Received: 17 February 2020 | Revised: 5 April 2020 | Accepted: 12 May 2020 DOI: 10.1111/jcmm.15434 ORIGINAL ARTICLE Defibrotide inhibits donor leucocyte-endothelial interactions and protects against acute graft-versus-host disease David García-Bernal1,2 | Marta Palomo3,4,5 | Carlos M. Martínez6 | José E. Millán-Rivero1,2 | Ana I. García-Guillén1 | Miguel Blanquer1,2 | Maribel Díaz-Ricart4,5 | Robert Sackstein7 | Enric Carreras3,5 | Jose M. Moraleda1,2 1 Hematopoietic Transplant and Cellular Therapy Unit, Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Virgen de la Arrixaca University Hospital, University of Murcia, Murcia, Spain 2 Internal Medicine Department, Medicine School, University of Murcia, Murcia, Spain 3 Josep Carreras Leukaemia Research Institute, Barcelona, Spain 4 Hematopathology, Department of Pathology, Centre de Diagnostic Biomedic (CBD), Hospital Clinic de Barcelona, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain 5 Barcelona Endothelium Team, Barcelona, Spain 6 Experimental Pathology Unit, Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Murcia, Spain 7 Department of Translational Medicine, and the Translational Glycobiology Institute, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA Correspondence David García-Bernal, Hematopoietic Abstract Transplant and Cellular Therapy Unit, Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective ther- Instituto Murciano de Investigación Biosanitaria-Arrixaca, Internal Medicine apy for the treatment of high-risk haematological malignant disorders and other Department, Medicine School, University life-threatening haematological and genetic diseases. Acute graft-versus-host dis- of Murcia, Spain. Avenida Buenavista s/n. El Palmar, Murcia, Spain. ease (aGvHD) remains the most frequent cause of non-relapse mortality following Email: david.garcia23@um.es allo-HCT and limits its extensive clinical application. Current pharmacologic agents Funding information used for prophylaxis and treatment of aGvHD are not uniformly successful and have Jazz Pharmaceuticals, Grant/Award Number: serious secondary side effects. Therefore, more effective and safe prophylaxis and IST-16-10355; German José Carreras Leukaemia Foundation, Grant/Award therapy for aGvHD are an unmet clinical need. Defibrotide is a multi-target drug Number: 03R/2019 and 11R/2016; Instituto successfully employed for prophylaxis and treatment of veno-occlusive disease/ de Salud Carlos III, Grant/Award Number: PIE15/00027 and RD16/0011/0001 sinusoidal obstruction syndrome. Recent preliminary clinical data have suggested some efficacy of defibrotide in the prevention of aGvHD after allo-HCT. Using a fully MHC-mismatched murine model of allo-HCT, we report here that defibrotide, either in prophylaxis or treatment, is effective in preventing T cell and neutrophil infiltra- tion and aGvHD-associated tissue injury, thus reducing aGvHD incidence and sever- ity, with significantly improved survival after allo-HCT. Moreover, we performed in vitro mechanistic studies using human cells revealing that defibrotide inhibits leuco- cyte-endothelial interactions by down-regulating expression of key endothelial ad- hesion molecules involved in leucocyte trafficking. Together, these findings provide Palomo and Martínez contributed equally to this work. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. J Cell Mol Med. 2020;24:8031–8044.  wileyonlinelibrary.com/journal/jcmm | 8031 8032 | GARCÍA-BERNAL et al. evidence that defibrotide may represent an effective and safe clinical alternative for both prophylaxis and treatment of aGvHD after allo-HCT, paving the way for new therapeutic approaches. KEYWORDS acute GvHD, defibrotide, hematopoietic stem cell transplantation 1 | I NTRO D U C TI O N of fully major histocompatibility complex (MHC)-mismatched al- lo-HCT to induce fulminant aGvHD, we report that administration Acute graft-versus-host disease (aGvHD) is the most frequent of defibrotide with same total daily dose employed for VOD in hu- life-threatening complication after allogeneic hematopoietic stem mans is also effective for both prophylaxis and treatment of murine cell transplantation (allo-HCT). The pathobiology of aGVHD results aGvHD. This effect is mediated via a significant early inhibition of from immunocompetent allogeneic T cells contained in the mar- donor alloimmune effector T cell and neutrophil tissue infiltration, a row graft that recognize the recipient as foreign and migrate and substantial decrease in aGvHD-associated tissue injury, and a rever- attack specific host tissues (typically the skin, gastrointestinal tract sal of plasma pro-inflammatory/anti-inflammatory cytokines ratios. and liver), resulting in significant immune/cytokine-mediated tis- These effects yield a significant increased survival. Mechanistic in sue injury, endothelial dysfunction, and high morbidity and mortal- vitro studies using human samples reveal that defibrotide inhibits ity.1-5 Different prophylactic and treatment regimens are currently peripheral blood leucocyte transendothelial migration by down-reg- employed to reduce the incidence and severity of aGvHD, most ulating expression of key endothelial adhesion molecules involved commonly based on corticosteroids, calcineurin inhibitors or other in leucocyte trafficking such as E-selectin, P-selectin, ICAM-1 and immunosuppressants. However, responses are unsatisfactory and VCAM-1. Collectively, these findings provide novel perspectives on aGvHD remains a significant problem. Thus, there is an unmet need the clinical applicability of this multi-target drug in aGvHD therapy for the development of newer strategies to prevent and treat this following allo-HCT. disease.6 Defibrotide is a polydisperse mixture of 90% single-stranded and 10% double-stranded