Role of TLR4 In Acute Gvhd After Allogenetic Hematopoietic Stem Cell Transplantation

Abstract 2538

Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation (HSCT). Lipopolysaccharide (LPS) has been implicated in the pathogenesis of GVHD. The toll-like receptor-4 (TLR4) has been identified as a major receptor for LPS. Here arises the question whether TLR4 mutations may increase risk of microbial infection and affect acute GVHD in allogeneic HSCT recipients. In order to clarify the role of TLR4 in the occurrence of acute GVHD, we detected the interaction of TLR4 mutations in recipient and donor cells and analyzed allogeneic lymphocyte infiltration in the liver, intestine and skin of host mice by immunohistochemistry after allogeneic HSCT. Wild type C57BL/6 (TLR4^+/+) and TLR4 knockout (TLR4^−/−) mice were received myeloablative total body irradiation, followed by tail vein injection of donor BALB/c bone marrow cells and splenocytes to induce acute GVHD. GVHD severity was assessed using clinical scores. In vivo the proliferation activity of allogeneic donor BALB/c T cells in TLR4^−/− and TLR4^+/+ transplanted mice was evaluated ex vivo by flow cytometry after labeling with CFSE. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the proliferation of allogeneic donor BALB/c T cells at different times of coculture with MHC class II antigen presenting cells (APCs) obtained from bone marrow of TLR4^+/+ or TLR4^−/− mice with or without LPS stimulation for 24 h. When myeloablative irradiated TLR4^−/− mice, instead of wild-type mice, were used as graft recipients, clinical score of acute GVHD severity were decreased and survival were increased (18/30 vs 9/30 mice still alive at day 30, GVHD clinical score 6.7 vs 4.5). The decreased mortality and morbidity in TLR4^−/− mice were associated with reduced proliferation of allogeneic donor cells transplanted in these mice.We evaluated the activation of spleen APCs in TLR4^+/+ or TLR4^−/− mice after myeloablative conditioning. Higher expression of CD80 and CD86 costimulatory molecules on MHC class II cells was detected in wide type strain at 3 d postirradiation. Ex vivo experiments CD80, CD86 and CD40 costimulatory markers on bone marrow APCs of C57BL/6 wild-type more significant up-regulation than TLR4^−/− mice after LPS stimulation 24 h. TLR4^−/− recipients receiving BALB/c donors developed significantly less GVHD as measured by liver, skin and intestinal of mice histopathology compared with TLR4^+/+ recipients. Cytokines IL-2/IFN-γexpression in TLR4^+/+ recipients mice serum was stronger but IL-4/IL-10 expression was weaker comparing to that in TLR4^−/− recipients. These results suggest that TLR-4 mutation in donor cells increases the expression of Th2-related cytokines and decreases the risk of GVHD after allogeneic bone marrow transplantation.These data reveal that TLR4 mutations in recipitents is crucial in the prevention of GVHD, while responsiveness of wide type mice APC to LPS may be an important risk factor for acute GVHD. Overall together, these results suggest that the function of TLR4 has influence on the occurrence of acute GVHD, which might provide methods to reduce this complication after allogeneic hematopoietic stem cell transplantation.