The Role Of TLR4 In GVHD After Hematopoietic Stem Cell Transplantation And The Underlying Mechanism

Hematopoietic stem cell transplantation (HSCT) has become one of the main strategies for the treatment of malignant hematological disease. However the full potential of HSCT has not been realized because of transplant related complication. Prominent among these complications is graft-versus-host disease (GVHD) which remains a major cause of morbidity and mortality after HSCT. During the past several years, Toll like receptors 4 (TLR4) has been showed to be a key molecule in the innate immunity and immune tolerance. The role of TLR4 in GVHD after HSCT was still unknown. The objective of this study is intended to elucidate the role and the mechanism of TLR4 in GVHD after HSCT. The project will try to determine the frequency of TLR4 mutations in a large cohort of HSCT recipients and their HLA-identical donors and to test the hypothesis that TLR4 mutations in either the recipient or donor influence the risk of acute GVHD in allogeneic HSCT recipients. To explore the mechanism of TLR4 in GVHD, the HSCT model with TLR4 deficient mouse will be employed. Not only the frequency of GVHD and survival term after HSCT, but also the function of DC and cytotoxicity of T lymphocyte will be investigated in this allograft mouse models. This project will probably acquire new insights into the pathphisology of GVHD and foster the development of new therapies to control GVHD after the hematopoietic stem cell transplantation.A few of controversial studies have not well identified the role of TLR4 activation on GVHD after HSCT. One retrospective study indicated a reduced risk of acute GVHD associated with TLR4 mutations in humans, whereas another study reported an increased risk for severe GVHD and intestinal GVHD or no significant influence of TLR4 on the incidence of GVHD. In order to clarify the role of TLR4 in the occurrence of acute GVHD after HSCT,we collected 208 samples from HSCT recipients and their HLA-identical donors to test the hypothesis that TLR4 polymorphils either in either the recipients or donors influence the risk of acute GVHD in allogeneic HSCT recipients. We examined the Asp299Gly and Thr339Ile polymorphism in all samples from post transplant GVHD patients and their donors using the TaqMan and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The products of PCR were sequenced to identify and characterize the SNPs, at the same time,.we used two restriction enzymes to randomly test samples, NcoⅠfor the PCR-RFLP assay of the Asp299Gly allele and Hinfl for the Thr339Ile allele. No homozygous or heterozygous variant alleles of the Asp299Gly or Thr339Ile polymorphisms were detected in any samples of our study.Our results demonstrate that the frequencies of variant alleles of TLR4 polymorphisms were variable among different ethnic groups. The frequency of TLR4 Asp299Gly polymorphism is higher in African and in Europeans.The highest mutation rate was reported amongst western Iranians. However the G allele or T allele of the Asp299Gly or Thr399Ile polymorphisms were very rare in other Asians populations.Our results demonstrated that the TLR4 Asp299Gly and Thr399Ile polymorphisms might be very rare in Chinese population, especially in transplant population of Zhejiang. The results of this study cannot confirm the role of TLR4 mutation in the pathogenesis of GVHD in humans.In followed studies, we evaluated the role of TLR4 in GVHD associated with allogeneic bone marrow transplantation by TLR4 knockout mice and found a obvious conclusion.We confirmed the hypothesis that TLR4 mutations in either the recipient or donor mice influence the risk of acute GVHD in allogeneic HSCT recipient mouse.In this study, we have found that TLR4 plays a critical role in the occurrence of acute GVHD using murine GVHD models. Donor T cell being activated by APCs is the most important one among the three stages described in acute GVHD, and then migrate to target organs that caused the recipient's target tissue damage. Dendritic cells (DCs), as immune cells, can recognize and present alloantigens to initiate the adaptive immune response and induce transplant rejection. People always believe that, DC is a kind of APC which having strongest antigen-presenting ability, and can unique activate naive T cell and leading to acute graft rejection mainly。