The Cell-specific Role Of MyD88in The Development Of Obesity-associated Inflammatory Diseases

Background:Low grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. A high fat diet (HFD) affects the gut-permeability, triggering the accumulation of systemic inflammatory stimuli, including exogenous and endogenous ligands for interlukin-1receptor (IL-1R)/Toll-like receptors (TLRs). Previous studies suggested a critical role for the EL-1R/TLRs in diet-induced systemic inflammation and obesity-associated inflammatory diseases.Objectives:In this study, we investigated how TLR-MyD88signaling in different cellular compartments coordinately participates in the initiation of HFD-induced systemic inflammation and metabolic inflammatory diseases.Methods:We generated endothelial cell-specific MyD88-deficient mice (MyD88EC-KO) and myeloid/macrophage-specific MyD88-deficient mice (MyD88MC-KO) by Cre-LoxP technology. MyD88EC-KO and MyD88MC-KOmice were crossed to ApoE-/-mice to generate MyD88EC-KO ApoE-/-and MyD88MC-KO ApoE-/-mice. We put these mice on chow diet or HFD (starting from6weeks old) for3months to establish diet-induced obesity model and atherosclerosis model. We checked the metabolic profile of these mice including body weight, fat mass, serum cholesterol/triglycerides level and fasting glucose/insulin concentration, performed glucose/insulin tolerance test (GTT/ITT) to evaluate insulin sensitivity. To test the insulin signaling in adipose tissue, muscle and liver, we injected the mice with insulin or saline by I.P., collected tissues and checked insulin stimulated IRS-1-PI3K-AKT signaling by western-blot. To examine HFD-induced systemic inflammation, protein level of pro-inflammatory cytokines in the serum will be analyzed by ELISA. Infiltration of macrophage in adipose tissue (ATM) from the experimental mice was analyzed by immunohisto-chemistry staining, and stromal vascular fraction (SVF) cells were stained followed by flow cytometry analysis. Under ApoE-/-background, enface (oil-red O staining) analysis will be done for the whole aorta tree of the experimental mice. Total mean lesion area will be quantified based on image-analysis by Photoshop software. The co-localization of granulocyte-macrophage colony-stimulating factor (GM-CSF) and endothelial cell surface marker CD31was dected by Immunostaining and examined by confocal microscopy. An adipocyte-macrophage co-culture system and FFA/LPS treatment were used to demonstrate cross-talk between adipocytes and macrophage. We used conditioned medium from endothelial cells treated with ox-LDL to culture monocyte/macrophage and treated GM-CSF primed macrophage with ox-LDL to dress the interplay between endothelial cells and macrophage. Tissue/celluar mRNA was analyzed by RT-PCR to measure the expression of inflammatory genes and markers for M1and M2macrophages. The activation of signal pathway was examined by western-blot.Results:MyD88deficiency in myeloid cells aborted a switch in ATM phenotype from M2to M1, resulting in substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. Using an adipocyte-macrophage co-culture system, we demonstrated the impact of MyD88-dependent cross-talk between adipocytes and macrophages on Ml macrophages. On the other hand, MyD88deficiency in endothelial cells also showed moderate reduction in diet-induced adipose M1macrophages, systemic inflammation and atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated systemic inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to Ml inflammatory macrophages.Conclusions:These results suggest that cross-talks among adipocytes, macrophages, and endothelial cells is necessary for establishment of the diet-induced inflammatory state through the production of pro-inflammatory mediators, which in turn cause insulin resistance, leading to inflammatory diseases associated with metabolic syndrome.