Figure 1. Neutrophil infiltration into the liver controls hepatic clock-gene expression.

(A) Flow cytometry analysis of the CD11b⁺Ly6G⁺ liver myeloid subset, isolated from C57BL6J mice at the indicated ZTs. Left, CD11b⁺Ly6G⁺ liver myeloid subset analyzed at 6 hr intervals and normalized by the tissue weight. Right, percentage of CD11b⁺Ly6G⁺ population analyzed at 4 hr intervals and normalized to ZT2 (n = 5). (B) Representative 3-D image of liver section showing the distribution on infiltrated neutrophils. Livers were stained with anti-S100A9 (Mrp14) (red) and vessels were stained with anti-CD31 and anti-endomucin (grey). Sizes of the liver sections are 510 x 510 x 28 µm and 160 x 160 x 28 µm, respectively. (C) qRT-PCR analysis of circadian clock-gene and nuclear-receptor mRNA expression in livers from C57BL6J mice at the indicated ZTs (n = 5). (D) Liver triglycerides and oil-red-stained liver sections prepared from C57BL6J mice at ZT2 and ZT14. Scale bar, 50 μm (n = 5). (E) qRT-PCR analysis of clock-gene mRNA in hepatocyte cultures exposed to freshly isolated FMLP-activated neutrophils (n = 4-6 wells of 3 independent experiments). (F) qRT-PCR analysis of clock-gene mRNA in hepatocyte cultures treated with 5 nM elastase (n = 3-4 wells of 3 independent experiments). (G) qRT-PCR analysis of clock-gene and nuclear-receptor mRNA expression in livers from control mice (Mrp8-Cre) and neutropenic mice (MCL1^Mrp8-KO) sacrificed at ZT2 (n = 5). (H) Hepatic triglycerides detected in livers from control mice (Mrp8-Cre) and neutropenic mice (MCL1^Mrp8-KO) at ZT2 (n = 5). Data are means ± SEM from at least 2 independent experiments. *p<0.05; **p<0.01; ***p<0.005 (A, left panel) One-way ANOVA with Tukey’s post hoc test. (A, right panel) Kruskal-Wallis test with Dunn’s post hoc test. (C) One-way ANOVA with Tukey’s post hoc test or Kruskal-Wallis test with Dunn’s post hoc test. (D to H) t-test or Welch’s test. ZT2 point is double plotted to facilitate viewing.

Figure 1—figure supplement 1. Neutrophils follow a circadian rhythm.

(A) Left, circulating neutrophils quantified at 4 hr intervals in whole blood of C57BL6J mice. Right, flow cytometry analysis at 6 hr intervals of the CD11b⁺Ly6G⁺ myeloid subset in bone marrow from C57BL6J mice. ZT2 point is double plotted to facilitate viewing (n = 5). (B) Representative 3-D image of liver section showing the distribution of Kupffer cells. Livers were stained with anti-Clec4F (green) and vessels were stained with anti-CD31 and anti-endomucin (grey). Sizes of the liver sections are 510 x 510 x 28 µm and 160 x 160 x 28 µm, respectively (n = 5-7). (C) qRT-PCR of Ccl3, Cxcl2, Cxcl12 and Cxcl1 chemokines mRNA expression at ZT2 and ZT14 and qRT-PCR of Cxcl1 mRNA expression at 6 hr intervals in livers from C57BL6J mice (n = 5). (D) qRT-PCR of Bmal1 mRNA expression in hepatocyte cultures exposed to freshly isolated T-lymphocytes, B-lymphocytes or bone-marrow derived macrophages (BMDM) and 1 µM FMLP; Bmal1 mRNA expression in hepatocyte cultures treated with 0.5 mg/mL collagenase (n = 3 wells of 2 to 3 independent experiments) (E) Left, flow cytometry analysis of number of liver Kupffer cells (KCs) in control Lyzs-Cre and MCL1^Lyzs-KO mice and in Mrp8-Cre and MCL1^Mrp8-KO mice normalized by tissue weight. Right, representative dot plots showing F4/80⁺Clec4F⁺ population gated on total intrahepatic CD45⁺CD11b⁺ leukocyte population (n = 4-6). (F) Flow cytometry analysis of the CD11b⁺ Gr-1^high liver myeloid subset isolated from control (Lyzs-Cre) and neutropenic (MCL1^Lyzs-KO) mice. The bar chart shows the CD11b⁺ Gr-1^high population as a percentage of the total intrahepatic CD11b⁺ leukocyte population (n = 7-10). Data are means ± SEM. *p<0.05; **p<0.01; ***p<0.005 (A, left) Kruskal-Wallis with Dunn’s post-hoc test. (A, right) One-way ANOVA with Tukey’s pots hoc test. (C, left) t-test. (C, right) Kruskal-Wallis with Dunn’s post-hoc test. (D) t-test. (E) One-way ANOVA with Tukey’s pots-hoc test. (F) t-test.

Figure 1—figure supplement 2. Neutrophil deficiency alters clock-gene expression.

(A) Representative dot plots showing the decrease in the CD11b⁺ Gr-1^high population in blood, bone marrow, and spleen from neutropenic mice (MCL1^Lyzs-KO) compared with control mice (Lyzs-Cre). Bar charts show the CD11b⁺ Gr-1^high population as a percentage of the total CD11b⁺ leukocyte population. (B) Blood levels of monocytes and neutrophils in control and neutropenic mice. (C) Myeloid cell populations in bone marrow and liver determined by flow cytometry and representative dot plots (CD11b⁺ Gr-1^neg as macrophages, CD11b⁺ Gr-1^int as monocytes and CD11b⁺ Gr-1^high as neutrophils). (D) qRT-PCR of clock genes in the livers from control (Lyzs-Cre) and neutropenic (MCL1^Lyzs-KO) mice. ZT2 point is double plotted to facilitate viewing (n = 5-7). (E) Left, flow cytometry analysis of the CD11b⁺ Ly6G⁺ lung myeloid subset of control (Lyzs-Cre) and neutropenic (MCL1^Lyzs-KO) mice at the indicated ZTs (n = 4). Right, qRT-PCR analysis of Bmal1 in lungs of control (Lyzs-Cre) and neutropenic (MCL1^Lyzs-KO) mice at the indicated ZTs (n = 4-6). Data are means ± SEM. *p<0.05; **p< 0.01; ***p<0.005. All tests are t-test or Welch’s test.