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Review

Targeting Acute Islet Inflammation to Preserve Graft Mass and Long-Term Function

Carly M. Darden, Srividya Vasu, Kenjiro Kumano, Bashoo Naziruddin, Michael C. Lawrence
Received: November 21, 2018; Published: January 29, 2019; doi:10.21926/obm.transplant.1901043

Abstract

Islet transplantation is a minimally invasive cell based replacement therapy to prevent or reverse diabetes or hypoglycemia through natural hormonal responses to regulate blood glucose. However, extending the islet graft functional lifespan remains a challenge that prevents long-term success and widespread use of the procedure. Islets are subject to stress and damage and undergo immunological assault during transplantation procedures. Current treatments to prevent immune reactivity toward the graft come with toxic side effects, and damage to islets and loss of graft function still occurs. Accumulating evidence suggests that acute inflammatory reactions contribute to a significant loss of islet cell mass early in the transplant process. Inflammatory reactions involving a blood coagulation cascade and communication between islet cells and immune cells can destroy more than half the islet mass. These cyclic events link innate and adaptive immune responses that lead to graft failure. In this review, we discuss key components and strategies to target islet cell inflammation and delink the progression of inflammatory islet-immune cell responses that contribute to islet graft destruction.

Keywords

Islet cell transplantation; innate immune response; adaptive immune response; acute inflammation; IBMIR

1. Introduction

Islet transplantation represents a promising cell replacement therapy to prevent or reverse diabetes mellitus and is currently performed in select patients with uncontrollable type 1 diabetes or chronic pancreatitis requiring pancreatectomy. It is less invasive with lower morbidity than pancreas transplantation and can alleviate requirements of insulin therapy. The primary advantage of this cell-based therapy over conventional insulin therapy is that islets can provide a more naturally balanced hormonal response to physiological nutrient and energy levels to moderate blood glucose and reduce glycosylated hemoglobin (HbA1C) without eliciting dangerous levels of hypoglycemia. Indeed, transplanted islets can reduce or prevent hypoglycemic episodes in cases of individuals requiring insulin therapy with hypoglycemic unawareness or brittle diabetes. Thus, islet transplantation not only provides a means to regulate blood glucose by replacing islet cells harboring natural responses to physiological stimuli, but is also an important therapeutic option for patients unable to sense hypoglycemia with increased risk of health and lifestyle complications from unstable fluctuations in blood glucose.

One of the major challenges of islet transplantation is long-term survival and function of the islet graft. Although significant improvements to procedures over the last couple of decades have increased success rates to achieve insulin independence, the rates significantly decline within 1-3 years after transplantation. Islets are subjected to damage and stress throughout transplantation procedures and there is attrition of islet mass and function following engraftment. This is largely attributed to inflammatory and an acute innate immune response. This early immune response is mediated in part by platelet and complement components of the blood that interact with the islet. The phenomenon has been referred to as the Instant Blood Mediated Inflammatory Reaction (IBMIR). As the blood reacts with the infused digested and exposed islet tissue, monocytes are recruited to the site to evoke an immune response. However, blood components and immune cells do not act alone in this process. The islet cells themselves produce chemokines and cytokines that exacerbate the response. We have referred to these islet cell cytokines as “isletokines” to distinguish their origin from immune cells. Stressed islets produce and release isletokines that can recruit immune cells to the graft and evoke a vicious cycle of immune reactivity (Figure 1). Thus, strategies for interfering with production of isletokines from islets and their communication with immune cells could potentially break the cycle of islet-immune cell interactions that exacerbate inflammatory damage and promote further downstream immune destruction of the islet graft. In this review, we discuss current pharmacological targets that have advanced immunosuppression regimens during islet transplantation and follow up with the novel strategies for targeting components of the acute innate response during the early post-transplant period that may reduce requirements of immunosuppression and preserve long-term islet cell mass and function.

