FLS are the major population in the synovium and play a critical role in arthritis pathogenesis. In this study, we investigated the effects of the arthritis-associated cytokines IL-17 and TNF-α on the migration and invasion of FLS. IL-17 and TNF-α are the primary focus in RA disease research, and combined blockade of both cytokines has been suggested as a novel therapy for patients who are unresponsive to selective TNF-α inhibition [24, 25]. Here, we revealed that NOX4 mediated a differential response to IL-17 and TNF-α stimulation in RA FLS compared to OA FLS. Specifically, NOX4 was increased by cytokine exposure and was required for RA FLS migration and invasion via increased expression of VCAM1 and VEGF. Our findings therefore highlight NOX4 as a potential treatment target in RA.
Interestingly, while RA FLS demonstrated a rapid and robust increase in migration and invasion following cytokine stimulation, FLS from OA donors showed little or no enhancement. The differential responses may be related to the different amounts of IL-17 and TNF-α in synovium. Indeed, IL-17 was detected at higher concentrations in RA synovium than in OA or control synovium [26, 27]. Although migration and invasion in OA FLS did not change with cytokine stimulation for 1 h, expression of VCAM1, ICAM1, NCAM1, and VEGF was increased 2-3-fold. Therefore, we concluded that the adhesion molecules and angiogenic factors were differentially regulated by these cytokines through a mechanism that is distinct from cell migration and invasion.
Next, we investigated whether IL-17 and TNF-α affected ROS levels in RA and OA FLS, because ROS have been shown to directly induce cell migration and invasion in the context of cancer . Mitochondria-specific ROS expression was increased by cytokines in RA FLS but not OA FLS, and similar results were found for invasiveness. Our findings strongly suggest that IL-17 and TNF-α induce a differential increase in ROS that leads to the distinct invasion capabilities between RA and OA.
The increased intracellular ROS in FLS may negatively influence RA-associated synovium changes, such as immune cell activation and pro-inflammatory cytokine secretion, as well as invasion of FLS into bone [29, 30]. In relation to cell migration, ROS-generating enzymes in the NOX family have been suggested as therapeutic target molecules in various diseases, such as cardiovascular disease, autoimmune disease, and inflammation [31, 32]. It was shown that RA FLS exhibited aggressive features, such as hypoxia, invasion, and inflammation, which are similar to cancer . NOX4 overexpression in human colorectal cancer was associated with poor prognosis and increased tumor migration and invasion . In vascular inflammatory disease, NOX4 knockdown mediated a significant decrease in inflammation [35, 36]. Although inhibition of excessive ROS has previously been suggested as an important target in RA treatment, few studies have investigated the role of NOX and RA FLS.
When mRNA expression in RA FLS was analyzed in response to IL-17 and TNF-α stimulation, NOX4 was increased most among the NOX family members. Following NOX4 inhibition in RA FLS, cytokine-induced ROS, VCAM1, VEGF, migration, and invasion were downregulated to levels observed for untreated controls. Together, these results indicate that IL-17- and TNF-α-mediated NOX4 expression activates a ROS-VCAM1-VEGF pathway that contributes to FLS migration and invasion in RA.
One limitation of this study is the use of ex vivo human FLS, which may not precisely reflect the in vivo response. Further study using an animal RA model is needed. In addition, the mRNA expression of NOX2 was also significantly increased in RA FLS, and therefore additional study of the role of NOX2 in RA is warranted.
In conclusion, we revealed that NOX4 was upregulated in RA FLS following IL-17 and TNF-α stimulation, leading to aggressive migration and invasion via a ROS-VCAM1-VEGF pathway. These findings suggest that NOX4 may be a critical factor in RA pathogenesis and that this enzyme may provide a therapeutic target for treatment of RA and other inflammatory diseases.