phosphodiester oligonucleotides derived from 2 | M ATE R I A L S A N D M E TH O DS controlled depolymerization of porcine intestinal mucosal DNA that has proved to be effective for prophylaxis and treatment of hepatic 2.1 | Mice veno-occlusive disease/sinusoidal obstruction syndrome (VOD/ SOS) following allo-HCT.7,8 It has been proposed to have a protective BALB/c (H-2Kd, CD45.2) and C57BL/6J (H-2Kb, CD45.2) mice were 9-11 effect on activated endothelial cells, and among others, pro-fi- purchased from Envigo Harlan (Huntingdon, Cambridgeshire, United brinolytic, anti-thrombotic and anti-inflammatory activities have Kingdom). At the time of allo-HCT, mice were aged 10-12 week. been reported.12 Defibrotide has been found to suppress hepara- Housing was maintained under specific pathogen-free conditions in nase gene expression, whose high levels have been postulated as the animal facilities of the University of Murcia. All animal protocols 13,14 a risk factor for aGvHD development, and it was initially iden- were approved by the Institutional Animal Care and Use Committee tified as an adenosine receptor agonist, whose activation inhibits at University of Murcia (protocol A13150201). T cell function.10,15,16 Importantly, preliminary evidence in recent clinical trials employing defibrotide for VOD suggests that it could also have a prophylactic effect on the occurrence of aGvHD after 2.2 | Induction and assessment of murine allo-HCT: patients who received VOD prophylaxis with defibrotide aGvHD and histopathology analysis had also a lower incidence and severity of aGvHD than untreated control group of patients, and it also did not seem to interfere with A model of fully MHC-mismatched allo-HCT was carried out by 17,18 a graft-versus-leukaemia effect. Based on these indirect data, transplanting bone marrow mononuclear cells from donor BALB/c defibrotide received an orphan designation for the prevention of mice into recipient C57BL/6J mice that were previously irradiated aGvHD. However, its efficacy for the prevention and treatment of by with a lethal dose of 10 Gy split into two equal doses of 5 Gy and aGvHD as the only drug without the administration of other immu- 24 hours apart (days −1 and +0). To induce aGvHD, on day +0 recipi- nosuppressant/s, as well as the molecular mechanisms mediating its ent C57BL/6J mice were injected intravenously with 1 × 107 bone aGvHD-protective effect, has not been previously addressed. marrow mononuclear cells from donor BALB/c mice and 1.5 × 107 Given such prior findings, we sought the mechanistic basis of BALB/c donor splenocytes. Mice survival post-transplant was defibrotide effects on evolving aGvHD. Here, using a murine model monitored daily, and clinical aGvHD was evaluated using a scoring GARCÍA-BERNAL et al. | 8033 system that generates a composite aGvHD score composed of in- Untreated controls were injected daily with same volume of sa- dividual scores for weight loss, posture, activity, skin integrity and line solution without defibrotide. fur texture.19 Histopathology analysis of aGvHD was performed in different sections of liver, intestine (colon), skin and oral mucosa (tongue) col- 2.4 | Determination of plasmatic cytokines by lected from the recipients 10 days after allo-HCT and analysed as enzyme-linked immunosorbent assays (ELISA) described previously19,20 by a single pathologist blinded to the treat- ment groups. Samples from the different organs were fixed in for- Murine IFNγ, TNFα, IL-6, IL-12, TGFβ and IL-10 were measured in malin and embedded in paraffin. After, three-micrometre sections blood plasma by ELISA (Diaclone, bioNova Cientifica, Madrid, Spain; were stained with haematoxylin and eosin stain for histopathologic and Elabscience, Bethesda, MD). All experimental samples and examination. In brief, the skin histopathologic lesions were grade 0 standards were assayed in triplicate according to the manufacturer's (normal), grade I (slight vacuolar degeneration of epidermal basal instructions. cells), grade II (scattered individual apoptotic epidermal basal cells, spongiosis), grade III (separation of dermoepidermal junction) and grade IV (diffuse and severe ulceration, with extensive destruction 2.5 | Cell cultures of epidermis). Colon aGvHD scores were either grade 0 (normal), grade I (scattered individual apoptotic cells, with inflammatory cell SK-Hep1, an immortalized human endothelial cell line derived infiltrate), grade II (crypt epithelial cell apoptosis, with villous blunt- from ascitic fluid, and primary human umbilical vein endothelial ing and/or exploding crypts), grade III (focal mucosal ulceration, with cells (HUVEC) were used to investigate defibrotide effects on en- moderate villous atrophy) and grade IV (diffuse and severe muco- dothelial adhesion molecule expression and human leucocyte ad- sal ulceration). Liver aGvHD was scored according to degree of bile hesion/transendothelial migration in vitro. SK-Hep1 was cultured ducts affected: 0 (normal), grade I (≤25% bile ducts affected), grade in Eagle's Minimum Essential Medium (Sigma-Aldrich) containing II (25%-49% bile ducts affected), grade III (50%-74% bile ducts af- 10% foetal bovine serum (Gibco, Carlsbad, CA), 1% L-glutamine fected) and grade IV (≥75% bile ducts affected). The scoring system (Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco) used for histopathologic evaluation of the tongue samples was based at 37ºC and 5% CO2. Cells were used between the tenth and fif- on intraepithelial inflammatory infiltrate as follows: grade 0 (ab- teenth passages. sence of inflammatory infiltrate), grade 1 (weak), grade 2 (moderate), HUVEC were isolated from human umbilical veins and cul- grade 3 (intense) and grade 4 (severe). The degree of T-lymphocyte tured in Endothelial Cell Growth Medium (PromoCell, Heidelberg, infiltrate or induction of apoptosis was evaluated by immunohisto- Germany) at 37ºC and 5% CO2 according to the manufactur- chemical labelling by using a commercial kit (EnVision Flex, Dako- er's instructions. Cells were used between the second and third Agilent Technologies, Barcelona, Spain). Briefly, tissue sections were passages. submitted to deparaffinization and rehydration, followed by antigen Human peripheral blood mononuclear cells (MNCs) were iso- demasking procedure and endogenous peroxidase inhibition. After, lated using Ficoll-Paque density gradient centrifugation from hep- sections were incubated with primary antibodies (anti–T-CD3, Dako) arinized blood samples obtained from healthy volunteers after for 1 hour at 37ºC, following by secondary anti-rabbit labelled poly- informed consent. In some experiments, pooled sera samples ob- mer incubation for 20 minutes at 37ºC. Immunoreaction was finally tained from three different patients with aGvHD on the day of revealed by incubation with 3-3´diaminobenzidine (Sigma-Aldrich, diagnosis, or from same number of healthy donors, were used. St. Louis, MO). Positive immunoreaction was identified as a dark- Hospital Virgen de la Arrixaca and Hospital Clinic de Barcelona brown pericellular staining. In addition, polymorphonuclear (PMN) Ethics Committees approved the protocols used to obtain and pro- neutrophils were identified on H&E-stained sections according to cess all human samples. morphologically evident segmented nuclei. All analyses were per- formed by using a standard direct-light microscope (Axio A10, Carl Zeiss, Barcelona, Spain). 2.6 | Parallel-plate flow chamber adhesion assays The effects of defibrotide on leucocyte adhesion on endothelial 2.3 | Defibrotide administration cells were analysed using a parallel-plate perfusion chamber. SK- Hep1 monolayers were treated previously with 100 µg/mL defib- Defibrotide solution was provided by Gentium SpA, Jazz rotide for 24 hours or left untreated and then grown for 48 hours Pharmaceuticals (Dublin, Republic of Ireland). Defibrotide was sus- in medium supplemented with 20% pooled sera of aGvHD patients pended in saline solution at a concentration of 2,5 µg/µL to reach (n = 15, Table 1) or of healthy donors (n = 15). Then, SK-Hep1 cells 25 mg/kg bodyweight, and 200-250 µL was injected intraperito- were perfused with control citrated blood from a healthy donor at neally (i/p) as a single dose as prophylaxis (day −2 to day +17 post- a shear rate of 300 s−1 for 10 minutes. After perfusion, coverslips allo-HCT) or treatment (day +7 to day +27 post-allo-HCT). were washed with PBS and fixed in 3% paraformaldehyde in PBS. 8034 TA B L E 1 Characteristics of acute GvHD patients and treatment | Date of Patient Number Birth Date Diagnostic (Stage at Treatment) Treatment transplantation GvHD Prophylaxis Donor Sk-HEP1 cells exposed during 48 h to a sera pool from three patients (P1, P2, P3) developing GvHD between day 7 and day 14. Sample from day 14. Patient 1 09-01-1971 Follicular Non-Hodgkin's lymphoma Cyclosporine A/Total Body 15-09-2008 Cyclosporine A/Methotrexate Non-related Irradiation Patient 2 02-01-1980 Hodgkin's disease: nodular sclerosis Fludarabine/Melphalan 21-08-2009 Cyclosporine A Non-related Patient 3 23-09-1988 Non-Hodgkin's lymphoma: Primary BEAM200 (BCNU-Etoposide100- 29-04-2009 Cyclosporine A/Methotrexate Non-related mediastinal large B cell Ara-C-Melphalan) Sk-HEP1 cells exposed during 48 h to a sera pool from three patients (P4, P5, P6) developing GvHD between day 15 and day 21. Sample from day 21. Patient 4 05-05-1975 Acute myeloid leukaemia subtype M5 Cyclosporine A/Total Body 28-08-2009 Cyclosporine A/Methotrexate Identical sibling (AML-M5) Irradiation Patient 5 23-09-1988 Non-Hodgkin's lymphoma: Primary BEAM200 (BCNU-Etoposide100- 29-04-2009 Cyclosporine A/Methotrexate Non-related mediastinal large B cell Ara-C - Melphalan) Patient 6 28-02-1983 Hodgkin's disease: nodular sclerosis Fludarabine/Melphalan 18-06-2008 Cyclosporine A/Mycophenolate mofetil Non-related Sk-HEP1 cells exposed during 48 h to a sera pool from three patients (P7, P8, P9) developing GvHD between day 7 and day 14. Sample from day 14. Patient 7 19-01-1959 Biphenotypic Leukaemia (Relapse) FLAG (Fludarabine- Ara-C -G-CSF)/ 10-03-2009 Cyclosporine A/Mycophenolate mofetil Non-related IDA (idarubicin)/Melphalan Patient 8 27-04-1957 Non-Hodgkin's lymphoma: BEAM200 (BCNU-Etoposide100- 16-12-2009 Cyclosporine A/Mycophenolate mofetil Non-related unclassified Ara-C-Melphalan Patient 9 17-03-1995 Chronic lymphocytic leukaemia BEAM200 (BCNU-Etoposide100- 20-07-2009 Cyclosporine A/Mycophenolate mofetil Non-related Ara-C-Melphalan Sk-HEP1 cells exposed during 48 h to a sera pool from three patients (P10, P11, P12) developing GvHD between day 14 and day 21. Sample from day 21. HUVEC cells exposed during 48 h to a sera pool from four patients (P10, P11, P12, P13) developing GvHD between day 7 and day 14. Sample from day 14. Patient 10 27-03-1968 Acute myeloid leukaemia subtype M5 FLAG (Fludarabine-Ara-C -GCSF)/ 31-10-2008 Cyclosporine A/Mycophenolate mofetil Identical sibling (AML-M5) IDA (idarubicin)/Melphalan Patient 11 19-12-1948 Acute myeloid leukaemia subtype M1 Fludarabine/Busulfan 22-04-2009 Cyclosporine A/Rapamycin Identical sibling (AML-M1) (1st remission) Patient 12 03-02-1988 Acute myeloid leukaemia subtype M3 Cyclosporine A/Total Body 31-10-2008 Cyclosporine A/Methotrexate Identical sibling (AML-M3) Irradiation Patient 13 22-04-1958 Diffuse large B cell lymphoma Fludarabine/Cyclosporine A/ 11-01-2019 Cyclosporine A/Tacrolimus/ Identical sibling Melphalan/Total Body Irradiation Mycophenolate mofetil Sk-HEP1 cells exposed during 48 h to a sera pool from 3 patients (P13, P14, P15) developing GvHD between day 22 and day 