Figure 2. Increased hepatic neutrophil infiltration alters clock-genes expression and augments triglyceride content in the liver.

(A–D) Control (Lyzs-Cre) (A–B) and control and neutropenic (MCL1^Lyzs-KO) mice (C–D) were housed for 3 weeks with a normal 12 hr: 12 hr light/dark cycle (Normal Cycle) or with the dark period extended by 12 hr every 5 days (JetLag). Samples were obtained at the indicated ZTs. (A) Left, flow cytometry analysis of the CD11b⁺Ly6G⁺ liver myeloid subset. Data represents the percentage CD11b⁺Ly6G⁺ normalized to Normal Cycle ZT2. Right, circulating neutrophils in whole blood. (n = 5-8). (B) Liver triglycerides and representative oil-red-stained liver sections at ZT14. Scale bar, 50 μm (n = 9-10). (C) Hepatic triglyceride content analyzed at 6 hr intervals, and representative oil-red-stained liver sections at ZT14. Scale bar, 50 μm (n = 4-6). (D) qRT-PCR analysis of Bmal1 mRNA in livers. (n = 5-8). (E) Flow cytometry analysis of the CD11b⁺Ly6G⁺ liver myeloid subset isolated at 6 hr intervals from C57BL6J mice fed a ND, a HFD (8 weeks) or a MCD (3 weeks). The chart shows the CD11b⁺Ly6G⁺ population as a percentage of the total intrahepatic CD11b⁺ leukocyte population normalized to ND group at ZT2 (n = 5 to 10). (F–I) Control mice (Lyzs-Cre) and neutropenic mice (MCL1^Lyzs-KO) or p38γ/δ^Lyzs-KO were fed a ND or the MCD diet for 3 weeks and sacrificed at ZT2. (F) Representative images of the infiltration of neutrophils in the liver stained with anti-Mrp14 (blue) and anti-NE (red); nuclei with Sytox Green. Scale bar, 50 μm (Top) and 25 μm (Bottom). (G) qRT-PCR analysis of clock-gene expression in livers (n = 6). (H) Liver triglycerides and representative oil-red-stained liver sections. Scale bar, 50 μm (n = 7-6). (I) qRT-PCR analysis of clock genes in livers at ZT2 (n = 9-17). Data are means ± SEM from at least two independent experiments. *p<0.05; **p<0.01; ***p<0.005 (A to D) t-test or Welch’s test. (E) Two-way ANOVA with Fisher’s post hoc test; p<0.05 ND vs HFD; p<0.0001 ND vs MCD. *p<0.05; ***p<0.005 (G to I) t-test or Welch’s test. ZT2 point is double plotted to facilitate viewing.

Figure 2—figure supplement 1. Defective neutrophil migration to the liver alters hepatic clock- gene expression and triglyceride content.

(A) Schematic representation of JetLag protocol with stepwise increases in the dark period of 12 h12h every 5 days (B) Flow cytometry analysis of the CD11b⁺Ly6G⁺ liver myeloid subset isolated from control (Lyzs-Cre) and neutropenic (MCL1^Lyzs-KO) mice housed for 3 weeks under a 12 hr:12 hr light/dark cycle (Normal Cycle) or Jetlag. The bar chart shows the percentage of CD11b⁺Ly6G⁺ total intrahepatic CD11b⁺ leukocyte population analyzed at 6-h intervals and normalized to Normal Cycle ZT2 (n = 5-7). Dot plots show CD11b⁺ Ly6G⁺ population at ZT14. (C–D) After bone -marrow (BM) reconstitution of irradiated WT mice using Mrp8-Cre (Mrp8-Cre BM) or CXCR2^Mrp8-KO (CXCR2^Mrp8-KO) mice as BM donors, mice were housed for 3 weeks under JetLag (n = 6-8) (C) qRT-PCR analysis of Bmal1 mRNA in livers at ZT14. (D) Hepatic triglyceride content and representative oil-red-stained liver sections at ZT14. Scale bar, 50 µm. (E–G) Control (Lyzs-Cre) and p38γ/δ^Lyzs-KO mice were housed for 3 weeks under JetLag (n = 6-7) (E) Flow cytometry analysis of the CD11b⁺ Ly6G⁺ liver myeloid subset analyzed at 6 hr intervals and normalized by the tissue weight. (F) qRT-PCR analysis of Bmal1 mRNA in livers at ZT14. (G) Hepatic triglyceride content and representative oil-red-stained liver sections at ZT14. Scale bar, 50 µm. Data are means ± SEM. *p<0.05; **p< 0.01; ***p<0.005 All tests are t-test or Welch’s test. ZT2 point is double plotted to facilitate viewing.

Figure 2—figure supplement 2. Neutrophil depletion alters hepatic clock-gene expression.

(A-C) Osmotic minipumps containing saline or Ly6G antibody were implanted subcutaneously in Lyzs-Cre mice. These animals were fed with a MCD diet for 3 weeks and sacrificed at ZT2. (A-B) Blood levels of neutrophils and monocytes in Lyzs-Cre after 3 weeks of MCD diet treated or not with Ly6G antibody. (C) qRT-PCR of clock genes in the liver (n = 7-9). Data are means ± SEM. *p<0.05; ***p<0.005. All tests are t-test or Welch’s test.