Figure 1 Factors contributing to acute islet inflammation and cyclical amplification of islet-immune cell reactivity. The acute inflammatory reaction highlighted in red consists of a thrombotic “IBMIR” response when isolated and damaged islet tissue is exposed to blood and initiates coagulation and complement cascades. Tissue factor (TF) produced by damaged islets promotes the coagulation by attracting platelets and leukocytes to produce a fibrin clot that contributes to islet hypoxia. Stressed islets release cytokines that recruit and activate neutrophils, macrophages (MΦ), and dendritic cells (DC) to the grafting site. Infiltrating and activated immune cells further promote islet inflammation and production of antigen-presenting cells (APC) that activate lymphocytes and induce an adaptive response. These cyclical and amplifying factors contribute to islet-immune cell mediated graft failure.

2. Inhibition of Calcineurin and mTOR for Global Immune Suppression

To date, the FK506 immunophilin binding protein macrolides, FK506 (tacrolimus) and rapamycin (sirolimus) have remained cornerstone compounds for lymphocytic immunosuppression in modern allotransplantation [1,2,3,4,5,6,7]. This is largely attributed to their potent pharmacological inhibition of intracellular signaling targets that induce lymphocyte activation and proliferation, respectively. Tacrolimus inhibits calcineurin, which in turn regulates the nuclear factor of activated T cells (NFAT) transcription factor required for T cell receptor (TCR) induction of key cytokines and receptors that mediate T cell activation. Sirolimus inhibits the mammalian target of rapamycin (mTOR) serine/threonine kinase complex 1 (mTORC1) which is part of the PI3K-AKT signaling arm required for lymphocyte cell proliferation and differentiation in response to cytokines. Thus, both tacrolimus and rapamycin can complement each other in synergistic effects of 1) suppressing cytokine expression and 2) preventing cytokine action on T and B cell expansion, respectively.

However, just as in the case with other allotransplant procedures, immunosuppression regimens involving long-term use of tacrolimus or rapamycin to protect islet graft mass and function come with increased risk of morbidity. The major clinical drawbacks of calcineurin and mTOR inhibition (CNI and mTORI) include significant side effects of nephrotoxicity, islet toxicity, and increased risk of infection resulting from global immunosuppression. As such, there has been intense interest in identifying equipotent alternatives to reduce significant long-term side effects observed from prolonged use of CNI and mTORI. Several approaches to avoiding or minimizing toxicities have been utilized and have had considerable success in utilizing lower doses of CNI or mTORI in combination with other less potent, but also less toxic immunosuppressants.

3. Selective Inhibition of Lymphocytes to Prolong Islet Graft Survival

3a) Mycophenolate mofetil (MMF), a prodrug morpholinoethyl ester of active compound mycophenolic acid (MPA), for example, has revealed a unique pathway for suppressing lymphocytes in allogeneic rejection [8,9] Its prime mechanism of action is to inhibit the intracellular enzyme inosine monophosphate dehydrogenase and deplete guanine nucleotide de novo synthesis. This, in turn, selectively suppresses phosphoribosyl pyrophosphate synthetase and DNA polymerase activity in lymphocytes, which is required for DNA replication and cellular proliferative responses to mitogenic stimuli [10]. Although not potent enough to provide adequate immunosuppression to prevent rejection alone, MMF has been successfully substituted for either tacrolimus or sirolimus in maintenance therapy withdrawal protocols to reverse nephrotoxicity and improve islet function [11,12]. These studies suggest that immunomodulation of events occurring in the early post-transplant period can subsequently reduce side effects associated with tacrolimus based long-term immunosuppression.

Major improvements in islet transplantation success rates have also come from advances in immunosuppression protocols involving antibody induction therapies in conjunction with maintenance therapy drugs to prevent lymphocyte activation and proliferation. This has primarily been achieved through selectively targeting lymphocyte surface receptors.

3b) Induction by monoclonal antibody daclizumab which binds to the CD25 alpha subunit of the IL-2 receptor on T cells helped provide landmark improvements in islet transplant outcomes when introduced with the Edmonton Protocol in 2000 [13]. Due to safety concerns of daclizumab, other lymphocyte receptor targets have been explored and have led to promising improvements in immunosuppressant regimens to extend islet graft survival with reduced toxicities [14].

3c) Anti-CD3 monoclonal antibodies (hOKT3γ1 Ala-Ala) or polyclonal anti-thymocyte immunoglobulin antibody preparation (thymoglobulin) to prevent expansion or deplete circulating effector T cells have extended islet graft survival with reduced requirements of tacrolimus or sirolimus [14].

