In this study we examined the expression of IL-17A in the human inflammatory joint. An ex vivo RA ST explant model, RASFCs, normal human cartilage and chondrocyte cultures were used to investigate the mechanistic role of IL-17A, alone and in combination, with TNF-α and OSM on matrix turnover and cartilage proteoglycan release. The effect of biologics therapy on IL-17A expression and matrix turnover was also examined in vivo in a previously described cohort.
In this study we have shown high expression of IL-17A in ST lysates with no significant difference between RA and PsA tissue. Furthermore, IL-17A expression was markedly higher in RA and PsA ST compared with OA. Our results are consistent with previous studies demonstrating increased IL-17A production from RA patients compared with OA [13
]; however, our data differ as samples are from patients with early active inflammation before commencing biologic therapy. We also report significantly higher IL-17A levels in SF and serum from inflammatory arthritis patients compared with OA and healthy controls. SF levels of IL-17A were significantly higher than matched serum suggesting that the cytokine is predominantly produced locally in the inflamed joint. Although previous reports showed elevated serum and SF IL-17A levels in RA patients compared with healthy controls [37
] these studies did not examine matched serum/SF samples as performed in this study. Interestingly, there was a strong correlation of SF IL-17A levels with CRP and disease duration. This finding is not surprising because previous animal model studies have suggested IL-17A drives disease activity and is associated with disease chronicity [27
]. Furthermore, IL-17A is a potent inducer of CRP from human smooth muscle cells and hepatocytes [38
]. We also demonstrated IL-17A expression in RA but not OA or healthy control synovium. IL-17A was expressed in 70% of RA patients examined, which is consistent with previous reports [39
A fine balance exists between active MMP and TIMP levels in normal tissue and cartilage. In inflammatory conditions such as RA, this balance shifts leading to an increase in the ratio of active MMP: TIMP [40
]. The effect of IL-17A on MMP expression was first examined in RASF and normal human chondrocyte cultures. IL-17A alone upregulated the expression of MMP-1 and MMP-13 in both chondrocytes and RASFCs. IL-17A combined with OSM synergistically upregulated MMP-1 production in both chondrocytes and RASFs. A similar effect was observed for chondrocyte MMP-13 production with a less potent effect on RASF MMP-13 production. IL-17A alone also caused an increase in matrix turnover in chondrocytes as seen by the increased MMP-9 activity. IL-17A combined with OSM had an additive effect on matrix turnover as seen by the increased MMP-9 activity compared with either cytokine alone. Our results are consistent with previous studies showing IL-17A induced MMP expression in human OA chondrocytes [41
] and in RA synoviocytes [18
]. In animal cells IL-17A has been previously shown to act synergistically with TNF-α and OSM in upregulating proinflammatory cytokine and chemokine expression and cartilage breakdown [18
]. In this study for the first time we have shown in human cells isolated from patients with inflammatory arthritis that IL-17A combined with OSM synergistically upregulates the expression of MMP-1, MMP-2, MMP-9 and MMP-13 in both chondrocytes and RASFs.
We also showed for the first time in human cartilage explants and a human ex vivo
synovial explant culture model, that IL-17A regulates MMP production and cartilage degradation, which is supported by previous animal studies [18
]. IL-17A alone significantly shifted the MMP-1: TIMP-1 ratio in favour of a destructive pattern. Significant matrix turnover was also demonstrated by increased MMP-2 and MMP-9 activity in response to IL-17A stimulation. Mild proteoglycan depletion was observed in response to IL-17A stimulation visualised by loss of safranin-O staining. However, when IL-17A was combined with other key pro-inflammatory cytokines it significantly potentiated the effect of TNF-α and OSM on MMP-1 and MMP-13 production. This had a profound effect on the MMP/TIMP ratios, which shifted dramatically in favour of matrix degradation. Furthermore, when human cartilage explants were incubated with IL-17A, TNF or OSM alone, mild proteoglycan depletion was observed; however, when IL-17A was combined with OSM or TNF, near complete proteoglycan depletion compared with either cytokine alone was demonstrated.
