Fig 2: Post-translational modification of TICAM-1 after IL-17A stimulation.(A) HeLa cells were stimulated with 100 ng/ml of IL-17A. Cells were collected at indicated time points, and whole cell extracts were prepared. Immunoprecipitation was performed with anti–TICAM-1 antibody. The proteins were subjected to SDS–PAGE and detected by Western blotting with indicated antibodies. (B) HeLa cells were stimulated with 100 ng/ml of IL-17A. Cells were collected at indicated time points, and WCE were prepared. The proteins were subjected to SDS–PAGE with 10% acrylamide gel (normal) or 0.05 mmol/l Phos-tag arylamide gel (Phos-tag), and then detected with indicated antibodies.

Fig 3: TICAM-1 regulates IL-17A–mediated activation of transcription factors.(A, B) WT or TICAM-1 KO HeLa cells were stimulated with IL-17A (50 ng/ml) for the indicated time, and whole cell extracts (WCEs) were blotted with anti-phospho-I?B (p-I?B), I?B, TICAM-1, and ß-actin (A) and anti-phospho-p65 (p-p65), p65, TICAM-1. and ß-actin (B) antibodies as indicated. Each band intensity of p-I?B and p-p65 was measured and normalized to that of total I?B and p65, respectively. Fold increases were calculated by dividing the normalized intensity at each time point with that at 0 min in WT HeLa cells. The data were representative of two independent experiments. (C) WT and TICAM-1 KO HeLa cells were transfected with a reporter plasmid containing the NF-?B promoter and Renilla luciferase vector (internal control). After 18 h, cells were stimulated with 50 ng/ml IL-17A. NF-?B reporter activities were determined 6 h after IL-17A stimulation. The results show ± SD means (n = 3 *P < 0.05; t test). (D) WT or TICAM-1 KO HeLa cells were stimulated with IL-17A (50 ng/ml) for the indicated time, and WCEs were blotted with anti-phospho-p38 (p-p38), p38, TICAM-1, and ß-actin antibodies as indicated. Each band intensity of phosphor-p38 (p-p38) was measured and normalized to that of total p38. Fold increases were calculated by dividing the normalized intensity at each time point with that at 0 min in WT HeLa cells. The data were representative of two independent experiments. (E) WT and Ticam-1 KO MEFs (E14) were stimulated with 50 ng/ml IL-17A for the indicated time. WCEs were blotted with anti-p-p65, p65, and ß-actin antibodies. Each band intensity of p-p65 was measured and normalized to that of total p65. Fold increases were calculated by dividing the normalized intensity at each time point with that at 0 min in WT MEFs. The data were representative of two independent experiments.

Fig 4: FOSL1 inhibits IFN-I response through modification of signaling molecules upstream of TBK1. (A) Immunoassay of 293T cell extracts transfected with plasmids encoding F-RIG-I, F-MAVS, F-TRAF3, F-TBK1, F-IRF3, and F-TRIF as well as HA-FOSL1, followed by immunoprecipitation (IP) with anti-FLAG beads and immunoblot analysis with anti-HA antibody. (B) Immunoassay of 293T cell extracts transfected with plasmids encoding F-TRAF2, F-TRAF3, F-TRAF5, and F-TRAF6 as well as HA-FOSL1, followed by IP with anti-FLAG beads and immunoblot analysis with anti-HA antibody. (C) IP and immunoblot analysis of THP-1 cell extracts treated with poly(I:C) or medium overnight. (D) Real-time qPCR analysis of IFN-ß and ISG56 expression in WT, FOSL1 KO, TRAF3 KO, and FOSL1/TRAF3 double-knockout BMDMs after stimulation with poly(I:C) for 16 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significantly different from control group (Student’s t test). Data are representative of results of three independent experiments (means ± SD in panel D).

Fig 5: Model of the C. acnes-induced intracellular signaling pathways. C. acnes recognition in human macrophages is carried out by membrane bound toll-like receptors with its adapter MyD88. This further leads to the activation of the NF-?B pathway and the subsequent expression of inflammatory cytokines. The TLR adapter TRIF shows importance during C. acnes-induced IFN-signaling. TRIF can be activated by specific TLR activation on the cell membrane or in the endosome and has also been shown to positively regulate the cGAS/STING pathway. Whether C. acnes is recognized by membrane-bound or endosomal TLR-TRIF or simply boosts the cGAS/STING pathway in a TRIF-dependent manner is not known.