The goal of this project is to characterize the mouse ortholog of Chemokine-Like Receptor 1 (CMKLR1) on macrophages and determine its role in obesity and atherosclerosis.
Aim 1 proposes to characterize the expression of CMKLR1 on developing and tissue resident macrophages. Bone marrow derived macrophages (BMDM) will be assayed for receptor expression over time. Various tissue resident macrophages (TRM) will also be assayed for receptor expression. BMDMs and TRMs will be stimulated with pro-inflammatory and anti-inflammatory factors and assayed for CMKLR1 expression in response to said factors (flow cytometry, immunohistochemistry [IHC]).
Aim 2 proposes to characterize the effect of functional CMKLR1 on basic macrophage effector functions either in the presence or absence of CMKLR1 ligand (chemerin). CMKLR1 KO or WT TRMs will be treated with or without chemerin and assayed for cell spreading, cytokine expression (ELISA or intracellular staining), nitric oxide production (Greiss assay), phagocytosis (FITC-labeled E. Coli), arginase activity (L-arginine metabolism), T cell antigen presentation ability (antigen T cell proliferation assay), and T cell costimulation ability (costimulation T cell proliferation assay) in response to various activating stimuli.
Aim 3 will focus on characterizing the role of CMKLR1 in mouse models of obesity and athersclerosis. For obesity, CMKLR1 WT and KO mice will be fed a high fat diet and their weights/food intake recorded over time. At various time points, whole body fat content will be assayed (dual-energy-X-ray-absorptiometry or quantitiative MRI). Mice will be assayed for glucose tolerance (intraperitoneal glucose tolerance assay), insulin tolerance (intraperitoneal insulin tolerance assay), and plasma lipid (Reflotron test strips). Adipose tissue will be assayed for macrophage content (flow cytometry, IHC), proinflammatory gene expression (quantitative RT-PCR, tissue macrophage intracellular cytokine expression), and chemerin expression (IHC). For atherosclerosis, CMKLR1 WT and KO mice deficient for ApoE will be fed a normal diet. Periodically, mice will be sacrificed and their aortas harvested along with blood. Plasma levels of lipids with be assayed and aortic atheromatous plaques quantitated (Oil-Red-O or Sudan V staining). Aortic macrophage content will be analyzed (IHC) and pro- inflammatory gene expression (qRT-PCR) will be assessed.
The proposed research is relevant to public health as macrophages play a large role in the development and maintenance of inflammatory diseases. The proposed research will endeavor to further elucidate the mechanisms by which macrophages infiltrate inflamed tissue, thus offering a potential therapeutic target.
| Ernst, Matthew C; Haidl, Ian D; Zúñiga, Luis A et al. (2012) Disruption of the chemokine-like receptor-1 (CMKLR1) gene is associated with reduced adiposity and glucose intolerance. Endocrinology 153:672-82 |
| Zúñiga, Luis A; Shen, Wen-Jun; Joyce-Shaikh, Barbara et al. (2010) IL-17 regulates adipogenesis, glucose homeostasis, and obesity. J Immunol 185:6947-59 |
| Goswami, Jaya; Hernández-Santos, Nydiaris; Zuniga, Luis A et al. (2009) A bone-protective role for IL-17 receptor signaling in ovariectomy-induced bone loss. Eur J Immunol 39:2831-9 |
| Zabel, Brian A; Nakae, Susumu; Zuniga, Luis et al. (2008) Mast cell-expressed orphan receptor CCRL2 binds chemerin and is required for optimal induction of IgE-mediated passive cutaneous anaphylaxis. J Exp Med 205:2207-20 |
