Pancreatic islet xenograft survival in mice is extended by a combination of alpha-1-antitrypsin and single-dose anti-CD4/CD8 therapy.

Clinical pancreatic islet transplantation is under evaluation for the treatment of autoimmune diabetes, yet several limitations preclude widespread use. For example, there is a critical shortage of human pancreas donors. Xenotransplantation may solve this problem, yet it evokes a rigorous immune response which can lead to graft rejection. Alpha-1-antitrypsin (AAT), a clinically available and safe circulating anti-inflammatory and tissue protective glycoprotein, facilitates islet alloimmune-tolerance and protects from inflammation in several models. Here, we examine whether human AAT (hAAT), alone or in combination with clinically relevant approaches, achieves long-term islet xenograft survival. Rat-to-mouse islet transplantation was examined in the following groups: untreated (n = 6), hAAT (n = 6, 60-240 mg/kg every 3 days from day -10), low-dose co-stimulation blockade (anti-CD154/LFA-1) and single-dose anti-CD4/CD8 (n = 5-7), either as mono- or combination therapies. Islet grafting was accompanied by blood glucose follow-up. In addition, skin xenografting was performed in order to depict responses that occur in draining lymph nodes. According to our results hAAT monotherapy and hAAT/anti-CD154/LFA-1 combined therapy, did not delay rejection day (11-24 days untreated vs. 10-22 day treated). However, host and donor intragraft inflammatory gene expression was diminished by hAAT therapy in both setups. Single dose T-cell depletion using anti-CD4/CD8 depleting antibodies, which provided 14-15 days of reduced circulating T-cells, significantly delayed rejection day (28-52 days) but did not achieve graft acceptance. In contrast, in combination with hAAT, the group displayed significantly extended rejection days and a high rate of graft acceptance (59, 61, >90, >90, >90). In examination of graft explants, marginal mononuclear-cell infiltration containing regulatory T-cells predominated surviving xenografts. We suggest that temporal T-cell depletion, as in the clinically practiced anti-thymocyte-globulin therapy, combined with hAAT, may promote islet xenograft acceptance. Further studies are required to elucidate the mechanism behind the observed synergy, as well as the applicability of the approach for pig-to-human islet xenotransplantation

T Helper Subsets, Peripheral Plasticity, and the Acute Phase Protein, α1-Antitrypsin

The traditional model of T helper differentiation describes the naïve T cell as choosing one of several subsets upon stimulation and an added reciprocal inhibition aimed at maintaining the chosen subset. However, to date, evidence is mounting to support the presence of subset plasticity. This is, presumably, aimed at fine-tuning adaptive immune responses according to local signals. Reprograming of cell phenotype is made possible by changes in activation of master transcription factors, employing epigenetic modifications that preserve a flexible mode, permitting a shift between activation and silencing of genes. The acute phase response represents an example of peripheral changes that are critical in modulating T cell responses. α1-antitrypsin (AAT) belongs to the acute phase responses and has recently surfaced as a tolerogenic agent in the context of adaptive immune responses. Nonetheless, AAT does not inhibit T cell responses, nor does it shutdown inflammation per se; rather, it appears that AAT targets non-T cell immunocytes towards changing the cytokine environment of T cells, thus promoting a regulatory T cell profile. The present review focuses on this intriguing two-way communication between innate and adaptive entities, a crosstalk that holds important implications on potential therapies for a multitude of immune disorders

Human α1-Antitrypsin Binds to Heat-Shock Protein gp96 and Protects from Endogenous gp96-Mediated Injury In vivo

The extracellular form of the abundant heat shock protein, gp96, is involved in human autoimmune pathologies. In patients with type 1 diabetes, circulating gp96 is found to be elevated, and is bound to the acute-phase protein, α1-antitrypsin (AAT). The two molecules also engage intracellularly during the physiological folding of AAT. AAT therapy promotes pancreatic islet survival in both transplantation and autoimmune diabetes models, and several clinical trials are currently examining AAT therapy for individuals with type 1 diabetes. However, its mechanism of action is yet unknown. Here, we examine whether the protective activity of AAT is related to binding of extracellular gp96. Primary mouse islets, macrophages and dendritic cells were added recombinant gp96 in the presence of clinical-grade human AAT (hAAT, GlassiaTM, Kamada Ltd, Israel). Islet function was evaluated by insulin release. The effect of hAAT on IL-1β/IFNγ-induced gp96 cell surface levels was also evaluated. In vivo, skin transplants were performed for examination of robust immune responses, and systemic inflammation was induced by cecal puncture. Endogenous gp96 was inhibited by gp96-inhibitory peptide (gp96i, Compugen Ltd., Israel) in an allogeneic islet transplantation model. Our findings indicate that hAAT binds to gp96 and diminishes gp96-induced inflammatory responses; e.g., hAAT-treated gp96-stimulated islets released less pro-inflammatory cytokines (IL-1β by 6.16-fold and TNFα by 2.69-fold) and regained gp96-disrupted insulin release. hAAT reduced cell activation during both skin transplantation and systemic inflammation, as well as lowered inducible surface levels of gp96 on immune cells. Finally, inhibition of gp96 significantly improved immediate islet graft function. These results suggest that hAAT is a regulator of gp96-mediated inflammatory responses, an increasingly appreciated endogenous damage response with relevance to human pathologies that are exacerbated by tissue injury

DLN response to human AAT monotherapy after skin xenografting.

<p>Mice were either SHAM operated (CT) or recipients of rat skin (Tx) in the absence or presence of human AAT monotherapy. (<b>A</b>) 14-day DLN. FACS analysis. Results expressed as fold change from CT, mean ± SEM from n = 10/group; **p<0.01, ***p<0.001. (<b>B</b>) 72-h DLN. RT-PCR. Results expressed as fold change from CT, mean ± SEM from <i>n</i> = 3/group; **p<0.01, ***p<0.001.</p

AAT treatment combined with debulking therapy; graft survival.

