Immunomodulation by juglone alleviates acute graft-versus-host disease without compromising the graft-versus-leukaemia activity in mice.

BACKGROUND AND PURPOSE: Acute graft-versus-host disease (GVHD) remains a major barrier to successful transplantation outcomes. Recent studies have shown that pharmacotherapy for GVHD should target both the innate and adaptive inflammatory immune responses. Juglone, a redox-active phytochemical found in walnuts, has shown potent anti-inflammatory effects in models of colitis and inflammatory bowel disease. However, its effects on T-cell-mediated immune responses remain largely unknown. Considering the overlapping mediators of inflammation in GVHD and the aforementioned conditions, we investigated the use of juglone as a prophylactic agent for GVHD. EXPERIMENTAL APPROACH: Immunomodulatory activity and mechanism of action of juglone were studied using murine splenic leukocytes in vitro. The GVHD prophylactic efficacy of orally administered juglone was evaluated using a murine model of allogeneic haematopoietic stem cell transplantation based on an MHC mismatch. KEY RESULTS: Juglone exhibited immunomodulatory activity by (i) inhibiting the activation of dendritic cells and CD4+ T-cells, (ii) inhibiting cytokine secretion and lymphocyte proliferation, and (iii) inducing exhaustion of CD4+ T-cells, as shown by increased expression of CTLA-4 (CD152) and Fas (CD95). Oral administration of juglone significantly reduced mortality and morbidity associated with GVHD while maintaining graft-versus-leukaemia activity. This was accompanied by a decrease in the number of naïve CD4+ cells, and an increase in the number of CD4+ and CD8+ central memory T-cells. CONCLUSION AND IMPLICATIONS: Juglone is a potent immunomodulator for GVHD prophylaxis. Our study is the first to provide a dosage framework for the oral administration of juglone that can be used for clinical development.

Withania Somnifera Extract Mitigates Experimental Acute Graft versus Host Disease Without Abrogating Graft Versus Leukemia Effect.

Acute graft versus host disease (aGvHD) is the major contributor of nonrelapse mortality in alloHSCT. It is associated with an inflammatory immune response manifesting as cytokine storm with ensuing damage to target organs such as liver, gut, and skin. Prevention of aGvHD while retaining the beneficial graft versus leukemia (GvL) effect remains a major challenge. Withania somnifera extract (WSE) is known for its anti-inflammatory, immune-modulatory, and anticancer properties, which are appealing in the context of aGvHD. Herein, we demonstrated that prophylactic and therapeutic use of WSE in experimental model of alloHSCT mitigates aGvHD-associated morbidity and mortality. In the prophylaxis study, a dose of 75 mg/kg of WSE offered greatest protection against death due to aGvHD (hazard ratio [HR] = 0.15 [0.03-0.68], P ≤ .01), whereas 250 mg/kg was most effective for the treatment of aGvHD (HR = 0.16 [0.05-0.5], P ≤ .01). WSE treatment protected liver, gut, and skin from damage by inhibiting cytokine storm and lymphocytic infiltration to aGvHD target organs. In addition, WSE did not compromise the GvL effect, as alloHSCT with or without WSE did not allow the leukemic A20 cells to grow. In fact, WSE showed marginal antileukemic effect in vivo. WSE is currently under clinical investigation for the prevention and treatment of aGvHD.

Mitochondrial-targeted curcumin inhibits T-cell activation via Nrf2 and inhibits graft-versus-host-disease in a mouse model.

