The Circadian Clock Controls Immune Checkpoint Pathway in Sepsis

Abstract

Sepsis and septic shock are associated with life-threatening organ dysfunction caused by an impaired host response to infections. Although circadian clock disturbance impairs the early inflammatory response, its impact on post-septic immunosuppression remains poorly elucidated. Here, we show that Bmal1, a core circadian clock gene, plays a role in the regulation of host immune responses in experimental sepsis. Mechanistically, Bmal1 deficiency in macrophages increases PKM2 expression and lactate production, which is required for expression of the immune checkpoint protein PD-L1 in a STAT1-dependent manner. Consequently, targeted ablation of Pkm2 in myeloid cells or administration of anti-PD-L1-neutralizing antibody or supplementation with recombinant interleukin-7 (IL-7) facilitates microbial clearance, inhibits T cell apoptosis, reduces multiple organ dysfunction, and reduces septic death in Bmal1-deficient mice. Collectively, these findings suggest that the circadian clock controls the immune checkpoint pathway in macrophages and therefore represents a potential therapeutic target for lethal infection.

Keywords: Bmal1; IL-7; Pd-l1; Pkm2; Stat1; checkpoint; circadian clock; macrophages; metabolism; sepsis.

Figure 1.. Altered Circadian and Sepsis Phenotype in Bmal1^Mye−/− Mice

(A) Western blot analysis of BMAL1 expression in PMs, livers, and brains from Bmal1 ^flox/flox and Bmal1^Mye−/− mice. (B) Ablation of Bmal1 in the myeloid cells results in disrupted circadian expression of clock genes Bmal1, Per1, Per2, and Per3 in PMs (n = 3 mice/group; data expressed as means ± SD). (C) Ablation of Bmal1 in myeloid cells does not disrupt circadian expression of clock genes Bmal1, Per1, Per2, and Per3 in liver (n = 3 mice/group; data expressed as means ± SD) (D−F) Indicated mice were subjected to CLP during ZT0–4 with syringe needles with gauges ranging from 27G (D; ‘low-grade sepsis’), 22G (E; ‘middle-grade sepsis’), to 17G (F; ‘high-grade sepsis’). Animal survival was assayed (n = 12–20 mice/group; *p < 0.05; Kaplan-Meier survival analysis). (G) Indicated mice were subjected to CLP-induced middle-grade sepsis at ZT0 or ZT12. Animal survival was assayed (n = 8–12 mice/group; *p < 0.05; Kaplan-Meier survival analysis).

Figure 2.. 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 Bmal1^Mye−/− 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 Bmal1^Mye−/− 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 Bmal1^Mye−/− 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 Bmal1^Mye−/− 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).

Figure 3.. BMAL1 Limits PD-L1 Expression in a Lactate-Metabolism-Dependent Manner

(A) Western blot analysis of BMAL1 and PKM2 protein expression in indicated PMs. (B) qPCR analysis of Bmal1, Per1, and Per2 mRNAs in indicated PMs (n = 3 mice/group; data expressed as means ± SD). (C) Indicated mice were subjected to CLP with 22G syringe needles (middle-grade sepsis), and animal survival was assayed (n = 20 mice/group; *p < 0.05; Kaplan-Meier survival analysis). (D–F) Analysis of serum lactate (D), Pd-l1 mRNA in PMs (E), and serum tissue function enzyme (F) in indicated middle-grade septic mice (n = 3 mice/group; data expressed as means ± SD). (G) Basal ECAR (indicative of glycolysis) and OCR (indicative of oxidative phosphorylation) were monitored at isolated PMs from indicated CLP (72 hr)-induced septic mice (n = 3 mice/group; *p < 0.05 versus Bmal1^Mye−/− group; data expressed as means ± SD). (H) Analysis of lactate production and Pd-l1 mRNA in indicated BMDMs following treatment with LPS (100 ng/mL) or HMGB1 (500 ng/mL) for 48 hr (n = 3; data expressed as means ± SD; *p < 0.05; ANOVA LSD test). (I) Bmal1 wild-type, but not I317D mutant, restored the inhibition of LPS (100 ng/mL)- or HMGB1 (500 ng/mL)-induced lactate production in Bmal1^Mye−/− BMDMs (n = 3; data expressed as means ± SD; *p < 0.05; ANOVA LSD test). (J) Indicated mice were subjected to LPS (15 mg/kg)-induced endotoxemia at ZT0 or ZT12. Animal survival was assayed (n = 10 mice/group; *p < 0.05; Kaplan-Meier survival analysis). *p < 0.05.

Figure 4.. BMAL1-Mediated PKM2 Expression Contributes to STAT1-Dependent PD-L1 Upregulation

(A) Binding of BMAL1 to Pd-l1 or Pkm2 promoter was analyzed by ChIP-qPCR in BMDMs following treatment with LPS (100 ng/mL) for 48 hr (n = 3; data expressed as means ± SD; *p < 0.05; t test). (B) Western blot analysis of indicated proteins in BMDMs following treatment with LPS (100 ng/mL) for 48 hr. (C) Immunoprecipitation analysis of indicated protein complexes in BMDMs following treatment with LPS (100 ng/mL) for 48 hr. (D) Western blot analysis of indicated proteins in BMDMs following treatment with LPS (100 ng/mL) for 48 hr. (E) Binding of PKM2, STAT1, or HIF1α to Pd-l1 promoter was analyzed using ChIP-qPCR in indicated BMDMs following treatment with LPS (100 ng/mL) for 48 hr (n = 3; data expressed as means ± SD; *p < 0.05; t test). (F) qPCR analysis of Pd-l1 mRNA in indicated BMDMs following treatment with LPS (100 ng/mL) for 48 hr (n = 3; data expressed as means ± SD; *p < 0.05; t test). (G) Western blot analysis of expression of indicated proteins in BMDMs following treatment with LPS (100 ng/mL) for 48 hr. (H) qPCR analysis of Pd-l1 mRNA in BMDMs and THP1 cells following treatment with lactate (10 mM) for 12–48 hr (n = 3; data expressed as means ± SD; *p < 0.05 versus untreated group; t test). (I) Knockdown of STAT1 and GPR81 inhibited lactate (10 mM; 24 hr)-induced PD-L1 protein expression in BMDMs (n = 3; data expressed as means ± SD; *p < 0.05 versus control shRNA group; t test). See also Table S1.

Figure 5.. The PD-L1 Pathway Contributes to Septic Death in Bmal1^Mye−/− Mice

(A) Repeated administration of anti-PD-L1 antibody (20 mg/kg) or IL-7 (5 mg/mice) at 3, 24, 48, and 72 hr after CLP (22G needle) significantly increased survival in indicated mice (n = 20 mice/group; *p < 0.05; Kaplan-Meier survival analysis). (B–G) In parallel, tissue H&E staining and injury score (day 3; scale bars, 200 αM; B), serum enzyme activity (C), bacterial loading (D), and caspase 3 activity in T cells (E) were assayed (n = 3–5 mice/group; data expressed as means ± SD; *p < 0.05; ANOVA LSD test). (F) Western blot analysis of cleaved-caspase 3 in indicated T cells. (G) Serum levels of IL-7 in indicated septic mice (n = 3 mice/group; data expressed as means ± SD; *p < 0.05; ANOVA LSD test).