Cathepsin E Deficiency Ameliorates Graft-versus-Host Disease and Modifies Dendritic Cell Motility

Microbial products influence immunity after allogeneic hematopoietic stem cell transplantation (allo-SCT). In this context, the role of cathepsin E (Ctse), an aspartate protease known to cleave bacterial peptides for antigen presentation in dendritic cells (DCs), has not been studied. During experimental acute graft-versus-host disease (GVHD), we found infiltration by Ctse-positive immune cells leading to higher Ctse RNA- and protein levels in target organs. In Ctse-deficient allo-SCT recipients, we found ameliorated GVHD, improved survival, and lower numbers of tissue-infiltrating DCs. Donor T cell proliferation was not different in Ctse-deficient vs. wild-type allo-SCT recipients in MHC-matched and MHC-mismatched models. Furthermore, Ctse- deficient DCs had an intact ability to induce allogeneic T cell proliferation, suggesting that its role in antigen presentation may not be the main mechanism how Ctse impacts GVHD. We found that Ctse deficiency significantly decreases DC motility in vivo, reduces adhesion to extracellular matrix (ECM), and diminishes invasion through ECM. We conclude that Ctse has a previously unrecognized role in regulating DC motility that possibly contributes to reduced DC counts and ameliorated inflammation in GVHD target organs of Ctse- deficient allo-SCT recipients. However, our data do not provide definite proof that the observed effect of Ctse−/− deficiency is exclusively mediated by DCs. A contribution of Ctse−/−-mediated functions in other recipient cell types, e.g., macrophages, cannot be excluded

Reduced Calcium Signaling Is Associated With Severe Graft-Versus-Host Disease: Results From Preclinical Models and From a Prospective EBMT Study

Despite its involvement in various immune functions, including the allogeneic activation of T-lymphocytes, the relevance of calcium (Ca2+) for GVHD pathobiology is largely unknown. To elucidate a potential association between Ca(2+)and GVHD, we analyzed Ca2+-sensing G-protein coupled receptor 6a (GPRC6a) signaling in preclinical GVHD models and conducted a prospective EBMT study on Ca(2+)serum levels prior alloSCT including 363 matched sibling allogeneic peripheral blood stem cell transplantations (alloSCTs). In experimental models, we found decreasedGprc6aexpression during intestinal GVHD. GPRC6a deficient alloSCT recipients had higher clinical and histopathological GVHD scores leading to increased mortality. As possible underlying mechanism, we found increased antigen presentation potential in GPRC6a(-/-)alloSCT recipients demonstrated by higher proliferation rates of T-lymphocytes. In patients with low Ca(2+)serum levels (≤ median 2.2 mmol/l) before alloSCT, we found a higher incidence of acute GVHD grades II-IV (HR = 2.3 Cl = 1.45-3.85p= 0.0006), severe acute GVHD grades III-IV (HR = 3.3 CI = 1.59-7.14,p= 0.002) and extensive chronic GVHD (HR = 2.0 Cl = 1.04-3.85p= 0.04). In conclusion, experimental and clinical data suggest an association of reduced Ca(2+)signaling with increased severity of GVHD. Future areas of interest include the in depth analysis of involved molecular pathways and the investigation of Ca(2+)signaling as a therapeutic target during GVHD

Endothelial damage and dysfunction in acute graft-versus-host disease

Altres ajuts: Deutsche Forschungsgemeinschaft (DFG) TRR221 (B11 Z02), TRR225 (B08)Clinical studies have suggested a potential involvement of endothelial dysfunction and damage in the development and severity of acute graft-versus-host disease (aGvHD). Accordingly, we found an increased percentage of apoptotic caspase 3 positive blood vessels in duodenal and colonic mucosa biopsies of patients with severe aGvHD. In murine experimental aGvHD, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGvHD target organs. During intestinal aGvHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. As recent data demonstrated an association of endothelium-related factors and steroid refractory aGvHD (SR-aGvHD), we analyzed human biopsies and murine tissues from SR-aGvHD. We found extensive tissue damage but low levels of alloreactive T-cell infiltration in target organs, providing the rationale for T-cell independent SR-aGvHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGvHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGvHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGvHD complementing current anti-inflammatory treatment options

TRAIL receptor I (DR4) polymorphisms C626G and A683C are associated with an increased risk for hepatocellular carcinoma (HCC) in HCV-infected patients

