C 176

CD4⁺ T cells were enriched from mice lymph nodes and spleen with anti-CD4 microbeads by using an AutoMACS magnetic cell sorter (Miltenyi Biotec) according to the manufacturer’s protocol. Thereafter, naive CD4⁺CD62L^highCD44^lowT cells were purified using a FACSAria III cell sorter (BD Biosciences). The sort-purified naive CD4 T cells were TCR-activated with plate-bound anti-CD3ε (4 μg/mL) and anti-CD28 (2 μg/mL) (BD Biosciences) in complete IMDM (supplemented with 10% FBS, L-glutamine, penicillin-streptomycin and β–mercaptoethanol). Skewing conditions were as follows: 1 ng/mL rhTGF-β1 (eBioscience) plus 25 ng/mL rmIL-6 (R&D Systems) for cT_H17; 25 ng/mL rmIL-6, 20 ng/mL rm-IL-1β and 30 ng/mL rmIL-23 (all from R&D Systems) for pT_H17; 1 ng/mL rhTGF-β1 for iT_reg; 20 ng/mL rmIL-12 and 20 ng/mL rmIL-2 (both from R&D Systems) for T_H1. When indicated, CH223191 (30 μM; Tocris), DMXAA (3–30 μM; Invivogen), C-176 (1 μM; Sigma-Aldrich), c-di-AM(PS)2 (Rp,Rp) (15 μM; Invivogen), c-di-AMP or c-di-GMP (both 30–100 μM; Invivogen) were used.

For the in vitro experiments, when the cells have grown to a density of 70%–80%, NBP (generously provided by Shijiazhuang Pharmaceutical Group, NBP Pharma Co., Ltd, China) or C-176 (GC33823, GLPBIO, USA) were diluted to 100 µMol/L (for NBP) or 10 µMol/L (for C-176) and incubated with the cells for 24 h. Rotenone (R8875, Sigma-Aldrich, St Louis, MO, United States) was diluted to 0.25 µMol/L incubated for 24 h.
Four groups were assigned for the in vivo studies: (1) control, (2) NBP, (3) Rotenone, and (4) NBP + Rotenone. Male mice were randomly selected (n = 12 mice per group). Rotenone was administered intragastrically for 8 consecutive weeks (from the first week to the eighth week) (Figure 4(a)). NBP (200 mg/kg/day) was administered intraperitoneally for 6 weeks (from the fifth and tenth weeks) (Figure 4(a)). Simultaneously, an equal volume of saline was administered to the mice in the control group.