Inflammatory and hypoxic stress-induced islet exosomes released during isolation are associated with poor transplant outcomes in islet autotransplantation.

Islets experience enormous stress during the isolation process, leading to suboptimal endocrine function after total pancreatectomy with islet autotransplantation (TPIAT). Our investigation focused on inducing isolation stress in islets ex vivo, where proinflammatory cytokines and hypoxia prompted the release of stress exosomes (exoS) sized between 50 and 200 nm. Mass spectrometry analysis revealed 3 distinct subgroups of immunogenic proteins within these exoS: damage-associated molecular patterns (DAMPs), chaperones, and autoantigens. The involvement of endosomal-sorting complex required for transport proteins including ras-associated binding proteins7A, ras-associated binding protein GGTA, vacuolar protein sorting associated protein 45, vacuolar protein sorting associated protein 26B, and the tetraspanins CD9 and CD63, in exoS biogenesis was confirmed through immunoblotting. Next, we isolated similar exoS from the islet infusion bags of TPIAT recipients (N = 20). The exosomes from infusion bags exhibited higher DAMP (heat shock protein family A [Hsp70] member 1B and histone H2B) levels, particularly in the insulin-dependent TPIAT group. Additionally, elevated DAMP protein levels in islet infusion bag exosomes correlated with increased insulin requirements (P = .010) and higher hemoglobin A1c levels 1-year posttransplant. A deeper exploration into exoS functionality revealed their potential to activate monocytes via the toll-like receptor 3/7: DAMP axis. This stimulation resulted in the induction of inflammatory phenotypes marked by increased levels of CD68, CD80, inducible nitric oxide synthase, and cyclooxygenase-2. This activation mechanism may impact the successful engraftment of transplanted islets.

Exosomes in transplantation: Role in allograft rejection, diagnostic biomarker, and therapeutic potential.

Exosomes are 50-200 nm-sized extracellular vesicles that are secreted by cells to transfer signals and communicate with other cells. Recent research has revealed that allograft-specific exosomes containing proteins, lipids, and genetic materials are released into circulation post-transplantation which are powerful indicators of graft failure in solid-organ and tissue transplantations. The macromolecular content of exosomes released by the allograft and the immune cells serve as potential biomarkers for assessing the function and the acceptance/rejection status of the transplanted grafts. Identifying these biomarkers could aid in the development of therapeutic strategies to improve graft longevity. Exosomes can be used to deliver therapeutic agonists/antagonists to grafts and prevent rejection. Inducing long-term graft tolerance has been demonstrated in many studies using exosomes from immunomodulatory cells such as immature DCs, T regulatory cells, and MSCs. The use of graft-specific exosomes for targeted drug therapy has the potential to reduce the unwanted side effects of immunosuppressive drugs. Overall, in this review, we have explored the critical role of exosomes in the recognition and cross-presentation of donor organ-specific antigens during allograft rejection. Additionally, we have discussed the potential of exosomes as a biomarker for monitoring graft function and damage, as well as their potential therapeutic applications in mitigating allograft rejection.

Assessment of Culture/Preservation Conditions of Human Islets for Transplantation.

Islet culture before clinical transplantation has been adopted by various centers, but its effect on the survival and function of islets relative to the culture conditions and media needs further assessment. Human islets were cultured or preserved under four different conditions and three media options. Parameters such as recovery, viability, function, islet damage, and gene expressions for markers of hypoxia, and inflammation were assessed after 48-h culture or preservation. Preservation of islets was performed at 4°C in Connaught's Medical Research Lab (CMRL) and University of Wisconsin (UW) media. Islets were cultured at 22°C, 37°C, and 37°C-22°C in CMRL and PRODO culture media. Islets preserved in UW solution had visually good morphology and exhibited higher recovery with less islet damage compared with the rest of the groups, whereas islets preserved in CMRL at 4°C resulted in poor morphology, recovery, viability, and function compared with the rest of the treatment conditions. Culture at 22°C and 37°C demonstrated an increase in the expression of inflammatory and hypoxia-related genes. In conclusion, islets preserved at 4°C in UW solution showed the best overall outcomes after 48 h compared with islets cultured at 22°C, 37°C, or 37°C-22°C in PRODO. Advancement in islet culture media is warranted to reduce inflammatory gene activation and improve recovery of islets for transplantation.

Withaferin A inhibits lymphocyte proliferation, dendritic cell maturation in vitro and prolongs islet allograft survival.

