Figure 5.

Cpd A attenuates IL-17-induced secondary responses in keratinocytes. (A) HaCaT keratinocytes were cultured for 24 h with supernatants derived from Cpd A treated Th17 cells either at a biologically inactive (0.13 nM) or at a pharmacologically active concentration (10 μM). To determine the effect of IL-17 in the conditioned supernatants on the HaCaT cell activation, half of the supernatants were pre-incubated with the IL-17 selective, neutralizing antibody Secukinumab (0.75 μg/ml). Alternatively, cells were left unstimulated or were exposed to Th17 cytokines that were used in the Th17 polarization assays. Keratinocytes were subjected to qRT-PCR analysis for determination of downstream IL-17-response genes, including NFKBIZ, DEFB4A, CCL20, and IL36G. (B,C) Defined IL-17A and TNFα concentrations which were measured in the Th17 cell supernatants originating from low-Cpd A-containing samples (IL-17: 0.6 ng/ml; TNFα 4 ng/ml) and from high-Cpd A-containing (IL-17: 0.15 ng/ml; TNFα: 4 ng/ml) or Secukinumab-treated samples were spiked into HaCaT cells (B) or alternatively into NHEK cell cultures originating from two donors (C). To determine the impact of IL-17 in this cellular system, IL-17 was neutralized by addition of Secukinumab (0.75 μg/ml). Graphs are representative from three experiments containing triplicate readings. Significance between low-Cpd A-containing supernatants and between high-Cpd A and Secukinumab-containing samples was determined by ANOVA followed by Dunnett's test (***p < 0.005; ****p < 0.0001). Error bars represent the SD.