3d) Alemtuzumab (Campath and Lemtrada), a humanized IgG1 monoclonal anti-CD52 antibody used to treat chronic lymphocytic leukemia and multiple sclerosis, has been observed to improve islet engraftment and rates of insulin-independence with improved tolerance [15]. Multiple mechanisms have been proposed for actions of alemtuzumab including antibody-dependent cell-mediated (ADCC) and complement-dependent (CDC) cytotoxicity toward T cells and B cells with high expression of glycosylphosphatidylinositol-linked surface protein CD52. It has sparing effects toward immature lymphocytes and innate immune cells having relatively low CD52 expression and preserves regulatory T cells that may contribute to graft survival [14,16,17]. Indeed, alemtuzumab treatment in a humanized mouse model showed that natural killer cells and neutrophils contribute to lymphocyte depletion independently of CDC. These studies suggest that the innate immune system also has a role in regulating the adaptive response under these conditions [18].

3e) Efalizumab, another humanized monoclonal antibody that targets the CD11a subunit of the leukocyte function antigen (LFA-1) showed promising results to prolong islet cell survival in single donor islet transplants without side effects observed by standard long-term tacrolimus and sirolimus [19]. Its unique action is to prevent binding of LFA-1 to intercellular adhesion molecule (ICAM) required for leukocyte extravasation to provide immunosuppression without T cell depletion. Unfortunately, long-term use of efalizumab for more than 4 years was associated with increased risk (~1 in 10,000) for progressive multifocal leukoencephalopathy (PML) in psoriasis patients, which led to its voluntary withdrawal from the global market by its sponsor. This consequently led to the finding that abatacept, the CD80 targeting CTLA-4 human Fc IgG1 fragment linked fusion protein, could sustain graft survival in islet transplant patients as a replacement for efalizumab when it was withdrawn from protocols during clinical trials.

3f) Belatacept, a second generation CTLA-4 fusion protein product with enhanced affinity toward both CD80 and CD86, could also provide insulin-independence when co-administered with sirolimus as a substitute for tacrolimus after thymoglobulin induction [20,21]. These CTLA-4 fusion proteins bind to CD80/CD86 and interfere with co-stimulatory requirements of APC antigens binding to CD28 to activate T cells. These clinical findings define yet another set of lymphocyte surface receptors that can be selectively targeted in CNI-sparing immunosuppressant regimens.

Collectively, these observations demonstrate that 1) multiple cell surface receptors can be effectively targeted on lymphocytes to prolong graft survival and 2) individual components of lymphocyte signaling can be selectively targeted to substantially inhibit lymphocyte activation and expansion. However, even when lymphocytes are selectively inhibited or depleted in the absence of tacrolimus or sirolimus, rejection still occurs. This suggests that lymphocyte inhibition alone is not sufficient to prevent alloimmune rejection and that other cellular mechanisms contribute to loss of islet graft mass and function. Indeed, components of innate immunity have been characterized to regulate the adaptive response in allo- and auto-immunity.

4. Non-Lymphocyte Cell Mediated Loss of Islet Graft Mass and Function

Mounting evidence indicates that innate immune cells not only initiate adaptive immune responses, but also directly contribute to allograft rejection [22,23,24,25]. Perhaps this is most evident in autologous islet transplantation where a large portion of the graft is lost in the absence of a T-cell mediated response [26]. Indeed, more than 50% of the islet graft is lost due to an acute innate response [27,28,29,30]. The initial islet cell mass is a key determinant of clinical islet transplant outcomes. Thus, the early post-transplant period during which an acute innate response occurs represents an important window of opportunity to extend the life of the graft.

Moreover, targeting innate immune cells may also alleviate subsequent priming or exacerbation of adaptive immune responses toward transplanted islets. Cells contributing to the acute innate response include platelets, neutrophils, macrophages, dendritic cells (DCs), natural killer (NK) cells and the islet endocrine cells themselves. Both cellular and humoral components, part of which have been described as IBMIR, produce coagulation and complement activation to produce an inflammatory environment that destroys up to half the graft before lymphocytes ever make it to the scene. Therefore, targeting acute islet inflammation in conjunction with lymphocyte alloimmune and autoimmune suppression must be taken into consideration when devising novel strategies to achieve highest islet transplant success rates.