The immunosuppressive regimen for clinical allogeneic islet transplantation uses beta cell-toxic compounds such as tacrolimus that cause islet graft loss. Previously we reported that the plant-derived steroidal lactone Withaferin A (WA) can protect islet grafts by inhibiting nuclear factor-kappa B (NF-κB). Since the NF-κB signaling pathway is essential for T-cell activation, we hypothesized that long-term WA administration may also provide an immunosuppressive effect. Treatment of BALB/c donor islets and C57BL/6N recipients with WA alone resulted in 80% islet graft long-term survival vs. 40% in low-dose FK506-treated mice. In vitro, WA significantly blocked mouse and human T-cell proliferation by CD3/CD28 bead stimulation and in mixed lymphocyte reaction assay. Treatment of immature dendritic cells with WA prevented their maturation in response to inflammatory stimuli, as seen by decreased expression of CD83 and human leukocyte antigen-DR isotype. Exosomes released by islets treated with WA contained significantly fewer proinflammatory molecules interleukin-6, interleukin-8, monocyte chemoattractant protein-1, interferon-gamma-induced protein-10, inducible nitric oxide synthase, and cyclooxygenase-2. In conclusion, WA treatment not only reduced inflammation but also prolonged allograft survival, possibly through suppression of dendritic cell maturation and T-cell proliferation. WA has the potential to inhibit both the innate and adaptive immune response to prolong allograft survival.

Differential expression and release of exosomal miRNAs by human islets under inflammatory and hypoxic stress.

AIMS/HYPOTHESIS: Pancreatic islets produce non-coding microRNAs (miRNAs) that regulate islet cell function and survival. Our earlier investigations revealed that human islets undergo significant damage due to various types of stresses following transplantation and release miRNAs. Here, we sought to identify and validate exosomal miRNAs (exo-miRNAs) produced by human islets under conditions of cellular stress, preceding loss of cell function and death. We also aimed to identify islet stress signalling pathways targeted by exo-miRNAs to elucidate potential regulatory roles in islet cell stress. METHODS: Human islets were subjected to proinflammatory cytokine and hypoxic cell stress and miRNA from exosomes was isolated for RNA sequencing and analysis. Stress-induced exo-miRNAs were evaluated for kinetics of expression and release by intact islets for up to 48 h exposure to cytokines and hypoxia. A subset of stress-induced exo-miRNAs were assessed for recovery and detection as biomarkers of islet cell stress in a diabetic nude mouse xenotransplant model and in patients undergoing total pancreatectomy with islet auto-transplantation (TPIAT). Genes and signalling pathways targeted by stress-induced exo-miRNAs were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and direct interactions of miRNAs with downstream signalling targets were validated in human islet cells using the miRNA Tests for Read Analysis and Prediction (MirTrap) system. RESULTS: Global exo-miRNA sequencing revealed that 879 miRNA species were released from human islets and 190 islet exo-miRNAs were differentially expressed in response to proinflammatory cytokines, hypoxia or both. Release of exo-miRNAs hsa-miR-29b-3p and hsa-miR-216a-5p was detected within 6 h of exposure to cytokines and hypoxia. The remaining subset of stress-induced exo-miRNAs, including hsa-miR-148a-3p and islet cell damage marker hsa-miR-375, showed delayed release at 24-48 h, correlating with apoptosis and cell death. Stress and damage exo-miRNAs were significantly elevated in the circulation in human-to-mouse xenotransplant models and in human transplant recipients. Elevated blood exo-miRNAs negatively correlated with post-transplant islet function based on comparisons of stress and damage exo-miRNA indices with Secretory Unit of Islet Transplant Objects (SUITO) indices. KEGG analysis and further validation of exo-miRNA targets by MirTrap analysis revealed significant enrichment of islet mRNAs involved in phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase signalling pathways. CONCLUSIONS/INTERPRETATION: The study identifies exo-miRNAs differentially expressed and released by islets in response to damage and stress. These exo-miRNAs could serve as potential biomarkers for assessing islet damage and predicting outcomes in islet transplantation. Notably, exo-miRNAs 29b-3p and 216a-5p could be detected in islets prior to damage-released miRNAs and indicators of cellular apoptosis and death. Thus, these stress-induced exo-miRNAs may have potential diagnostic value for detecting early islet stress prior to progressive loss of islet cell mass and function. Further investigations are warranted to investigate the utility of these exo-miRNAs as early indicators of islet cell stress during prediabetic conditions.

Islet damage during isolation as assessed by miRNAs and the correlation of miRNA levels with posttransplantation outcome in islet autotransplantation.

High-quality pancreatic islets are essential for better posttransplantation endocrine function in total pancreatectomy with islet autotransplantation (TPIAT), yet stress during the isolation process affects quality and yield. We analyzed islet-enriched microRNAs (miRNAs) -375 and -200c released during isolation to assess damage and correlated the data with posttransplantation endocrine function. The absolute concentration of miR-375, miR-200c, and C-peptide was measured in various islet isolation steps, including digestion, dilution, recombination, purification, and bagging, in 12 cases of TPIAT. Posttransplantation glycemic control was monitored through C-peptide, hemoglobin A1c , insulin requirement, and SUITO index. The amount of miR-375 released was significantly higher during enzymatic digestion followed by the islet bagging (P < .001). Mir-200c mirrored these changes, albeit at lower concentrations. In contrast, the C-peptide amount was significantly higher in the purification and bagging steps (P < .001). Lower amounts of miR-375 were associated with a lower 6-month insulin requirement (P = .01) and lower hemoglobin A1c (P = .04). Measurement of the absolute quantity of miRNA-375 and -200c released during islet isolation is a useful tool to assess islet damage. The quantity of released miRNA is indicative of posttransplantation endocrine function in TPIAT patients.