Anti-inflammatory and immune suppressive agents are required to moderate hyper-activation of lymphocytes under disease conditions or organ transplantation. However, selective disruption of mitochondrial redox has not been evaluated as a therapeutic strategy for suppression of T-cell-mediated pathologies. Using mitochondrial targeted curcumin (MitoC), we studied the effect of mitochondrial redox modulation on T-cell responses by flow cytometry, transmission electron microscopy, transcriptomics, and proteomics, and the role of Nrf2 was studied using Nrf2- /- mice. MitoC decreased mitochondrial TrxR activity, enhanced mitochondrial ROS (mROS) production, depleted mitochondrial glutathione, and suppressed activation-induced increase in mitochondrial biomass. This led to suppression of T-cell responses and metabolic reprogramming towards Treg differentiation. MitoC induced nuclear translocation and DNA binding of Nrf2, leading to upregulation of Nrf2-dependent genes and proteins. MitoC-mediated changes in mitochondrial redox and modulation of T-cell responses are abolished in Nrf2- /- mice. Restoration of mitochondrial thiols abrogated inhibition of T-cell responses. MitoC suppressed alloantigen-induced lymphoblast formation, inflammatory cytokines, morbidity, and mortality in acute graft-versus-host disease mice. Disruption of mitochondrial thiols but not mROS increase inculcates an Nrf2-dependent immune-suppressive disposition in T cells for the propitious treatment of graft-versus-host disease.

Withaferin-A alleviates acute graft versus host disease without compromising graft versus leukemia effect.

Acute graft versus host disease (aGvHD) contributes to a significant proportion of non-relapse mortality and morbidity in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Withaferin-A (WA), a phytomolecule obtained from Withania somnifera (Ashwagandha), is known to have anti-inflammatory, anti-proliferative and immunomodulatory properties. The efficacy of WA for the prevention and treatment of aGvHD was evaluated using a murine model of alloHSCT. Prophylactic administration of WA to mice mitigated the clinical symptoms of aGvHD and improved survival significantly compared to the GvHD control [HR = 0.07 (0.01-0.35); P < 0.001]. Furthermore, WA group had better overall survival compared to standard prophylactic regimen of CSA + MTX [HR = 0.19 (0.03-1.1), P < 0.05]. At the same time, WA did not compromise the beneficial GvL effect. In addition, WA administered to animals after the onset of aGvHD could reverse the clinical severity and improved survival, thus establishing its therapeutic potential. Our findings suggest that WA reduced the systemic levels of Th1, Th2 and Th17 inflammatory cytokine and increased the anti-inflammatory cytokine IL-10 levels significantly (P < 0.05). WA also inhibited lymphocytes migration to gut, liver, skin and lung and protected these organs from damage. Ex-vivo, WA inhibited proliferation of human peripheral blood mononuclear cells (hPBMCs), modulated immune cell phenotype and decreased cytokine release. In addition, WA inhibited pJAK2 and pSTAT3 protein levels in mouse splenocytes and hPBMCs. In conclusion, our study demonstrates the utility of WA for the prevention and treatment of aGvHD, which should be further evaluated in a clinical setting.

Safety, toxicity and pharmacokinetic assessment of oral Withaferin-A in mice.

Withaferin-A (WA) is the principle component of Withania somnifera (Ashwagandha). It has several biological activities including anti-cancer, anti-diabetic, neuroprotective, hepatoprotective and immune-modulatory properties. The acute and sub-acute toxicity of oral WA was investigated in mice. In the acute toxicity study, up to 2000 mg/kg of WA was well tolerated without any signs of toxicity or death. In the sub-acute toxicity study, mice were orally administered 10, 70 and 500 mg/kg of WA respectively, daily for 28 days. Upon physiological, serum chemistry, hematology and histopathogical examination, no features suggestive of drug-induced toxicity were observed at any dose levels, thereby confirming the No-Observed Adverse Effect Level (NOAEL) to be at least 500 mg/kg. Furthermore, the oral bioavailability of WA was evaluated using single intravenous and oral doses of 10 mg/kg and 70 mg/kg respectively using sparse sampling strategy. Bioanalysis was carried out using a validated LC-MS/MS method. The AUC of WA was found to be 3996.9 ± 557.6 ng/mL*h and 141.7 ± 16.8 ng/mL*h for the intravenous and oral routes of administration respectively. The oral bioavailability was determined to be 1.8%. To conclude, WA was found to be extremely safe even at high doses, with a low oral bioavailability.

Acute and sub-acute oral toxicity assessment of 5-hydroxy-1,4-naphthoquinone in mice.

