Hashimoto (Yokohama City University) for generously providing the ApoE KO mice. development. In contrast, disruption of the intestinal microbiota by a broad-spectrum antibiotic cocktail (AVNM) led to the attenuation of atherosclerosis by suppressing B2 cells, despite the persistence of serum lipid abnormalities. Furthermore, pharmacological depletion of B2 cells with an anti-B2-cell surface CD23 antibody also attenuated commensal microbe-induced atherosclerosis. Moreover, expression analysis of TLR-signaling-related genes in the activated B2 cell subsets, assessed using the Toll-Like Receptor Signaling Pathway RT2 Profiler PCR Array, confirmed activation of the B2-cell autoantibody-production axis, which was associated with an increased capacity of B2 cells to bind to intestinal microbiota. Together, our findings reveal the critical role of commensal microbe-specific activation of B2 cells in the development of atherogenesis through lipid metabolism-independent mechanisms. and were dramatically reduced (Table 1). These findings strongly suggest that Raddeanin A the observed changes resulted from the elimination of the intestinal microbiota, which promotes atherosclerosis via activation of B2-cell TLR signaling pathway. B2-cell TLR signaling thus mediates microbiota-driven atherosclerosis. Taken together, these findings support a specific role of TLR signaling in B2 cells during microbiota-driven atherosclerosis. Open in a separate window Fig. 3 Distinct gene expression profiles associated with TLR signaling pathway in B2 cells following WD and AT. Messenger RNA preparations of sorted FO B cells from PVAT and spleen and MZ B cells from spleen were analyzed by mouse toll-like receptor signaling C5AR1 pathway RT2 Profiler PCR arrays. Gene expression reportedly associated with TLR signaling pathway was compared among the indicated FO B2 cell groups (A) and indicated MZ B2 cell groups (B), respectively. Results are displayed as heat maps. Red, max (magnitude of gene expression); green, min (magnitude of gene expression). Table 1 Relative fold Raddeanin A changes in the expression of genes relevant to TLR signaling pathway in FO B cells.

Gene Fold regulation (compared with spleen FO B cells of WD group)

FO B cells of spleen
(WD-AT group) FO B cells of PVAT
(WD group)

Ccl2??1.148117.8381Cd141.9937.189Cebpb3.23311.1982Fos1.726??3.5108Hspa1a??5.11669.3761Il1b??1.03073.3433Il1r11.26163.5637Jun1.2743??2.16Nfkbib2.80251.5068Ptgs2??2.34993.9493Tnfrsf1a1.58272.7597 Open in a separate window Table 2 Up-down regulation in the expression of genes relevant to TLRs signaling pathway in MZ B cells in the WD group versus WD?+?AT group.

Gene Fold regulation in MZ B cells
(compared with WD group)

Chuk3.8971Fos2.6863Hspa1a??2.3842Ikbkb2.6361Irf12.0659Mapk82.6917Mapk8ip36.4522Mapk93.1227Tlr5??2.4133Tollip2.1339Tradd3.121Traf63.3725 Open in a separate window 3.4. B2-cell Deficiency Attenuates MicrobiotaCinduced Atherosclerosis Because intestinal microbiota depletion may influence the development of atherosclerosis by reducing the number of activated B2 cells, we further investigated whether B2-cell deficiency might afford protection against microbiota-induced atherosclerosis. A cohort of WD-fed mice was pretreated with a B2-cellCdepleting agent, anti-mouse CD23 antibody. Intraperitoneal injections of anti-CD23 antibody were started 1?week before the development of atherosclerosis. The control group for these experiments comprised mice pretreated with saline. As expected, mice that received the mouse-specific CD23 antibody had far fewer B2 cells in their spleens and PVAT than did mice treated with saline (Fig. 4ACB). There were no changes in other cell populations (Supplementary Fig. S4). Furthermore, we found that WD-fed mice treated with anti-CD23 antibody gained Raddeanin A weight in association with increased visceral and subcutaneous fat and serum lipid levels, similar to the WD-fed controls (Fig. 4CCI). However, after 8?weeks of WD, we compared plaque in WD-fed mice versus WD-fed plus anti-CD23 antibody-treated mice. WD-fed plus anti-CD23 antibody-treated mice exhibited a marked reduction in plaque formation as compared with that in WD-fed mice (Fig. 4JCK). At the same time, serum IgG and IgG3 levels were found to be elevated only in WD-fed mice not treated with antibody (Fig. 4LCM). These results confirmed that potential triggering of atherosclerosis by microbiota requires initial help from B2 cells. Altogether, these Raddeanin A data indicate that microbiota aggravates atherosclerosis by stimulating activated B2-cell production and shifting the host response toward TH1-associated immunity. Open in a separate window Fig. 4 Pharmacological depletion of B2 cells protects mice from atherosclerosis. (A and B) Representative flow cytometric plots of B2 cell numbers in the PVAT (A) and spleens Raddeanin A (B) of mice treated with a mouse specific CD23 antibody or saline (n?=?6 per group). (C) Body weights measured at the end of 8?weeks of a Western diet in mice treated with a mouse specific CD23 antibody or saline. Values are presented as means??SEM from n?=?6 experiments. (D and E) Micro-CT of total percent of visceral fat volume (D) and subcutaneous fat volume (E). Data are representative as means??SEM. n?=?6 for each group. (FCI) Serum levels of total cholesterol (F), LDL (G) and HDL (H) and triglyceride (I) were assessed. LDL: low-density lipoprotein; HDL: high-density lipoprotein. Results are presented as means??SEM. n?=?6 per group. (J and K) Effect of an anti-mouse CD23 antibody on plaque formation is shown in JCK. (J) Representative images and quantification of oil red.