These results suggest that in the inflamed joint environment, which has a complex milieu of pro-inflammatory cytokines, that in addition to exerting its effects alone, IL-17A appears to have an important role in dramatically potentiating the destructive effects of other pro-inflammatory cytokines, such as TNF-α and OSM. Increased MMP expression has been observed in human chondrocytes in response to IL-17A stimulation [41
] but no previous studies has examined its effect on RA whole tissue synovial explants or human cartilage explants. Whole tissue explant cultures more closely mimic the joint environment, as the architecture, cell-cell interactions and ECM remains intact, which can result in expression of genes and proteins to stimuli that differ from monolayer cultures. Thus the combination of mono-culture and whole tissue explant culture in this study dissects the cellular responses such as MMP expression to specific stimuli more effectively [44
Finally, we examined IL-17A and MMP/TIMP production in patients pre/post biologic therapy on IL-17A. We demonstrate for the first time that IL-17A serum levels are reduced in inflammatory arthritis patients following TNF blockade in vivo
. We show that serum IL-17A levels are modulated by biologic therapy with 80% of patients showing a decrease in IL-17A three months post therapy. Significantly, this reduction was demonstrated in patients who showed a clinical response, while non-responders showed an increase. Change in IL-17A also correlated with change in CRP and A-SAA, markers of systemic inflammation. Previous studies have shown synovial membrane mRNA levels of IL-17A may predict joint damage progression in RA [45
] and IL-17A serum levels correlate with disease severity in psoriasis patients [46
]. IL-23 (a key factor in Th17 differentiation) has been genetically linked to increased susceptibility to psoriasis [46
]. Furthermore, disease resolution in psoriasis patients following TNF blockade correlates with reduced Th17 responses [48
Furthermore, we demonstrate a significant difference in MMP/TIMP ratios from baseline to three months post therapy in IL-17A-negative patients, with no significant difference in IL-17A-positive patients. However, interpretation of this data is complex. IL-17A-positive patients may exhibit a sustained stimulation of MMPs; however, as the IL-17A levels do reduce after three months in most patients, it may suggest other pro-inflammatory mediators such as TNF-α, IL-1β and OSM are driving MMP expression. Indeed, this is supported by our in vitro
data and the results of other studies [34
] demonstrating IL-17A acts in synergy with TNF-α, OSM and other cytokines leading to increased MMP activity.
Numerous studies have associated MMP serum levels in particular MMP-3 levels to disease activity and radiographic progression [49
]. SNPs in the IL-17A gene have been associated with radiographic progression [56
]. Furthermore, one of the main drivers of IL-17A production, IL-23, is present in higher levels in patients with bone erosions than those without erosions [57
]. The observation of higher CS846 levels in IL-17A-positive sera may be relevant radiographically. Increased serum levels of CS846 are an indicator of increased turnover of newly formed matrix, as part of an attempt to repair cartilage degradation [58
]. Furthermore, patients with rapid radiographic progression have been shown to have higher CS846 epitope levels than slow progressors [59
Overexpression of IL-17A by injection or gene expression significantly increased inflammatory infiltrate and resulted in extensive joint destruction [22
]. Furthermore, in studies that blocked IL-17A or in IL-17R-/- mice reduced inflammation and bone damage was observed [61
]. In murine studies IL-17A has been shown to act independently of TNF-α [27
]. A number of studies have shown TNF inhibition has no effect on IL-17A or IL-23 expression [63
]. Furthermore, the IL-17A/TNF-α ratio pre-treatment was shown to be lower in responders to anti-TNF treatment [29
]. This data and previous studies suggest that IL-17A synergises with other pro-inflammatory cytokines but can also enhance inflammation and destruction independently and would propose IL-17A as a potential target in the treatment of RA.