Figure 2. myBmal-KO mice show decreased and circadian time-dependent susceptibility to LPS.

(A) Circadian mortality in myBmal-KO mice. Mice kept in DD (n=10–14 per group) were challenged with half-lethal doses of LPS (30 mg/kg, i.p.) at indicated time points. Mortality was assessed 60 hr after LPS injection. Statistics were performed as in Figure 1C, (p=0.0009; gray shaded area indicates 95% confidence interval). (B) LPS dose-mortality curves of mice challenged at 4–8 time points across 24 hr in constant dark conditions. About threefold decrease of susceptibility to LPS in myBmal-KO mice (blue circles) as compared to wild-type mice (gray circles – re-plotted from Figure 1B kept in DD). Gray lines were calculated by fitting an allosteric model to each group. (C) Reduced mean mortality in myBmal-KO mice (n=103) compared to control strains LysM-Cre (n=39) or C57Bl/6 wild-type (WT, n=40). All mice were kept in DD and challenged with 30 mg/kg LPS i.p. Mean values and 95% confidence intervals from WT and myBmal-KO mice were calculated from experiments shown in Figure 1C (WT) and (A) (myBmal-KO)(** p=0.0021, *** p¡0.0001). (D) Constant dark conditions render mice more susceptible to LPS (30 mg/kg LPS) independent of Bmal1 in myeloid lineage cells (n=40 (LD) and n=103 (DD)). ( ** p<0.01, *** p<0.001).

Figure 2—figure supplement 1. Molecular characterization of myBmal-KO mice.

Molecular characterization of myBmal-KO mice. (A) Western blots of whole cell lysates from peritoneal cavity cells or liver of control (Bmal1^flox/flox) and myBmal-KO mice (n=3). (B) Prototypical genotyping results for Bmal1^flox/flox mice. A 600 bp band indicates successful recombination - likely from immune cells of myeloid origin residing in skin. (C) Relative mRNA levels of selected clock genes in peritoneal macrophages from Bmal1^flox/flox (light blue circles) or myBmal-KO (blue circles) mice at indicated circadian times. Phase and amplitude information are depicted in (D) as analyzed by Chronolyse. Non-significant circadian expression (p>0.05) are indicated by gray shaded circles for both myBmal-KO and LysM-cre control. (E) Kaplan-Meier survival curve of endotoxic shock experiments shown in Figure 2C. Mice kept in DD and challenged with LPS (30 mg/kg, i.p.). Gray line refers to C57Bl/6 mice (wild-type control, n=40), brown line to LysM-Cre control (n=39) and purple line to myBmal-KO (n=103). Data shown have been aligned to time after challenge with LPS, irrespective of time of day of challenge (same experiment as shown in Figure 1C and Figure 2A, respectively). Survival curves of wild-type and myBmal-KO as well as LysM-Cre and myBmal-KO differ significantly (p<0.0001 and p=0.0031, respectively, log-rank tests).