Faisal Kunnathodi

Inflammatory Response in Islet Transplantation

Islet cell transplantation is a promising beta cell replacement therapy for patients with brittle type 1 diabetes as well as refractory chronic pancreatitis. Despite the vast advancements made in this field, challenges still remain in achieving high frequency and long-term successful transplant outcomes. Here we review recent advances in understanding the role of inflammation in islet transplantation and development of strategies to prevent damage to islets from inflammation. The inflammatory response associated with islets has been recognized as the primary cause of early damage to islets and graft loss after transplantation. Details on cell signaling pathways in islets triggered by cytokines and harmful inflammatory events during pancreas procurement, pancreas preservation, islet isolation, and islet infusion are presented. Robust control of pre- and peritransplant islet inflammation could improve posttransplant islet survival and in turn enhance the benefits of islet cell transplantation for patients who are insulin dependent. We discuss several potent anti-inflammatory strategies that show promise for improving islet engraftment. Further understanding of molecular mechanisms involved in the inflammatory response will provide the basis for developing potent therapeutic strategies for enhancing the quality and success of islet transplantation.

Alleviation of instant blood-mediated inflammatory reaction in autologous conditions through treatment of human islets with NF-κB inhibitors.

Background The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA), an inhibitor of nuclear factor (NF) &kgr;B, was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-&kgr;B inhibitors affected IBMIR using an in vitro human autologous blood islet model. Methods Human islets were pretreated with or without NF-&kgr;B inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide, proinsulin, thrombin-antithrombin (TAT) complex, and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry. Results Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group, but not in the NF-&kgr;B inhibitor–treated groups, whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr, but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (P<0.001) and CAY10512 (P<0.001) groups. Conclusions A miniaturized in vitro tube model can be useful in investigating IBMIR. The presence of NF-&kgr;B inhibitor could alleviate IBMIR, thus improving the survival of transplanted islets. Protection of islets in the peritransplant phase may improve long-term graft outcomes.

NFAT targets signaling molecules to gene promoters in pancreatic β-cells.

Nuclear factor of activated T cells (NFAT) is activated by calcineurin in response to calcium signals derived by metabolic and inflammatory stress to regulate genes in pancreatic islets. Here, we show that NFAT targets MAPKs, histone acetyltransferase p300, and histone deacetylases (HDACs) to gene promoters to differentially regulate insulin and TNF-α genes. NFAT and ERK associated with the insulin gene promoter in response to glucagon-like peptide 1, whereas NFAT formed complexes with p38 MAPK (p38) and Jun N-terminal kinase (JNK) upon promoters of the TNF-α gene in response to IL-1β. Translocation of NFAT and MAPKs to gene promoters was calcineurin/NFAT dependent, and complex stability required MAPK activity. Knocking down NFATc2 expression, eliminating NFAT DNA binding sites, or interfering with NFAT nuclear import prevented association of MAPKs with gene promoters. Inhibiting p38 and JNK activity increased NFAT-ERK association with promoters, which repressed TNF-α and enhanced insulin gene expression. Moreover, inhibiting p38 and JNK induced a switch from NFAT-p38/JNK-histone acetyltransferase p300 to NFAT-ERK-HDAC3 complex formation upon the TNF-α promoter, which resulted in gene repression. Histone acetyltransferase/HDAC exchange was reversed on the insulin gene by p38/JNK inhibition in the presence of glucagon-like peptide 1, which enhanced gene expression. Overall, these data indicate that NFAT directs signaling enzymes to gene promoters in islets, which contribute to protein-DNA complex stability and promoter regulation. Furthermore, the data suggest that TNF-α can be repressed and insulin production can be enhanced by selectively targeting signaling components of NFAT-MAPK transcriptional/signaling complex formation in pancreatic β-cells. These findings have therapeutic potential for suppressing islet inflammation while preserving islet function in diabetes and islet transplantation.

Pancreatic Beta Cell-derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these “isletokines” in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ–induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet–specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti–IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell–induced inflammatory loss of graft function after islet cell transplantation.

Pancreatic Ductal Perfusion at Organ Procurement Enhances Islet Yield in Human Islet Isolation

Objective Pancreas preservation is a major factor influencing the results of islet cell transplantation. This study evaluated the effects of 2 different solutions for pancreatic ductal perfusion (PDP) at organ procurement. Methods Eighteen human pancreases were assigned to 3 groups: non-PDP (control), PDP with ET-Kyoto solution, and PDP with cold storage/purification stock solution. Pancreatic islets were isolated according to the modified Ricordi method. Results No significant differences in donor characteristics, including cold ischemia time, were observed between the 3 groups. All islet isolations in the PDP groups had more than 400,000 islet equivalence in total islet yield after purification, a significant increase when compared with the control (P = 0.04 and P < 0.01). The islet quality assessments, including an in vivo diabetic nude mice assay and the response of high-mobility group box protein 1 to cytokine stimulation, also showed no significant differences. The proportion of terminal deoxynucleotidyl transferase dUTP nick-end labeling–positive cells showing apoptosis in islets in the PDP groups was significantly lower than in the control group (P < 0.05). Conclusions Both ET-Kyoto solution and cold storage/purification stock solution are suitable for PDP and consistently resulted in isolation success. Further studies with a larger number of pancreas donors should be done to compare the effects of the PDP solutions.

