Fig 1: The Circadian Clock Limits PD-L1 Expression in Activated Macrophages and Monocytes(A) qPCR analysis of expression of indicated genes in PMs from middle-grade septic Bmal1 flox/flox and Bmal1Mye-/- mice (n = 3 mice/group; data expressed as means ± SD; *p < 0.05; ANOVA LSD test).(B) Western blot analysis of expression of indicated proteins in PMs at day 3 from middle-grade septic Bmal1 flox/flox and Bmal1Mye-/- mice (n = 3 mice/group).(C) qPCR analysis of Pd-l1 and Pd-l2 mRNA expression in indicated BMDMs treated with LPS (100 ng/mL) or HMGB1 (500 ng/mL) for 24–48 hr (n = 3; data expressed as means ± SD; *p < 0.05; t test).(D) Western blot analysis of PD-L1 and PD-L2 protein expression in indicated BMDMs treated with LPS (100 ng/mL) or HMGB1 (500 ng/mL) for 48 hr.(E) Western blot analysis of BMAL or CLOCK protein expression in indicated knockdown THP1 cells.(F) Knockdown of Bmal1 or Clock increased LPS (100 ng/mL)- or HMGB1 (500 ng/mL)-induced Pd-l1 mRNA expression in THP1 cells (n = 3; data expressed as means ± SD; *p < 0.05; t test).(G) Bmal1 wild-type, but not I317D mutant, restored the inhibition of LPS (100 ng/mL)- or HMGB1 (500 ng/mL)-induced Pd-l1 mRNA expression in Bmal1Mye-/- BMDMs (n = 3; data expressed as means ± SD; *p < 0.05; t test).(H) Wild-type PMs were harvested at ZT0 and ZT12 and treated immediately with LPS (100 ng/mL) for 24 hr and analyzed for Pd-l1 mRNA expression (n = 3; data expressed as means ± SD; *p < 0.05; t test).(I) Indicated neutrophils harvested from middle-grade septic Bmal1 flox/flox and Bmal1Mye-/- mice were exposed to phorbol 12-myristate 13-acetate (PMA; 0.08 µM) for 30 min and analyzed for myeloperoxidase (MPO) release (n = 3; data expressed as means ± SD).