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. Author manuscript; available in PMC: 2015 Feb 1.
Published in final edited form as: Nat Med. 2014 Jul 27;20(8):919–926. doi: 10.1038/nm.3599

Figure 3.

Figure 3

Loss of the club cell clock results in enhanced neutrophil responses to LPS. (a) Neutrophil recruitment after aerosolized LPS challenge in CCSP-Bmal−/− and wild-type littermates at CT0 (n=8/genotype) and CT12 (n=8 wild-type, n=10 CCSP-Bmal−/−) (two-way ANOVA and post hoc Bonferroni; scale bar, 100 μm). (b) IgM levels in BAL fluid after saline (n = 6/genotype) or LPS exposure (n=11 CCSP-Bmal−/−, n = 9 wild-type littermates) (two-way ANOVA and post hoc Bonferroni). (c) Myeloperoxidase (MPO) activity levels in lung tissue from CCSP-Bmal−/− (n = 10) and wild-type littermates (n = 8) mice challenged with LPS at CT12. (d) Confirmation of a quantitative PCR (qPCR) array, measuring transcription of Cxcl5, Ccl20 and Ccl8 in CCSP-Bmal−/− (n = 12) and wild-type littermate (n = 7) lung tissue 2 h after LPS challenge, qPCR data normalized to wild-type littermate controls and values expressed as relative quantification (RQ) to wild-type levels, Student’s t-test). (e) Diurnal variation in LPS-induced cytokine secretion in CCSP-Bmal−/− mice (n = 8 at CT0, n = 10 at CT12) and wild-type littermates (n = 7/time point) (two-way ANOVA and post hoc Bonferroni; genotype differences ***P ≤ 0.005, time-of-day differences †P ≤ 0.05 and ††P ≤ 0.01,). (f) Quantification of BAL neutrophils at timed intervals after aerosolized LPS at ZT4 in CCSP-Bmal−/− mice (0h: n = 3 and 2h-96h: n = 4) and wild-type littermates (0h–10h: n = 4 and 24-96h: n=5); two-way ANOVA and post hoc Bonferroni). (g) H&E staining of histological sections of lung collected 10h after LPS illustrating neutrophil infiltration (arrow) (representative of n = 4; scale bar, 100 μm). (h) Cxcl5 transcript and CXCL5 protein levels in LPS-stimulated lungs from CCSP-Bmal−/− mice (0h: n = 3 and 2h-24h: n = 4)) and wild-type litttermates (n = 4); qPCR data normalized to wild-type naive lung; two-way ANOVA and post hoc Bonferroni). (i) Bacterial load in the lung and blood, and neutrophil counts in BAL after infection with S. pneumonia in CCSP-Bmal−/− mice (8h – 24h: n = 9; 48h: n = 19) and wild-type littermates (8h: n = 8; 24h: n = 10; and 48h: n = 14), (median values marked, Mann-Whitney U-test). (j) CXCL5 levels in BAL fluid from naïve, and 24h post bacterial infection in CCSP-Bmal−/− mice (naïve: n = 4 and infected: n = 6) and wild-type littermates (n = 5), two-way ANOVA and post hoc Bonferonni). Data are expressed as mean ± s.e.m and *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005.