Abstract 2037 The Role of Intestinal ApoA-I in Mouse Models of Diet-Induced Inflammation and Atherosclerosis

Introduction: Systemic inflammation is a major underlying risk factor for the development of atherosclerosis. The inflammatory condition is often initiated or exacerbated by a western-style diet (WD) high in calories and fats. To mitigate this, anti-inflammatory lipoproteins, such as high-density lipoprotein (HDL), function to bind and clear inflammatory lipids. Apolipoprotein A-I (ApoA-I), the major protein component of HDL, is synthesized equally in both the liver (HA-I) and the intestine (IA-I). Although HA-I has been studied extensively, little is known about IA-I and its role in HDL biology, systemic inflammation, or atherosclerosis. Methods and Results: To determine the physiological role of IA-I, and to examine whether the tissue of origin dictates the ability of ApoA-I to protect against systemic inflammation and atherosclerosis, we first utilized the ApoA-I whole-body knockout mice crossed onto a hyperlipidemic Ldlr-/- background: ApoA-I-/- Ldlr-/- mice. After 8 weeks on a WD, ApoA-I-/- Ldlr-/- mice gained significantly more weight compared to Ldlr-/- mice, and exhibited higher levels of IL-6 in the jejunum tissues. We established a method to isolate pure enterocytes from jejunum tissues using FACS by gating for EpCam+ cells and excluding dead and aggregated cells, and cells positive CD45 (lymphocytes), CD44 (crypts), CD31 (endothelial cells), F4/80 (macrophages), and LYVE1(lymphatic endothelial cells). To explore the major pathways associated with ApoA-I in the enterocytes from the small intestine, we performed RNA-Seq on the FACS-sorted jejunal enterocytes from both genotypes and on both diets. We identified significant changes between Ldlr-/- and ApoA-I-/- Ldlr-/- enterocytes in anti-microbial and anti-inflammatory gene expression, with Reg3g, Defa4, Muc4, and Apex1 being upregulated, and Gfi1 and ApoA-IV being downregulated in ApoA-I-/- Ldlr-/- mice. To further explore how ApoA-I deletion in the intestine affects anti-microbial and anti-inflammatory pathways, we have generated a novel inducible intestine-specific knockout of ApoA-I in C57BL/6 mice on an Ldlr-/- background: ApoA-Ifl/fl iVilCre+ Ldlr-/- mice. Conclusions and Future Directions: We utilized a whole-body deletion of ApoA-I to show increased markers of systemic inflammation and altered anti-microbial and anti-inflammatory gene expression in the small intestine of ApoA-I-/- Ldlr-/-mice. Future studies will seek to validate phenotypes observed in the whole-body knockout in the ApoA-Ifl/fl iVilCre+ Ldlr-/- mice. Additional experiments include shotgun proteomics on HDL isolated via ultracentrifugation from ApoA-Ifl/fl iVilCre+ Ldlr-/- mice, to assess whether the loss of IA-I alters the levels of pro- and anti-inflammatory proteins on HDL. We will also perform single cell RNA-Seq on FACS-sorted jejunal enterocytes from ApoA-Ifl/fl iVilCre+ Ldlr-/- mice to explore gene expression changes in individual enterocytes upon deletion of ApoA-I. This work was supported in part by Ruth L. Kirschstein National Research Service Award T32HL069766 (to S.D.), and by NIEHS research grant R01 ES033660 (to S.R.).