Fig 2: Feeding LDLR-null mice WD resulted in increased mRNA levels for IFN-a, IFN-ß, and CH25H in the duodenum and jejunum of the mice described in Fig. 2, and adding Tg6F and ezetimibe to WD prevented the increase. IFN-a, IFN-ß, and CH25H mRNA levels in the duodenum and jejunum were determined by RT-qPCR as described in Materials and Methods. A: Results for IFN-a are shown. B: Results for IFN-ß are shown. C: Results for CH25H are shown. In each case, the left panel shows the results for the duodenum, and the right panel shows the results for the jejunum. The results are mean ± SEM. Abbreviations are the same as in the Fig. 2 legend.

Fig 3: Spp1 (osteopontin) mRNA and Spp1 protein expression were reduced in Tg6F fed mice. Total RNA and protein were isolated from the lungs of the BALB/c mice described in Fig. 1. (a) Spp1 mRNA expression was quantified by RT-qPCR (left side of panel) and protein lysates from lungs were analyzed by immunoblotting for Spp1 (right side of panel) as described in Materials and Methods. Each lane in the immunoblot is from a separate mouse. (b) Spp1 mRNA expression was reduced in the intestines of Tg6F fed mice. Total RNA was isolated from the jejunum of the BALB/c mice described in Fig. 1. Spp1 mRNA expression was quantified by RT-qPCR as described in Materials and Methods. The data shown are Mean ± SEM. (c) Tg6F decreased Spp1 levels in the plasma as measured by ELISA as described in Materials and Methods. The data shown are Mean ± SEM. (d) Cholesterol 25-hydroxylase (CH25H) protein expression in the villi of the jejunum was quantified by immunohistochemistry as described in Materials and Methods. The results shown are Mean ± SEM. (e) 25-hydroxycholesterol (25-OHC) levels in jejunum were determined by LC-MS/MS as described in Materials and Methods. The results shown are Mean ± SEM.

Fig 4: A hypothesis to explain how WD induces inflammation in the jejunum that Tg6F and ezetimibe ameliorate. WD provides cholesterol, triglycerides, and phospholipids [phosphatidylcholine (PC)]. In the lumen of the small intestine, phospholipase A2 group 1B (PLA2G1B) removes the fatty acid moiety from the sn-2 position of PC, yielding LysoPC. The triglycerides are acted upon by pancreatic lipase to yield two fatty acids and a monoglyceride (data not shown). Also not shown is the action of bile in forming micelles. The enterocytes of the small intestine take up the dietary lipids together with small amounts of ligands for TLRs. This induces IFN-ß in the enterocytes, which in turn induces CH25H, which contributes to increased levels of 25-OHC in the enterocytes. Subsequently, 25-OHC enters the lamina propria of the villi of the small intestine, where it acts on immune cells. This leads to increased levels of inflammatory cytokines [e.g., 25-OHC is known to induce increased levels of IL-6, IL-8 (mouse functional homologs MIP2 and KC), and M-CSF], which induce inflammation in the villus of the small intestine. Additionally, IFN-ß can induce the expression of autotaxin that increases the conversion of unsaturated LysoPC to unsaturated LPA, which further stimulates inflammation and leads to dyslipidemia as discussed in refs. 14, 16, and 25. Intestinal inflammation leads to systemic inflammation as measured by increased levels of plasma SAA (data not shown). Ezetimibe and Tg6F act to decrease levels of IFN-ß, CH25H, and 25-OHC, thus partially ameliorating inflammation and dyslipidemia in LDLR-null mice fed WD.

Fig 5: WD increased levels of CH25H in the enterocytes of the jejunum of the mice described in Fig. 2, which was better ameliorated by the novel method for adding Tg6F and ezetimibe compared with adding the same doses of Tg6F and ezetimibe to WD by the combined formulation. The induction of CH25H by WD in the enterocytes of the jejunum was determined by IHC and ELISA as described in Materials and Methods. A: An example of IHC showing induction of CH25H expression by WD and amelioration by the novel method. B: Quantification of IHC was performed as described in Materials and Methods. C: The results of IHC were confirmed by ELISA as described in Materials and Methods. The results shown are mean ± SEM. Abbreviations are the same as in the Fig. 2 legend.