The Exploration Of Roles Of TLR4 In The Thrombosis In A Murine Model Of Antiphospholipid Syndrome

Objective:Antiphospholipid (aPL)/anti-β2-glycoprotein I (β2GPI) antibodies are considered to play a pivotal pathogenic role in antiphospholipid syndrome (APS) by inducing an intracellular signaling and procoagulant/proinflammatory phenotype that leads to thrombosis. There is increasing evidence that toll-like receptor 4 (TLR4) could serve as an important molecule for β2GPI/anti-β2GPI recognition on target cells. However, few studies have focused on the effects of TLR4 in in vivo models. In the present study, we investigated the role of TLR4 in the pathogenic effects of aPL/anti-β2GPI more precisely using TLR4-intact (C3H/HeN) and TLR4-defective (C3H/HeJ) mice. The study may provide a new way for prevention and treatment of thrombotic issues in APS.Methods:(1) The peptides from the NH2-terminal 35th-51th amino acids of P2GPI were synthesized by standard Fmoc, and then used to immunize New Zealand white rabbit after coupling with keyhole limpet hemocyanin (KLH). The polyclonal antibody specific to a β2GPI domain I in the rabbit serum was purified by Protein G column, and identify its specificity and pathogenic effects.(2) The peritoneal macrophages separated from C3H/HeN and C3H/HeJ mice were treated with/without β2GPI/anti-β2GPI in vitro. In order to observe the role of TLR4 in p2GPI/anti-p2GPI induced macrophage expression of TF and inflammatory cytokines, the mRNA expression of TF, TNF-α, IL1β and IL6 in macrophages after stimulation was measured by real-time quantitative PCR (RT-qPCR), the protein expression of TF, TNF-α, IL1β and IL6 in macrophages culture supernatant was detected by Western blot.(3) In order to further analyze the effects of TLR4 in expression of TF and inflammatory factor in vivo, the experimental antiphospholipid antibody syndrome (EAPS) mice model were established using C3H/HeN and C3H/HeJ mice by intraperitoneal injection of anti-β2GPI (100 μg) or IgG-APS purified from the APS patients (500 μg) in 0 h and 48 h, respectively. Carotid arteries dissected from both sides and peritoneal macrophages in each animal were collected at 72 h after the first injection. Carotids and macrophages were homogenized by sonication and TF activity was measured through its ability to enhance the FVIIa-catalyzed activation of factor X. TF expression in femoral artery endothelial cells was detect by Immunohistochemistry. The protein expression of TF, TNF-α, IL1β and IL6 in macrophages was examined by immunofluorescence.(4) Aortas were collected from EAPS mice, and the mRNA and protein expression of VCAM-1, ICAM-1 and E-selectin in aortas were detected by RT-qPCR and Western blot, respectively, in order to analyze the effects of TLR4 on anti-β2GPI and IgG-APS induced expression of adhesion molecular.(5) EAPS mouse model was established using C3H/HeN and C3H/HeJ mice by intraperitoneal injection of anti-β2GPI (100 μg). Filter paper soaked in 3.5% FeCl3 was placed on the adventitial surface of the artery and femoral vein to induced thrombosis, then thrombus size was observed by HE staining. For visualizing the size of thrombus formation in artery by fluorescence microscopy, CF633-labeled CD41 Fab fragments were administered intravenously (0.25 μg/g) before FeCl3 treatment.Results:(1) The anti-β2GPI peptide antibody against both human and mouse β2GPI has been successfully prepared. The antibody can specifically recognize β2GPI cryptic epitope in domain I and have the pathogenic effect, which provides an important tool for further study on the roles of β2GPI and anti-β2GPI antibodies in the mechanism of APS.(2) Compared with C3H/HeJ mice, mRNA and protein expression of TF, TNF-α, IL1β and IL6 was significantly increased in peritoneal macrophages from C3H/HeN mice after stimulation by anti-β2GPI/β2GPI complex in vitro.(3) In the EAPS model mice, IgG-APS or anti-β2GPI-induced carotid artery and peritoneal macrophage tissue factor activity was significantly lesser in C3H/HeJ than in C3H/HeN mice. TF protein expression in macrophages and endothelial cells from C3H/HeN mice was also significantly upregulated. The protein expression of TNF-α, IL1β and IL6 on macrophage surface from C3H/HeN mice was also enhanced.(4) The IgG-APS or anti-β2GPI induced expression of VCAM-1, ICAM-1, and E-selectin in the aorta of C3H/HeJ mice was significantly reduced compared to C3H/HeN mice.(5) Following anti-β2GPI antibody injections and vascular injury by FeCl3, thrombus formation in both the carotid artery and femoral vein was markedly reduced in C3H/HeJ mice when compared with C3H/HeN mice.Conclusions:(1) TLR4 not only mediates anti-β2GPI/β2GPI complexes induced expression of TF, TNF-α, IL1β and IL6 on peritoneal macrophage in vitro, but also contributes to TF and inflammatory factor expression in EAPS mice treated with anti-β2GPI or IgG-APS, indicating that TLR4 play an important role in aPL-induced prothrombotic and proinflammatory phenotype.(2) TLR4 is involved in expression of adhesion molecule VCAM-1, ICAM-1 and E-selectin in the aorta of EAPS mice, suggesting that TLR4 plays an important role in anti-β2GPI or IgG-APS-induced proadhesive and proinflammatory phenotype in endothelial cells.(3) TLR4 plays a role not only in arterial thrombosis, but also in the venous thrombus induced by anti-β2GPI antibody, indicating that TLR4 might be an appropriate target’for the development of efficient treatments or therapies for better management of APS.