Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.
Key Points• IL-33 and ST2 expression are increased post-conditioning and with GVHD, resulting in increased T-cell activation via the IL-33/ST2 axis.• Infusion of ST2-Fc protein exploits sST2's function as a negative regulator of acute GVHD inhibiting proinflammatory cytokines.Interleukin (IL)-33 binding to the receptor suppression of tumorigenicity 2 (ST2) produces pro-inflammatory and anti-inflammatory effects. Increased levels of soluble ST2 (sST2) are a biomarker for steroid-refractory graft-versus-host disease (GVHD) and mortality. However, whether sST2 has a role as an immune modulator or only as a biomarker during GVHD was unclear. We show increased IL-33 production by nonhematopoietic cells in the gastrointestinal (GI) tract in mice post-conditioning and patients during GVHD. Exogenous IL-33 administration during the peak inflammatory response worsened GVHD. Conversely, GVHD lethality and tumor necrosis factor-a production was significantly reduced in il33 2/2 recipients. ST2 was upregulated on murine and human alloreactive T cells and sST2 increased as experimental GVHD progressed. Concordantly, st2 2/2 vs wild-type (WT) donor T cells had a marked reduction in GVHD lethality and GI histopathology. Alloantigen-induced IL-18 receptor upregulation was lower in st2 2/2 T cells, and linked to reduced interferon-g production by st2 2/2 vs WT T cells during GVHD. Blockade of IL-33/ST2 interactions during allogeneic-hematopoietic cell transplantation by exogenous ST2-Fc infusions had a marked reduction in GVHD lethality, indicating a role of ST2 as a decoy receptor modulating GVHD. Together, these studies point to the IL-33/ST2 axis as a novel and potent target for GVHD therapy. (Blood. 2015;125(20):3183-3192)
Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic receptor P2X7 that a noninfectious damage has occurred. A second ATP-activated purinergic receptor involved in inflammatory diseases is P2Y2. In this study, we used P2y2 2/2 mice to test the role of this receptor in GvHD. P2y2recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanistically, P2y2 2/2 inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2 + cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD.
The IL-1 superfamily member IL-33 is produced in barrier tissues. IL-33 binds to the receptor suppression of tumorigenicity 2 (ST2), expressed on stromal cells, regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), and macrophages. IL-33 has both anti-inflammatory and pro-inflammatory properties. It is not known if IL-33 plays a role in acute GvHD, and if so what properties it exerts. By immunohistochemistry staining of gut tissues, IL-33 production by non-hematopoietic cells was increased in mice post-conditioning and in patients during GvHD. To determine whether IL-33 could augment GvHD via a host signaling mechanism, we compared st2-/-to wildtype (wt) hosts and observed decreased GvHD lethality (Figure 1A). Additionally, IL-33-/- versus wt hosts had a marked decrease in GvHD lethality and reduced TNFα production. Conversely, IL-33 administration during the peak inflammatory response worsened GvHD. Previous studies have shown increased levels of the soluble form of ST2 (sST2) are a biomarker for steroid-refractory GvHD (Vander Lugt, NEJM, 2013). We hypothesized that sST2 acted not only as an indicator of tissue injury and biomarker of GvHD but also as an immune modulator during GvHD. In rodents, we found that ST2 was upregulated on alloreactive T cells and sST2 increased as GvHD progressed. St2-/-versus wt donor T cells had a marked reduction in GvHD lethality (Figure 1B) without compromise of graft-vs-leukemia responses. Comparable data was seen in 2 different strain combinations. Alloantigen-induced IL-18 receptor upregulation was significantly lower in the absence of ST2, which was linked to significantly reduced IFNγ production by st2-/- vs wt CD4 and CD8 T cells during GvHD. Similarly, sST2 transgenic hosts and wt recipients given exogenous sST2-Fc fusion protein infusions (Figure 1C) to block ST2/IL-33 interaction each had significantly reduced GVHD lethality, establishing the functional role of ST2 as a decoy receptor modulating GVHD. During the peak of the GvHD inflammatory response, IL-33 signalling of either donor or host cells promoted activation of donor T cells, while the use of exogenous sST2-Fc protein to prevent IL33/ST2 engagement ameliorates disease. Together, these studies point to targeting of the IL-33/ST2 axis as a novel and potent target for GvHD therapy. Disclosures Warncke: Novartis Pharma AG: Employment. Junt:Novartis Pharma AG: Employment.
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