TLR signaling leads to the induction of costimulatory molecules and cytokines necessary for activation and differentiation of T lymphocytes. In an initial effort to identify the effect of DC from TLR4 knockout (TLR4-/-) mice on allograft immune tolerance, we used the mice model of allogeneic Bone Marrow Transplantation (BMT), and analyzed the related immunological mechanisms. DC can affect donor T cells' proliferation, differentiation and induce graft rejection. We have clearly demonstrated that TLR4 plays an essential role in the course of mice GVHD.BALB/c and C57BL/6 mice are purebred and congenic mice strains with different major and minor histocompatibility antigen (MHC-Ⅰ,Ⅱ) that are available for graft rejection study. TLR4-/- refers to TLR4 knockout mice which have the same background of C57BL/6 mice (TLR4+/+). It was reported that both host-derived and donor-derived APCs were present in secondary donor lymphoid tissue after allo-BMT. both host-derived and donor-derived APCs are present in secondary lymphoid organs. The T-cell receptor (TCR) of the donor T cells can not only recognize alloantigens on host APCs (direct presentation) but also donor APCs (indirect presentation).To gain insight into the effect of TLR4-/- derived DC in GVHD between direct presentation and indirect presentation, we designed mice allo-BMT model with TLR4+/+ and TLR4-/- mice as the donor, and injected with bone marrow cells and splenocytes obtained from BALB/c recipient. In subsequent reverse direction experiments, in which BALB/c as the recipient, then TLR4+/+ and TLR4-/- mice as the donor. We want to learn the different expression of GVHD on TLR4-/- compare with TLR4+/+ recipient mice when TLR4-/- derived DC on direct and indirect presenting alloantigens. The results was demonstrated that TLR4-/- mice as either donor or recipient, the chimera have slightly GVHD and lower clinic score, less weight loss, minor liver and small intestine damage. These indicated that TLR4-/- mice can induce tolerance against specific antigens, resulting immune tolerance in allograft rejection. In addition, we also observed that TLR4-/- mice as the donor compared with TLR4-/- mice as the recipient, the chimera showed later time on the occurrence of GVHD(median time:16.1 days vs 12.3 days), rare bleeding blot on surface of liver and small intestine. We believed that it was due to different proportion of both host-derived and donor-derived DC on recipient lymphoid tissues after transplantation. With TLR4-/- mice as the donor, provided DC were the major DC in the chimera lymphoid tissue after allo-BMT 14 days. Because of its weaker ability on activating T cells by presenting allogeneic antigen, the degree of GVHD occurred is slightly.In next study, we compared the antigen presenting ability of TLR4-/- mice derived DC, the MLR response with allogeneic T cell both in vivo and vitro, inhibited the differentiation of Thl subsets and induced hyporesponsiveness of alloantigen-specific T cells in vitro to explain the immunological mechanisms of transplant tolerance. Recent studies suggest that DCs express the highest concentration of MHC Class II molecules, such as CD86, CD80, and CD40 on their surface, play a major role in the activation of naive T lymphocytes. However, this is not enough to cause activation and maturation of T cells, anergizing and leading to apoptosis, if the antigen presenting cell can not express enough costimulatory molecules。We also realized that TLR4-/- could protect immature DC from mature by exogenous LPS in vitro. Our data from FACS demonstrated that Day-5-DCs from TLR4+/+ mice were stimulated by exogenous LPS (1μg/ml) for 24h, expressed high levels of CD80, CD86, CD40 and MHC-Ⅱmolecules, showing mature DC phenotypes, increased IL-12 secretion levels. While TLR4-/-derived Day-5-DC were still maintain immature phenotype and IL-12 secretion of DC have no difference with or without LPS stimulation, making T cell unreactive to activation.Interleukin 12(IL-12), as mainly produced by dendritic cells in response to lipopolysaccharide, can induce interferon-γ(IFN-γ) production and trigger CD4+ T cells to differentiate into Thl phenotype, The proliferation of T cells will inhibited by lacking of IL-12. The immature phenotype of TLR4-/- derived DCs significantly decreased the ability of antigen-presenting after LPS treated, reduced MLR between DC and allogeneic T cells, induced weaker T cell proliferation, leading to reduced the occurrence of GVHD. In vivo experiment, the expression of costimulatory molecules CD80 and CD86 in DCs obtained