Tolerogenic IL-10–positive CCR7-positive dendritic cells (DC) promote T regulatory (Treg) cell differentiation upon CCR7-dependent migration to draining lymph nodes (DLN). Indeed, in human DC deficiencies, Treg levels are low. α-1 antitrypsin (AAT) has been shown to reduce inflammatory markers, promote a semimature LPS-induced DC phenotype, facilitate Treg expansion, and protect pancreatic islets from alloimmune and autoimmune responses in mice. However, the mechanism behind these activities of AAT is poorly understood. In this study, we examine interactions among DC, CD4+ T cells, and AAT in vitro and in vivo. IL-1β/IFN-γ–mediated DC maturation and effect on Treg development were examined using OT-II cells and human AAT (0.5 mg/ml). CCL19/21-dependent migration of isolated DC and resident islet DC was assessed, and CCR7 surface levels were examined. Migration toward DLN was evaluated by FITC skin painting, transgenic GFP skin tissue grafting, and footpad DC injection. AAT-treated stimulated DC displayed reduced MHC class II, CD40, CD86, and IL-6, but produced more IL-10 and maintained inducible CCR7. Upon exposure of CD4+ T cells to OVA-loaded AAT-treated DC, 2.7-fold more Foxp3+ Treg cells were obtained. AAT-treated cells displayed enhanced chemokine-dependent migration and low surface CD40. Under AAT treatment (60 mg/kg), DLN contained twice more fluorescence after FITC skin painting and twice more donor DC after footpad injection, whereas migrating DC expressed less CD40, MHC class II, and CD86. Intracellular DC IL-10 was 2-fold higher in the AAT group. Taken together, these results suggest that inducible functional CCR7 is maintained during AAT-mediated anti-inflammatory conditions. Further studies are required to elucidate the mechanism behind the favorable tolerogenic activities of AAT.

Mitochondria mediate dual metabolic and Ca2+ shuttling activities. While the former is required for Ca2+ signalling linked to insulin secretion, the role of the latter in β cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca2+ transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na+/Ca2+ exchanger that is linked to Ca2+ signalling in MIN6 and primary β cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX) or of its activity, by a dominant negative construct (dnNCLX), enhanced mitochondrial Ca2+ influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca2+. Importantly, NCLX controlled the rate and amplitude of cytosolic Ca2+ changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca2+ signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na+/Ca2+ exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca2+ signals thereby regulating the temporal pattern of insulin secretion in β cells.

Alpha-1-antitrypsin (AAT) is the primary circulating serine protease inhibitor, and is known to exert potent anti-inflammatory effects and to inhibit the progression of several autoimmune diseases. In this study, transgenic mice that over-express surfactant-driven human (h)AAT on the C57BL/6 background were evaluated for resistance to MOG-35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), compared to WT C57BL/6 control mice. According to the results, sustained levels of circulating hAAT profoundly inhibited induction of clinical signs, inflammatory lesions and demyelination observed in WT mice with EAE, concomitant with enhanced levels of CD4+FoxP3+ Treg cells, reduced secretion of MOG peptide-induced pro-inflammatory cytokines, IL-17, IL-1β & IL-6, diminished expression of caspase-1 and enhanced expression of CCR6. These results implicate hAAT as a potent immunoregulatory agent worthy of further investigation as a potential therapy in human autoimmune diseases including multiple sclerosis.

Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmidderived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated "EF") alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline-injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure-function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.

Although islet transplantation for individuals with type 1 diabetes has been shown to yield superior blood glucose control, it remains inadequate for long-term control. This is partly due to islet injuries and stresses that can lead to beta cell loss. Inhibition of excess IL-1b activity might minimize islet injuries, thus preserving function. The IL-1 receptor antagonist (IL-1Ra), an endogenous inhibitor of IL-1b, protects islets from cytokine-induced necrosis and apoptosis. Therefore, an imbalance between IL-1b and IL-1Ra might influence the courses of allogeneic and autoimmune responses to islets. Our group previously demonstrated that the circulating serine-protease inhibitor human alpha-1-antitrypsin (hAAT), the levels of which increase in circulation during acute-phase immune responses, exhibits anti-inflammatory and islet-protective properties, as well as immunomodulatory activity. In the present study, we sought to determine whether the pancreatic islet allograft-protective activity of hAAT was mediated by IL-1Ra induction. Our results demonstrated that hAAT led to a 2.04-fold increase in IL-1Ra expression in stimulated macrophages and that hAAT-pre-treated islet grafts exhibited a 4.851-fold increase in IL-1Ra transcript levels, which were associated with a moderate inflammatory profile. Unexpectedly, islets that were isolated from IL-1Ra-knockout mice and pre-treated with hAAT before grafting into wild-type mice yielded an increase in intragraft IL-1Ra expression that was presumably derived from infiltrating host cells, albeit in the absence of hAAT treatment of the host. Indeed, hAAT-pre-treated islets generated hAAT-free conditioned medium that could induce IL-1Ra production in cultured macrophages. Finally, we demonstrated that hAAT promoted a distinct phosphorylation and nuclear translocation pattern for p65, a key transcription factor required for IL-1Ra expression.