Alpha 1-antitrypsin reduces inflammation and enhances mouse pancreatic islet transplant survival

Abstract

The promise of islet cell transplantation cannot be fully realized in the absence of improvements in engraftment of resilient islets. The marginal mass of islets surviving the serial peritransplant insults may lead to exhaustion and thereby contribute to an unacceptably high rate of intermediate and long-term graft loss. Hence, we have studied the effects of treatment with alpha 1-antitrypsin (AAT) in a syngeneic nonautoimmune islet graft model. A marginal number of syngeneic mouse islets were transplanted into nonautoimmune diabetic hosts and islet function was analyzed in control and AAT treated hosts. In untreated controls, marginal mass islet transplants did not restore euglycemia. Outcomes were dramatically improved by short-term AAT treatment. Transcriptional profiling identified 1,184 differentially expressed transcripts in AAT-treated hosts at 3 d posttransplantation. Systems-biology-based analysis revealed AAT down-regulated regulatory hubs formed by inflammation-related molecules (e.g., TNF-α, NF-κB). The conclusions yielded by the systems-biology analysis were rigorously confirmed by QRT-PCR and immunohistology. These data suggest that short-term AAT treatment of human islet transplant recipients may be worthy of a clinical trial.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

AAT treatment preserves the insulin contents of islet transplants. Insulin content of 200 islets in preparations prepared for transplantation was compared with those harvested 3 d posttransplantation in treated and control recipients. Results are expressed using bar graphs representing the mean and SEM values in the groups (****P < 0.0001, by one-way ANOVA, Bonferroni’s multiple comparison test).

Fig. 2.

Transcriptional profiling of islets from AAT-treated and control recipient mice day 3 posttransplantation. Heat map of the top differentially expressed genes in islets from AAT-treated compared with control hosts at day 3 posttransplantation is shown. Genes were identified in a supervised analysis using an LCB value >1.2 and fold change >1.5. Each column presents data from an individual AAT-treated or control mouse and each row represents a gene. Gene expression is shown by pseudocolor scale (−3 to 3), with red denoting high expression level and green denoting low expression level of a gene. Top chemokine receptors and cytokines down-regulated by AAT treatment are marked with an asterisk (*). Transcriptional profiling was performed on total RNA obtained from at least three separately conducted experiments.

Fig. 3.

Pathway enrichment and interactive network analysis from AAT-treated recipient mice 3 d posttransplantation. (A) Pathway enrichment analysis of the modulations induced by AAT treatment. Each bar represents an enriched pathway with significance determined using Benjamini–Hochberg hypothesis-corrected P values (shown on x axis). About 70–100% of the genes impacted by AAT in these pathways are down-regulated by AAT treatment. (B) Interactive network of top 20 focus gene hubs from AAT-treated recipient mice. The Ingenuity Pathway Analysis (IPA) tool was used to generate the networks from the differentially expressed genes. The focus gene hubs were ranked in the merged network on the basis of degree of connectivity. The intensity of the node color indicates the degree of up-regulation (red) and down-regulation (green) in AAT-treated mice compared with controls. (C) Validation of target genes using QRT-PCR in islets ± AAT on day 3. The graph presents the statistical analyzes of relative mRNA levels after normalization by 18S RNA levels. Results are expressed as mean ± SD of two sets of islets.

Fig. 4.

Histology analysis of syngeneic islets ± AAT on 3 d posttransplantation. The experiment was performed three times by obtaining islets from control and AAT-treated mice. Following resection of the islet graft on day 3 posttransplantation, histological analysis of syngeneic islets of control and AAT-treated mice was undertaken. Immunohistology was performed using (i) H&E 10×, (ii) antiinsulin, (iii) anti-CD3, (iv) anti-F4/80, and (v) caspase. H&E 10× and immunoperoxidase (IMP) for insulin 10× staining shows numerous islets with insulin+ β cells in AAT-treated islets vs. controls. Using anti-CD3 and F4/80 staining, the gross infiltration of T cells and activated macrophages and monocytes into the islets was observed only in control islets. Caspase staining shows that AAT treatment reduces the numbers of apoptotic islets considerably as compared to control islets.