(A) WT and Per2^Brdm1 thioglycolate-elicited peritoneal macrophages were challenged with PAMPs targeting different TLRs. TNF-α and IL-12 levels in culture supernatants were determined by ELISA 16 h after challenge from 4 independent experiments; *, significantly different from WT. (B) Circadian oscillations of Nr1d1 and Per2 mRNA expression in serum-entrained peritoneal macrophages. *, significantly different from time points showing the lowest mRNA expression. (C) Circadian oscillations of Tlr mRNA expression in serum-entrained peritoneal macrophages, significantly different from T10 and T30 time points. (D) Impaired serum-induced Tlr9 expression in Per2^Brdm1 peritoneal macrophages, significantly different from T6. (E) ChiP analysis of CLOCK and BMAL1 binding to the Tlr9 promoter. Tlr9 (top) and Gapdh (bottom) promoter specific sequences were amplified by qPCR from DNA-protein complexes pulled-down with specific antibodies. (F) Impaired Tlr9 expression after siRNA-mediated knockdown of Clock, significantly different from T6. (A–D, F) Bars represent means ± s.e.m. *P < 0.05; significantly different as per two-sided t test. See also Figure S1.

Daily oscillations of Tlr mRNA levels in (A) spleen, (B) macrophages, (C) B cells, (D) DCs. Relative mRNA levels at each time point were calculated as the percentage of the maximum value over the 24-h period. Data are mean ± s.e.m. (A) n = 5 mice per time point (B) n = 10 animals per time point, compiled from 2 independent experiments (C) n = 15 animals per time point, compiled from 3 independent experiments (D) n = 15 animals per time point, compiled from 3 independent experiments. Oscillations of Tlr9 mRNA levels under DD in (E) spleen, (F) macrophages (G) B cells (H) DCs. Data are mean ± s.e.m. (E–H) n = 5 mice per time point. (A–H) * P < 0.05, ** P < 0.01, significantly different from lowest level of mRNA (nadir) by one-way ANOVA with the Dunnett posttest. Increased TLR9 protein levels at ZT19 in (I) spleen (J) splenic CD11b+ cells (K) splenic CD19+ cells. TLR9 protein amounts were determined by immunoblot and actin levels were used as normalizing factor. Relative protein amounts were calculated as the percentage of the maximum value. (I) n = 3 animals per time point (J) n = 10 animals per time point, compiled from 2 independent experiments (K) n = 15 animals per time point, compiled from 3 independent experiments. (L) TLR9 levels determined by flow cytometry in MHC-II+ cells (top) and CD19+ cells (bottom). n = 5 animals per time point. (I–L) Data are mean ± s.e.m. * P < 0.05, significantly different from ZT7 as per two-sided t test. See also Figure S2.

(A) Increased TLR9-mediated cytokine response at ZT19. Cytokine and co-stimulatory molecule mRNA amounts were determined 2 h after CpG challenge, n = 10 mice per time point, compiled from 2 independent experiments. (B) TLR9-mediated cytokine and co-stimulatory response in WT and Per2^Brdm1 mice. Cytokine and co-stimulatory molecule mRNA amounts in the spleen were determined 2 h after CpG. CD86 mRNA amount plotted as the percentage of the maximum value, n = 10 animals per time point, compiled from 2 independent experiments. (C) Increased splenic Tlr9 mRNA expression in Per2^Brdm1 mice at ZT19. Relative mRNA amounts were calculated as the percentage of the maximum value, n = 8 animals per group compiled from 2 independent experiments. (D) Decreased Tlr9 mRNA expression in splenic macrophages isolated from Per2^Brdm1 mice at ZT19, n = 5. (E) Increased TLR9 protein amounts in splenic CD19+ cells in Per2^Brdm1 at ZT19, n = 5. (C–D) TLR9 protein amounts were determined by immunoblot. Actin amounts were used as normalizing factor. (A–E) Data are mean ± s.e.m. * P < 0.05, significantly different as per two-sided t test. See also Figure S3.

(A,E) Enhanced lymphocyte proliferation in total lymph node cell cultures obtained from mice immunized with CpG-OVA at ZT19 compared to ZT7 or WT and Per2^Brdm1 mice at ZT19. Bars represent the average of 4 replicates from lymphocyte cultures pooled from 5 mice per treatment and time point. Results were reproduced in multiple independent experiments. (B–D) Enhanced OVA-induced IFN-γ production in lymphocyte cultures obtained from mice challenged with CpG-OVA at ZT19 compared to ZT7. Mice were immunized with OVA-CpG (B) OVA-control ODNs or OVA-CpG ODNs (C) and OVA-Pam3cys or OVA-CpG (D) at the indicated time points. Bars represent the average of 4 replicates from lymphocyte cultures pooled from 5 mice per treatment and time point. Results were reproduced in 3 independent experiments.

A more severe sepsis pathophysiology ensued in mice that underwent CLP at ZT19 vs. ZT7 including: (A) earlier mortality; (B) worse disease score (disease was scored as follows: 0 = bright, alert, responsive; 1 = slightly lethargic; 2 = lethargic and hunched; 3 = very lethargic, hunched and shaky; 4 = dead) (C) more severe hypothermia; (D) higher concentration of serum creatine kinase; higher concentration of serum inflammatory mediators including (E) IL-6; (F) Il-12/23p40 and (G) MCP-1 and (H) higher amounts of bacterial CFU in the peritoneum.