28. Sample from day 28. Patient 14 20-10-1981 Hodgkin's disease: nodular sclerosis Fludarabine/Melphalan 01-12-2009 Cyclosporine A/Rapamycin/Melphalan Identical sibling Patient 15 06-01-1968 Acute lymphoblastic leukaemia: T cell Cyclosporine A/Total Body 17-07-2008 Cyclosporine A/Methotrexate Non-related Irradiation Patient 16 14-04-1979 Multiple myeloma Fludarabine/Melphalan 03-03-2009 Cyclosporine A/Mycophenolate mofetil Non-related GARCÍA-BERNAL et al. GARCÍA-BERNAL et al. | 8035 For leucocyte immunostaining, coverslips were treated with 1% 3 | R E S U LT S BSA, permeabilized with 0.1% Triton-X and stained with an anti- human CD45 antibody (Abcam), followed by Alexa488-conjugated 3.1 | Defibrotide aGVHD treatment reduces aGvHD anti-rabbit secondary antibody (Life Technologies, Carlsbad, CA), clinical manifestations and reduces level of donor T and PE-conjugated anti-human CD3 (BD Biosciences, San Jose, CA) cell and PMN neutrophil infiltrates in aGvHD-target and DAPI (Sigma-Aldrich) for nuclei counterstaining. Finally, cover- organs slips were visualized by fluorescent microscopy (Leica-DM4000B) through a video camera (Leica-DFC310FX) and analysed using To test the hypothesis that defibrotide has direct effects in aGvHD ImageJ software (National Institutes of Health, Bethesda, MD). The onset and/or evolution, mice with ongoing aGvHD (from day +7 number of adherent leucocytes and T cells was expressed as per- after allo-HCT) were treated with a daily i/p infusion of 25 mg/ centage related to the total number of endothelial cells (number of kg of defibrotide for 20 days, following the same total daily dose leucocytes per 100 endothelial cells). used in human VOD/SOS patients (schematic diagram shown in Figure 1A). Control mice receiving allo-HCT (BM transplant, that is receiving donor BALB/c bone marrow cells w/o splenocytes) and 2.7 | Transwell migration assays and flow mice receiving syngeneic transplant (ie transplanted with bone cytometry analysis marrow cells and splenocytes from donor C57BL/6J) did not de- velop aGvHD, whereas mice receiving allo-HCT plus donor BALB/c HUVEC in culture were exposed to medium containing 20% pooled splenocytes without defibrotide treatment (ie ‘untreated’ group) sera of aGvHD patients (n = 4, Table 1) or of healthy donors (n = 4) showed a rapid severe aGvHD occurrence, which caused the early for 48 hours and incubated with 100 µg/mL defibrotide (treat- death in all mice (Figure 1C). However, daily administration of defi- ment), or alternatively, by previous exposure to defibrotide for brotide showed a significantly lower aGvHD-related mortality than 24 hours followed by continuous incubation (100 µg/mL, added untreated mice group (P < .001) (60% survival in defibrotide group every 24 hours) before exposition to sera (prophylaxis). Afterwards, versus 0% survival in untreated group at day +60 post-transplan- 7.5 × 10 4 HUVEC were seeded onto the upper chamber of Transwell tation, P < .001) (Figure 1C). Also, defibrotide-treated mice signifi- filters with 5-µm pores (NUNC, Roskilde, Denmark). Then, 3 × 105 cantly improved their clinical aGvHD score compared to untreated peripheral blood MNCs or T-CD3 cells (obtained by magnetic selec- mice group, being significantly lower from the onset of the disease tion using Pan human T cell isolation kit (Miltenyi Biotec, Bergisch (P < .001) (Figure 1D). To evaluate whether defibrotide had effects Gladbach, Germany)) were resuspended in 100 µL of DMEM me- in the prophylaxis of aGVHD, mice were treated with a daily intra- dium (Sigma-Aldrich) containing 0.3% bovine serum albumin (BSA) peritoneal infusion of 25 mg/kg of defibrotide from 2 days before (Sigma-Aldrich) and added to the upper chamber. The lower chamber allo-HCT to 17 days after (schematic diagram shown in Figure 1B). contained DMEM medium supplemented with 0.3% BSA and 20% Remarkably, mice receiving daily prophylaxis with defibrotide dis- pooled sera of aGvHD patients/healthy donors as chemoattractant played a high survival advantage over the untreated mice group stimulus. Next, cells were allowed to migrate for 16 hours at 37ºC. (P < .001) (87.5% survival in defibrotide group versus 0% survival Finally, the extent of cell migration was analysed using an automated in untreated group at day +60 post-transplantation) (Figure 1E), cell counter (Bio-Rad TC20, Hercules, CA). All experimental condi- also showing a significant reduction in aGvHD clinical score dur- tions were analysed in triplicate. ing aGvHD evolution compared to untreated group of animals For flow cytometry analysis, HUVEC were detached with tryp- (P < .001) (Figure 1F). These in vivo results suggest that defibrotide sin-EDTA (Gibco), resuspended in PBS containing 1% BSA and in- can mitigate the severity of early-stage aGvHD, both as the only cubated with fluorescence-labelled anti-human antibodies specific prophylaxis or treatment drug. for E-selectin, P-selectin, VCAM-1 and ICAM-1 (all from eBioscience, To determine the impact of defibrotide on reversal of aGvHD, San Diego, CA), or their control isotype antibodies for 30 minutes in a histopathology analysis was performed in the main aGvHD-tar- the dark at 4ºC. Finally, cells were washed twice and acquired in a get organs (skin, liver, colon and tongue) in animals receiving pro- FACSCanto flow cytometer (BD Biosciences, San Jose, CA). phylactic defibrotide infusions after allo-HCT (Figure 2). On day +10 post-transplantation, untreated mice displayed skin vacuolar degeneration and scattered apoptotic epidermal basal cells (grade 2.8 | Statistical analysis 0-I), whereas there were no patent lesions on the skin of the de- fibrotide-treated, BM transplant or syngeneic (Syn)-HCT animals. Results are expressed as mean ± standard deviation. Student's paired Histological