In the following, we first reevaluate what is currently known about the mechanistic contributions of innate immunity to adaptive alloimmune responses in transplantation. In addition, we discuss potential targets based on recent and ongoing studies in animal models and clinical trials that may further advance our methods to improve clinical islet transplant outcomes.

4.1 Regulation of the Adaptive Response by Innate Immune Cells

The founding concept that the innate immune system could sense non-self antigens by receptors was first introduced by Charles Janeway, Jr. who proposed that pattern recognition receptors (PRRs) recognize bacterial or viral molecules termed pathogen-associated molecular patterns (PAMPs) [31,32,33]. Innate immune recognition toward conserved components of microbial non-self antigens induced expression of co-stimulatory molecules to activate lymphocytes with reactivity toward the presented antigen. However, this model did not adequately describe how innate immunity could confer adaptive responses toward non-self tissue allografts and tumors or reactivity toward self antigens in autoimmune diseases in the absence of microbes in an otherwise “sterile” environment. Thus, the concept was further developed by Polly Matzinger who later introduced the “danger hypothesis” to describe innate immune cells recognizing self-antigen molecules from stressed or damaged cells as an associated danger and subsequently activating defenses and repair mechanisms [34,35,36].

This broad array of molecules was referred to as danger associated molecular patterns (DAMPs) which include reactive oxygen species (ROS), ATP, uric acid crystals, high mobility group protein b1 (HMGB1), nucleic acids, fibronectin, hyaluronic acid, and heparan sulfate among many others. DAMPs are recognized by a diverse set of PRRs including Toll like receptors (TLR) and IL-1 receptor (IL-1R) of the Toll/IL-1R (TIR) superfamily to initiate signaling pathways that induce an inflammatory response in stressed or damaged tissue. Upon binding to DAMPs, TLR and IL-1R recruit myeloid differentiation primary response 88 (MyD88) adapter protein and IL-1R-associated kinase (IRAK) family kinases to form a receptor complex that activates E3 ubiquitin ligases TRAF6 and Pellinos. This results in formation of Lys63- and Met1- linked ubiquitin chain co-recruitment of TAK1 and canonical IκB kinase (IKK) master kinase complexes, respectively. TAK1 activates MAP kinase kinase (MKK) cascades that switch on p38 and c-Jun N terminal kinase (JNK) MAP kinases while the IKKβ component of IKK activates the nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) transcription factor. MAP kinases and NF-κB coordinately regulate several cytokine and chemokine genes that induce proliferation and mediate inflammation at the site of damage.

As innate immune cells are recruited to the site of inflammation, more cytokines and chemokines are produced to activate and recruit cells required to repair and regenerate damaged tissue. Overstimulation or chronic activation of inflammatory signaling pathways in target cells results in ER stress and can induce apoptosis and cell death. If damage signals are not appropriately resolved or the tissue cannot be restored to a normal non-stressed physiological state, innate immune cells proceed to destroy and phagocytose tissue at the site. Immune cells then circulate back to lymphoid cells for antigen presentation and acquire memory for further targeted destruction of the recalcitrant threat. In this way, the innate immune system activates and tailors the adaptive response toward dangerous conditions or invasive cells that may threaten homeostasis or the physiological integrity of the host.

4.2 DAMP Signaling and IBMIR in Acute Islet Inflammation

In the case of islet transplantation, the graft is subjected to damage and insults that produce DAMPs during multiple steps throughout the transplantation process. DAMPs are already being produced and released by stressed and damaged tissue in cadaveric donors due to traumas related to brain death or by ischemia and organ recovery in the case of live tissue donors. In addition, virtually all graft tissue undergoes some forms of stress insults and damage during procurement and transport. These include temperature changes, ROS, hypoxia, and ischemia-reperfusion injury. During the islet isolation process, the pancreas is also subjected to enzymatical digestion, hypoxia, and mechanical stress [37]. This further produces DAMPs that can directly induce production and release of inflammatory cytokines by activating TLRs on both innate immune cells and islet endocrine cells ex vivo. These cytokines accumulate with DAMPs in a vicious cycle over the duration of the isolation process and are reintroduced into the recipient host at the time of islet infusion.

