Interleukin‐6 neutralization and regulatory T cells are additive in chondroprotection from IL‐1β‐induced inflammation

Abstract Anti‐inflammatory Regulatory T cells (Tregs) are enriched in the joints of patients with osteoarthritis (OA) compared to healthy joints. Tregs maintain homeostasis through secretion of anti‐inflammatory cytokines and cell‐to‐cell interactions including immune checkpoint signaling. Interleukin‐6 (IL‐6) is a pleiotropic cytokine secreted by inflamed synoviocytes and chondrocytes that can inhibit or alter Treg function. This study tested the hypothesis that neutralization of IL‐6 would enable Treg anti‐inflammatory function to resolve inflammation and catabolism elicited by IL‐1β in an equine chondrocyte/synoviocyte/Treg tri‐culture OA model. Synoviocyte/chondrocyte co‐cultures were stimulated with IL‐1β, and treated with αIL‐6 neutralizing antibody. Activated Tregs secreting IL‐10 were added in direct contact with synoviocytes to create a tri‐culture. Neutralization of IL‐6 partially restored Treg anti‐inflammatory functions and, in combination, reduced IL‐1β‐stimulated synoviocyte MMP13 expression to control levels and restored Acan expression in chondrocytes. IL‐6 neutralization alone decreased Il6 expression in chondrocytes and synoviocytes, mitigating IL‐6 positive feedback loop. Although Tregs were the primary producers of anti‐inflammatory IL‐10 and IL‐4, they also produced pro‐inflammatory IL‐17A, as detected by ELIA, which may have been responsible for incomplete rescue of synoviocyte/chondrocyte homeostasis following IL‐1β stimulation. Treg secretion of IL‐10, IL‐4, and IL‐17A was not altered by tri‐culture conditions or presence of αIL‐6, therefore, it was unlikely that Treg phenotype instability occurred. The significant effect of chondrocyte/synoviocyte donor, but not Treg donor, on gene expression and IL‐6 concentration in conditioned media, indicated that personalized therapy considering the patient's OA status might be needed for successful implementation of immunotherapy in the context of OA.