5-hydroxy-1,4-naphthoquinone (5NQ) or juglone is a bioactive molecule found in walnuts and has shown therapeutic effects in various disease models. Limited information is available regarding the toxicity of 5NQ, thereby limiting the clinical development of this drug. In the present study, oral acute (50, 300 and 2000 mg/kg) and sub-acute toxicity (5, 15 and 50 mg/kg) was assessed in mice to evaluate the safety of 5NQ. The acute toxicity study identified 118 mg/kg as the point-of-departure dose (POD) for single oral administration of 5NQ using benchmark dose modeling (BMD). Repeated administration of 5NQ at doses of 15 and 50 mg/kg/day caused reduction in food consumption and body weight of mice along with alterations in liver and renal function. Histopathological assessment revealed significant damage to hepatic and renal tissues at all doses in the acute toxicity study, and at higher doses of 15 and 50 mg/kg in the sub-acute toxicity study. We observed dose dependent mortality in sub-acute toxicity study and the no observed adverse effect level (NOAEL) was established as < 5 mg/kg/day. Modeling the survival response in sub-acute toxicity study identified 1.74 mg/kg/day as the POD for repeated administration of 5NQ. Serum levels of aspartate aminotransferase (AST) were most sensitive to 5NQ administration with a lower limit of BMD interval (BMDL) of 1.1 × 10-3 mg/kg/day. The benchmark doses reported in the study can be further used to determine a reference dose of 5NQ for human risk assessment.

Comparison of different autoanalyzers for the determination of lymphocyte and neutrophil counts in mouse blood.

OBJECTIVES: Autoanalyzers are used in clinical haematology for analysis of blood samples in clinical as well as in nonclinical studies. The results from these analyzers vary from machine to machine. In this study, we compared the lymphocyte and neutrophil count of mouse blood between ADVIA 2120i, Horiba Yumizen H2500 and CellaVision analyzers against manual counting as gold standard. METHODS: Blood samples from 28 female BALB/c mice were collected and analyzed. Agreement between different autoanalyzers and manual counting were determined by Bland-Altman method. RESULTS: A high level of agreement was found between CellaVision and manual technique for lymphocyte (bias=4.75, 95% limits of agreement -14 to 24) and neutrophil count (bias=0.68 [-17 to 19]). Agreement in lymphocyte count was also observed between ADVIA and manual counting, but to a lesser extent compared to CellaVision (bias=13.9 [-10.45 to 38.27]). However, no agreement was observed for ADVIA (Neutrophils), Horiba (lymphocytes and neutrophils) with manual counting. CONCLUSIONS: Our data suggests that CellaVision could be used for the differential counting of neutrophil and lymphocytes in mouse blood sample.

Prevention of acute graft-versus-host-disease by Withaferin a via suppression of AKT/mTOR pathway.

Acute Graft versus Host Disease (aGVHD) is a frequent and serious complication in patients receiving allogeneic bone marrow transplantation (allo-BMT) and often requires rigorous prophylaxis. The current treatment regimens for aGVHD are associated with several side effects which necessitates the development of novel interventions that prevent aGVHD without precluding graft-versus-tumor effects. In the present study, we show that treatment of donor graft with plant steroidal lactone Withaferin A (WA) prior to transplantation markedly reduced aGVHD mediated damage in target organs without compromising the graft-versus.-tumor activity of the transplanted lymphocytes. WA abrogated post-transplant cytokine storm associated with allo-activation of donor lymphocytes. This was attributed to the ability of WA to inhibit early signaling events in T-cell activation including lymphoblast formation and activation of AKT/mTOR pathway. Mortality and morbidity related to allo-transplantation was significantly reduced in recipients of WA treated donor splenocytes compared to recipient of vehicle treated donor splenocytes. Further, WA treatment did not have any effect on reconstitution of lymphoid and myeloid lineages in recipients, resulting in stable and complete donor chimerism. In agreement with previous reports showing the effectiveness of WA in a mouse model of partial chimerism, our data further establishes that WA is able to attenuate aGVHD in an MHC-mismatched high dose chemo-conditioned murine model without compromising engraftment. This study provides compelling scientific basis for possible application of WA for prevention and treatment of aGVHD in patients receiving allo-BMT.