The combination of DAMPS, cytokines, and blood components interacting with islet tissue proteins and receptors upon infusion culminate into an acute thrombotic reaction known in the field of islet transplantation as IBMIR. Bennet and colleagues first described this event by characterizing cellular interactions between freshly isolated human islets and ABO compatible allogeneic blood [38]. Engagement of IgG and IgM antibodies with islet cell surface collagen and laminins play a key role in initiating the complement cascade. In parallel, the glycoprotein CD142 tissue factor, expressed on the cell surface and released by damaged and stressed islets, binds to factor VII to catalyze the coagulation cascade [39,40,41,42]. Platelets activated during this process produce a fibrin mesh clot surrounding the islets resulting in their embolic occlusion within the hepatic sinusoids of the liver [43,44]. This in turn deprives the graft of oxygen and nutrients leading to increased apoptosis and necrosis further contributing to increased DAMPs, proinflammatory cytokines, and immune cell infiltration.

4.3 Islet Contributions to Acute Graft Inflammation

In addition to expressing proteins that interact with blood components to initiate complement and coagulation cascades, islets express isletokines that contribute to initiating and exacerbating acute inflammation. Several cytokines have been identified in islets during diabetes and in islet grafts [45,46]. Originally, it was assumed that infiltrating immune cells were sole producers of proinflammatory cytokines in islets. However, flow cytometry, immunohistochemistry, and laser capture analysis studies revealed that the islet endocrine cells also produce proinflammatory mediators that can elicit immune cell responses [47,48,49,50,51,52,53,54,55]. In fact, the islet cells produce and release dozens of isletokines that can be detected in the blood circulation upon infusion that correlate with transplant outcomes [55,56]. In particular, HMGB1, MCP-1 and IP-10 are highly expressed and released in damaged or stressed islets and play a key role in initiating acute inflammation in transplanted islets [55,57,58,59,60,61,62].

Induction of isletokine genes in the endocrine component of islet cells is conferred by DAMP and cytokine-induced signaling. In pancreatic beta cells, DAMPs can activate TLR/IL-1R signaling to induce activation of MAP kinases and NF-κB. In addition, CN and NFAT signaling can be activated by TLR/IL-1R [63]. The culmination of MAP kinases and NF-κB and NFAT signaling results in the induction of multiple cytokine genes in beta cells, including IL-1β, TNF-α, HMGB1 and IP-10. IL-1β and TNF-α can further stimulate NF-κB- and NFAT-mediated isletokine gene expression in beta cells. Moreover, HMGB1 and IP-10 not only function as chemokines to recruit immune cells, but also act as DAMPs which can bind to and activate TLR4 [64]. Thus, DAMP and cytokine-induced signaling can produce a vicious cycle of damage and inflammation if not resolved by damage-repair mechanisms. In the case of islet transplantation, the islet cells are stripped of their natural niche within the pancreas and exposed to a new location in a damaged state. Sustained expression and release of isletokines by beta cells out of their natural context results in amplified proinflammatory and adaptive immune responses contributing to graft destruction (Figure 1).

4.4 Hypoxia and Oxidative Stress in Islets

Pancreatic beta cells are enriched with GLUT2 glucose transporters, allowing them to dynamically uptake and utilize glucose in the blood as one of the most metabolically active tissues [65]. Oxygen is required to maintain beta-cell function through the tricarboxylic acid (TCA) cycle, which links synthesis of intracellular ATP to the production and release of insulin [66]. ROS generated through production of ATP by the TCA cycle also contribute to signaling mechanisms that regulate glucose-induced insulin secretion [67,68]. Thus, a sensitive and carefully balanced redox system allows beta cells to sense nutrient load and appropriately produce insulin upon physiological demand. This is achieved in part by a significantly lower expression of ROS-depleting enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in beta cells compared to other tissues. These inherent characteristics of metabolic sensing make beta cells particularly vulnerable to redox imbalance and oxidative stress [67,69,70,71].

