The Role Of TLR4 In The Activation Of B Cells In β₂GPI-Immunized Mice Models

Objective: High titers of anti-β2-glycoprotein I antibodies(anti-β2GPI Ab) are regarded as an independent risk factor in serum of antiphospholipid syndrome(APS) patients by increasing the risk of thrombosis and recurrent abortion in APS patients. It is well known that anti-β2GPI antibodies play an active role in the pathogenic mechanisms of APS by inducing the production of tissue factors, adhesion molecule, tumor necrosis factors and multiple cytokines in monocytes and endothelial cells, so as to causing a stage of proinflammatory and hypercoagulability. Our previous research suggests that TLR4 is involved in β2-glycoprotein I(β2GPI)- induced mice bone marrow-derived dendritic cells(BMDCs) maturation and β2GPI may induce Th2 cell bias polarization through TLR4. However, the role of TLR4 in the autoimmune response to B cells and the production of APS-related antibodies still need further exploration. Based on the previous studies and related literature, we compared the change of serum anti-β2GPI Ab level, B cell activation markers and related inflammatory molecules as well as spleen germinal centers between β2GPI- immunized C3H/He N mice(TLR4 intact) and C3H/He J mice(TLR4 defective), to investigate the immune mechanism of Toll- like receptor 4(TLR4) in the activation of B cells in the spleen of mice immunized with β2GPI.Methods:(1) C3H/He N mice and C3H/He J mice were immunized with β2GPI, BSA(bovine serum albumin) and NS(normal saline). The serum from above mice was collected and detected the titer of anti-β2GPI antibodies by ELISA.(2) The changes of splenic germina l centers in C3H/He N mice and C3H/He J mice immunized with β2GPI, BSA and NS were observed by HE staining and the photographs were obtained by light microscopy.(3) The expression of CD40 L in the splenic germinal centers in C3H/He N mice and C3H/He J mice immunized with β2GPI, BSA and NS were detected by immunohistochemistry and the photographs were obtained by light microscopy. (4) The m RNA level of IL-6 and IL-10 of spleen B cells in C3H/He N mice and C3H/He J mice after β2GPI or BSA immunization or saline injection were measured by RT-q PCR.(5) In order to further investigate the effects of TLR4 in expression of BAFF, The m RNA level of BAFF in mice spleens was measured by RT-q PCR, and the protein level of BAFF was measured by Western blot in C3H/He N and C3H/He J mice immunized with β2GPI, BSA or NS.(6) In order to investigate the effects of TLR4 in β2GPI- immunized B cell activation, the lymphocytes were extracted from mice spleens of C3H/He N mice and C3H/He J mice immunized with β2GPI, BSA or NS, and the expression of CD80, CD86 and MHCII were detected by flow cytometry.Results(1) After immunization with β2GPI, high titer of anti-β2GPI Ab was measured both in the serum of C3H/He N mice and C3H/He J mice. While compared with β2GPI-immunized C3H/He J mice(1:100,000), higher titer of anti-β2GPI Ab in β2GPI-immunized C3H/He N mice was measured(>1:100,000). BSA and NS treated mice could not induce the anti-β2GPI Ab.(2) The spleen germinal centers in β2GPI- immunized mice were bigger both in the size and number compared with BSA- immunized and NS-immunized mice. Moreover, the number and the size of β2GPI- immunized C3H/He N mouse germinal centers are higher than those in β2GPI- immunized C3H/He J mice.(3) Compared with β2GPI- immunized C3H/He J mice, both m RNA and protein of BAFF are expressed higher in β2GPI- immunized C3H/He N mice(p<0.01, p<0.05).(4) CD40 and CD40 L expression in the spleen from β2GPI- immunized C3H/He N mice was significantly increased in β2GPI-immunized C3H/He N mice, compared to β2GPI-immunized C3H/He J mice( p<0.05 for three).(5) The expression of CD80, CD86 and MHC II was significantly upregulated on the surface of CD19+ B cells from β2GPI- immunized C3H/He N mice compared with β2GPI-immunized C3H/He J mice. (6) Following β2GPI injections, IL-6 and IL-10 secreted by B cells in spleens from C3H/He N mice was higher when compared with C3H/He J mice.Conclusions:(1) The β2GPI immunization can induce the specific anti-β2GPI antibody. All the indexes for B cell activation in β2GPI- immunized mice were higher than those in BSA- immunized mice.(2) TLR4 promotes the production of anti-β2GPI antibodies. After β2GPI immunization, higher titer of anti-β2GPI Ab was obtained in the serum of C3H/He N mice compared with the same treatment of C3H/He J mice.(3) TLR4 promotes the activation of B cells in β2GPI- immunized mice. Following β2GPI injections, the level of anti-β2GPI antibody in the serum of C3H/He N mice was gradually increased and the number and size of germinal centers in the spleen were also significantly increased when compared with C3H/He J mice. Moreover, the β2GPI-induced expression of C D40 L, CD40, CD80, C D86 and MHCII in C3H/He N mice was significantly higher than that in C3H/He J mice. Furthermore, the β2GPI-induced expression of B cell activating factor(BAFF) in the spleen and IL-6 and IL-10 in B cells from C3H/He N mice was also significantly increased compared to C3H/He J mice.(4) TLR4 and Breg cells may serve as an ideal target for prevention and treatment of APS. Our study results suggested that down-regulation of TLR4 expression or enhancing Bregs response might reduce the anti-β2GPI Ab production in vivo, so as to prevent and remedy APS.