Highlighting TH-1 Rather Than TH-17 Cytokine Network in Acute Cutaneous Gvhd

Abstract

Graft versus Host disease (GVHD) is a major lethal complication of allogeneic hematopoietic stem cell transplantation (HSCT). As skin is one of the major organs involved and the most accessible, it serves as an ideal organ site to further understand the pathogenesis and progression of acute GVHD and could serve as a window into pathophysiology of other organ sites. Cytokine network aberrations are a central feature of the pathogenesis of acute cutaneous GVHD, terminating in the hallmark of premature apoptosis of keratinocytes. The purpose of this study is to delineate the cytokine network aberrations at the molecular and cellular level in affected skin from patients with acute GVHD with the goal of developing novel therapeutic approaches to defined targets with the goal of preventing or reducing acute GVHD. We performed immuno-phenotypic profiling of acute cutaneous GVHD biopsies determining relative numbers of various types of immunocytes including T cells, dendritic cells, and macrophages. Cytokine expression (IFNγ and TNFα) was determined by RT-PCR on RNA extracted from eight different lesional skin and two non-lesional biopsies. We analyzed for TH-1 (IFN γ and TNFα), TH-2 (IL-4) and TH-17 (IL-17A, IL-22) cytokine mRNAs. We observed that TH-1 cytokine mRNAs were detected in 7 out of 8 different lesional skin biopsies but none of two nonlesional skin biopsies. Interestingly TH-2 and TH-17 cytokine mRNAs were not detectable in acute cutaneous GVHD lesions or nonlesional skin biopsies. RNA extracted from a 2-way mixed lymphocyte reaction (MLR) served as a positive control for these RT-PCR results. Given recent data that suggested that Notch-1 regulated TH-1 polarization by transcriptionally controlling the Tbx gene (MinterLM et al 2005), we hypothesized that inhibiting Notch signaling might be of potential therapeutic importance given these results of TH-1 polarization in acute skin GVHD. To block Notch signaling we utilized several pharmacological gamma secretase inhibitors (which may have therapeutic applications), and supplemented this pan-Notch receptor inhibition approach by use of a Notch-1 specific siRNA. Preliminary studies suggest that both approaches reduce IFN γ levels in allogeneic T cell responses in-vitro. Further experiments are in progress to confirm these findings and to determine the molecular mechanism. This study begins to define the cytokine network operative within acute cutaneous GVHD lesions highlighting the finding that TH-1 rather than TH-17 cytokines may be the more important target.