Extreme conditions of oxygen availability, oxygen consumption, and metabolic stress can offset redox balance and result in oxidative stress. Chronic exposure of beta cells to high glucose and increased metabolic flux favors mishandling of excess electrons via NADH and FADH2 and the mitochondrial electron transport chain, which transfer and reduce O2 to superoxide free radicals and further generate ROS [71]. In the absence of appropriate oxygen tension, i.e. hypoxia, NADH and FADH2 can also accumulate with inefficient aerobic respiration, allowing electrons to escape and produce excessive ROS [71]. Uncontrolled generation of ROS disrupts redox signaling and leads to oxidative damage of cellular proteins, lipids, and DNA [72,73].

The hepatic portal system site of transplantation offers lower parenchymal oxygen tension and vasculature compared to pancreatic portal system. At lower cellular oxygen concentrations, prolyl-4-hydroxylases (PHDs) are inhibited which consequently allows increased expression and activation of hypoxia-inducible factor-1 alpha (HIF-1α) and NF-κB [74,75,76,77]. These transcription factors acutely regulate genes involved in hypoxic and metabolic adaptation. However, they concomitantly also induce cytokines that induce inflammation [78,79]. Cytokines can contribute to ER stress, nitric oxide (NO) and ROS generation to further activate NF-κB, p38 and JNK MAP kinase pathways that regulate uncoupled protein response and inflammatory genes [80,81,82]. When hypoxic and inflammatory stress are sustained, these pathways activate apoptotic programming [83,84]. Thus, hypoxia can induce and exacerbate conditions of oxidative and inflammatory stress in islets by overlapping pathways leading to immune destruction and beta-cell death.

5. Strategies to Prevent Islet-Immune Cell Mediated Loss of Islet Graft Mass and Function

Attempts to inhibit immune cell function by pharmacological agents have provided invaluable information regarding requirements and specificity of molecular targets for induction of immune responses to allografts. For example, selective inhibition of intracellular targets CN and mTOR have shown most potent effects to prevent alloimmune rejection. Their mode of action is primarily attributed to suppression of cytokine production and responses in lymphokines, respectively. However, accumulating evidence indicates that blocking these intracellular targets is not specific to lymphocyte cell types and that CN and mTOR also regulate cytokines in innate immune cells and islet endocrine cells [54,85,86,87,88]. This is further complicated by the findings that islets require CN and mTOR pathways for physiological adaptation and cellular function [89,90,91,92].

Because most intracellular signaling pathways are shared among multiple immune and non-immune cell types with diverse functions, their inhibition often results in adverse off-target effects. Indeed, sustained blockade of CN and mTOR contributes to side effects beyond immunosuppression, most notably damage to kidney and islet cell function. Moreover, CN/NFAT signaling has been shown to have a role in limiting DC priming of T cells and regulating Treg-mediated immune tolerance [93,94]. mTOR also regulates cytokine production in myeloid cells in response to TLR signaling and induces tolerogenic DC phenotypes [95]. Thus, although CN and mTOR inhibition has potent effects to break islet-immune cell interactions by modulating cytokine signaling in beta cells and lymphocytes, the benefits are offset by toxicity to other tissue functions and impairment of immune tolerance. This prompted the field to explore alternative, more cell-selective targets for immune suppression.

As new molecular targets have been identified that are uniquely expressed by subsets of cells, novel pharmaceutical approaches have been pursued that selectively effect cell-specific mechanisms involved in pathogenic processes. As reviewed above, several monoclonal antibodies and soluble mediators have been successful in meeting these criteria by targeting unique surface molecules or receptor-mediated cell signaling. Although there have been considerably less side effects in several trial regimens focusing on select targets of inflammation and immune responses, it is becoming increasingly clear that there are multiple components throughout transplant procedures that need to be targeted to prolong graft survival. The key components include 1) the lymphocytic response that directly erodes islet cell mass and function, 2) the acute inflammatory response that destroys a large portion of the graft during the early peri-transplant period, and 3) cytokine signaling between the islets and immune cells that perpetuate and amplify innate and adaptive immune responses.