<p><b>(A–C)</b> Rat islets were grafted into mice that were treated with anti-CD4/CD8 depleting antibodies, in the absence of AAT therapy (<i>n</i> = 7) or with added AAT therapy (<i>n</i> = 5). (<b>A</b>) CD45⁺CD3⁺ cells from peripheral blood, as monitored by FACS analysis. Results presented as the percent out of initial amount prior to injection. Representative follow-up out of 10 mice. (<b>B</b>) Islet xenograft survival curve. ***p<0.001 between DB and BD/AAT. (<b>C</b>) Glucose follow-up. Representative mouse. Milestones indicated: <i>hAAT treatment stopped,</i> therapy withdrawn followed by glucose follow-up; <i>nephrectomy</i>, graft explantation followed by glucose follow-up; <i>second xenograft</i>, rat islets grafted into the right renal subcapsular space followed by glucose follow-up.</p

Human AAT monotherapy during pancreatic islet xenotransplantation.

<p>Rat pancreatic islets were grafted into the renal subcapsular space of hyperglycemic mice. Recipients were treated with saline (CT) or human AAT throughout the experiment. (<b>A</b>) Islet graft survival curve (<i>n</i> = 6/group). (<b>B</b>) Graft histology. Representative day-seven explanted grafts from CT and hATT-monotreated mice (<i>n</i> = 3/group). <i>Black arrows</i>, remains of rat pancreatic islets. (<b>C</b>) Mouse gene expression at graft site. Grafts were explanted at indicated times after transplantation. Mean ± SEM from <i>n</i> = 3 grafts/group; *p<0.05, **p<0.01, ***p<0.001. (<b>D</b>) Rat gene expression at graft site. Grafts were explanted at indicated times after transplantation. Mean ± SEM from <i>n</i> = 3 grafts/group; **p<0.01.</p

AAT treatment combined with debulking therapy; histology and gene expression.

<p>Rat islets were grafted into mice that were treated with anti-CD4/CD8 depleting antibodies, in the absence of AAT therapy (<i>n</i> = 7) or with added AAT therapy (<i>n</i> = 5), as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063625#pone-0063625-g003" target="_blank">Figure 3B</a>. (<b>A</b>) Graft site histology. <i>K</i>, kidney tissue; <i>G</i>, graft site. From <i>left</i> to <i>right</i>, representative syngeneic mouse islet graft (day 35), xenograft (debulking therapy alone, day 25), <i>black arrows</i> indicate immune cell mononuclear infiltration, xenograft (debulking therapy combined with AAT, day 11 after rejection) and xenograft (debulking therapy combined with AAT, day 90). (<b>B</b>) Treg cell content in xenograft sites. Immunofluorescent staining. DB, debulking therapy alone (rejected graft); DB/AAT, combined debulking and AAT therapy (rejected and accepted grafts). <i>Green</i>, foxp3; <i>blue</i>, DAPI nuclear counterstaining. Representative images. (<b>C</b>) Mouse (recipient) gene expression profiles. RT-PCR. CT vs. AAT monotherapy, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063625#pone-0063625-g001" target="_blank">Figure 1C</a>, shown over gray background, next to day 90 explants from mice treated by the combination of debulking therapy and AAT (DB/AAT). Results expressed as fold change from CT, mean ± SEM from <i>n</i> = 3/group; *p<0.05, **p<0.01, ***p<0.001. (<b>D</b>) Rat (donor) insulin expression profile. RT-PCR. CT vs. AAT monotherapy, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063625#pone-0063625-g001" target="_blank">Figure 1D</a>, are shown over gray background, next to day 90 explants from mice treated by the combination of debulking therapy and AAT (DB/AAT). Results expressed as fold change from CT, mean ± SEM from <i>n</i> = 3/group; **p<0.01.</p

Point Mutation of a Non-Elastase-Binding Site in Human α1-Antitrypsin Alters Its Anti-Inflammatory Properties

IntroductionHuman α1-antitrypsin (hAAT) is a 394-amino acid long anti-inflammatory, neutrophil elastase inhibitor, which binds elastase via a sequence-specific molecular protrusion (reactive center loop, RCL; positions 357–366). hAAT formulations that lack protease inhibition were shown to maintain their anti-inflammatory activities, suggesting that some attributes of the molecule may reside in extra-RCL segments. Here, we compare the protease-inhibitory and anti-inflammatory profiles of an extra-RCL mutation (cys232pro) and two intra-RCL mutations (pro357cys, pro357ala), to naïve [wild-type (WT)] recombinant hAAT, in vitro, and in vivo.MethodsHis-tag recombinant point-mutated hAAT constructs were expressed in HEK-293F cells. Purified proteins were evaluated for elastase inhibition, and their anti-inflammatory activities were assessed using several cell-types: RAW264.7 cells, mouse bone marrow-derived macrophages, and primary peritoneal macrophages. The pharmacokinetics of the recombinant variants and their effect on LPS-induced peritonitis were determined in vivo.ResultsCompared to WT and to RCL-mutated hAAT variants, cys232pro exhibited superior anti-inflammatory activities, as well as a longer circulating half-life, despite all three mutated forms of hAAT lacking anti-elastase activity. TNFα expression and its proteolytic membranal shedding were differently affected by the variants; specifically, cys232pro and pro357cys altered supernatant and serum TNFα dynamics without suppressing transcription or shedding.ConclusionOur data suggest that the anti-inflammatory profile of hAAT extends beyond direct RCL regions. Such regions might be relevant for the elaboration of hAAT formulations, as well as hAAT-based drugs, with enhanced anti-inflammatory attributes