<p>Abstract</p> <p>Background</p> <p>Tumour surveillance via induction of TRAIL-mediated apoptosis is a key mechanism, how the immune system prevents malignancy. To determine if gene variants in the TRAIL receptor I (<it>DR4</it>) gene affect the risk of hepatitis C virus (HCV)-induced liver cancer (HCC), we analysed <it>DR4 </it>mutations C626G (rs20575) and A683C (rs20576) in HCV-infected patients with and without HCC.</p> <p>Methods</p> <p>Frequencies of <it>DR4 </it>gene polymorphisms were determined by LightSNiP assays in 159 and 234 HCV-infected patients with HCC and without HCC, respectively. 359 healthy controls served as reference population.</p> <p>Results</p> <p>Distribution of C626G and A683C genotypes were not significantly different between healthy controls and HCV-positive patients without HCC. <it>DR4 </it>variants 626C and 683A occurred at increased frequencies in patients with HCC. The risk of HCC was linked to carriage of the 626C allele and the homozygous 683AA genotype, and the simultaneous presence of the two risk variants was confirmed as independent HCC risk factor by Cox regression analysis (Odds ratio 1.975, 95% CI 1.205-3.236; p = 0.007). Furthermore HCV viral loads were significantly increased in patients who simultaneously carried both genetic risk factors (2.69 ± 0.36 × 10⁶ IU/ml vs. 1.81 ± 0.23 × 10⁶ IU/ml, p = 0.049).</p> <p>Conclusions</p> <p>The increased prevalence of patients with a 626C allele and the homozygous 683AA genotype in HCV-infected patients with HCC suggests that these genetic variants are a risk factor for HCC in chronic hepatitis C.</p

Endothelial damage and dysfunction in acute graft-versus-host disease

Clinical studies suggested that endothelial dysfunction and damage could be involved in the development and severity of acute graft-versus-host disease (aGVHD). Accordingly, we found increased percentage of apoptotic Casp3+ blood vessels in duodenal and colonic mucosa biopsies of patients with severe aGVHD. In murine experimental aGVHD, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGVHD target organs. During intestinal aGVHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. Because recent data demonstrated an association of endothelium-related factors and steroid refractory aGVHD (SR-aGVHD), we analyzed human biopsies and murine tissues from SR-aGVHD. We found extensive tissue damage but low levels of alloreactive T cell infiltration in target organs, providing the rationale for T-cell independent SR-aGVHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGVHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGVHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGVHD complementing current anti-inflammatory treatment options

