Impact of Macrophage Inflammatory Protein-1α Deficiency on Atherosclerotic Lesion Formation, Hepatic Steatosis, and Adipose Tissue Expansion

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR−/−) mice were transplanted with bone marrow from CCL3−/− or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3−/− marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3−/− recipients after 6 weeks and in male CCL3−/− recipients after 12 weeks of WD feeding (P<0.05). Surprisingly, male CCL3−/− recipients had a 50% decrease in adipose tissue mass after WD-feeding, and plasma insulin, and leptin levels were also significantly lower. These results were specific to CCL3, as LDLR−/− recipients of monocyte chemoattractant protein−/− (CCL2) marrow were not protected from the metabolic consequences of high fat feeding. Despite these improvements in LDLR−/− recipients of CCL3−/− marrow in the bone marrow transplantation (BMT) model, double knockout mice, globally deficient in both proteins, did not have decreased body weight, plasma lipids, or atherosclerosis compared with LDLR−/− controls. Finally, there were no differences in myeloid progenitors or leukocyte populations, indicating that changes in body weight and plasma lipids in CCL3−/− recipients was not due to differences in hematopoiesis. Taken together, these data implicate a role for CCL3 in lipid metabolism in hyperlipidemic mice following hematopoietic reconstitution

Absence of macrophage inflammatory protein-1α does not impact macrophage accumulation in adipose tissue of diet-induced obese mice

Macrophages and T-lymphocytes are known to accumulate in the white adipose tissue (WAT) of obese mice and humans, but the factors that cause this infiltration are not yet determined. Chemokines, which attract leukocytes to inflammatory sites, are candidates for this process. Macrophage inflammatory protein-1α (MIP-1α) expression is significantly elevated in WAT of obese mice and humans and positively correlates with fasting plasma insulin, but its potential role in leukocyte recruitment to WAT is unknown. MIP-1α-deficient, heterozygous, and wild-type mice were fed a Western diet (WD) for 16 wk. Plasma lipids, adipose tissue mass, energy expenditure, food intake, liver triglyceride content, and inflammatory cytokine expression were not different among genotypes. Gene expression of macrophage markers F4/80 and CD68, as well as T-lymphocyte marker CD3ε was increased in perigonadal WAT of obese WD-fed mice but was not influenced by MIP-1α expression level. Immunohistochemical analysis of WAT also showed no effect of MIP-1α on macrophage content. Two related chemokines, MIP-1β and RANTES, had reduced expression in obese male MIP-1α-deficient mice compared with wild-type controls (P ≤ 0.05). In mice fed the WD for 6 wk, WAT macrophage content was unchanged; however, CD8+ T-lymphocytes accumulated to a lesser extent in the MIP-1α-null mice. Therefore, expression of MIP-1α, as well as that of MIP-1β and RANTES, increases as a consequence of weight gain, but these chemokines may not be required for the recruitment of monocytes to WAT during diet-induced obesity in mice and may impact T-lymphocyte recruitment only at early time points after WD feeding

Recipients of CCL3^−/− marrow have reduced lesional T lymphocytes.

<p>Sections from the aortic root of male and female CCL3^+/+→LDLR^−/− and CCL3^−/−→LDLR^−/− mice were used for immunofluorescent analysis of CD4 positive T-cells according to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031508#s4" target="_blank">Methods</a> section. For each mouse, data from the three valves was combined. Total dapi stained nuclei (Panel A) and CD4 positive cells were quantified (Panel B). The percent of cells that were CD4 T cells was calculated by dividing the total cells by the CD4 positive cells (Panel C). Data represent the mean ± SEM of 3–6 mice per group. * <i>P</i><0.01 compared to male CCL3^+/+→LDLR^−/−.</p

Adipose tissue mass and gene expression.

<p>Male LDLR^−/− mice were transplanted with C57BL/6 or CCL3^−/− bone marrow. At 4 weeks post-BMT, mice were placed on WD for either 6 or 12 weeks. (A–C) Body weight, total body lean mass, and total body adipose tissue were measured by NMR pre-BMT, 4 weeks post-BMT, and at 6 and 12 weeks post-WD. Data from pre-BMT, 4 weeks post-BMT, and 6 weeks post-WD are combined for the 6 week and 12 week groups. The 12 week post-WD data represent only the mice sacrificed at that time point. (D–F) Perigonadal adipose tissue was collected from the mice fed WD for 12 weeks. RNA was isolated and used for real-time RT-PCR analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031508#s4" target="_blank">Methods</a> section. Data are the mean ± SEM of the relative gene expression for 12–14 mice per group. Images are from Toluidine Blue O stained sections processed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031508#s4" target="_blank">Methods</a> section. * <i>P</i><0.05 ** <i>P</i><0.01 *** <i>P</i><0.0005 CCL2 = monocyte chemoattractant protein-1 TNF-α = tumor necrosis factor-α Man Rec C = mannose receptor C Mgl = macrophage galactose N-acetyl-galactosamine specific lectin.</p

Stem cell progenitors in CCL3^−/−→LDLR^−/− mice.

<p>Male LDLR^−/− mice were transplanted with C57BL/6 or CCL3^−/− bone marrow. At 6 weeks post-WD, bone marrow cells were collected and analyzed by flow cytometry. (A) Quantification of flow cytometry analysis of HSC cells which were identified as lineage negative (Lin⁻) and expressing high levels of c-kit and Sca-1 (Lin⁻c-kit^highSca-1⁺). (B) CMP cells were identified as Lin⁻c-kit^highCD34⁺FcγRII/III^lo. (C) MEP cells were defined as Lin⁻c-kit^highCD34⁻FcγRII/III^lo. (D) GMP cells were defined as Lin⁻c-kit^highCD34⁺FcγRII/III^high. Data represent the mean± SEM of 5–6 mice per group. *<i>P</i><0.05 compared to C57BL/6^−/− groups.</p

Body and tissue weights in CCL3^−/−→LDLR^−/− mice.

<p>LDLR^−/− mice were transplanted with C57BL/6 or CCL3^−/− bone marrow. At 4 weeks post-BMT, mice were placed on WD for either 6 or 12 weeks. Body weight as well as total body lean and fat mass were measured at each time point. There were no differences in values between the two transplantations cohorts at baseline; thus, these values are presented for all mice together. Six week and 12 week values represent only the mice sacrificed at that time point. Data are the mean ± SEM. N/D = not determined.</p><p>*<i>P</i><0.05.</p>†<p><i>P</i><0.01.</p>‡<p><i>P</i><0.005.</p>§<p><i>P</i><0.0005.</p>∥<p><i>P</i><0.0001.</p

Plasma parameters in C67BL/6→LDLR^−/−, CCL3^−/−→LDLR^−/−, and CCL2^−/−→LDLR^−/− mice at 6 weeks post WD-feeding.

<p>Male LDLR^−/− mice were transplanted with C57BL/6, CCL3^−/− or CCL2^−/− bone marrow. At 4 weeks post-BMT, mice were placed on WD for 6 weeks. Data are the mean ± SEM of 6–7 mice per group.</p

Lesion complexity does not differ with CCL3 bone marrow genotype.

<p>LDLR^−/− mice were transplanted with C57BL/6 or CCL3^−/− bone marrow. At four weeks post-BMT, mice were placed on WD for 12 weeks. ORO (4×), Trichrome, MOMA-2, and CD4 stained lesion (10×) are in columns as indicated on figure. Gender and donor marrow are indicated on the left of the figure. Sections were chosen from images representing mice with lesion areas close to the mean of their respective groups.</p