Christoph Dürr

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X7R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X7R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X7R blockade or genetic deficiency of P2X7R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X7R could lead to the development of tolerance without the need for intensive immunosuppression.

Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity

Allergic contact hypersensitivity (CHS) is a T cell–mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rβ2–deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12–independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) β2, or both, we show that the concomitant absence of TLR4 and IL-12Rβ2, but not the absence of TLR4 or IL-12Rβ2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rβ2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12–independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12–competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rβ2–deficient mice, but not in germ-free TLR4/IL-12Rβ2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Contact sensitizers induce skin inflammation via ROS production and hyaluronic acid degradation.

Background Allergic contact dermatitis (ACD) represents a severe health problem with increasing worldwide prevalence. It is a T cell-mediated skin disease induced by protein-reactive organic and inorganic chemicals. A key feature of contact allergens is their ability to trigger an innate immune response that leads to skin inflammation. Previous evidence from the mouse contact hypersensitivity (CHS) model suggests a role for endogenous activators of innate immune signaling. Here, we analyzed the role of contact sensitizer induced ROS production and concomitant changes in hyaluronic acid metabolism on CHS responses. Methodology/Principal Findings We analyzed in vitro and in vivo ROS production using fluorescent ROS detection reagents. HA fragmentation was determined by gel electrophoresis. The influence of blocking ROS production and HA degradation by antioxidants, hyaluronidase-inhibitor or p38 MAPK inhibitor was analyzed in the murine CHS model. Here, we demonstrate that organic contact sensitizers induce production of reactive oxygen species (ROS) and a concomitant breakdown of the extracellular matrix (ECM) component hyaluronic acid (HA) to pro-inflammatory low molecular weight fragments in the skin. Importantly, inhibition of either ROS-mediated or enzymatic HA breakdown prevents sensitization as well as elicitation of CHS. Conclusions/Significance These data identify an indirect mechanism of contact sensitizer induced innate inflammatory signaling involving the breakdown of the ECM and generation of endogenous danger signals. Our findings suggest a beneficial role for anti-oxidants and hyaluronidase inhibitors in prevention and treatment of ACD.

Impact of Mammalian Target of Rapamycin Inhibition on Lymphoid Homing and Tolerogenic Function of Nanoparticle-Labeled Dendritic Cells following Allogeneic Hematopoietic Cell Transplantation

Dendritic cells (DC) play a major role in the pathogenesis of graft-vs-host disease (GvHD). Directed modification of surface molecules on DC that provide instructive signals for T cells may create a tolerogenic DC phenotype that affects GvHD severity. To investigate the impact of the mammalian target of rapamycin (mTOR) inhibitor rapamycin (RAPA) on in vivo migratory capacities, tolerogenic function, and B7 superfamily surface expression on DC following allogeneic hematopoietic cell transplantation (aHCT), we generated a platform for magnetic resonance imaging and bioluminescence imaging based cell trafficking studies. Luciferase transgenic DC were labeled with superparamagnetic iron oxide nanoparticles bound to a murine IgG Ab that allowed for Fc-γR-mediated endocytosis. Locally injected luc+ DC could be tracked within their anatomical context by bioluminescence imaging and magnetic resonance imaging after aHCT, based on stable intracellular localization of superparamagnetic iron oxide-IgG complexes. RAPA preconditioned DC (DC-R) displayed reduced expression of MHC class II, B7-1 (CD80), and B7-2 (CD86) but not B7-H4 whose ligation of T cells has a profound inhibitory effect on their proliferation and cytokine secretion. DC-R of recipient genotype reduced GvHD severity that is compatible with their tolerogenic phenotype. CCR5, CCR7, and CD62L expression was not affected by mTOR inhibition, which allowed for DC-R in vivo trafficking to secondary lymphoid compartments where immunregulation is required. This study is the first to delineate the impact of RAPA on DC migration and tolerogenic function after aHCT. Modification of the DC phenotype by mTOR inhibition may have therapeutic potential in an attempt to reduce GvHD following aHCT.

The Sphingosine 1-Phosphate Receptor Agonist FTY720 Potently Inhibits Regulatory T Cell Proliferation In Vitro and In Vivo

CD4+CD25+ regulatory T cell (Treg) entry into secondary lymphoid organs and local expansion is critical for their immunosuppressive function. Long-term application of the sphingosine-1 phosphate receptor agonist FTY720 exerts pleiotropic anti-inflammatory effects, whereas short-term FTY720 boosts antiviral immunity. In this study, we provide evidence that FTY720 potently inhibits Treg proliferation in vitro and in vivo without affecting their viability, phenotype, or in vitro immunosuppression. In contrast, adoptively transferred Treg exposed ex vivo to FTY720 lost their protective effects in murine models of acute glomerulonephritis and acute graft-vs-host disease. On a cellular level, FTY720 inhibits IL-2-induced STAT-5 phosphorylation, paralleled by a loss of FoxP3 expression during Treg expansion in vitro. Notably, loss of in vivo immunosuppression is not due to impaired migration to or localization within secondary lymphoid organs. We could even show a selective trapping of adoptively transferred Treg in inflammatory lymph nodes by FTY720. Finally, Treg isolated from animals systemically exposed to FTY720 also exhibit a significantly impaired proliferative response upon restimulation when compared with Treg isolated from solvent-treated animals. In summary, our data suggest that sphingosine-1 phosphate receptor-mediated signals induced by FTY720 abrogate their in vivo immunosuppressive potential by blocking IL-2 induced expansion, which is indispensable for their in vivo immunosuppressive activity.