Pancreatic ß-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 ß-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 ß-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 ß-cells and reveal IP-10 as a primary therapeutic target to prevent ß-cell-induced inflammatory loss of graft function after islet cell transplantation.

MiR-375 and miR-200c as predictive biomarkers of islet isolation and transplantation in total pancreatectomy with islet autotransplantation.

BACKGROUND: Total pancreatectomy with islet autotransplantation (TPIAT) is a promising treatment for refractory chronic pancreatitis. Predictable biomarkers for the endocrine function after transplantation would be helpful in selecting patients for TPIAT. This study aims to identify novel biomarkers for predicting the outcome of islet isolation and transplantation in TPIAT patients. METHODS: This paper studied microRNA of 31 TPIAT patients and 11 deceased donors from plasma samples before TPIAT. MiR-7, miR-200a, miR-200c, miR-320, and miR-375 were analyzed along with patient characteristics and the outcomes of islet isolation and transplantation via univariate and multivariate regression analysis. RESULTS: MiR-375 before TPIAT showed a significant correlation with ∆C-peptide (r = -0.396, P = 0.03) and post-digestion islet count (r = -0.372, P = 0.04). And also miR-200c was significantly correlated with insulin requirement, C-peptide, and SUITO index at 1 year after transplantation. Moreover it was confirmed that miR-200c was a predictable factor of endocrine outcome in multi regression analysis (coefficient = -7.081, P = 0.001). CONCLUSIONS: We concluded that miR-375 and miR-200c could potentially serve as novel biomarkers in predicting the islet yield in islet isolation and the metabolic function after transplantation for chronic pancreatitis patients.

Autologous and Allogenous Antibodies in Lung and Islet Cell Transplantation.

The field of organ transplantation has undoubtedly made great strides in recent years. Despite the advances in donor-recipient histocompatibility testing, improvement in transplantation procedures, and development of aggressive immunosuppressive regimens, graft-directed immune responses still pose a major problem to the long-term success of organ transplantation. Elicitation of immune responses detected as antibodies to mismatched donor antigens (alloantibodies) and tissue-restricted self-antigens (autoantibodies) are two major risk factors for the development of graft rejection that ultimately lead to graft failure. In this review, we describe current understanding on genesis and pathogenesis of antibodies in two important clinical scenarios: lung transplantation and transplantation of islet of Langerhans. It is evident that when compared to any other clinical solid organ or cellular transplant, lung and islet transplants are more susceptible to rejection by combination of allo- and autoimmune responses.

Telmisartan attenuated LPS-induced neuroinflammation in human IMR-32 neuronal cell line via SARM in AT1R independent mechanism.

AIM: The aim of this study was to find the protective role of Telmisartan (TS) in LPS intoxicated neuronal cells and elucidate the possible neuroprotective mechanism of action. MAIN METHODS: TLR4 and AT1R specific primers were designed and used in rtPCR to confirm the receptor expression in IMR-32 and Neuro2A cell lines. The protective effect of TS was assayed by MTT assay. The mechanism of action of TS was elucidated by assessing the expression and activation of TLR4 specific adaptor proteins SARM and MyD88, phosphorylated NFκB, PPARγ, MAPK p38, c-JNK, ERK by Western blotting. Selective PPARγ antagonist GW9662 was used to confirm the link between PPARγ activation and TLR4 mediated NFκB inflammatory mechanisms. The pro-inflammatory cytokines TNFα, IL1ß, and IL-6 and anti-inflammatory cytokine IL10 release were measured by ELISA. KEY FINDINGS: IMR-32 cells expressed TLR4 receptor and Neuro2A cells expressed both AT1R and TLR4 receptors. TS significantly protected both the cell lines from LPS intoxication. TS significantly suppressed the TLR4 mediated inflammatory response by PPARγ and SARM protein activation and the effect was reversed significantly by selective PPARγ antagonist GW9662, confirming the existence of link between PPARγ activation and TLR4 mediated inflammation via SARM. SIGNIFICANCE: LPS intoxicated human neuronal IMR-32 cells can be a good in vitro model to study inflammatory mediated neurodegeneration due to TLR4 receptor expression. Our study strongly recommends that the PPARγ activating pleiotropic effect of TS is responsible for the protective effect in LPS induced TLR4 mediated inflammation via SARM adaptor protein in the IMR-32 cell line.