synovial inflammation and vascularity during anti-TNF treatment.

Anti-TNF treatment led to a significant and sustained improvement in clinical measures of disease activity and morphological improvement in synovial thickening and vascularity determined by grey scale and PDUS during 12 weeks of treatment. We observed strong positive correlations between DAS28, a composite measure of disease activity, and synovial vascularity score by PDUS, a more objective and quantitative measure of synovitis in the limited set of joints assessed. These findings are in agreement with previous studies [14–16, 30–33]. There was a clear difference between anti-TNF EULAR good responders and non-responders in the change in ultrasound measures of synovial thickening and vascularity during anti-TNF treatment. Responders demonstrated a significant improvement in synovial thickening and vascularity after 1, 4 and 12 weeks on treatment, whereas there were no significant changes in the non-responder group. The ultrasound measures of synovial vascularity were better able to discriminate between responder and non-responder groups compared to synovial thickening, which has also been shown by others [19, 29, 31, 34].

Synovial thickness and vascularity scores improved during anti-TNF treatment in EULAR good responders, but interestingly they exhibited different kinetics of change, with synovial vascularity showing earlier and more marked improvement compared with synovial thickening scores. PDUS signal has been shown to reflect vascularisation of pannus in RA and to correlate with histological changes of synovitis and synovial membrane microvascular density [32, 33]. One of the mechanisms of action of anti-TNF agents is through reduction of neovascularisation and angiogenesis of the synovial tissue by reducing expression of vascular endothelial growth factor (VEGF) [35]. Thus, anti-TNF appears to act rapidly to reduce synovial vascularity and therefore inflammation, which is reflected by improvement in ultrasound measures of vascularity. The reduction in synovial thickness assessed by grey scale ultrasonography is a slower process as it is likely to represent a decrease in swelling and inflammation of the synovium, which is likely a combination of reduction in infiltration of inflammatory cells in the joints, reduced expression of inflammatory cytokines and chemokines and reduction in synovial vascularity [36–38].

Using Elispot and intra-cellular cytokine staining, we demonstrated an increase in circulating Th17 cells during anti-TNF treatment in patients with RA. These results were obtained using two different but complementary techniques for assessing cellular immune responses and were consistent, thus strengthening our findings. The increase in circulating Th17 cells during anti-TNF treatment has been indicated in two small studies but these have evaluated Th17 cells using flow cytometry only, or by measurement of IL17 production by stimulated PBMC using ELISA at one time point on treatment [24, 25].

We found significant negative correlation between the change in numbers of Th17 cells from baseline to 12 weeks on treatment and the change in ultrasound scores for synovial thickening and vascularity from baseline to 12 weeks. Thus, as the frequency of Th17 cells increases in peripheral blood during anti-TNF treatment, there is a corresponding improvement in synovial thickening and vascularity. Our results suggest that the increase in Th17 cells in peripheral blood during treatment is associated with improvement in synovial thickening and vascularity.

This is the first study to link changes in T cell immunopathology assessed by cellular assays with the morphological changes in inflamed joints assessed by PDUS during anti-TNF treatment. These correlations are consistent with the mechanism of action of anti-TNF agents. One of the key mechanisms of action through which anti-TNF has been shown to lead to improvement in arthritis is through reduction in trafficking of inflammatory cells to joints through reduced synovial expression of chemokines and adhesion molecules and also reduced angiogenesis and synovial VEGF expression [35, 37, 38]. In patients with RA, infliximab causes a reduction in the cellularity of inflamed synovial tissue, with significant reductions in the number of intimal and sublining macrophages, plasma cells and T cells which parallels the rapid reduction in swollen joints as early as 48 hours after infliximab infusion [39]. Furthermore, a study in patients with RA demonstrated that the frequency of Th17 cells in synovial fluid from inflamed knee joints correlated with positive PDUS signal of the knee joint and increased levels of synovial fluid VEGF, suggesting that the presence of PDUS signal in the joints may therefore be a surrogate marker for the presence of Th17 cells [7]. Thus, the negative correlation between the increase in peripheral blood Th17 cells during anti-TNF treatment and the decrease in synovial thickness and vascularity on ultrasound suggests that anti-TNF treatment may induce redistribution of inflammatory cells from joints, leading to improvement in joint swelling and inflammation.

Another possible mechanism through which anti-TNF may cause an increase in circulating Th17 cells is through an increase in the p40 subunit shared between IL12 and IL23, the key cytokines involved in differentiation of Th1 and Th17 cells, respectively. In a study by our group using the CIA mouse model of RA, anti-TNF therapy ameliorated arthritis by decreasing numbers of Th1 and Th17 cells in arthritic joints, but also caused an increase in Th1 and Th17 cells in draining lymph nodes [23]. By using knockout mice, the increase in Th1 and Th17 cells was shown to occur through signalling via the TNFp55 receptor, which increased expression of the p40 subunit shared between IL12 and IL23. A similar mechanism was found to occur in a mouse model of reactive arthritis, where Yersinia-induced reactive arthritis in mice lacking TNFp55 receptor was associated with more severe disease. Increased levels of IL17, IL23 and IL12p70 were found in the arthritic joints of these mice and antibody blockade of IL17 was shown to reduce arthritis severity. The increase in Th17 responses in the TNFRp55-/- mice was shown to be mediated by an increase in IL12/23p40 [40]. TNF?-mediated inhibition of IL12/23p40 may also occur in human disease. A study by our group in patients with RA treated with anti-TNF showed that the increase in circulating Th17 cells up to 12 weeks on anti-TNF was accompanied by an increase in IL12/23p40 production in supernatants from PBMC stimulated with lipopolysaccharide (LPS) and also in the plasma layer of whole blood stimulated with LPS [25]. Taken together, these findings suggest that anti-TNF agents may act through several mechanisms to increase circulating Th17 cells during treatment.

Another interesting finding to emerge from this study is that anti-TNF non-responders showed a trend towards a higher baseline frequency of Th17 cells compared to responders and this trend was observed using results from both Elispot and intra-cellular cytokine staining. Two other studies also point to an association between higher baseline levels of IL17 or a higher frequency of Th17 cells and poor anti-TNF treatment response in RA; although in these studies this relationship has been investigated using clinical measures of disease activity only and at a single time point on treatment, rather than longitudinally [25, 41]. We investigated this hypothesis by exploring relationships between ultrasonographic and T cell immunological changes during anti-TNF therapy to determine if a higher frequency of IL17-producing cells at baseline was associated with poor treatment response. Indeed, we found significant correlation between higher numbers of Th17 cells at baseline and a smaller improvement in synovial vascularity on ultrasound at 1 week, and a smaller improvement in synovial thickening at 1, 4 and 12 weeks after anti-TNF initiation This suggests that a higher frequency of Th17 cells at baseline is associated with poor anti-TNF treatment response.

We have used two different but complementary techniques (Elispot and intracellular cytokine staining) to assess the frequency of Th17 cells prior to anti-TNF initiation and using both methods, we found a significant relationship between a higher baseline frequency of Th17 cells and poor treatment response assessed by ultrasonographic measures. If these associations are also confirmed in larger patient cohorts and in patients with other types of inflammatory arthritis aside from RA, the characterisation of Th17 cells as a marker of anti-TNF non-response raises the possibility of being able to tailor biologic therapy of inflammatory arthritis according to individual patient immunological profiles. Further investigation is warranted into whether patients with higher baseline frequencies of circulating Th17 cells may have more IL17-driven disease and whether these patients may derive greater benefit from treatment with a combination of anti-IL17 and anti-TNF agents.