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. 2010 Apr;59(4):916-25.
doi: 10.2337/db09-1403. Epub 2010 Jan 26.

C-C chemokine receptor 2 (CCR2) regulates the hepatic recruitment of myeloid cells that promote obesity-induced hepatic steatosis

Amrom E Obstfeld  1 , Eiji Sugaru, Marie Thearle, Anne-Marie Francisco, Constance Gayet, Henry N Ginsberg, Eleanore V Ables, Anthony W Ferrante Jr
Affiliations

C-C chemokine receptor 2 (CCR2) regulates the hepatic recruitment of myeloid cells that promote obesity-induced hepatic steatosis

Amrom E Obstfeld et al. Diabetes. 2010 Apr.

Abstract

Objective: Obesity induces a program of systemic inflammation that is implicated in the development of many of its clinical sequelae. Hepatic inflammation is a feature of obesity-induced liver disease, and our previous studies demonstrated reduced hepatic steatosis in obese mice deficient in the C-C chemokine receptor 2 (CCR2) that regulates myeloid cell recruitment. This suggests that a myeloid cell population is recruited to the liver in obesity and contributes to nonalcoholic fatty liver disease.

Research design and methods: We used fluorescence-activated cell sorting to measure hepatic leukocyte populations in genetic and diet forms of murine obesity. We characterized in vivo models that increase and decrease an obesity-regulated CCR2-expressing population of hepatic leukocytes. Finally, using an in vitro co-culture system, we measured the ability of these cells to modulate a hepatocyte program of lipid metabolism.

Results: We demonstrate that obesity activates hepatocyte expression of C-C chemokine ligand 2 (CCL2/MCP-1) leading to hepatic recruitment of CCR2(+) myeloid cells that promote hepatosteatosis. The quantity of these cells correlates with body mass and in obese mice represents the second largest immune cell population in the liver. Hepatic expression of CCL2 increases their recruitment and in the presence of dietary fat induces hepatosteatosis. These cells activate hepatic transcription of genes responsible for fatty acid esterification and steatosis.

Conclusions: Obesity induces hepatic recruitment of a myeloid cell population that promotes hepatocyte lipid storage. These findings demonstrate that recruitment of myeloid cells to metabolic tissues is a common feature of obesity, not limited to adipose tissue.