Initiierung der akuten Graft-versus-Host Krankheit durch Angiogenese

For a variety of malignant diseases of the hematopoietic system, the allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment option. Accordingly, the number of allo-HSCTs performed worldwide has increased greatly in the past decades. However, the mortality after allo-HSCT is high: more than half of the patients die within the first two years because of graft-versus-host disease (GVHD) or GVHD-associated complications as tumor relapse or fatal infections. Acute GVHD is a systemic inflammatory disease caused by alloreactive T cells primarily affecting liver, skin and intestines; so that current therapeutic approaches aim at supressing these effector T cells. This has the significant disadvantage of creating a secondary immune deficiency with increased risk for the above named complications; and most GVHD-related deaths are due to treatment failure or significant toxicities of the used immunosuppressive agents. Therefore, there is an urgent medical need for alternative therapies, which do not attenuate the immune system. Recent work identified such a novel approach: the inhibition of pathologic angiogenesis, which is involved in GVHD as well as in tumor growth after allo-HSCT. The crosstalk between angiogenesis and inflammation is well-established and used in anti-angiogenic treatment strategies. However, it is still not clear if angiogenesis is a consequence or the cause of inflammation. This obstacle and missing suitable targets that are differentially regulated during pathologic and physiologic angiogenesis limit the efficacy of current anti-angiogenic therapies and hinder the development of novel therapeutic approaches. This cumulative thesis consists of two publications, aiming to understand GVHD-initiating mechanisms related to angiogenesis and to provide potential new therapeutic targets being involved in early GVHD while aiming at the endothelium. For a better translation of experimental results into the human setting of allo-HSCT, I first established a clinical relevant, acute GVHD mouse model; as the most commonly used ones exhibit significant clinical limitations. The features of our novel chemotherapy-based, major histocompatibility complex (MHC)-matched GVHD model included profound engraftment, typical clinical features of GVHD, and systemic and target organ-specific inflammation. The clinical pattern and timing of GVHD closely resembled the clinical situation of human leukocyte antigen (HLA)-matched allo-HSCT with GVHD prophylaxis, providing a suitable tool to better understand pathogenic mechanisms in GVHD as well as to develop and translate new treatment approaches. Second, I provided novel evidence on a primary involvement of angiogenesis in the initiation of tissue inflammation in GVHD and found that during initial angiogenesis classical inflammation- associated endothelial activation signs were absent, but metabolic and cytoskeletal alterations occurred, resulting in enhanced migratory and proliferative potential of endothelial cells. I identified potential novel targets for pursuing mechanistic studies and the development of anti- inflammatory therapies aiming at angiogenesis. This study helps to amend the knowledge on the interplay between the vasculature and inflammation and opens a new window to develop novel therapeutic strategies targeting the endothelium after allo-HSCT.Für verschiedene maligne Erkrankungen des hämatopoetischen Systems stellt die allogene hämatopoetische Stammzelltransplantation (allo-HSZT) die einzige kurative Therapieoption dar, sodass die Anzahl der weltweit durchgeführten allo-HSZTs in den letzten Jahrzehnten deutlich angestiegen ist. Allerdings ist die Mortalität nach allo-HSZT sehr hoch. Mehr als die Hälfte der Patienten versterben in den ersten zwei Jahren an der graft-versus-host Krankheit (GVHD) oder an GVHD-assoziierten Komplikationen wie Tumorrezidive oder tödlich verlaufende Infektionen. Die akute GVHD ist eine systemische Entzündungskrankheit bei der alloreaktive T-Zellen in die Leber, Haut und den Gastrointestinaltrakt einwandern und diese Organe schädigen. Die aktuellen GVHD Therapien sind daher auf eine Unterdrückung dieser T-Zellfunktionen ausgerichtet, wodurch sich allerdings häufig kritische sekundäre Immundefizienzen entwickeln, die mit einem erhöhten Risiko eine der oben genannten Komplikationen zu entwickeln, einhergehen. Fast alle GVHD Todesfälle lassen sich auf ein Therapieversagen oder auf erhebliche Nebenwirkungen der eingesetzten Immunsuppressiva zurückführen, was einen dringenden medizinischen Bedarf an alternativen therapeutischen Ansätzen, die nicht das Immunsystem schwächen, aufzeigt. Vorarbeiten konnten solch einen innovativen therapeutischen Ansatz identifizieren: die Hemmung der pathologischen Gefäßneubildung (Angiogenese), die sowohl bei Tumorwachstum als auch während der GVHD nach allo-HSZT involviert ist. Antiangiogene Therapiestrategien bauen auf der weithin etablierten Beobachtung auf, dass Angiogenese und Entzündung zwei eng miteinander verknüpfte Prozesse sind. Allerdings ist nicht geklärt, ob die Angiogenese ein initiales oder sekundäres Ereignis gegenüber der Entzündung darstellt. Dies und das bisherige Fehlen von therapeutischen Zielstrukturen, die bei pathologischer und physiologischer Angiogenese unterschiedlich reguliert werden, behindern zurzeit die effektive therapeutische Nutzung der Hemmung des Blutgefäßwachstums. In der vorliegenden Arbeit wurde untersucht, ob die Angiogenese einen initialen Mechanismus bei der Entstehung der GVHD darstellt. Zusätzlich sollten neue potentielle therapeutische Ansatzpunkte am Endothel identifiziert werden, die in der frühen Entstehungsphase der GVHD eine Rolle spielen. Dieser kumulativen Arbeit liegen zwei eigene Publikationen zugrunde. Für eine bessere translationale Übertragung der experimentell gewonnenen Daten, wurde zunächst ein neues Mausmodell der akuten GVHD entwickelt, welches im Gegensatz zu den zurzeit am häufigsten genutzten Mausmodellen, die klinische Situation der allo-HSZT besser abbildet. Dieses neue Chemotherapie-basierte, Haupthistokompatibilitätskomplex (MHC)-kompatible GVHD Modell ist charakterisiert durch ein stabiles Anwachsen der Knochenmarksstammzellen, einen klinisch-vergleichbaren GVHD-Phänotyp sowie durch eine systemische als auch Zielorgan-spezifische Entzündungsreaktion. Die GVHD-Pathogenese im Mausmodell ist eng vergleichbar mit dem klinischen GVHD-Verlauf nach einer humanen Leukozyten-Antigen (HLA)-kompatiblen allo-HSZT mit GVHD-Prophylaxe. Daher stellt dieses Modell ein geeignetes Werkzeug dar, um pathogene Mechanismen der GVHD und translationale Therapieansätze zu untersuchen. In diesem Mausmodell konnte erstmalig gezeigt werden, dass die Angiogenese ein initiales Ereignis während der GVHD-Pathogenese darstellt und der charakteristischen Entzündungsreaktion vorausgeht. Während dieser initialen Angiogenese zeigte sich keine entzündungs-assoziierte Endothelzellaktivierung. Stattdessen traten signifikante metabolische und zytoskeletale Veränderungen in den Endothelzellen auf, die zu einem erhöhten Migrations- und Proliferationspotenial führten. Neue Kandidatengene und Proteine wurden identifiziert, die in Endothelzellen während der pathologischen Angiogenese nach allo-HSZT unterschiedlich reguliert waren. Diese sollen in weiterführenden mechanistischen Studien untersucht werden. Insgesamt konnte die Arbeit neue Erkenntnisse zur Entstehung der pathologischen Angiogenese bei entzündlichen Erkrankungen wie der GVHD liefern und sie leistet einen substantiellen Beitrag für die translationale Entwicklung der therapeutischen Hemmung der pathologischen Angiogenese nach allo-HSZT