First and foremost, resetting auto- and alloimmune reactivity by T cell and B cell depletion induction therapy in the host/recipient appears to be a prerequisite to achieving long-term allograft survival and function [14]. Thymoglobulin and alemtuzumab induction has been shown to have beneficial effects in reducing the rate of acute rejection when combined with maintenance regimens. MMF and belatacept have thus far been most promising in helping maintain suppressed immune reactivity after lymphocyte depletion with reduced side effects attributed to CNI and mTORI. However, MMF and belatacept only delay loss of allograft function, and rejection is accelerated in the absence of tacrolimus or sirolimus.

The next important step to improving islet transplantation is to selectively reduce the acute innate inflammatory response in islets occurring during the early transplant period. Benefits in graft survival and function have been observed from combinatorial approaches of lymphocyte depletion and anti-inflammatory soluble mediators etanercept and anakinra to block TNF-α and IL-1β signaling, respectively. These clinical findings indicate that early blockade of key cytokines during the induction period can improve long-term clinical outcomes. Double blockade by both etanercept and anakinra improves the response [96]. There is also strong evidence to suggest that chemokines MCP-1 and IP-10 play key roles in initiating inflammatory responses in islet transplantation [55,57]. It should also be noted that IP-10 is a key isletokine expressed by beta cells in insulitic lesions in type 1 diabetes [97,98,99]. The use of anti-IP-10 neutralization antibody after T-cell depletion by anti-CD3 antibody prevented reinfiltration of islet-specific T cells and resulted in remission of diabetes [100]. Based on these observations, it is conceivable that a combinatorial approach to resetting the adaptive response by lymphocyte depletion and neutralizing key isletokines, which evoke islet inflammation, could break the cycle of innate immune activation of adaptive responses toward islets.

Interestingly, transgenic animal studies show that whereas islet donor-derived IP-10 induces graft-damaging inflammation upon transplantation, islet donor-derived MCP-1 is not required for graft failure [101]. Detrimental effects on donor islets were only observed when MCP-1 production was intact in graft recipients and IP-10 was intact in donor islets. In either case, blocking MCP-1 or IP-10 signaling reduced islet inflammation and anti-IP-10 neutralizing monoclonal antibody prolonged graft survival. Together, these studies indicate that graft damage observed by MCP-1 is propagated by the recipient immune system in response to donor-selective IP-10-induced inflammation. They also provide proof of concept that blocking IP-10-induced inflammation by a monoclonal antibody could prevent early loss of islet grafts and improve islet transplant outcomes. Further study needs to be performed to determine if a trio of soluble mediators targeting TNF-α, IL-1β, and IP-10 could provide additional benefit for blocking the acute innate inflammatory response to warrant its use in clinical islet transplantation.

To achieve highest efficacy in targeting acute islet inflammation, it must also be recognized that inflammatory events are already occurring in donor islets prior to infusion, beginning as early as donor trauma and organ procurement. Thus, approaches to alleviate inflammatory stress could be implemented as early as time of procurement and throughout the islet isolation process during which islets are stressed and producing proinflammatory isletokines. These isletokines are released at their highest level upon islet infusion and remain high during the IBMIR response. Thus, the early procedures of the islet cell transplant process are potential key target areas for suppressing acute inflammation and preventing large losses of islet cell mass.

Strategies for complete protection of islets from the acute inflammatory response will require further upstream procedural approaches to include therapeutic interventions in isolation of the recipient. These pre-transplant interventions would allow blockade of intracellular stress response signals and could be performed ex vivo with minimal exposure of pharmacological agents to the host. For example, efforts to block CN/NFAT signaling or other cellular targets required for cytokine production could be performed during organ procurement and islet isolation procedures ex vivo without adverse effects associated with recipient long-term systemic use.

Although there are multiple reports indicating direct effects of CNI to inhibit beta cell proliferation, insulin transcription, and GSIS, we and others have shown it to have minimal effects on beta cell viability and apoptosis within short durations of in vitro exposure [102,103]. Moreover, short-term administration of tacrolimus has been shown to have no effect on islet secretion in healthy human subjects and improves beta cell viability and function when islets are under stress [102,104,105]. Indeed, acute exposure of tacrolimus prevents apoptosis in islets exposed to cytokines in vitro and can restore insulin gene transcription in islets chronically exposed to high glucose within a 48 h window [106,107]. Furthermore, short-term peptide inhibition of the CN downstream target NFAT in islets was shown to prolong graft function in an allogeneic mouse transplant model. Thus, the use of tacrolimus during pre-transplant and induction procedures may provide benefits to inhibit the acute innate response, thereby reducing its requirements for maintenance therapy.