Regulation of different inflammatory diseases by impacting the mevalonate pathway

The 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase inhibitors (statins) interfere with the mevalonate pathway. While initially developed for their lipid‐lowering properties, statins have been extensively investigated with respect to their impact on autoantigen and alloantigen driven immune responses. Mechanistically it was shown that statins modify immune responses on several levels, including effects on dendritic cells, endothelial cells, macrophages, B cells and T cells. Several lines of evidence suggest that statins act in a disease‐specific manner and are not effective in each immune disorder. This review discusses possible modes of action of statins in modulating immunity towards autoantigens and alloantigens.

CXCL12 Mediates Immunosuppression in the Lymphoma Microenvironment after Allogeneic Transplantation of Hematopoietic Cells

Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen-presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)-based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12-dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT.

Inhibition of protein geranylgeranylation and farnesylation protects against graft-versus-host disease via effects on CD4 effector T cells.

Despite advances in immunosuppressive regimens, acute graft-versus-host disease remains a frequent complication of allogeneic hematopoietic cell transplantation. Pathogenic donor T cells are dependent on correct attachment of small GTPases to the cell membrane, mediated by farnesyl- or geranylgeranyl residues, which, therefore, constitute potential targets for graft-versus-host disease prophylaxis. A mouse model was used to study the impact of a farnesyl-transferase inhibitor and a geranylgeranyl-transferase inhibitor on acute graft-versus-host disease, anti-cytomegalovirus T-cell responses and graft-versus-leukemia activity. Treatment of mice undergoing allogeneic hematopoietic cell transplantation with farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor reduced the histological severity of graft-versus-host disease and prolonged survival significantly. Mechanistically, farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor treatment resulted in reduced alloantigen-driven expansion of CD4 T cells. In vivo treatment led to increased thymic cellularity and polyclonality of the T-cell receptor repertoire by reducing thymic graft-versus-host disease. These effects were absent when squalene production was blocked. The farnesyl-transferase inhibitor and geranylgeranyl-transferase inhibitor did not compromise CD8 function against leukemia cells or reconstitution of T cells that were subsequently responsible for anti-murine cytomegalovirus responses. In summary, we observed an immunomodulatory effect of inhibitors of farnesyl-transferase and geranylgeranyl-transferase on graft-versus-host disease, with enhanced functional immune reconstitution. In the light of the modest toxicity of farnesyl-transferase inhibitors such as tipifarnib in patients and the potent reduction of graft-versus-host disease in mice, farnesyl-transferase and geranylgeranyl-transferase inhibitors could help to reduce graft-versus-host disease significantly without having a negative impact on immune reconstitution.

High expression of GCLC is associated with malignant melanoma of low oxidative phenotype and predicts a better prognosis

Reactive oxygen species (ROS) are strongly implicated in melanoma development, and treatment with antioxidants has shown efficacy in suppressing malignant transition and progression. Here, we investigated the significance of the glutamate-l-cysteine ligase catalytic subunit (GCLC) expression, a key regulator of glutathione synthesis, for malignant melanoma. A large set of melanoma cell lines (n = 36) was analyzed, and higher GCLC levels were associated with lower presence of intracellular ROS and interestingly also lower rates of cell proliferation. Moreover, treatment with the antioxidant N-acetylcysteine efficiently reduced the growth speed of several investigated malignant cells. In addition GCLC expression was significantly linked to a prominent set of cellular antioxidants, accounting for the observed lower basal levels of oxidative stress and higher antioxidative capacity. Key attributes defining the malignant phenotype of melanoma cells including survival, invasiveness, and switch from E-cadherin to N-cadherin expression were more prominent in cells with lower GCLC expression. Our findings were further corroborated by observations in Rag2−/−γc−/−mice, in which melanoma cells with lower GCLC expression depicted a dramatically stronger tumor growth. Furthermore, prognostic significance of GCLC expression was investigated in patients (n = 28) with advanced malignant melanoma. High tumor immunoreactivity for GCLC was a significant determinant for better 5-year overall survival. Conclusively, we show for the first time that GCLC may serve a dual role, as a surrogate marker for cellular redox state as well as malignant potential of melanoma cells. These promising results regarding its prognostic significance as well as its potential as a pharmacological target require further in-depth investigations.

Graft‐versus‐host disease reduces regulatory T‐cell migration into the tumour tissue

The therapeutic principle of allogeneic haematopoietic cell transplantation (allo‐HCT) is based on an active donor immune system that eliminates host‐derived tumour cells. We hypothesized that in addition to the alloantigen‐driven anti‐tumour response, disruption of the immunological microenvironment within the tumour is responsible for its elimination after allo‐HCT. We observed that induction of graft‐versus‐host disease (GvHD) significantly reduced the abundance of luc+ FoxP3+ regulatory T (Treg) cells in the tumour tissue, which is indicative of impaired or over‐ridden tumour recruitment signals towards Treg cells. Analysis of the intestines and liver revealed chemokines and purine nucleotides as candidates for attracting Treg to these sites of inflammation. Despite its expression on tissue‐residing Treg cells, the chemokine receptor CCR3 was not critical for Treg‐cell function following allo‐HCT. Extracellular ATP can attract immune cells via P2Y2. P2Y2 was found to be expressed on Treg cells, and we found a partial reduction of GvHD prevention when P2Y2−/− rather than P2Y2+/+ Treg cells were given. Exogenous local inflammation reduced Treg‐cell accumulation in the tumour, suggesting a potential clinical approach to prevent Treg‐cell‐mediated tumour escape. In conclusion, we demonstrate that GvHD‐related inflammation reduced Treg‐cell numbers at the tumour sites, which may in turn help to explain the observation that patients with GvHD have a lower risk of tumour relapse.