Ocular Graft-versus-Host Disease in a Chemotherapy-Based Minor-Mismatch Mouse Model Features Corneal (Lymph-) Angiogenesis

Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic window of opportunity is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool

Spatio-Temporal Bone Remodeling after Hematopoietic Stem Cell Transplantation

The interaction of hematopoietic cells and the bone microenvironment to maintain bone homeostasis is increasingly appreciated. We hypothesized that the transfer of allogeneic T lymphocytes has extensive effects on bone biology and investigated trabecular and cortical bone structures, the osteoblast reconstitution, and the bone vasculature in experimental hematopoietic stem cell transplantations (HSCT). Allogeneic or syngeneic hematopoietic stem cells (HSC) and allogeneic T lymphocytes were isolated and transferred in a murine model. After 20, 40, and 60 days, bone structures were visualized using microCT and histology. Immune cells were monitored using flow cytometry and bone vessels, bone cells and immune cells were fluorescently stained and visualized. Remodeling of the bone substance, the bone vasculature and bone cell subsets were found to occur as early as day +20 after allogeneic HSCT (including allogeneic T lymphocytes) but not after syngeneic HSCT. We discovered that allogeneic HSCT (including allogeneic T lymphocytes) results in a transient increase of trabecular bone number and bone vessel density. This was paralleled by a cortical thinning as well as disruptive osteoblast lining and loss of B lymphocytes. In summary, our data demonstrate that the adoptive transfer of allogeneic HSCs and allogeneic T lymphocytes can induce profound structural and spatial changes of bone tissue homeostasis as well as bone marrow cell composition, underlining the importance of the adaptive immune system for maintaining a balanced bone biology

Initiation of acute graft-versus-host disease by angiogenesis

The inhibition of inflammation-associated angiogenesis ameliorates inflammatory diseases by reducing the recruitment of tissue-infiltrating leukocytes. However, it is not known if angiogenesis has an active role during the initiation of inflammation or if it is merely a secondary effect occurring in response to stimuli by tissue-infiltrating leukocytes. Here, we show that angiogenesis precedes leukocyte infiltration in experimental models of inflammatory bowel disease and acute graft-versus-host disease (GVHD). We found that angiogenesis occurred as early as day12 after allogeneic transplantation mainly in GVHD typical target organs skin, liver, and intestines, whereas no angiogenic changes appeared due to conditioning or syngeneic transplantation. The initiation phase of angiogenesis was not associated with classical endothelial cell (EC) activation signs, such as Vegfa/VEGFR112 upregulation or increased adhesion molecule expression. During early GVHD at day12, we found significant metabolic and cytoskeleton changes in target organ ECs in gene array and proteomic analyses. These modifications have significant functional consequences as indicated by profoundly higher deformation in real-time deformability cytometry. Our results demonstrate that metabolic changes trigger alterations in cell mechanics, leading to enhanced migratory and proliferative potential of ECs during the initiation of inflammation. Our study adds evidence to the hypothesis that angiogenesis is involved in the initiation of tissue inflammation during GVHD