Protein-Based Identification of Inflammatory Signaling Pathways in Graft-Versus-Host-Disease (GVHD): Evidence for STAT1 Pathway as a Key Regulator in GVHD.

The pathogenesis of GVHD is influenced by the immunogenetic disparities between donor and recipient, the presence of host-derived antigen-presenting cells, the inflammatory reaction in response to conditioning-induced tissue damage and cytokine secretion. Intestinal damage and subsequent translocation of LPS into the circulation have been shown to be central to the subsequent pathogenetic events occurring in development of GVHD. Interfering with this proinflammatory response by targeted therapy might be an attractive new approach for inhibiting the development of GVHD. We have therefore started to comprehensively analyze the inflammatory signaling pathways, which are activated during the induction phase of GVHD in order to identify key molecular regulators of GVHD, which might serve as targets for future therapy. For this purpose GVHD was in induced in the fully MHC-mismatched BALB/c to B6 strain combination following lethal irradiation with 9.5 Gy. Spleens as well as GVHD target organs liver, skin and small bowel and colon were harvested on days 1, 3, and + 6 post-BMT to study activation of JAK-STAT, MAPK, PI3-Kinase and NF-κB signaling transduction pathways. Studies were performed using western blot, multiplex assays for analysis of phoshotyrosine proteins and transcription factor binding activity and also standard gel shift assays. Furthermore, GVHD liver tissue was subjected to microarray gene expression profiling. Most strikingly, in contrast to syngeneic and allogeneic controls induction of GVHD was associated with a strong activation of JAK-STAT pathway as determined by Tyr701-specific STAT1 phosphorylation and also Tyr705-specific STAT3 phosphorylation in the liver and spleen as early as on day +1 post-BMT and remained elevated until day +6 post-BMT. In line with these results we observed a significant increase of STAT1 dependent gene expression (e.g. CXCL10 5.6-fold, IRF7 4.5-fold, CXCL9 4.2-fold) as studied by microarray gene expression profiling in the liver. Furthermore, multiplex DNA-binding and standard gel shift studies in the liver revealed that GVHD led to an increased DNA binding activity of most prominently NF-κB but also AP-2, EGR1 and NF1. In conclusion we provide evidence that induction of GVHD is associated with strong activation of the STAT1 pathway in the spleen and liver as early as on day +1 post-BMT in additon to activation of the NF-κB pathway. Therefore this study might help to identify candidate molecules to create more specific therapies targeting the inflammatory component of GVHD and thus reducing the development of GVHD without mitigating the GVL response.