br Methods br Results br Discussion The intriguing

Methods
Results
Discussion The intriguing relationship between commensal microbes and atherosclerosis has received increasing attention over the past few years. However, the specific mechanisms whereby commensal microbes regulate the development of atherosclerosis are just beginning to be elucidated (Brown and Hazen, 2015; Koren et al., 2011; Serino et al., 2014; Tang and Hazen, 2014). Recently, Spence et al. found that a number of metabolites from proteins/amino acids in the diet, including p-cresyl sulfate, indoxyl sulfate, and others (Spence et al., 2016), might contribute to development of cardiovascular disease (CVD). Previous studies have identified the pathway linking dietary lipid intake, intestinal microflora, and atherosclerosis. These studies indicated that increasing the dietary intake of precursors of toxic metabolic products of the intestinal microbiome, such as the trimethylamine-N-oxide from phosphatidylcholine and other forms of choline and carnitine (Koeth et al., 2013; Tang et al., 2013; Wang et al., 2011), was associated with CVD. The pathway of the metabolism of these toxic metabolic products of the intestinal microbiome represents a unique additional nutritional contribution to the pathogenesis of CVD. In fact, numerous studies have shown that commensal microbe-derived metabolites can act as hormones modulating CVD risk (Brown and Hazen, 2015). Metabolism-independent pathways, in particular the role of the immune system in commensal microbe-derived atherosclerosis, remain largely unexplored. Our study showed that serum lipid levels were significantly increased in WD-fed mice regardless of AT or not, which is in line with previous observations (Fu et al., 2015; Le Chatelier et al., 2013; Velagapudi et al., 2010). However, it remains unclear how the gut microbiota affects serum lipid metabolism and systemic lipid metabolism in adipose tissue. It is thought that hyperlipidemia (especially LDL-C) generates an adaptive immune response mediated by autoantibody that is produced by activated rgs protein and then induces atherosclerosis (Hermansson et al., 2010; Hilgendorf et al., 2014). However, in the present study, there was no correlation between lipid levels and atherosclerosis development. Therefore, we determined whether B2-cell activation is mediated by microbiota rather than by hyperlipidemia in atherosclerosis. Here, we confirmed, by eliminating the intestinal microbiota and depleting B2 cells in the WD-fed mice, that hyperlipidemia did not directly potentiate atherosclerosis by altering B2-cell activation. Our results provide novel evidence that B2 cells are causally related to microbiota-induced atherosclerosis. Our findings also indicate that proatherogenic effects do not solely depend on hypercholesterolemia-induced immune response. Clinically, these findings may explain why only control of lipids is not effective preventive therapy in some patients with atherosclerosis. B cells play a complex role in the development of atherosclerosis via antibody production. In particular, the role of B2 cells in atherosclerosis is highly debatable (Kyaw et al., 2010; Tsiantoulas et al., 2014). As such, understanding the impact of B2 cells on atherosclerosis and elucidating factors that regulate their activity are important. In the present study, our observed differences in FO B cells of PVAT between the WD group and WD-AT group suggest that FO B cells are missing from the PVAT in the WD-AT group, potentially due to commensal microbe deletion by broad-spectrum antibiotics. However, it remains controversial whether commensal microbes directly induce B2-cell activation during the development of atherosclerosis and whether activated B2 cells play a critical role in this process. Thus, future studies are needed to determine whether B2 cells are essential for commensal microbe-derived atherogenesis and to elucidate potential atherogenic pathways. It is known that one of the early consequences of B2 cell activation is the up-regulation of costimulatory molecules, such as MHC class II molecules, which can serve to enhance B cell interactions and present antigen (Jiang et al., 2013; Jin et al., 2014). Another major outcome of B2-cell activation is the production of large amounts of specific antibodies (Shapiro-Shelef and Calame, 2005). The results of our study apparently showed that a greater proportion of B2 cells expressed MHC class II molecules in the spleen of WD-fed mice. We also detected elevated levels of IgG. In particular, the IgG subclass IgG3 displayed the highest activity in serum, whereas AT significantly reduced the expression of MHC class II molecules and the levels of IgG and IgG3. These results support the concept that microbiota-induced atherosclerosis is associated with B2-cell activation.