Elisa Cantarelli

CXCR1/2 inhibition enhances pancreatic islet survival after transplantation

Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1/2 axis in mice improved intrahepatic islet engraftment and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation.

Rapamycin unbalances the polarization of human macrophages to M1

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon‐γ or interleukin‐4 (IL‐4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor‐β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL‐6, tumour necrosis factor‐α and IL‐1β release but reduced CD206 and CD209 expression and IL‐10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll‐like receptor 4‐stimulated peripheral blood mononuclear cells showed a clear shift to an M1‐like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2‐related diseases through mTOR inhibitor treatment.

Alternative transplantation sites for pancreatic islet grafts.

The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being an ideal site because of immunologic, anatomic, and physiologic factors leading to a significant early graft loss. A huge amount of alternative sites have been used for islet transplantation in experimental animal models to provide improved engraftment and long-term survival minimizing surgical complications. The pancreas, gastric submucosa, genitourinary tract, muscle, omentum, bone marrow, kidney capsule, peritoneum, anterior eye chamber, testis, and thymus have been explored. Site-specific differences exist in term of islet engraftment, but few alternative sites have potential clinical translation and generally the evidence of a post-transplant islet function better than that reached after intraportal infusion is still lacking. This review discusses site-specific benefits and drawbacks taking into account immunologic, metabolic, and technical aspects to identify the ideal microenvironment for islet function and survival.

Autologous Pancreatic Islet Transplantation in Human Bone Marrow

The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans.

Bone marrow as an alternative site for islet transplantation

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.

Role of CCL2/MCP-1 in islet transplantation.

High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes. The aim of our work was to determine whether CCL2 secreted by the islet has independent proinflammatory effects that influence engraftment and graft acceptance. Both in mice and humans CCL2 is significantly positively associated with other cytokines/chemokines, in particular with the highly released “proinflammatory” IL-6 and CXCL8 or CXCL1. Transplantation of CCL2-/- islets into syngenic recipients did not improve the transplant function. Transplantation of islets into CCL2-/- syngenic recipients led to a significant improvement of transplant function and partial abrogation of local hepatic inflammation. When evaluated in human islets CCL2 release was strongly related to the immediate local inflammatory response in the liver and impacted short-term human islet function dependently by the induced inflammatory response and independently by the immunosuppressive therapy. The data showed that islet CCL2 release is a sign of “inflamed” islets without having a direct role in graft failure. On the other hand, a causal effect for developing detrimental proinflammatory conditions after transplant was proved for recipient CCL2. Strategies to selectively decrease recipient, but not donor, CCL2 release may increase the success of islet transplantation.

Anti-Inflammatory Strategies to Enhance Islet Engraftment and Survival

Early innate inflammatory reaction strongly affects islet engraftment and survival after intrahepatic transplantation. This early immune response is triggered by ischemia-reperfusion injury and instant blood mediated inflammatory reaction (IBMIR) occurring hours and days after islet infusion. Evidence in both mouse model and in human counterpart suggest the involvement of coagulation, complement system, and proinflammatory chemokines/cytokines. Identification and targeting of pathway(s), playing a role as “master regulator(s)” in post-transplant detrimental inflammatory events, is now mandatory to improve islet transplantation success. This review will focus on inflammatory pathway(s) differentially modulated by islet isolation and mainly associated with the early post-transplant events. Moreover, we will take into account anti-inflammatory strategies that have been tested at 2 levels: on the graft, ex vivo, during islet culture (i.e., donor) and/or on the graft site, in vivo, early after islet infusion (i.e., recipient).

The state of the art of islet transplantation and cell therapy in type 1 diabetes

In patients with type 1 diabetes (T1D), pancreatic β cells are destroyed by a selective autoimmune attack and their replacement with functional insulin-producing cells is the only possible cure for this disease. The field of islet transplantation has evolved significantly from the breakthrough of the Edmonton Protocol in 2000, since significant advances in islet isolation and engraftment, together with improved immunosuppressive strategies, have been reported. The main limitations, however, remain the insufficient supply of human tissue and the need for lifelong immunosuppression therapy. Great effort is then invested in finding innovative sources of insulin-producing β cells. One old alternative with new recent perspectives is the use of non-human donor cells, in particular porcine β cells. Also the field of preexisting β cell expansion has advanced, with the development of new human β cell lines. Yet, large-scale production of human insulin-producing cells from stem cells is the most recent and promising alternative. In particular, the optimization of in vitro strategies to differentiate human embryonic stem cells into mature insulin-secreting β cells has made considerable progress and recently led to the first clinical trial of stem cell treatment for T1D. Finally, the discovery that it is possible to derive human induced pluripotent stem cells from somatic cells has raised the possibility that a sufficient amount of patient-specific β cells can be derived from patients through cell reprogramming and differentiation, suggesting that in the future there might be a cell therapy without immunosuppression.

CXCR1/2 Inhibition Blocks and Reverses Type 1 Diabetes in Mice

Chemokines and their receptors have been associated with or implicated in the pathogenesis of type 1 diabetes (T1D), but the identification of a single specific chemokine/receptor pathway that may constitute a suitable target for the development of therapeutic interventions is still lacking. Here, we used multiple low-dose (MLD) streptozotocin (STZ) injections and the NOD mouse model to investigate the potency of CXCR1/2 inhibition to prevent inflammation- and autoimmunity-mediated damage of pancreatic islets. Reparixin and ladarixin, noncompetitive allosteric inhibitors, were used to pharmacologically blockade CXCR1/2. Transient blockade of said receptors was effective in preventing inflammation-mediated damage in MLD-STZ and in preventing and reversing diabetes in NOD mice. Blockade of CXCR1/2 was associated with inhibition of insulitis and modification of leukocytes distribution in blood, spleen, bone marrow, and lymph nodes. Among leukocytes, CXCR2+ myeloid cells were the most decreased subpopulations. Together these results identify CXCR1/2 chemokine receptors as “master regulators” of diabetes pathogenesis. The demonstration that this strategy may be successful in preserving residual β-cells holds the potential to make a significant change in the approach to management of human T1D.

Murine animal models for preclinical islet transplantation No model fits all (research purposes)

Advances in islet transplantation research have led to remarkable improvements in the outcome in humans with type 1 diabetes. However, pitfalls, mainly linked both to early liver-specific inflammatory events and to pre-existing and transplant-induced auto- and allo-specific adaptive immune responses, still remain. In this scenario research into pancreatic islet transplantation, essential to investigate new strategies to overcome open issues, needs very well-designed preclinical studies to obtain consistent and reliable results and select only promising strategies that may be translated into the clinical practice. This review discusses the main shortcomings of the mouse models currently used in islet transplantation research, outlining the main factors and variables to take into account for the design of new preclinical studies. Since several parameters concerning both the graft (i.e., islets) and the recipient (i.e., diabetic mice) may influence transplant outcome, we recommend considering several critical points in designing future bench-to-bedside islet transplantation research.