Circadian gene Bmal1 regulates diurnal oscillations of Ly6C(hi) inflammatory monocytes

Abstract

Circadian clocks have evolved to regulate physiologic and behavioral rhythms in anticipation of changes in the environment. Although the molecular clock is present in innate immune cells, its role in monocyte homeostasis remains unknown. Here, we report that Ly6C(hi) inflammatory monocytes exhibit diurnal variation, which controls their trafficking to sites of inflammation. This cyclic pattern of trafficking confers protection against Listeria monocytogenes and is regulated by the repressive activity of the circadian gene Bmal1. Accordingly, myeloid cell-specific deletion of Bmal1 induces expression of monocyte-attracting chemokines and disrupts rhythmic cycling of Ly6C(hi) monocytes, predisposing mice to development of pathologies associated with acute and chronic inflammation. These findings have unveiled a critical role for BMAL1 in controlling the diurnal rhythms in Ly6C(hi) monocyte numbers.

Fig. 1. Diurnal variation of Ly6C^hi monocytes

(A) Quantitative RT-PCR analysis of clock controlled genes (Arntl, Nr1d1, and Dbp) in blood monocytes during a 12 hour light-dark cycle (n = 3-4 samples per time point). (B and C) Ly6C^hi monocyte numbers in blood (B) and spleen (C) during a 12 hour light-dark cycle (n=5 mice per time point). (D and E) Recruitment of Ly6C^hi monocytes to inflamed peritoneum. Ly6C^hi monocyte number (D), and concentration of IL1β (E) were quantified in the peritoneal fluid 2 hours after elicitation with thioglycollate (n = 5 mice per time point). Pooled data (A) or representatives (B to E) of two independent experiments are shown as mean ± SEM. Two-tailed Student's t-tests (A) and one-way ANOVA (B-E) are used for statistical analyses (comparisons were made between the acrophase and other time points). *P<0.05; **P<0.01; ***P<0.001.

Fig. 2. Diurnal variation in the pathogenicity of Listeria monocytogenes

(A to C) Mice kept under a 12 hour light-dark cycle were intraperitoneally inoculated with 1×10⁶ L. monocytogenes at ZT0 and ZT8, and colony forming units (CFUs) recovered from the peritoneal cavity (A), spleen (B), and liver (C) were quantified 2 dpi (n = 14-15 mice per time point). (D to F) Numbers of iNOS⁺CD11c⁺ and TNFα⁺CD11c⁺ cells in peritoneal cavity (D), spleen (E), and liver (F) of mice 2 dpi with 1×10⁶ L. monocytogenes at ZT0 and ZT8 (n = 15 mice per time point). (G) Concentration of chemokines and cytokines in peritoneal fluid 2 dpi with 1×10⁶ L. monocytogenes at ZT0 and ZT8 (n = 15 mice per time point). (H) Survival curves of mice after infection with 1×10⁷ L. monocytogenes at ZT0 and ZT8 (n = 25 mice per time point). (I) Serum concentration of chemokines and cytokines 2 dpi with 1×10⁷ L. monocytogenes at ZT0 and ZT8 (n = 10 mice per time point). Pooled data from two or three independent experiments are presented as mean ± SEM. Statistical significance (*P<0.05; **P < 0.01; ***P < 0.001) was assessed using two-tailed Student's t-test (A to G, and I), and log-rank test (H).

Fig. 3. BMAL1 regulates rhythmic oscillations of Ly6C^hi monocytes in a cell autonomous manner

(A) Quantitative RT-PCR kinetic analysis of Nr1d1 mRNA in blood monocytes isolated from Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice kept on a 12 hour light-dark cycle (n = 3-4 samples per genotype and time point). (B to D) Ly6C^hi monocyte numbers in blood (B), spleen (C), and bone marrow (D) of Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice kept under a 12 hour light-dark cycle at various ZTs. (n = 5 mice per genotype and time point). (E) Survival curves of Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice after infection with 1×10⁶ L. monocytogenes at ZT0 and ZT8 (n = mice 10-11 per genotype and time point). (F to I) Serum concentrations of IL1β (F), IL6 (G), IFNγ (H), and CCL2 (I) in Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice 2 dpi with 1×10⁶ L. monocytogenes at ZT0 and ZT8 (n = mice 4-6 per genotype and time point). Pooled data (A and E) and representative (B to D) of two to three independent experiments are shown as mean ± SEM and analyzed using two-tailed Student's t-tests (A to D, and F to I), and log-rank test (E). *P<0.05; **P<0.01; ***P<0.001.

Fig. 4. BMAL1 recruits PRC2 to repress expression of Ccl2

(A) Quantitative RT-PCR analysis of Ccl2 expression in blood monocytes of Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice kept on a 12 hour day-ligh cycle (n = 3-4 samples per genotype and time point). (B) ChIP analysis of BMAL1 binding to the Ccl2 promoter (n = 4 samples per genotype and time point). (C) Coimmunoprecipitation of BMAL1 and with members of PRC2. Nuclear lysates from serum shocked BMDMs were immunopercipiated with BMAL1 antibody, and immunoblotted for BMAL1, CLOCK, EZH2, EED, and SUZ12. (D and G) ChIP analysis for the recruitment of EZH2 (D) and Pol II (G) to the proximal promoter of the Ccl2 gene (n = 4 samples per genotype and time point). (E and F) ChIP analysis for H3K27Me3 (E) and H3K4Me3 (F) at the proximal promoter of Ccl2 gene (n = 4 samples per genotype and time point). (H) Ly6C^hi monocyte numbers in the blood of wild type or Ccr2^−/− mice during a 12 hour light-dark cycle. Wild type mice were intraperitoneally injected with PBS (Veh) or CCL2 (20 μgkg⁻¹) 24 hours prior to quantification of Ly6C^hi monocytes (n = 4-5 mice per genotype/treatment and time point). Pooled data (A to G) from two independent experiments are shown as mean ± S.E.M and analyzed using two-tailed Student's t-tests (A and B; D to G) and two-way ANOVA (H). *P<0.05; **P<0.01; ***P<0.001 represent comparison between Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre or between wild type treated Veh vs. CCL2 or Ccr2^−/− at each time point.

Fig. 5. Myeloid cell-specific deletion of BMAL1 exacerbates metabolic disease

(A to D) Body weight (A), adiposity (B), tissue weights (C) and oxygen consumption (D) in Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice kept on a 12 hour light-dark cycle fed high fat diet for 19 weeks (n = 4-5 mice per genotype). (E) Total and Ly6C^hi macrophage content in eWAT of Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice fed high fat diet (n = 5 mice per genotype). (F and G) Glucose (F) and insulin tolerance (G) tests of Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre mice fed high fat diet (n = 5-8 mice per genotype). (H) Immunoblots of total and phosphorylated AKT (pAKT) in eWAT of obese Arntl^LoxP/LoxP and Arntl^LoxP/LoxPLyz2^Cre administered intraportal insulin. Representative data of two to four independent experiments (A to C, E to H) are shown as mean ± S.E.M and analyzed using two-tailed Student's t-tests. *P<0.05; **P<0.01; ***P<0.001.