from spleen cells of TLR4-/-→BALB/c mice after BMT 21 day were obviously decreased compared to TLR4+/+→BALB/c, TLR4 gene deficiency will help DC maintain immature with low sensitivity of T cells.It has been suggested that the Thl/Th2 polarization of T helper cell subsets plays an important role in the development of GVHD. Thl switch to Th2 cells can change the local graft immune response, inhibit cell-mediated rejection. The Thl response is characterized by the production of IFN-y, IL-2 and characteristics of the Th2 response is the production of IL-4 and IL-10. IL-17 plays a critical role in prevention of extracellular pathogens as the third subset of T cell, which deficiency may lead to the progression of inflammation and severe autoimmune diseases.To determine the differentiate effect on allogeneic CD4+ T cells with TLR4-/-mice derived DC on MLR, we have tested and analyzed the expression of Th cytokines in the supernatant of MLR culture media. We concluded that the levels of cytokines produced by Thl subsets, IFN-y, IL-2, significantly decreased after immature DC from TLR4-/-mice and CD4+ T cell from BALB/C mice in MLR (p<0.01). While the levels of cytokines produced by Th2 subsets, IL-10, IL-4, also significantly decreased at the same time, interestingly (p<0.05). Results indicate that DC from TLR4-/- mice, can inhibit the proliferation of Thl subsets, without inducing Thl switch to Th2 subsets. Besides the expression of IL-17 in the supernatant of MLR from TLR4+/+mice was significantly higher than TLR4-/-mice, which means IL-17 has promoted effect to the GVHD progression. In MLR experiments we found TLR4-/- derived DC had attenuated effect on allogeneic T cell activation compared to TLR4+/+ group, detected by FCS and CFSE. The results indicate that TLR4 gene deficiency in MLR response in vitro will keep DC in immature state, leading to alloantigen hyporesponsiveness of T cells without fully activation.The cytokines produced by Thl subsets are associated with the severity GVHD and mortality. To investigate the variance level of cytokines produced by Th1/Th2 subset after transplantation in murine BMT model, to speculate on the direction of allogeneic T cells differentiation under the influence of TLR4-/- derived DC in vivo. We used ELISA method to detect the level of cytokines in the serum of day 7 post-transplantation chimeric mice, found the levels of IL-2 as representation of Thl subset from TLR4-/-donor mice were significantly lower. The interesting thing is the level of IFN-y in reverse, which in the serum of TLR4-/-→BALB/c chimeric mice is significantly higher than TLR4+/+→BALB/c mice. It is probability because of other cells such as NK, APC in addition to T cells also secreted more amount of IFN-γ. This result consistent with other scholars view that IFN-γcan reduce the occurrence of GVHD, negatively regulate alloreactive T cells via inhibiting cell differentiation, promoting cell death, and to prevent host tissue from damage. Studies found that Th2 cytokines can be associated with down-regulation cell-mediated immune responses, to antagonistic the effect of Thl cytokines, and to reduce GVHD. TLR4-/- mice as donor, the serum of recipient BALB/c mice were analyzed on day 7 post-transplant for cytokine, the production of IL-10 as the characteristic of the Th2 response showed significantly increased, suggesting that mice can inhibit the occurrence of GVHD after transplantation. In addition, the maximum number of mature DCs emigrates to host lymphoid tissue on day 7 after transplantation. To some extent, this explained why the level of serum cytokines from chimeric mice after transplantation is highest on day 7.There are many factors affect on the GVHD, which have formed a complex network of immune interactions in vivo, As a result, it does ultimately affect the occurrence and severity of GVHD. Signal transduction pathway of LPS/TLR4 plays an important role in immune response, which is mediated and priming by allogeneic T lymphocytes. Based on these, we take our interest in the role of TLR4 in GVHD and finally concluded T cells from TLR4-/- knockout mice can not be effectively activated and then proliferate under the stimulation by APCs. On the contrary, it can induce tolerance for specific antigen, leading to immune tolerance of allogeneic organ transplantation and thus significantly reduce GVHD of host. In future, the development of new targeted therapy for TLR4 gene will provides a new treatment idea.