Presentations

Abstract

Introduction:

Atherosclerosis is a lipid-driven inflammatory disease caused by plaque buildup in blood vessel linings. Both innate and adaptive immune responses are involved in the development and progression of atherosclerosis. Previously, atheroprotective and pro-atherogenic functions of B cell subsets have been observed. Anergy, a state of unresponsiveness to antigens, is responsible for silencing many self-reactive B cells to prevent autoimmune-associated immune responses. Currently, it is not known how B cell anergy is affected in atherosclerosis. To date, it is also not quite clear to what extent anergic B cells can respond to stimuli other than the BCR engagement. It has been recently shown that TLR4 engagement induces B1a cell differentiation into innate response activator (IRA) B cells that play a pro-atherogenic role. The goal of this study was to test to what extent anergic B cells can respond to TLR4 activation using a model of IRA- B cell differentiation under homeostatic conditions.

Methods:

Ars/A1 transgenic model, where anergic B cells express a dual-reactive antigen receptor that binds, in addition to a self-antigen, the hapten p-azophenylarsonate (Ars) and control C57BL/6 mice were used in this study. Additionally, as an additional BCR-transgenic mouse model (not anergic B cells), we used the MD4 transgenic mice that express a BCR that recognize hen egg lysozyme (HEL). Mice were administered 10 μg of LPS daily by intraperitoneal injection (i.p.) for 4 days. Controls received PBS alone. After 4 days of injections, the mice were euthanized and immune cells from the peritoneum and spleen were isolated, stained with Abs for B1 and IRA B cells and analyzed by Flow Cytometry.

Results:

Our data demonstrates that i.p. injection of LPS induced a significant differentiation of IRA+ B cells in the spleen of C57BL/6 mice. Interestingly, LPS injection into MD4 transgenic mice induced similar levels of IRA+ B cells in the MD4-transgenic mice, suggesting that MD4 B cells respond normally to TLR4 stimulation. In contrast, i.p. injection of LPS into ARS/A1 recipients did not induce generation of IRA+ B cells in the spleen or peritoneal cavity of the ARS/A1 mice.

Conclusion:

Several reports suggested that ARS/ A1 anergic B cells respond normally to a TLR simulation in vitro. Our experiments further tested this important question using in vivo assays of the generation of IRA+ B cells. The obtained results demonstrate that peritoneal ARS/A1 B cells do not respond to TLR4 stimulation in vivo and do not develop IRA+B cells. Further studies will be focused on testing effects of TLR stimulations on antibody production and release of pro-inflammatory cytokines in the in vivo assays.