examination of liver and colon displayed significant t test or one-way ANOVA followed by Bonferroni's post hoc com- differences in the aGvHD severity between both experimental parison tests was used for statistical comparisons between groups. and control groups of animals. Whereas livers of untreated ani- Survival curves were plotted using Kaplan-Meier estimates and sta- mals displayed an extensive epithelial damage, abundant peripor- tistically analysed using the Mantel-Cox log-rank test. P values < .05 tal inflammatory infiltrate and destruction of the majority of bile were considered statistically significant. ducts (grade II-III), defibrotide-treated group showed much lower 8036 | GARCÍA-BERNAL et al. aGvHD-related hepatic epithelium damage (grade 0-I). Also, colon at day +10 after allo-HCT. Significantly, we observed that livers, of untreated mice showed moderate villous atrophy, interstitial colon and tongues of untreated allo-HCT recipients displayed sig- inflammatory cell infiltrate and crypt epithelial cell apoptosis nificant higher levels of T-CD3+ infiltrates than BM transplanted or (grade II-III), whereas those from animals receiving prophylactic syn-HCT animals. However allo-HCT mice that received defibrot- defibrotide only displayed a slight inflammatory infiltrate and ide showed a substantial decrease in T-CD3+ infiltrates compared scattered apoptotic cells (grade I). In addition, the tongue was also to their untreated counterparts, mainly in the colon (P < .001) analysed as a representative tissue of oral mucosa, which is also (Figure 3A,B). On the other hand, skin sections of mice among the affected during aGvHD progression (Figure 2). Tongue dermis of two groups of animals only displayed scattered infiltrates of donor untreated animals displayed a weak or moderate inflammatory in- T cells, which correlated with no observable lesions in this tissue at filtrate (grade 0-I), which was absent in defibrotide-treated mice. this time post-transplantation. These observations suggest that the improved survival observed Apart from tissue trafficking of T cells, it has been previously in the group of animals receiving defibrotide was correlated with described that neutrophils can also infiltrate the aGvHD-target a substantially reduced degree of tissue-specific destruction. organs, being considered as a bad prognostic marker of early al- Tissue damage in aGvHD-target organs is largely related with lo-HCT–associated mortality. 24 As such, we also examined the the ability of alloreactive donor T cells to traffic and infiltrate spe- level of neutrophil infiltrates in aGvHD-target organs of all groups cifically these tissues. 21-23 Thus, we next investigated levels of of animals. Although at day +10 post-transplant mice remain still T-CD3+ cell infiltrates in skin, liver, colon and tongue of untreated neutropenic, there were significantly reduced neutrophil infiltrates animals as compared to animals receiving prophylactic defibrotide in skin, liver and colon of mice receiving prophylactic defibrotide F I G U R E 1 Defibrotide treatment or prophylaxis reduces the incidence and severity of aGvHD in mice undergoing a fully MHC- mismatched allo-HCT. After aGvHD onset (from day +7 after allo-HCT), mice were treated with a daily administration of 25 mg/kg of defibrotide for 20 d or with 25 mg/kg of prophylactic defibrotide from 2 d before allo-HCT to 17 days after. Schematic diagrams of the experimental protocols of defibrotide treatment (A) or prophylaxis (B) after allo-HCT are shown. Untreated mice received same doses and volume of saline solution. C,D, Kaplan-Meier survival curves and aGvHD clinical score of recipient C57BL/6J treated with defibrotide after aGvHD onset or (E,F) receiving prophylactic defibrotide are shown, respectively. Survival or aGvHD clinical score was significantly higher compared to untreated group, ***P < .001. Results are representative of n = 3 separate experiments, n = 8 mice per experimental group GARCÍA-BERNAL et al. | 8037 compared to untreated counterparts after allo-HCT, P < .001 3.2 | Prophylactic administration of defibrotide (Figure 3C,D). reverses ratios of plasma pro-inflammatory and anti- Importantly, control experiments revealed that peripheral blood inflammatory cytokines lymphocytes and neutrophils counts in untreated mice and mice re- ceiving defibrotide were parallel, indicating that the different degrees To further evaluate the effects of defibrotide administration on of tissue infiltration of both immune populations were not a conse- aGvHD development, we analysed the release of several pro- quence of a different leucocyte engraftment after allo-HCT (data not inflammatory cytokines described to have key roles during the shown). effector phase of aGVHD. 25 Mice with ongoing aGvHD after F I G U R E 2 Histopathological assessment of aGvHD in mice receiving prophylactic defibrotide. Main histopathological aGvHD signs were analysed in skin, liver, colon and tongue of all groups of transplanted mice: BM transplant (w/o splenocytes), syngeneic transplant (syn-HCT) and allo-HCT mice, this last group including untreated mice or animals receiving prophylactic defibrotide at day +10 post- transplantation. Skin: vacuolar degeneration and scattered apoptotic epidermal basal cells (yellow arrowhead); liver: 50%-75% of bile ducts surrounded by inflammatory cell infiltrate and epithelial damage (black arrowhead), or >75% of unaffected bile ducts (red arrowhead); colon: moderate villous atrophy (+), interstitial inflammatory cell infiltrate (yellow asterisk), crypt epithelial cell apoptosis (black asterisk), or slight inflammatory infiltrate and scattered apoptotic cells (yellow arrowhead); and tongue: weak or moderate inflammatory cell infiltrate (yellow asterisk). H&E staining images