Essential phospholipids prevent islet damage induced by proinflammatory cytokines and hypoxic conditions

The pancreatic islet damage that occurs through an inflammatory response and hypoxia after infusion is a major hurdle in islet transplantation. Because essential phospholipids (EPL) have been shown to exhibit anti‐inflammatory properties in liver disease, we analysed their protective effect on islets in inflammatory or hypoxic conditions.

Implication of IFNγ Production in Clinical Allogeneic Islet Transplantation in Contrast to Autologous Transplantation.: Abstract# 599

599 Implication of IFNγ Production in Clinical Allogeneic Islet Transplantation in Contrast to Autologous Transplantation. M. Takita,1 R. Shahbazov,1 F. Kunnathodi,1 M. Kanak,2 M. Lawrence,1 H. Ueno,3 B. Naziruddin,4 M. Levy.4 1Islet Cell Laboratory, Baylor Research Institute, Dallas, TX; 2Institute of Biomedical Studies, Baylor University, Waco, TX; 3Baylor Institute for Immunology Research, Dallas, TX; 4Baylor Annette C. and Harold C. Simmons Transplant Institute, Dallas, TX. Introduction: We have shown that the autoimmune response to GAD65 peptide is a crucial cause of graft dysfunction in clinical islet transplantation for type 1 diabetes and a signifi cant IFNγ release from CD4+ or CD8+ T cells which corresponded with the autoimmune response. In this study, the serum IFNγ levels in allogeneic islet recipients were assessed, in comparison with autologous recipients. Methods: Six allogeneic and 6 autologous islet recipients were included. Patients in both groups matched in basic characteristics. There was no difference in transplanted islet dose between both groups: 9,393±707 and 7,949±672 IEQ/kg of body weight, respectively (p=0.17). All recipients were administered with both etanercept and anakinra from Day 0 to 10. Autoimmune response was investigated with EpiMax assay after stimulation of peripheral blood mononuclear cells by GAD65 peptide clusters. Cytokine levels were measured using Luminex assay. Results: Signifi cant IFNγ production from both CD4+ and CD8+ T cells was observed at peri-transplant in allogeneic islet recipients (Fig. A). Signifi cant elevation of serum IFNγ levels were also seen immediately after islet infusion in allo-recipients and the elevation were sustained for 7 days (Fig. B). Autologous recipients, however, did not show signifi cant increase in IFNγ levels. Conclusions: Signifi cant difference in serum IFNγ levels was observed between allogeneic and autologous islet recipients even when similar dose of islets were transplanted. Further characterization of IFNγ production may help to determine the role of autoimmune response in clinical islet transplantation. Figure. An example of IFNγ production in EpiMax assay in an allogeneic recipient (A) and serum IFNγ levels in islet transplantation (B) are shown. Abstract# 600 The Instant Blood Mediated Inflammatory Reaction in a Dual Transplant Model of Islet Transplantation. K. Samy,1 B. Martin,1 M. Lowe,1 P. Thompson,1 A. Anderson,1 J. Cano,1 M. Song,1 F. Leopardi,1 E. Strobert,2 A. Kirk.1 1Emory Transplant Center; 2Yerkes National Primate Research Center, Atlanta, GA. Purpose: Signifi cant islet loss following intraportal infusion is attributed to a poorly defi ned Instant Blood Mediated Infl ammatory Reaction (IBMIR). We developed a nonhuman primate (NHP) dual transplant model to study factors contributing to early posttransplant alloand xeno-islet loss. Methods: Isolated islets were infused intraportally into rhesus macaques, with comparator islet preps separated into the right and left hemiliver. We assessed rhesus alloislets(AI) vs wild-type neonatal pig islets(WT)(n=4) and α-1,3-galactosyl transferase knockout neonatal pig islets(GKO) vs inert microspheres(MS)(n=6). Digital immunohistological analysis using Aperio Imagescope® software staining quantifi cation was performed on protocol liver sections taken at 1 and 24 hours post-transplant. Results: Comparing AI vs WT islets, galactose-α-1,3-galactose staining was distinct at 1h and 24h(p<0.05) indicating successful islet type segregation. C4d, IgG, IgM, macrophages, neutrophils, platelets, and caspase 3 were similar between islet phenotypes at 1 and 24 hours. Complement and antibody deposition unexpectedly increased over 24 hours in AI but not WT islets. Macrophage staining increased over 24 hours in WT(p=0.02) and AI(p=0.07) with increasing infi ltration observed on histology. Interestingly, inert MS vs GKO at one hour had no difference in staining of C4d, IgG, IgM, macrophages, or neutrophils(p>0.05). At 24 hours, GKO islets stained stronger than MS for C4d(p=0.06), neutrophils(p=0.04), macrophages(p=0.02), and platelets(p=0.02). Conclusions: These data suggest that complement is a signifi cant component of both alloand xeno-specifi c responses. Similarly, antibody deposition occurs in both and is initially non-specifi c. Macrophage infi ltration also plays a signifi cant role in the response to both islet phenotypes. The embolic phenomenon in the portal sinusoids also contributes to IBMIR, although this may be self-contained as infused cell antigenicity seemed to propagate the reaction. Thus, early IBMIR is likely a largely innate response initially driven by non-specifi c features of portal vein embolization, with subsequent infl ammatory responses common to both alloand xeno-islet preps. Differential alloor xeno-specifi c interactions likely manifest beyond 24 hours.