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Figures

FIG. 1.
FIG. 1.
Obesity induces a chemotactic program in the liver. A: Body mass correlates with hepatic expression of Ccl2 and Ccr2 among mice with varying body mass due to genetic mutations, sex, and diet (n = 45, P < 0.01 for Ccr2 and Ccl2). B: Obesity induces hepatic expression of genes involved in myeloid cell recruitment (n = 8–9). □, C57BL/6J Lep+/+; ■, Lepob/ob. C: Hepatic immune cells (CD45+) but not hepatocytes express Ccr2 (n = 4). D: Hepatocyte expression of Ccl2 is induced in obese leptin-deficient mice compared with lean mice (n = 4). *P < 0.05; **P < 0.01; ***P < 0.005. AU, arbitrary units.
FIG. 2.
FIG. 2.
Obesity induces hepatic recruitment of a novel myeloid cell population. A: Strategy used to identify hepatic cell populations after collagenase digestion. B: Representative scatter plots depict relative cell surface expression of F4/80 (x-axis) and CD11b (y-axis) among hepatic CD45+ cells from lean (C57BL/6J Lep+/+) and obese (C57BL/6J Lepob/ob) mice. KCs were identified by high expression of the F4/80 (right gate), but a distinct population of CD11b+ F4/80dim cells (upper left gate) was also identified. Cataloging of hepatic immune cells reveals a decrease in the percent of KCs and an increase in CD11b+ F4/80dim in obese leptin-deficient (C57BL/6J Lepob/ob) (C) and HFD-fed obese (D) mice (n = 5–10) (□, lean; ■, obese). E: Liver RMC content is associated with body mass in a set of 23 male mice with varying degrees of adiposity resulting from dietary and genetic alterations (r = 0.611, P < 0.01). Data represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.005.
FIG. 3.
FIG. 3.
Hepatic RMCs are morphologically, functionally, and transcriptionally distinct from KCs. A: Representative histogram plot depicting the distribution of side (indicating relative granularity, left panel) and forward (indicating relative size, right panel) light scatter values obtained from FACS analysis of RMCs and KCs from lean (Lep+/+) and leptin-deficient obese (Lepob/ob) mice. B: KCs are larger than RMCs as seen in hematoxylin and eosin (H&E)-stained isolated cells (bar equals 10 microns). C: Relative expression of Ccr2 was more than 100× higher in RMCs than in KCs (KC expression is set at 100 arbitrary units [AU]) (n = 5–6). D: The relative expression of genes associated with classically (M1) or alternatively (M2) activated cells is not consistently associated with either RMCs or KCs isolated from livers of obese mice (Lepob/ob) mice (□, KC Lepob/ob; ■, RMC Lepob/ob) (n = 5–6). For C and D, data represent mean ± SD. *P < 0.05; ***P < 0.005. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 4.
FIG. 4.
Hepatic recruitment of RMCs is CCR2 chemokine receptor dependent. A: Timeline of experiments in which C57BL/6J CD45.1 mice were irradiated and reconstituted with bone marrow from either C57BL/6J Ccr2−/− (CCR2KO/WT) or wild-type CD45.2 siblings (CCR2WT/WT). B: Body mass, fasting blood glucose concentrations, and serum insulin concentrations of CCR2WT/WT and CCR2KO/WT after 4 weeks on an HFD were not different (P > 0.05). C: The size of the RMC population was reduced and KC population increased in mice that lacked hematopoietic Ccr2 (CCR2KO/WT) (□, CCR2WT/WT; ■, CCR2KO/WT). n = 7 for WT/WT and n = 5 for KO/WT. **P < 0.01.
FIG. 5.
FIG. 5.
Hepatic recruitment of RMCs is enhanced by CCL2. A: Timeline of experiments in which human CCL2 (hCCL2) or LacZ was induced through use of adenoviral expression vectors. B: Growth chart of mice over the course of the study. C: Serum concentration of hCCL2 before injection and 5, 14, and 21 days after Ad-hCcl2 injection (results are mean of 6–8 animals ± SD). D: hCCL2 expression in liver, heart, and perigonadal adipose tissue (PGAT) of Ad-hCcl2–infected and control animals (top panel) 5, 14, and 21 days after Ad-hCcl2 injection. E: The RMC population of cells is increased in mice by hepatic expression of CCL2 (Ad-hCcl2) for 1 week in both LFD- and HFD-fed mice (□, Ad-LacZ; ■, Ad-hCcl2). Data represent mean ± SD. *P < 0.05.
FIG. 6.
FIG. 6.
RMCs regulate hepatic TG accumulation in the presence of excess lipid. Hepatic expression of hCCL2 and expansion of the RMC content did not alter insulin sensitivity (A) or liver mass (B) of mice on LFD or HFD (n = 4). C: Increased recruitment of RMCs did not increase hepatic TG in LFD-fed mice but did induce steatosis in mice with excess lipid intake (□, Ad-LacZ; ■, Ad-hCcl2). D: Hepatic expression of hCCL2 and recruitment of RMCs increased histologically visible hepatic steatosis and lipid droplets in HFD-fed mice (bar = 50 microns). *P < 0.05.
FIG. 7.
FIG. 7.
RMCs promote expression of lipogenic genes. A: Increased recruitment of RMCs through hepatic expression of hCCL2 increased lipogenic gene expression in HFD-fed mice (□, Ad-LacZ; ■, Ad-hCcl2). B: Deficiency of CCR2 in weight-matched mice fed an HFD for 9 weeks reduced lipogenic gene expression (□, CCR2+/+; ■, CCR2−/−) (n = 13). Data represent mean ± SD. *P < 0.05; **P < 0.01. AU, arbitrary units.
FIG. 8.
FIG. 8.
RMCs promote expression of lipogenic genes. RMCs from obese HFD-fed mice induce in hepatocytes from lean mice a program that would favor fatty acid esterification (□, control; ■, RMCs). Data represent mean ± SD. *P < 0.05; ***P < 0.005. AU, arbitrary units.
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