from the different organs shown (magnification ×200) are representative of n = 8 animals per group. Scale bar: 50 µm 8038 | GARCÍA-BERNAL et al. allo-HCT and without defibrotide administration (ie untreated anti-inflammatory TGFβ and IL-10 (Figure 4). However, defibrotide animals) displayed significant increases in plasma pro-inflam- prophylactic daily administration induced a marked decrease in matory cytokines such as IFNγ, TNFα, IL-6 and IL-12 on day +10 these pro-inflammatory mediators (P < .01, P < .001) and signifi- post-transplantation compared to syn-HCT control group (w/o cant increased levels of the anti-inflammatory cytokines TGFβ and aGvHD) (P < .001), with simultaneous reduced concentrations of IL-10 (P < .01) (Figure 4). F I G U R E 3 Defibrotide prophylactic administration significantly decreases donor T cell and PMN neutrophil infiltration in aGvHD-target organs after allo-HCT. Tissues from the different groups of animals, BM transplant (BMT), syn-HCT (SYN) or allo-HCT (either untreated (UNT) or receiving prophylactic defibrotide (DF)), were isolated 10 d after transplant. A, B, T cell infiltrates were detected by standard ABC anti-CD3 immunohistochemistry procedure. C, D, Polymorphonuclear (PMN) neutrophils were identified in the different tissue sections on the basis to its morphological features (nuclear segmentation). Representative images of the skin, liver, colon and tongue from the different mice groups showing T-CD3+ or PMN neutrophil infiltrates (red arrows) are shown (magnification ×200 or ×400, respectively) (n = 8 animals per group, n = 3 separate experiments). Scale bar: 50 µm. Absolute T-CD3+ cell or PMN neutrophil counts are presented as mean ± SD per high-power field from counts relative to 10 high-power fields (magnification ×200 or ×400, respectively). T-CD3+ cell or PMN neutrophil infiltrates were significantly increased compared to syn-HCT group, **P < .01, ***P < .001, or decreased compared to untreated allo-HCT group, ∆∆∆ P < .001, respectively GARCÍA-BERNAL et al. | 8039 F I G U R E 4 Defibrotide prophylaxis modifies the systemic profile of secreted plasma pro-inflammatory and anti-inflammatory cytokines on mice with ongoing aGvHD after allo-HCT. Plasma concentrations of the pro-inflammatory cytokines IFNγ, TNFα, IL-6 and IL-12, and anti- inflammatory TGFβ and IL-10 were determined on the different groups of animals, BM transplant (BMT), syn-HCT (SYN) or allo-HCT (either untreated (UNT) or receiving prophylactic defibrotide (DF)), on day +10 post-transplant. Data are presented as mean ± SD of n = 5 animals per group and n = 3 separate experiments. Levels of each cytokine were significantly up-regulated compared to SYN group, ***P < .001, or significantly down- or up-regulated compared to SYN group, ∆∆∆ P < .001, respectively 3.3 | Defibrotide impairs human leucocyte subsequent Transwell migration assays showed that when HUVEC trafficking and down-regulates endothelial adhesion were exposed to aGvHD sera and treated with defibrotide after- molecules expression: mechanism of action involved wards (Figure 5A) or before (Figure 5B), transendothelial migration in the protective effect of defibrotide. of MNCs and T-CD3 cells was significantly inhibited as compared to the levels of migration observed using untreated HUVEC (P < .001). Acute GvHD is associated with the up-regulation of several endothe- Also, parallel-plate perfusion chamber experiments showed that lial cell adhesion molecules such as E-selectin, P-selectin, VCAM-1 previous exposure of endothelial SK-Hep1 cells to defibrotide signif- and ICAM-1 in the affected organs in response to the effect of pro- icantly prevented MNCs and T-CD3 rolling interactions and subse- inflammatory cytokines, favouring donor alloreactive leucocytes to quent firm adhesion under hemodynamic flow conditions compared engage the endothelium and extravasate to the target tissue(s). 26-28 to untreated endothelial cells (P < .01 and P < .05, respectively) In order to extrapolate the findings obtained in the aGvHD murine (Figure 5C,D). model to the human disease, we first analysed whether defibrotide The observed decreased interactions between leucocytes and interferes with peripheral blood MNC or T cell adhesion and migra- endothelial cells suggest that defibrotide could modulate expres- tion on endothelial HUVEC and SK-Hep1 cell lines. As previously re- sion of key adhesion molecules involved in the sequential cascade 11 ported, defibrotide does not interact with peripheral blood MNCs, of events that control migration of leucocytes from vascular to ex- and when leucocytes were directly incubated with defibrotide, travascular compartments. To this end, we analysed expression of leucocyte-endothelial rolling adhesive interactions under hemody- E-selectin, P-selectin, VCAM-1 and ICAM-1 on HUVEC previously namic flow conditions were not affected (data not shown). However, treated or not with defibrotide and after being stimulated with the 8040 | GARCÍA-BERNAL et al. aGvHD sera. Whereas untreated HUVEC displayed a significant to hypothesize that defibrotide may be an effective agent for aGvHD marked up-regulation of all these adhesion molecules (P < .001), pre- prevention and/or treatment. vious incubation of HUVEC with defibrotide significantly down-reg- Our findings indicate that daily infusions of defibrotide employ- ulates expression of E-selectin, P-selectin, VCAM-1 and ICAM-1 ing the same total daily dose as in human VOD/SOS patients, given (P < .01, P < .001) (Figure 5E,F). Collectively, these results indicate prior to allo-HCT (prophylaxis) or once aGvHD has already devel- that defibrotide protective function in endothelium includes the oped (treatment), significantly dampened the severity of aGvHD, down-regulation of expression of endothelial adhesion molecules resulting in a much lower aGvHD clinical score and a markedly highly overexpressed during aGvHD, which could prevent the sub- improved survival. Importantly, histological analysis demonstrates sequent in vivo donor leucocyte extravasation to target tissue(s). that infusions of defibrotide markedly restrain aGvHD-target organ damage, particularly in the liver and colon compared to those that did not receive any treatment. Nevertheless, this allo-HCT model 4 | D I S CU S S I O N did not allow evaluating the efficacy of defibrotide in reversing skin aGvHD. Skin manifestations are a frequent early complica- Allogeneic HCT provides a replacement of the diseased marrow tion during human evolving aGvHD. 