Fig 2: Altered Circadian and Sepsis Phenotype in Bmal1Mye−/− Mice(A) Western blot analysis of BMAL1 expression in PMs, livers, and brains from Bmal1 flox/flox and Bmal1Mye−/− 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).

Fig 3: BMAL1 null mice shows altered disc morphologyA.-D. Representative images of H&E and alcian blue staining of sagittal sections of lumbar disc from WT and KO mice. Compared to that of WT animals (A, B) NP tissue in null animals show decreased accumulation of matrix (lower matrix-to-cell ratio) (C, D), Scale bars: A, C: 50 μm, B, D: 10 μm. Disc height on images is outlined with dotted line. E.-H. Representative image of Picosirius red staining of WT and KO under bright field (E, G) and polarized light (F, H). Annulus fibrosus (AF) tissue in KO animals showed hyperplasia and increased thickening (white arrow). Scale bars: 100 μm.

Fig 4: Hnf4a knockout alters the genome-wide epigenetic landscape.a, b Heatmap of H3K4me1 (a) or H3K27ac (b) ChIP-seq signals at ZT6 in control (left) or HKO (right) liver centered at all peaks in control liver. Peaks are ordered vertically by signal strength. c Motif analysis of HKO-deprived H3K4me1 sites. Known consensus motifs are shown with corresponding enrichment significance values. d H3K4me1 peaks in control and HKO livers were partitioned into three categories with DiffBind (the HKO-enriched group has only 23 peaks and could not be plotted), and then the corresponding HNF4A occupancy (at ZT16) at each H3K4me1 site was plotted. Each horizontal line represents a single H3K4me1 site. Peaks were ordered vertically by the strength of the H3K4me1 ChIP signal in control liver. e Motif analysis of all H3K4me1 marked sites in the control liver. Known consensus motifs are shown with corresponding enrichment significance values. f Metaplot showing the average intensity of BMAL1, H3K4me1, and H3K27ac ChIP-seq signals (all at ZT6) in control or HKO livers surrounding HKO-unchanged (upper panel) or HKO-reduced (lower panel) BMAL1 peak centers.

Fig 5: Deletion of BMAL1 in macrophages protects against pneumococcal infection. (A) LysM-Bmal1−/− and (B) CX3CR1-Bmal1−/− mice (knockout [KO]) and floxed littermate controls (WT) were infected with S. pneumoniae at ZT12. The bacterial load in lung and peripheral blood at 48 was quantified (n = 8 to 12, Mann–Whitney U test *P < 0.05). (C) Immune cells were recovered from the lung tissue in A neutrophils, alveolar macrophages (AMs), and interstitial macrophages (IMs). (D) Weight loss after 48 h (Mann–Whitney U test *P < 0.05). (E) Representative lung histology with H&E staining. (Scale bar, 50 µm.) SPN, Streptococcus pneumoniae. (F) Cytokine analysis of lung tissue from A was performed using a NanoString inflammatory gene panel (see also SI Appendix, Fig. S1F). Each of the 248 genes is marked, and those that statistically differ by genotype are marked in red, with gene abbreviation. (G) Multiplex cytokine analysis of tissue from A (n = 8, t test, exact P value marked, and *P ≤ 0.05).