Project Summary/Abstract The increase in childhood obesity/Type 2 diabetes is paralleling a worldwide increase in obesity-associated metabolic syndrome. Maternal obesity is one of the key drivers tightly associated with the incidence of offspring obesity and metabolic disorders. However, the mechanisms underlying the impacts of maternal obesity on offspring metabolic disorders are not fully understood. Our recent work has led to the discovery that CRIg+ macrophages protect host cells from the gut microbial DNA-containing extracellular vesicle (mEV)-induced cellular abnormalities, whereas CRIg+ macrophages are absent in the context of obesity in both humans and mice, accompanied with enrichment of bacterial DNAs in host cells and worsen insulin sensitivity or insulin secretion. Intestinal mEVs can readily pass through the gut barrier of obese mice and further deliver microbial DNAs into key metabolic tissues. We further demonstrated the critical role of CRIg+ macrophages in clearing gut mEVs from bloodstream, as evidenced by a robust accumulation of microbial DNAs within key metabolic tissues in NCD CRIg-/- mice after intravenously injected with gut mEVs. By contrast, CRIg+ cells in NCD WT mice blocked the infiltration of gut mEVs through a C3-mediated mechanism, thus preventing the enrichment of bacterial DNAs in host cells. Recovery of CRIg+ cells efficiently attenuated tissue inflammation and metabolic disorders in obese mice. Depletion of microbial DNAs blunted the pathogenic effects of gut mEVs, while accumulation of microbial DNAs triggered the activation of cGAS/STING pathway for host cell abnormalities. By contrast, knockout of cGAS prevented microbial DNA- induced cellular disorders. These results lead to the conclusion that CRIg+ macrophages protect host cells from the pathogenesis of gut mEVs. We also found a significant reduction in CRIg+ macrophage population in maternal liver and placenta in obese pregnancy, concomitant with bacterial DNA enrichment in embryos. By contrast, lean pregnant mice harbored high abundance of CRIg+ macrophages in placenta, and no 16s rRNAs were detected in their embryos. Maternal gut mEVs can reach and deliver microbial DNAs into embryos in obese pregnant mice or lean CRIg-/- mice, but not in lean WT mice. Thus, this proposal seeks to reveal the important roles of CRIg+ macrophages in pregnancy and the impacts of maternal obesity-induced embryonic microbial DNA enrichment on offspring metabolic responses. We will further determine: 1) critical roles of maternal CRIg+ macrophages in blocking the infiltration of gut mEVs into embryos; 2) effects of maternal obesity on the development of embryonic CRIg+ macrophages; 3) effects of maternal obesity- induced embryonic microbial DNA accumulation on offspring tissue inflammation and metabolic phenotypes.