Benefits of inhibition of TLR/IL-1R and downstream activation of NF-κB and p38 and JNK MAP kinase signaling pathways to reduce cytokine production and apoptosis in islets in vivo have also been observed [108,109,110,111,112,113]. Ex-vivo surface modification of islets with a slow-releasing TLR4-selective antagonist TAK242 improved islet graft survival in a syngeneic mouse model. Moreover, the use of TAK242 during pancreatic ductal perfusion and digestion steps of the islet isolation process reduced activation of MAPK stress kinase signaling and expression of proinflammatory cytokines to prolong islet viability and function. TLR4 is one of several mechanisms to activating MAPKs and NF-kB in islets and other targets should be explored during pre-transplantation procedures. For example, it would be predicted that blockade of IL-1R signaling by anakinra in addition to TLR blockade during these steps would further prevent cytokine and DAMP-mediated inflammatory reactions in islets.

TLR activation not only augments the acute innate response, but contributes to the adaptive immune response through effects on antigen-presenting cells (APCs) and activation of T cells [114,115,116]. In APCs, TLRs drive maturation as well as production of IL-12 that results in T helper cells (Th1) response [114]. MyD88 dependent TLR signaling inhibition resulted in reduced numbers of activated CD4+ and CD8+ T cells and increased mortality rates of mice [117]. TLR-knockout murine models are evidence of the initiation and development of an adaptive host resistance to pathogens that is dependent on the TLR/IL-1R activation of MyD88 signaling pathways described above [114,118,119]. Most notably, TLR-stimulated islets were shown to induce islet graft failure by a CD8+ T cell-dependent mechanism [115]. These findings indicate that selective inhibition of TLR signaling in islets could potentially alleviate or prolong onset of lymphocyte-mediated destruction of the islet graft.

Lastly, anticoagulants have been shown to reduce the acute inflammatory response [120]. This is evident from reports on inhibition of thrombin by melagatran or heparin in vitro [38,121]. Despite routine use of systemic heparin in the clinical setting, the formation of micro and macrothrombi is not completely blocked, leading to the observed loss of islet graft mass in the days immediately after transplantation. Interestingly, short-term administration of acute phase reactant serine protease inhibitor alpha 1-antitrypsin (AAT) could dramatically improve survival of human, monkey, and mouse islet grafts in animal models [122,123]. This was attributed to suppression of blood-mediated coagulation pathways, inhibition of NF-κB signaling, downregulation of inflammatory mediators, and reduced CD3+ T cells and F4/80+ activated monocytes in grafted islets. These findings indicate that AAT has useful properties for both anti-coagulation and anti-inflammatory therapies in islet transplantation. Multiple clinical trials are investigating the effects of AAT ARALAST NP during islet processing, culture, and patient treatment pre- and post- transplant on the acute inflammatory response in both allogeneic and autologous islet transplantation.

6. Conclusions

Islet cell destruction during transplantation results from complex cell signaling communication between islet cells and immune cells, linking islet inflammatory stress responses to innate and adaptive immune responses. Damaged and stressed islets release inflammatory mediators that recruit and activate immune cells, which attempt to resolve or remove damaged, foreign, or maladaptive tissue. Delinking this process can break an amplifying cycle of inflammation and immune sensitization toward transplanted islets. Because an acute innate inflammatory response largely affects islet graft mass and dictates further downstream adaptive immune events, it follows that early intervention of islet-immune cell interactions may be key to improving islet transplant outcomes. Hence, the most successful islet transplant regimens going forward will likely be interventions targeting both islet-induced inflammation and activation of innate immune cells in addition to T-cell mediated immune events.

Author Contributions

CMD and MCL contributed to manuscript writing and figure design; SV, KK, and BN contributed feedback, proofreading, and editing for the manuscript.

Competing Interests

The authors have declared that no competing interests exist.

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