α-1-antitrypsin gene delivery reduces inflammation, increases T-regulatory cell population size and prevents islet allograft rejection - PubMed (original) (raw)
α-1-antitrypsin gene delivery reduces inflammation, increases T-regulatory cell population size and prevents islet allograft rejection
Galit Shahaf et al. Mol Med. 2011 Sep-Oct.
Abstract
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 plasmid-derived 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.
Figures
Figure 1
Production of hAAT in mice by HD injection of pEF-hAAT. Expression of hAAT in mouse liver: Heterozygote hAAT transgenic mice were administered either PBS (HD-PBS, n = 3) or pEF-hAAT (HD-pEF-hAAT, 100 μg, n = 3) via HD tail-vein injection (1.8 mL/6 s). (A) Twenty-four hours later, animals were sacrificed and liver samples were examined for hAAT expression by RT-PCR of extracted liver RNA using primers specific for hAAT (top). Western blot analysis of extracted liver protein using an anti-hAAT antibody is shown (bottom). Protein size and specificity are compared to clinical-grade hAAT (Aralast™). (B) Immunohistochemistry (blue, nuclear DAPI stain; red, hAAT antibody).
Figure 2
Circulating pEF-hAAT–derived hAAT protects islet allografts from acute rejection. Healthy heterozygote hAAT transgenic mice were either non-HD tail-vein–injected animals (CT, n = 20) or administered PBS (n = 11), pEF-hAAT (n = 11) or a truncated modified plasmid pEF-Δ-hAAT (n = 6) via HD tail-vein injection, rendered diabetic by a single streptozotocin (STZ) injection and then grafted with allogeneic islets. (A) Representative follow-up of blood glucose and circulating hAAT levels. pEF-hAAT, pEF-Δ-hAAT and PBS administration (indicated by HD injection arrowhead [▴]) was followed by STZ injection (indicated by STZ arrowhead [▴]) before transplantation of allogeneic islets (day 0, indicated by an up arrow [↑]). hAAT levels are indicated by the black dashed line. Blood glucose levels were assessed periodically in pEF-hAAT (black solid line), pEF-Δ-hAAT (gray dashed line) and PBS (gray solid line) groups. Nephrectomy, removal of the graft-containing kidney. (B) Graft survival curve. The day of islet graft failure is defined as the time after transplantation in which blood glucose levels exceed 300 mg/dL. CT, islet graft recipient mice that were not HD tail-vein injected. (C) Graft histology. Accepted allografts were removed from mice for staining (30–100 d; representative stain of a 72-d graft explant). Graft, islets, noninvasive “cuff” and Treg cells are indicated. Red, insulin staining; blue, DAPI nuclear staining; green, foxp3 Treg cells inside the noninvasive mononuclear “cuff.”
Figure 3
Cytokine expression profile in explanted islet allografts. Mice were grafted with allogeneic islets and graft mRNA expression levels were assessed. Renal tissue (CT, n = 8) was obtained from the opposite pole to islet grafts of non-HD tail-vein–injected animals, to represent background gene expression levels. Graft tissue: PBS (n = 8, PBS administered by HD tail-vein injection, islets grafted 17 d later and grafts explanted at 48 h); pEF-hAAT (n = 8, pEF-hAAT administered by HD tail-vein injection, islets grafted 17 d later and grafts explanted 30–100 d later). The results are presented as arbitrary units (AU) of mean ± SEM normalized to β-actin mRNA expression; *P < 0.05.
Figure 4
Circulating pEF-hAAT–derived hAAT modifies posttransplantation foxp3 Treg cell population. (A) Mice were grafted intraperitoneally with allogeneic skin tissue, and splenic Treg cell population size was assessed 7 d after transplantation. Control (nontransplanted mice): CT, background Treg cell population size in mice that were not injected by HD tail-vein injection (n = 6); pEF-hAAT, Treg cell population size from mice that were HD tail-vein injected with pEF-hAAT (n = 6). Transplantation (skin-tissue graft): PBS, allo-geneic skin-tissue transplantation into mice that were HD tail-vein injected with PBS (n = 6). pEF-hAAT, allogeneic skin-tissue transplantation into mice that were HD tail-vein injected with pEF-hAAT (n = 6). Treg cell population size was assessed by FACS after CD4-positive T-cell enrichment. Mean ± SEM; *P < 0.05. (B) Representative FACS analysis images (y axis: forward scatter [FSC]; x axis: Foxp3-GFP).
Figure 5
Macrophages exhibit an antiinflammatory response and a reduced maturation profile in the presence of circulating pEF-hAAT–derived hAAT. (A) RAW 264.7 cells were stimulated with LPS (1 ng/mL) in the presence of mouse serum (33% volume) from the following sources: nonstimulated and CT (serum from noninjected mice, n = 8); serum + hAAT (serum from noninjected mice plus 160 μg/mL hAAT, n = 8); serum pEF-hAAT (serum from plasmid-injected mice expressing 500 μg/mL hAAT, n = 8); and serum PBS (serum from PBS-injected mice, n = 8). The 48-h supernatant cytokine levels were analyzed. (B) Surface levels of CD40 and MHCII in peritoneal cell populations directly obtained from islet-grafted mice. The peritoneal cell population (1 × 106 cells per sample) from CT nongrafted mice (black dashed line, n = 6) or islet-grafted mice that were administered PBS (gray solid line, n = 6) or pEF-hAAT (thick black solid line, n = 6) via HD tail-vein injection was assessed for the expression of CD40 (upper panels) and MHCII (lower panels) in total cells (left) and CD11bHI peritoneal cells (middle) by FACS (representative images are shown). Bar graphs depict events from CD11bHI peritoneal cells, presented as fold from CT. Mean ± SEM; *P < 0.05, **P < 0.01.
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