Characterization of Distinct Subpopulations of Hepatic Macrophages in HFD/Obese Mice
Article Figures & Tables
Figures
- Figure 1
Isolation and characterization of NPCs from lean and obese mouse liver. A: Schematic diagram of isolation of NPCs from NC-fed lean vs. HFD-fed obese mouse liver. B: FACS analysis of NPCs from lean (NC) and obese (HFD) mouse liver with CD11b and F4/80 gating. NC mouse liver R1 population is labeled in blue, HFD mouse liver R2 population is labeled in green, and R3 population is labeled in red. The scattergram is representative of five to six independent mice from each group. C: Representative histogram plots depict the distribution of values of side (indicating relative granularity, left panel) and forward (indicating relative size, right panel) light scatter obtained from FACS analysis of R2 and R3 population from panel B. D: The relative gene expression from R1, R2, and R3 cell populations obtained from FACS sorting is shown. Data represent mean ± SEM (n = 5–6). *P < 0.05 compared with R2 vs. R3. E: TNF-α, F4/80, and CD11b protein expression in lean and obese mouse liver analyzed by immunohistochemistry. The image is representative of six independent mice from each group. Scale bar indicates 100 μm.
- Figure 2
RHM isolation with PKH26 fluorescent labeling. A: Schematic diagram of RHM isolation and characterization with PKH26+ fluorescent-labeled monocytes. B: FACS analyses of PKH26+ cells isolated from lean or obese mouse livers 1, 5, or 16 days after injection with PKH26+-labeled monocytes. Cells were first gated out of total NPCs for PKH26 and then plotted for F4/80+ and CD11b+ fluorescence. F4/80low/CD11b+ (blue-gated) and F4/80high/CD11b+ (red-gated) fraction, similar to the R3 and R2 cells observed in Fig. 1B. The scattergram is representative of four to five independent mice from each group. C: Average number of PKH26+ cells from lean and obese mouse liver was analyzed by FACS and then plotted as fold induction ± SEM from three independent experiments (n = 4 in each group). *P < 0.05 compared with NC vs. HFD. D: FACS analysis of the average number of PKH26+ cells isolated from HFD-fed recipient mice after injection PKH26+-labeled monocytes from WT or CCR2 KO donor mice. Mean ± SEM from three independent experiments (n = 5 in each group). *P < 0.05 compared with WT vs. CCR2 KO. E: TNF-α+ (left) and interleukin-6+ (right) cells were gated out of PKH26+ cells from NPCs of lean and obese mouse liver by FACS and then plotted as the mean ± SEM from three independent experiments (n = 5 in each group). *P < 0.05 compared with NC vs. HFD. F: Western blot analysis of TNF-α protein expression in PKH26+ cells from lean and obese mouse NPCs. PKH26+ cells from lean and obese mouse NPCs were sorted by FACS and then cultured overnight before protein isolation. HSP90 was used as the loading control. The image is representative from three independent Western blots (n = 2 for each group per Western blot).
- Figure 3
Identification of KCs in mouse liver. A: Schematic diagram of the irradiation model used to distinguish KC and RHM. i.v., intravenous. B: FITC+ KCs and PKH26+ newly infiltrating macrophages from HFD mice were plotted in forward and side scatter. The scattergram is representative of five independent mice. C: Whole-liver FITC+ KC numbers from mice fed NC were compared with mice after 12 weeks of HFD feeding. Data represent mean ± SEM (n = 5–6). D: MCP-1 and CCR2 mRNA expression were measured from FACS-sorted KCs and RHMs isolated from NC and HFD mice. Data represent mean ± SEM (n = 5). *P < 0.05 compared with KC vs. RHM. E: Hepatic glucose production assay in mouse hepatocytes. Hepatocytes were cultured with conditioned media from FACS-sorted KCs and RHMs from NC (N) and HFD (H) mice. Data represent mean ± SEM (n = 5). *P < 0.05 compared with glucagon and insulin in RPMI.