38 However, many studies with healthy hematopoietic progenitor cells derived from the donor. point out that the choice of mouse strains to be used as donors Moreover, allo-HCT is an important type of adoptive cell immuno- and recipients of allo-HCT greatly affects the development of the therapy in which donor alloreactive immune cells confer a potent subsequent clinical manifestations of aGvHD due to differences in graft-versus-malignancy effect. However, it can also induce immu- their immune triggering mechanisms (ie mismatches in major and nopathological processes such as aGvHD, which is related to the im- minor histocompatibility complex molecules, effector T cell sub- munologic recognition and destruction of specific host tissues that populations involved or different production of pro-inflammatory notably increase the mortality of the procedure. 29 mediators). 39,40 Thus, these specific differences in spatio-tempo- In the present study, we provide evidence on the therapeutic ef- ral GvHD-target organ involvement depend largely on the murine ficacy of defibrotide in prevention and treatment of aGvHD after model of HCT employed. allo-HCT. Although two preliminary clinical studies have suggested During the effector phase of aGvHD, donor effector T cells at- indirect evidence that defibrotide prophylaxis might decrease the tack the recipient after recognizing the host tissues as antigenically risk of aGvHD in paediatric and adult patients receiving allo-HCT,17,18 foreign. 23,41 As expected, animals receiving allo-HCT that devel- this is the first comprehensive preclinical study demonstrating that oped aGvHD displayed high levels of T cell infiltrates in aGvHD-tar- administration of defibrotide as a single agent, without other con- get organs at early time post-allo-HCT (by day +10), mainly in liver comitant treatment, is sufficient to effectively prevent and/or treat and colon. However, mice receiving defibrotide prophylaxis before this immunopathological complication. transplant displayed a marked and significant reduction of T cell Defibrotide has been previously described as a multi-target infiltrates. drug, showing anti-thrombotic/thrombolytic, anti-ischaemic, pro-fi- After T cell recruitment, aGvHD-target organs are also infiltrated brinolytic and anti-angiogenic effects.30-34 But defibrotide has also by other immunocompetent cells of the myeloid lineage, that is neu- demonstrated important anti-inflammatory properties and a pro- trophils, in a complementary innate pathway in which cognate T cell- tective effect on endothelial cells from HCT conditioning.12,35,36 MHC interactions are not needed. Previous studies have revealed These findings and the fact that aGvHD occurrence is associated that an abundance neutrophil infiltrates in host tissues contribute with higher activation and dysfunction of endothelial cells37 led us synergistically to increase aGvHD severity and related mortality by F I G U R E 5 Defibrotide inhibits human peripheral blood MNC and T cell migration and down-regulates endothelial adhesion molecules expression. A, HUVEC were exposed to culture medium containing pooled sera of aGvHD patients or from healthy donors (control) and treated with defibrotide, or (B) incubated with defibrotide before adding sera (prophylaxis). Then, peripheral blood MNC and T-CD3 cell transendothelial migration towards aGvHD or control sera were analysed in Transwell assays in the presence or absence (‘untreated’, ie UNT) of defibrotide. Data are expressed as percentages of migrated cells related to the total number of MNCs or T-CD3 cells added to the upper chamber. Migration was significantly increased in the aGvHD condition, ***P < .001, or significantly inhibited after defibrotide incubation, ∆∆∆ P < .001, respectively. C, Also, effect of defibrotide on the MNC or T-CD3 cell rolling and firm adhesion on SK-Hep1 cells was analysed using a parallel-plate perfusion chamber under dynamic flow conditions. Adhesion was significantly increased in the aGvHD condition, **P < .01, ***P < .001, and significantly inhibited after defibrotide incubation, ∆ P < .05, ∆∆ P < .01, respectively. D, Representative micrographs showing adherent leucocytes (green staining with Alexa488-conjugated anti-human CD45, red staining with PE-conjugated anti-CD3 and blue staining with DAPI) obtained in the different experimental conditions are shown (magnification ×40). After, expression of endothelial adhesion molecules on HUVEC exposed to pooled aGvHD sera with DF or without prophylactic defibrotide (UNT) was analysed by flow cytometry. E, Representative histograms showing E-selectin, P-selectin, VCAM-1 and ICAM-1 expression on HUVEC in the different experimental conditions are shown. F, Relative expression of the different adhesion molecules was normalized to untreated HUVEC (ie w/o prophylactic defibrotide) incubated with healthy donor pooled sera (UNT control). Expression was up-regulated in the aGvHD condition compared to UNT control, ***P < .001, or down-regulated compared to UNT aGvHD, ∆∆ P < .01, ∆∆∆ P < .001, respectively GARCÍA-BERNAL et al. | 8041 8042 | GARCÍA-BERNAL et al. production of reactive oxygen species that leads to T cell activation In summary, our findings indicate that defibrotide, used as a and subsequent potentiation of the tissue damage, mainly in the gas- concomitant prophylactic or therapeutic drug to the usual stan- trointestinal tract. 