Role of monocyte-induced development of Th17 cells, the heat shock protein 90 and proinflammatory S100 proteins in the pathogenesis of graft-versus-host disease

Abstract

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for patients with hematologic malignancies, aplastic anaemia, and congenital immunodeficiency disorders. One of the major serious morbidities associated with HCT is the development of acute or chronic graft-versus-host disease (GvHD). The pathophysiology of GvHD is complex and not fully understood. The role of Th17 cells during GvHD is discussed controversially and still remains unclear. In this study, the induction of Th17 cells by monocytes of patients with GvHD in vitro was analysed demonstrating that monocytes isolated from patients with acute skin and intestinal GvHD stage I-IV or chronic GvHD induce significantly increased levels of Th17 cells compared to patients without GvHD after HCT and healthy controls. Several studies suggest using the determined levels of regulatory (Treg) cells and the ratio of Th17 cells to Treg cells in the peripheral blood of patients as diagnostic markers for GvHD. However, the data of the present study have demonstrated that the determined percentages of Treg cells in peripheral blood mononuclear cells (PBMCs) isolated from patients with acute GvHD do not differ from the assessed percentages of Treg cells in PBMCs of healthy donors and patients without GvHD after HCT. By contrast, the percentages of Treg cells in PBMCs from patients with extensive chronic GvHD seem to be increased in comparison to the healthy controls and the non-GvHD group. The results of the present work further indicate that the calculated ratios of Th17 cells to Treg cells are not altered in patients with acute or chronic GvHD compared to patients without GvHD after HCT. Development and progression of GvHD is mediated by multiple cellular and inflammatory effectors. However, several of these molecules are still unknown. Previous studies have demonstrated that S100 proteins act as innate amplifier of inflammation and play an important role in many inflammatory diseases such as inflammatory bowel disease or rheumatoid arthritis. These proinflammatory S100 proteins belong to the group of Damage Associated Molecular Pattern (DAMP) molecules and are released by activated or damaged phagocytes under conditions of cell stress during infections and autoimmune diseases. Therefore, expression levels of S100 proteins in monocytes and the presence of S100 proteins in the stool, serum and bowel tissue were investigated in patients with acute or chronic GvHD and compared to healthy controls and patients without GvHD after HCT. Additionally, the influence of S100 proteins on monocyte-mediated induction of Th17 cells was analysed. The data of this study demonstrate that the expression of S100 proteins is increased in monocytes from patients with GvHD compared to the controls. Overall, elevated levels of S100 proteins can be detected in the serum, stool and bowel tissue of patients with GvHD demonstrating the release of these phagocyte-specific proteins during GvHD. Furthermore, S100 proteins were found to bind to toll-like receptor 4 (TLR4) on monocytes resulting in the promotion of monocyte-induced Th17 development. These data emphasize the role of S100 proteins in Th17 triggered inflammation. Additionally, it was investigated if the induction of Th17 cells is mediated by proinflammatory cytokines released by monocytes or by cell contact between monocytes and CD4+ T cells. The data of the present study have revealed that monocyte-mediated Th17 development occurs in a cell-cell-contact dependent manner with the involvement of proinflammatory cytokines secreted by in vitro or in vivo activated monocytes. A further part of this thesis examined the influence of heat shock protein 90 (Hsp90) in monocyte-mediated induction of Th17 cells. Hsp90 is a ubiquitously expressed molecular chaperon that is known to play an important role in signal transduction, transcription regulation and survival of the cell. The data of this work have demonstrated that Hsp90 inhibition in in vivo activated monocytes by the geldanamycin derivative 17-DMAG decreases Th17 responses. Further results have shown that the stimulatory effects of proinflammatory S100 proteins on monocyte-induced Th17 development can be blocked by chemical inhibition of Hsp90 using 17-DMAG or by specific siRNA-mediated knockdown of the stress-inducible Hsp90α in monocytes. In contrast to dexamethasone which is a potent synthetic member of the glucocorticoid class of steroids that are widely used drugs for the treatment of acute and chronic GvHD, the chemical Hsp90 inhibitor 17-DMAG does not seem to induce the development of proinflammatory Th17 cells expressing the multi-drug resistance protein 1 (MDR1). MDR1 is an ATP-dependent efflux pump that plays a crucial role in the bioavailability of a wide range of drugs and xenobiotics. Altogether, the results of the present work indicate that levels of proinflammatory S100 proteins are increased in the serum, stool and bowel tissue of patients with GvHD and might promote monocyte-induced development of Th17 cells during GvHD. Specific inhibition of Hsp90 might prevent the induction of inflammation-promoting Th17 cells. Therefore, Hsp90 could be a novel, critical target for the treatment of GvHD.