Yonghua Sheng

Glycemic control in diabetes is restored by therapeutic manipulation of cytokines that regulate beta cell stress

In type 2 diabetes, hyperglycemia is present when an increased demand for insulin, typically due to insulin resistance, is not met as a result of progressive pancreatic beta cell dysfunction. This defect in beta cell activity is typically characterized by impaired insulin biosynthesis and secretion, usually accompanied by oxidative and endoplasmic reticulum (ER) stress. We demonstrate that multiple inflammatory cytokines elevated in diabetic pancreatic islets induce beta cell oxidative and ER stress, with interleukin-23 (IL-23), IL-24 and IL-33 being the most potent. Conversely, we show that islet-endogenous and exogenous IL-22, by regulating oxidative stress pathways, suppresses oxidative and ER stress caused by cytokines or glucolipotoxicity in mouse and human beta cells. In obese mice, antibody neutralization of IL-23 or IL-24 partially reduced beta cell ER stress and improved glucose tolerance, whereas IL-22 administration modulated oxidative stress regulatory genes in islets, suppressed ER stress and inflammation, promoted secretion of high-quality efficacious insulin and fully restored glucose homeostasis followed by restitution of insulin sensitivity. Thus, therapeutic manipulation of immune regulators of beta cell stress reverses the hyperglycemia central to diabetes pathology.

Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease

Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell–dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed 51Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.

Colonic microbiota can promote rapid local improvement of murine colitis by thioguanine independently of T lymphocytes and host metabolism.

Objective Mercaptopurine (MP) and pro-drug azathioprine are ‘first-line’ oral therapies for maintaining remission in IBD. It is believed that their pharmacodynamic action is due to a slow cumulative decrease in activated lymphocytes homing to inflamed gut. We examined the role of host metabolism, lymphocytes and microbiome for the amelioration of colitis by the related thioguanine (TG). Design C57Bl/6 mice with or without specific genes altered to elucidate mechanisms responsible for TGs actions were treated daily with oral or intrarectal TG, MP or water. Disease activity was scored daily. At sacrifice, colonic histology, cytokine message, caecal luminal and mucosal microbiomes were analysed. Results Oral and intrarectal TG but not MP rapidly ameliorated spontaneous chronic colitis in Winnie mice (point mutation in Muc2 secretory mucin). TG ameliorated dextran sodium sulfate-induced chronic colitis in wild-type (WT) mice and in mice lacking T and B lymphocytes. Remarkably, colitis improved without immunosuppressive effects in the absence of host hypoxanthine (guanine) phosphoribosyltransferase (Hprt)-mediated conversion of TG to active drug, the thioguanine nucleotides (TGN). Colonic bacteria converted TG and less so MP to TGN, consistent with intestinal bacterial conversion of TG to so reduce inflammation in the mice lacking host Hprt. TG rapidly induced autophagic flux in epithelial, macrophage and WT but not Hprt−/− fibroblast cell lines and augmented epithelial intracellular bacterial killing. Conclusions Treatment by TG is not necessarily dependent on the adaptive immune system. TG is a more efficacious treatment than MP in Winnie spontaneous colitis. Rapid local bacterial conversion of TG correlated with decreased intestinal inflammation and immune activation.

MUC13 protects colorectal cancer cells from death by activating the NF-κB pathway and is a potential therapeutic target

MUC13 is a transmembrane mucin glycoprotein that is over produced by many cancers, although its functions are not fully understood. Nuclear factor-κB (NF-κB) is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently upregulating BCL-XL. MUC13 promoted tumor necrosis factor (TNF)-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NF-κB essential modulator. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signaling in response to both TNF and DNA-damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to killing by cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers.

Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation

Current immunosuppressive/anti-inflammatory agents target the responding effector arm of the immune response and their nonspecific action increases the risk of infection and malignancy. These effects impact on their use in allogeneic haematopoietic cell transplantation and other forms of transplantation. Interventions that target activated dendritic cells (DCs) have the potential to suppress the induction of undesired immune responses (for example, graft versus host disease (GVHD) or transplant rejection) and to leave protective T-cell immune responses intact (for example, cytomegalovirus (CMV) immunity). We developed a human IgG1 monoclonal antibody (mAb), 3C12, specific for CD83, which is expressed on activated but not resting DC. The 3C12 mAb and an affinity improved version, 3C12C, depleted CD83+ cells by CD16+ NK cell-mediated antibody-dependent cellular cytotoxicity, and inhibited allogeneic T-cell proliferation in vitro. A single dose of 3C12C prevented human peripheral blood mononuclear cell-induced acute GVHD in SCID mouse recipients. The mAb 3C12C depleted CMRF-44+CD83bright activated DC but spared CD83dim/- DC in vivo. It reduced human T-cell activation in vivo and maintained the proportion of CD4+ FoxP3+ CD25+ Treg cells and also viral-specific CD8+ T cells. The anti-CD83 mAb, 3C12C, merits further evaluation as a new immunosuppressive agent in transplantation.