24,42 In this study, we also found that similarly to dard immunosuppressants employed for aGvHD, is effective in what was observed with T cells, mice treated with prophylactic in- ameliorating the inflammatory response and tissue damage associ- fusions of defibrotide showed markedly lower neutrophils infiltrates ated with this immunopathological disease, reducing its incidence than allo-HCT untreated mice. and severity and significantly improving survival after allo-HCT. The pathogenesis of aGvHD is closely linked to cytokines pro- In addition, defibrotide is not associated with relevant secondary duced by T cells and other immune cells that infiltrate aGvHD-tar- side effects associated with other established treatments such get organs.43,44 Here, we found that mice receiving prophylactic as with corticosteroids. Therefore, defibrotide might represent a defibrotide displayed significant decreased systemic concentra- promising option for the prophylaxis and treatment of aGvHD in tions of a variety of pro-inflammatory cytokines (ie IFNγ, TNFα, patients receiving allo-HCT. IL-6 and IL-12) as compared to allo-HCT untreated animals, to- gether with an increased production of anti-inflammatory TGFβ AC K N OW L E D G M E N T S and IL-10. These data reveal that the administration of defibrotide This study has been funded by Instituto de Salud Carlos III (ISCIII) prior to the aGvHD onset is sufficient to attenuate the systemic through the Spanish Network of Cell Therapy (TerCel), RETICS inflammation associated with aGvHD. This ‘shift’ in systemic cyto- subprogram of the I + D + I 2013-2016 Spanish National Plan, kine levels from a pro-inflammatory to an anti-inflammatory profile project ‘RD16/0011/0001' funded by ISCIII and co-founded by has been described to have an important role in the improvement ERDF ‘una manera de hacer Europa', Jazz Pharmaceuticals Plc of the systemic inflammatory condition in several immune-me- (IST-16-10355), German José Carreras Leukaemia Foundation diated pathologies, favouring the achievement of peripheral im- (Grant 11R/2016 and 03R/2019) and ISCIII, Spanish Government munotolerance.45,46 Moreover, modulation of systemic cytokines (PIE15/00027). could down-regulate the expression of endothelial adhesion mole- cules involved in the trafficking of alloreactive immune cells to the C O N FL I C T S O F I N T E R E S T aGvHD-target tissues.47,48 This research has been partially supported by Jazz Pharmaceuticals Extravasation of circulating leucocytes across vascular wall in- plc/Gentium Inc M.P and M.D-R declare conflict of interest with Jazz volves a multistep adhesive interactions mediated by several mole- Pharmaceuticals plc/Gentium Inc in the form of speaker's fee for cules: a) selectins that mediate first tethering contacts and rolling; b) symposia. E.C declares conflict of interest with Jazz Pharmaceuticals chemokines that mediate inside-out integrin activation and c) inte- plc/Gentium Inc as consultant and in the form of speaker's fee for grins that mediate firm adhesion, crawling and subsequent transen- symposia. The remaining authors declare no competing commercial dothelial migration. It has been demonstrated in in vitro experiments or financial interests. and in vivo animal models that defibrotide decreases leucocyte ex- travasation by down-regulating expression of endothelial P-selectin AU T H O R C O N T R I B U T I O N S or by interfering with the interaction between the integrin LFA-1 and D.G-B., JMM, RS, MB and E.C conceptualized and designed the study, 49-52 its endothelial ligand ICAM-1. Recent findings have also revealed and D.G-B., MP, JEM-R. and AIG-G. performed all the experiments. that defibrotide suppresses the up-regulated expression of VCAM-1, CMM carried out and analysed all aGvHD histopathology experi- ICAM-1, VE-cadherin and von Willebrand factor in endothelial cells ments. D.G-B., MP, CMM and RS analysed data. MB, RS, JMM, E.C, exposed to sera from aGvHD patients.53 These reported findings, M.D-R. and DGB developed the overall concept, designed and su- together with the observed results in our in vivo model of allo-HCT pervised all research, discussed the data and wrote the manuscript. and aGvHD, led us to hypothesize that the protective mechanism of action of defibrotide could be related to interruption of adhesive in- DATA AVA I L A B I L I T Y S TAT E M E N T teractions between circulating lymphocytes and endothelium. With The data that support the findings of this study are available from the purpose of extrapolating our in vivo results to what would occur the corresponding authors upon reasonable request. in the human aGvHD condition, we studied whether defibrotide may affect cell-surface expression of adhesion receptors on human en- ORCID dothelial cells, as well as its effects on human leucocyte adhesion David García-Bernal https://orcid.org/0000-0001-6610-8442 and migration. Our results provide direct evidence that defibrotide down-regulates the expression of critical endothelial molecules in- REFERENCES volved in leucocyte recruitment into tissues (ie P-selectin, E-selectin, 1. Teshima T, Ordemann R, Reddy P, et al. Acute graft-versus-host dis- VCAM-1 and ICAM-1) during aGvHD. This mechanism, together with ease does not require alloantigen expression on host epithelium. Nat Med. 2002;8(6):575-581. the observed in vivo impairment in leucocyte adhesion and migration 2. 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