Depletion of B2 but Not B1a B Cells in BAFF Receptor-Deficient ApoE−/− Mice Attenuates Atherosclerosis by Potently Ameliorating Arterial Inflammation

We have recently identified conventional B2 cells as atherogenic and B1a cells as atheroprotective in hypercholesterolemic ApoE−/− mice. Here, we examined the development of atherosclerosis in BAFF-R deficient ApoE−/− mice because B2 cells but not B1a cells are selectively depleted in BAFF-R deficient mice. We fed BAFF-R−/− ApoE−/− (BaffR.ApoE DKO) and BAFF-R+/+ApoE−/− (ApoE KO) mice a high fat diet (HFD) for 8-weeks. B2 cells were significantly reduced by 82%, 81%, 94%, 72% in blood, peritoneal fluid, spleen and peripheral lymph nodes respectively; while B1a cells and non-B lymphocytes were unaffected. Aortic atherosclerotic lesions assessed by oil red-O stained-lipid accumulation and CD68+ macrophage accumulation were decreased by 44% and 50% respectively. B cells were absent in atherosclerotic lesions of BaffR.ApoE DKO mice as were IgG1 and IgG2a immunoglobulins produced by B2 cells, despite low but measurable numbers of B2 cells and IgG1 and IgG2a immunoglobulin concentrations in plasma. Plasma IgM and IgM deposits in atherosclerotic lesions were also reduced. BAFF-R deficiency in ApoE−/− mice was also associated with a reduced expression of VCAM-1 and fewer macrophages, dendritic cells, CD4+ and CD8+ T cell infiltrates and PCNA+ cells in lesions. The expression of proinflammatory cytokines, TNF-α, IL1-β and proinflammatory chemokine MCP-1 was also reduced. Body weight and plasma cholesterols were unaffected in BaffR.ApoE DKO mice. Our data indicate that B2 cells are important contributors to the development of atherosclerosis and that targeting the BAFF-R to specifically reduce atherogenic B2 cell numbers while preserving atheroprotective B1a cell numbers may be a potential therapeutic strategy to reduce atherosclerosis by potently reducing arterial inflammation

Low expression of adhesion molecule, VCAM-1, is associated with reduction in immature and mature dendritic cells in atherosclerotic lesions of BAFF-R^−/− ApoE^−/− mice.

<p>(<b>A</b>) VCAM-1 expression decreased in atherosclerotic lesions by disruption of BAFF-R gene was accompanied by (<b>B</b>) decreased immature dendritic cells as assessed by anti-CD11c antibody and (<b>C</b>) mature dendritic cells as assessed by anti-CD83 antibody in <i>BaffR.ApoE</i> DKO mice. Open bar = <i>ApoE</i> KO; Black bar = <i>BaffR.ApoE</i> DKO; n = 9–11 mice; scale bar = 100 µm; *: <i>p</i>&lt;0.05, ***: <i>p</i>&lt;0.001.</p

T cell infiltrates and cellular proliferative activity are decreased in atherosclerotic lesions of <i>BaffR.ApoE</i> DKO.

<p>(<b>A and B</b>) T cell infiltrates as assessed by anti-CD4 and anti-CD8 antibodies and (<b>C</b>) cellular proliferative activity as assessed by PCNA antibody were reduced by deficiency of BAFF-R. Open bar = <i>ApoE</i> KO; Black bar = <i>BaffR.ApoE</i> DKO; n = 9–11 mice; scale bar = 100 µm; *: <i>p</i>&lt;0.05, **: <i>p</i>&lt;0.01.</p

Non-B lymphocyte populations in spleen (×10⁶).

<p>At the end of 8 week HFD, spleen cells are analyzed for non-B lymphocyte populations (n = 9–11 mice).</p

BAFF-R deficiency responsible for loss of B2 cells affects loss of B cells and IgG deposits in atherosclerotic lesion and attenuates plasma IgG levels.

<p>(<b>A</b>) CD22+ B cells and (<b>B–D</b>) immunoglobulins deposits (IgG1, IgG2a and IgM) found in the atherosclerotic lesion of <i>ApoE</i> KO mice were not detected in <i>BaffR.ApoE</i> DKO mice. (<b>E</b>)The plasma IgG1, IgG2a and IgM levels are also significantly decreased in <i>BaffR.ApoE</i> DKO but to a lesser extent than in lesions. Open bar = <i>ApoE</i> KO; Black bar = <i>BaffR.ApoE</i> DKO; n = 9–11 mice; scale bar = 100 µm; **: <i>p</i>&lt;0.01, ***: <i>p</i>&lt;0.001.</p

BAFF-R deficiency attenuates selectively conventional B2 cells, not peritoneal B1a cells.

<p>(<b>A</b>) PCR analysis of BAFF-R gene disruption was performed using DNA extracted from tails of BAFF-R^−/− ApoE^−/−, BAFF-R^+/+ ApoE^−/− and BAFF-R^+/+ ApoE^+/+ mice. (<b>B</b>) <i>BaffR.ApoE</i> DKO mice showed that CD22⁺ B cells were lowered in peripheral blood, peritoneal cavity, spleen and peripheral lymph nodes. (<b>C</b>) FACS analysis on peritoneal cavity revealed that depleted B cells were conventional CD22+ CD5- B2 cells, not CD22+ CD5+ B1a cells comparing to <i>ApoE</i> KO mice. (<b>D</b>) A representative FACS analysis of peritoneal cavity showed that only conventional CD22+ CD5- B2 cell population (left upper quadrant) was decreased in <i>BaffR.ApoE</i> DKO and peritoneal CD22+ CD5+ B1a cell (right upper quadrant) was unaffected. Open bar = <i>ApoE</i> KO; Black bar = <i>BaffR.ApoE</i> DKO; n = 9–11 mice; **: <i>p</i>&lt;0.01, ***: <i>p</i>&lt;0.001.</p

Real-time PCR analysis of proinflammatory cytokines.

<p>RNAs extracted from aortic arches were analysed for proinflammatory cytokines, TNF-α, IL1-β, MCP-1 and IFN-γ. <i>BaffR.ApoE</i> DKO showed reduced expression of proinflammatory cytokines in aortic arches compared to <i>ApoE</i> KO. Open bar = <i>ApoE</i> KO; Black bar = <i>BaffR.ApoE</i> DKO; n = 9–11 mice; *: <i>p</i>&lt;0.05.</p

Factors contributing in atherosclerosis pathogenesis.

<p>At the end of 8 week HFD, body weight and plasma lipids were analyzed (see text) (n = 9–11 mice).</p

Deficiency of BAFF-R attenuates atherosclerosis.

<p>After feeding a HFD for eight weeks, <i>BaffR.ApoE</i> DKO showed decreased atherosclerosis at aortic sinus as assessed by (<b>A</b>) oil-red O stained lipid accumulation and (<b>B</b>) CD68+ macrophage accumulation compared to <i>ApoE</i> KO. Open bar = <i>ApoE</i> KO; Black bar = <i>BaffR.ApoE</i> DKO; n = 9–11 mice; scale bar = 100 µm; *: <i>p</i>&lt;0.05, **: <i>p</i>&lt;0.01.</p