MUC13 overexpression in renal cell carcinoma plays a central role in tumor progression and drug resistance

Metastatic renal cell carcinoma is a largely incurable disease, and existing treatments targeting angiogenesis and tyrosine kinase receptors are only partially effective. Here we reveal that MUC13, a cell surface mucin glycoprotein, is aberrantly expressed by most renal cell carcinomas, with increasing expression positively correlating with tumor grade. Importantly, we demonstrated that high MUC13 expression was a statistically significant independent predictor of poor survival in two independent cohorts, particularly in stage 1 cancers. In cultured renal cell carcinoma cells MUC13 promoted proliferation and induced the cell cycle regulator, cyclin D1, and inhibited apoptosis by inducing the anti‐apoptotic proteins, BCL‐xL and survivin. Silencing of MUC13 expression inhibited migration and invasion, and sensitized renal cancer cells to killing by the multi‐kinase inhibitors used clinically, sorafenib and sunitinib, and reversed acquired resistance to these drugs. Furthermore, we demonstrated that MUC13 promotion of renal cancer cell growth and survival is mediated by activation of nuclear factor κB, a transcription factor known to regulate the expression of genes that play key roles in the development and progression of cancer. These results show that MUC13 has potential as a prognostic marker for aggressive early stage renal cell cancer and is a plausible target to sensitize these tumors to therapy.

The Monoclonal Antibody 3C12C, Targeting CD83 Is a T Cell Sparing, Potential New Immunosuppressive Agent

