Coexpression of IL-17RA and IL-17RC improves responses to IL-17A and IL-17F in an NF-κB reporter gene assay

Rachel Moxley, Emma Hickman, Carol E Jones. Novartis Horsham Research Centre, RH12 5AB, UK.

IL-17A and IL-17F have been implicated in a variety of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, asthma and psoriasis (1). Activated human CD4+ T cells express both IL-17A and IL-17F homodimers, as well as an IL-17A/F heterodimer. The IL-17 receptor family consists of 5 members (IL-17RA, RB, RC, RD, and RE). The IL-17RA receptor subunit binds IL-17A, IL-17F and the IL-17A/F heterodimer with differing binding affinities whereas all three cytokines bind IL-17RC with comparable affinities (2). The aim of this study was to develop an assay to examine the receptor requirements for IL-17A and IL-17F induced NF-κB signalling and for profiling antibodies which block the IL-17 response. IL-17RA and IL-17RC were transfected individually or in combination into an NF-κB reporter stable cell line and the expression of these receptors was shown by western blot analysis. For analysis of NF-κB signalling in response to either IL-17A or IL-17F, NIH-3T3-NF-κB-luc cells were transiently transfected with IL-17RA, IL-17RC or a combination of IL-17RA and IL-17RC and luciferase responses measured after 18 h of stimulation with cytokine. IL-17A treatment of cells transfected with IL-17RA, IL-17RC or IL-17RA/RC induced a dose-dependent increase in luciferase activity with similar EC50’s of 2.1, 2.5 and 2.3 nM, respectively. However, the presence of both IL-17RA and IL-17RC induced higher magnitude of response to IL-17A compared to transfection with IL-17RA alone. In the case of IL-17F, a dose-dependent increase in luciferase activity was observed in cells which expressed IL-17RA or co-expressed both IL-17RA and IL-17RC with EC50’s of 9.7 and 1.3 nM, respectively. Data showing the effects of the IL-17A/F heterodimer in the same NF-κB reporter assay system will also be described.

In conclusion, IL-17A and IL-17F induce NF-κB signalling in NIH-3T3-NF-κB-luc cells co-transfected with IL-17RA and IL-17RC subunits. In the case of IL-17A, transfection with both IL-17RA and IL-17RC increased the response of the cells to the cytokine compared to transfection with IL-17RA alone. This represents a new cell-based assay to study IL-17 signalling and the effects of antibodies which inhibit IL-17 mediated effects.

1. Ouyang W et al., (2008) The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity; 28, 454-465.

2. Wright J et al., (2008). The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex. J. Immunol; 181, 2799-2805.