Project description:Expression profiling of rat major histocompatibility complex and natural killer complex genes in skin explant assays reveals genes that are regulated in graft versus host disease. The major histocompatibility complex (MHC) is the most important genomic region that contributes to the genetic risk of graft rejection and graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Therefore, MHC matching is most essential for the success of clinical transplantations. However, the MHC contains in addition to MHC class I and class II genes that are genotyped for selection of donors further so far unidentified genes that also contribute significantly to the risk to develop acute GVHD. It is difficult to identify these MHC genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes that might be involved in the pathohysiology of GVHD by expression profiling. To reduce the complexity of our model, we used genetically well-defined inbred rat strains (PVG and BN) and skin explant assays, an in-vitro-model of graft versus host reaction (GVHR), to analyse the expression of MHC and natural killer complex (NKC) genes in a cutaneous GVHR by a custom microarray. A higher percentage of genes in these immunologically highly important genomic regions were regulated than in the rest of the genome. We observed a statistically significant regulation of 25 MHC and 6 NKC genes and 168 other genes (i.e., 4.9%, 14.0%, and 6.6%, respectively) in rat skin explants cultured in the presence of pre-stimulated allogeneic lymphocytes compared to control samples cultured in the presence of syngeneic lymphocytes. Seven MHC and 3 NKC genes were selected for analysis by quantitative real-time polymerase chain reaction PCR. Most of the results of the microarray were confirmed in the same experimental set that was used for the microarray analysis and in a second independent experimental set of skin explant samples. In addition, GVHD-affected skin lesions of transplanted rats were analysed and similar regulations of most of the selected MHC and NKC genes were observed. Thus, our skin explant model of GVHR is informative for the gene regulation during GVHD. Interestingly, the human homologues of several of the regulated genes are polymorphic and could therefore contribute to the genetic risk of GVHD. These genes include RT1-Dmb, C2, Aif1, Spr1, Ubd, and Olr1. The human homologues of these genes might be useful for risk assessment and diagnosis of GVHD in patients. Two-condition experiment, Stimulated vs. NonSimulated cells. 12 Biological replicates, 2 Technical Replicates (Dye Swap) per Biological Replicate.

Project description:During inflammatory diseases, infiltrating immune cells can induce endothelial activation and angiogenesis by cytokines and other mediators. The inhibition of inflammation-associated angiogenesis ameliorates inflammatory diseases by reducing the recruitment of tissue infiltrating leukocytes. However, there is limited evidence on initial mechanisms of both processes. We show in experimental models that angiogenesis precedes leukocyte infiltration during graft-versus-host disease (GVHD). GVHD, a systemic inflammatory disease caused by host-reactive T cells primarily attacking the epithelial cells of the GVHD target organs liver, skin and intestines, leads to substantial morbidity and mortality after allogeneic hematopoetic stem cell transplantation. A key feature of GVHD is the incompletely understood organ tropism to skin, liver and the intestines. We demonstrate that angiogenesis initiates GVHD in target organs whereas in non-target organs no angiogenesis and no subsequent inflammation occurred. To shed light into the initiation phase of angiogenesis we looked at classical endothelial cell activation signs, such as upregulation of the Vegfa/VEGFR1+2 pathway or adhesion molecules, but could not find an association. However, endothelial cells in early GVHD showed profoundly higher deformation in real-time deformability cytometry suggesting enhanced migratory and proliferating potential of endothelial cells during the initiation of angiogenesis in GVHD target organs. We used microarrays to detect alternative pathways in endothelial cells of the GVHD target organ colon that could be responisble for initial angiogenesis in GVHD. We identified several up-and down-regulated genes during this process. Strikingly, we found several cytoskeleton as well as metabolic-associated and related gene alterations which may explain the enhanced migration and proliferation of endothelial cells in initiating GVHD. Our study adds evidence to the hypothesis that angiogenesis can initiate inflammation and provides novel insight in pathophysiology and tropism of GVHD.

Project description:Expression profiling of rat major histocompatibility complex and natural killer complex genes in skin explant assays reveals genes that are regulated in graft versus host disease. The major histocompatibility complex (MHC) is the most important genomic region that contributes to the genetic risk of graft rejection and graft versus host disease (GVHD) after haematopoietic stem cell transplantation. Therefore, MHC matching is most essential for the success of clinical transplantations. However, the MHC contains in addition to MHC class I and class II genes that are genotyped for selection of donors further so far unidentified genes that also contribute significantly to the risk to develop acute GVHD. It is difficult to identify these MHC genes by genetic association studies alone due to linkage disequilibrium in this region. Therefore, we aimed to identify MHC genes that might be involved in the pathohysiology of GVHD by expression profiling. To reduce the complexity of our model, we used genetically well-defined inbred rat strains (PVG and BN) and skin explant assays, an in-vitro-model of graft versus host reaction (GVHR), to analyse the expression of MHC and natural killer complex (NKC) genes in a cutaneous GVHR by a custom microarray. A higher percentage of genes in these immunologically highly important genomic regions were regulated than in the rest of the genome. We observed a statistically significant regulation of 25 MHC and 6 NKC genes and 168 other genes (i.e., 4.9%, 14.0%, and 6.6%, respectively) in rat skin explants cultured in the presence of pre-stimulated allogeneic lymphocytes compared to control samples cultured in the presence of syngeneic lymphocytes. Seven MHC and 3 NKC genes were selected for analysis by quantitative real-time polymerase chain reaction PCR. Most of the results of the microarray were confirmed in the same experimental set that was used for the microarray analysis and in a second independent experimental set of skin explant samples. In addition, GVHD-affected skin lesions of transplanted rats were analysed and similar regulations of most of the selected MHC and NKC genes were observed. Thus, our skin explant model of GVHR is informative for the gene regulation during GVHD. Interestingly, the human homologues of several of the regulated genes are polymorphic and could therefore contribute to the genetic risk of GVHD. These genes include RT1-Dmb, C2, Aif1, Spr1, Ubd, and Olr1. The human homologues of these genes might be useful for risk assessment and diagnosis of GVHD in patients.

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Screening regulated pathway and genes expression in intestinal GVHD patients

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