A42 Immunosuppressive Capacities of Human Renal Tubular Epithelial Cells; a Role for Indoleamine 2,3-Dioxygenase? M. Demmers, C. Baan, M. Roemeling-van Rhijn, T. van den Bosch, M. Hoogduijn, M. Betjes, W. Weimar, A. Rowshani. Internal Medicine, Section Nephrology and Transplantation, Erasmus MC University Medical Center, Rotterdam, Netherlands. Introduction Renal tubular epithelial cells (TECs) are one of the main targets of T cell attack during acute cellular rejection. We hypothesize that TECs modulate the outcome of allo-immunity in a bi-directional way executing immunosuppressive effects and dampening the local infl ammation. Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme inhibiting T-cell proliferation. TECs express cytoplasmic IDO during acute rejection. We studied whether TECs possess immunosuppressive capacities and if IDO might play a role suppressing T-cell alloactivity. Materials and Methods Anti CD3/CD28 activated peripheral blood mononuclear cells were cocultured with IFN-γ/TNF-α activated TECs for 3 days. We analysed CD4+ T-cell and CD8+ T-cell proliferation response in the absence or presence of IDO inhibitor 1-L-MT. Next we analysed early and late apoptosis as increased IDO acitivity is associated with increased apoptosis. Further we examined whether inhibition of T cell proliferation was cell-cell contact dependent using transwell membrane experiments. Results We found that TECs dose-dependently inhibited CD4+ T-cell and CD8+ T-cell proliferation. TEC mRNA analysis and supernatant L-kynurenine showed that activated TECs express IDO mRNA expression and signifi cantly upregulated L-kynureninen, which was signifi cantly downregulated using 1-L-MT. Transwell experiments showed that TEC-mediated immunosuppression is cell-cell contact dependent. Downregulated CD4+ T-cell proliferation was partly recovered after addition of 1-L-MT, while CD8+ T-cell proliferation was not affected by 1-L-MT. Activated TECs increased early and late apoptosis of proliferating CD4+ T-cells, 1-L-MT abrogated both early and late CD4+ T-cell apoptosis. Discussion Our data show that TECs possess immunosuppressive capacities and inhibit the allo-reactive T cell proliferation that can partly be explained by indoleamine 2,3-dioxygenase immune regulation. Abstract# A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. A43 Introduction of a New Cell Model of Biopsy-Derived Human Proximal Tubule Cells to Study the Role of Pharmacogenetics in CNIAssociated Nephrotoxicity. N. Knops,1,2 D. Kuypers,3 R. Masereeuw,4 E. Levtchenko,1,2 L. Van den Heuvel.2 1Pediatric Nephrology and Solid Organ Transplantation, University Hospital Leuven, Leuven, Belgium; 2Labarotory for Pediatrics, Dept of Development & Regeneration, KU Leuven, Leuven, Belgium; 3Nephrology, University Hospital Leuven, Leuven, Belgium; 4Pharmacology and Toxicology, Radboud University, Nijmegen, Netherlands. Background: Calcineurin inhibitors (CNI) constitute the basis of immunosuppressive regimes in transplantation, but are associated with the development of histological lesions leading to kidney failure. CNI’s are metabolized by CYP3A and excreted by Pgp (ABCB1) in the gut and liver but also in proximal tubular cells (PTC). Clinical studies demonstrated a relation between common variants of CYP3A5/ ABCB1 genes and CNI-associated nephrotoxicity (CNIT). The mechanism is unknown. We established a model of human PTC that can be used to study the pathogenesis of CNIT. Methods: A technique was developed to culture cells from a protocol biopsy in renal allograft recipients. Primary cells were transfected with SV40T and hTERT virus for conditional immortalization and differentiation. Subclones were selected based upon specifi c PTC markers (AQP1 and CD13) using Western Blot (WB) and FACS. Light and scanning electron microscopy were performed to detect PTC morphology. PCR and sequencing was used to assess genotype. Quantative RT-PCR, WB and immunohistochemistry was performed for CYP3A5 an ABCB1 expression. CYP3A5 activity was assessed by differential midazolam(MDZ) hydroxylation using LC-MS and Pgp activity by calcein effl ux. Results: From 27 out of 38 biopsies cell lines were generated. Based upon genotype 11 subclones with PTC biomarkers were selected. In vitro PTC morphology with brush border microvilli was observed. We confi rmed CYP3A5 and Pgp mRNA and protein expression. CYP3A5*1 carriers had increased 1OH/4OH MDZ formation vs.*3/*3 (1,44 vs 0,7; p<0,05). Pgp activity was confirmed by 39% calcein accumulation(95% CI:33-44), but not related ABCB1 3435CT genotype. Tacrolimus disappearance was 49 times higher in CYP3A5*1 vs.*3/*3 carriers, but again not related to ABCB1 3435CT genotype. Conclusion: PTC cell lines can be generated from a kidney biopsy and demonstrate functional expression of genes involved in CNI metabolism after immortalization. Differences in protein function were detected for CYP3A5 genotype. This in vitro model can be used to study the role of pharmacogenetic variation in CNIT. DISCLOSURES: Knops, N.: Grant/Research Support, Astellas. Kuypers, D.: Grant/ Research Support, Astellas. Levtchenko, E.: Grant/Research Support, Astellas. Abstract# A44 Angiogenin Promotes Cell Survival During Cyclosporine-Induced Endoplasmic Reticulum Stress. I. Mami,1 N. Bovier,1 S. Pezet,1 P. Beaune,1,2 N. Pallet,1,2 E. Thervet.1,3 1INSERM U-775, INSERM, Paris, France; 2Service de Biochimie, Hopital Europeen Georges Pompidou, Paris, France; 3Service de Nephrologie, Hopital Europeen Georges Pompidou, Paris, France. Background Calcineurin inhibitors nephrotoxicity promotes chronic kidney injury, and contributes to chronic allograft nephropathy. We have demonstrated previously that cyclosporine is an ER stress inducer, ER stress mediates its nephrotoxicity. ER stress contributes to kidney disease, and constitutes a progression factor. Recent studies suggest that Angiogenin (ANG), a stress-activated and secreted ribonuclease, cleaves tRNA to generate fragments called tiRNA. These tiRNA contribute to stress-induced translational repression, indicating that ANG and tiRNA help to reprogram protein translation during stress, and are previously unappreciated components of the stress response. The implication of tiRNA in the ER stress-induced translational repression is unknown. Objectives Our hypothesis is that cyclosporine regulates the production and activation of ANG during the Unfolded Protein Response (UPR), the adaptive program activated in response to ER stress, in the kidney epithelium. That ANG promotes cellular adaptation during stress, mediated by tiRNA integrated in the UPR-induced translational repression. The purpose of this study is to characterize the mechanisms of ANG synthesis, cellular localization and biological functions, during ER stress activated by cyclosporine. Results In a model of human epithelial cells, we have demonstrated that ANG expression is induced during ER stress, that ANG production depends on IRE1a, and that ANG expression is regulated by the transcription factor sXBP1 and NF-kB. ER stress promotes a nucleo-cytoplasmic transfert of ANG which localizes in part in stress granules. ANG inhibits ER stress-indu