- Figure 4
RNA-seq analysis of KCs and RHMs from NC- and HFD-fed mice. A: Principal component analysis of expressed genes in KCs and RHMs from NC- or HFD-fed mice. Samples representing three biological replicates cluster together (KC-NC, red; KC-HFD, blue; RHM-NC, green; RHM-HFD, black). B: Bar chart shows the number of differentially expressed genes between cell types (KC vs. RHM) and diets (NC vs. HFD). C: Venn diagram shows common and distinct differentially expressed genes induced by the HFD within cell types. D: Heat map shows differentially expressed inflammatory response genes between KCs and RHMs from mice fed NC and the HFD in a four-way comparison. Gene counts were adjusted for cell number as KC increase 1.17-fold on HFD and RHMs increase 5.8-fold. Upregulated genes are colored red, downregulated genes are colored blue, and darker colors reflect higher fold changes. RNA-seq studies used three biological replicates per each group.
Tables
- Table 1
Cell surface markers to distinguish between KCs and RHMs*
Symbol Description NC HFD KC RHM f(KC/RHM) KC RHM f(KC/RHM) KC marker† F8 Coagulation factor VIII 20,355 4,238 5 12,074 1,198 10 Kit Kit oncogene 11,300 2,186 5 5,430 492 11 Wnt2 Wingless-related MMTV integration site 2 4,836 818 6 2,580 233 11 Prelp Proline arginine-rich end leucine-rich repeat 3,485 699 5 2,383 234 10 Masp1 Mannan-binding lectin serine peptidase 1 2,396 450 5 1,915 182 11 Vwf Von Willebrand factor homolog 2,320 416 6 787 121 7 Wnt9b Wingless-type MMTV integration site 9B 1,149 156 7 288 21 14 Selp Selectin, platelet 973 178 5 1,006 87 12 Cdh13 Cadherin 13 929 114 8 982 64 15 Dkk3 Dickkopf homolog 3 (Xenopus laevis) 911 156 6 447 49 9 Tnxb Tenascin XB 828 154 5 577 41 14 Col3a1 Collagen, type III, α 1 752 100 8 711 46 15 Fbln2 Fibulin 2 355 61 6 397 32 12 Pdgfd Platelet-derived growth factor, D polypeptide 343 66 5 231 23 10 Col1a2 Collagen, type I, α 2 381 59 6 379 29 13 Col1a1 Collagen, type I, α 1 357 50 7 423 20 21 RHM marker† C1qa Complement component 1, q subcomponent, α polypeptide 15,635 80,760 5 39,139 107,329 3 C1qc Complement component 1, q subcomponent, C chain 15,459 74,157 5 34,129 98,710 3 Apoc1 Apolipoprotein C-I 2,510 12,325 5 2,734 10,236 4 C4b Complement component 4B (Chido blood group) 2,361 7,773 3 2,798 8,656 3 Gbp2b Guanylate binding protein 2b 1,156 5,569 5 3,824 13,625 4 C6 Complement component 6 927 8,118 9 1,142 6,303 6 Kif23 Kinesin family member 23 912 2,633 3 1,257 3,436 3 Cpq Carboxypeptidase Q 661 2,884 4 1,571 4,243 3 C4a Complement component 4A (Rodgers blood group) 221 869 4 224 780 3 Kcp Kielin/chordin-like protein 75 471 6 63 231 4 Slc1a3 Solute carrier family 1 (glial high affinity glutamate transporter), member 3 36 220 6 72 228 3 *Differentially expressed genes between KC and RHMs that were classified within the GO cellular component term “extracellular region part” are listed.
†The top 16 KC marker genes listed are expressed at least fivefold higher in KC vs. RHM from NC- or HFD-fed mice. The top 11 RHM markers are all expressed at least threefold higher in RHMs vs. KCs. All markers are expressed at 200 counts per gene or more. RNA-seq studies used three biological replicates per each group.
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