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The role of apoptosis in immune hyporesponsiveness following AAV8 liver gene transfer.

ABSTRACT: Gene therapy provides a significant opportunity to treat a variety of inherited and acquired diseases. However, adverse immune responses toward the adeno-associated virus (AAV) antigens may limit its success. The mechanisms responsible for immunity or tolerance toward AAV-encoded transgene products remain poorly defined. Studies in mice demonstrate that AAV2/8 gene transfer to liver is associated with immunological hyporesponsiveness toward both AAV vector and antigenic transgene product. To evaluate the role of activation-induced cell death (AICD) and cytokine withdrawal (intrinsic cell death) in the deletion of mature T lymphocytes, we compared immunological responses in hepatic AAV2/8 transfer in murine recipients lacking the Fas receptor, and recipients overexpressing Bcl-xL, to WT murine counterparts. Prolonged transgene expression was dependent on both Fas signaling and Bcl-xL-regulated apoptosis in T cells. Abrogation of intrinsic cell death enhanced Th1 responses, whereas AICD functioned to limit neutralizing antibody production toward AAV2/8. In addition, immune hyporesponsiveness and stable transgene expression was dependent on upregulation of FasL expression on transduced hepatocytes and a corresponding apoptosis of infiltrating Fas (+) cells. These data provide evidence that both AICD and apoptosis due to cytokine withdrawal of lymphocytes are essential for immune hyporesponsiveness toward hepatic AAV2/8-encoded transgene product in the setting of liver gene transfer.

SUBMITTER: Faust SM

PROVIDER: S-EPMC3863804 | biostudies-other | 2013 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundApoptosis plays important roles in a variety of functions, including immunity and response to environmental stress. The Inhibitor of Apoptosis (IAP) gene family of apoptosis regulators is expanded in molluscs, including eastern, Crassostrea virginica, and Pacific, Crassostrea gigas, oysters. The functional importance of IAP expansion in apoptosis and immunity in oysters remains unknown.ResultsPhylogenetic analysis of IAP genes in 10 molluscs identified lineage specific gene expansion in bivalve species. Greater IAP gene family expansion was observed in C. virginica than C. gigas (69 vs. 40), resulting mainly from tandem duplications. Functional domain analysis of oyster IAP proteins revealed 3 novel Baculoviral IAP Repeat (BIR) domain types and 14 domain architecture types across gene clusters, 4 of which are not present in model organisms. Phylogenetic analysis of bivalve IAPs suggests a complex history of domain loss and gain. Most IAP genes in oysters (76% of C. virginica and 82% of C. gigas), representing all domain architecture types, were expressed in response to immune challenge (Ostreid Herpesvirus OsHV-1, bacterial probionts Phaeobacter inhibens and Bacillus pumilus, several Vibrio spp., pathogenic Aliiroseovarius crassostreae, and protozoan parasite Perkinsus marinus). Patterns of IAP and apoptosis-related differential gene expression differed between the two oyster species, where C. virginica, in general, differentially expressed a unique set of IAP genes in each challenge, while C. gigas differentially expressed an overlapping set of IAP genes across challenges. Apoptosis gene expression patterns clustered mainly by resistance/susceptibility of the oyster host to immune challenge. Weighted Gene Correlation Network Analysis (WGCNA) revealed unique combinations of transcripts for 1 to 12 IAP domain architecture types, including novel types, were significantly co-expressed in response to immune challenge with transcripts in apoptosis-related pathways.ConclusionsUnprecedented diversity characterized by novel BIR domains and protein domain architectures was observed in oyster IAPs. Complex patterns of gene expression of novel and conserved IAPs in response to a variety of ecologically-relevant immune challenges, combined with evidence of direct co-expression of IAP genes with apoptosis-related transcripts, suggests IAP expansion facilitates complex and nuanced regulation of apoptosis and other immune responses in oysters.

Project description:AimsAlthough transforming growth factor-beta (TGF-beta) is believed to stimulate intimal hyperplasia after arterial injury, its role in remodelling remains unclear. We investigate whether Smad3, a TGF-beta signalling protein, might facilitate its effect on remodelling.Methods and resultsUsing the rat carotid angioplasty model, we assess Smad3 expression following arterial injury. We then test the effect of arterial Smad3 overexpression on the response to injury, and use a conditioned media experimental design to confirm an Smad3-dependent soluble factor that mediates this response. We use small interfering RNA (siRNA) to identify this factor as connective tissue growth factor (CTGF). Finally, we attempt to replicate the effect of medial Smad3 overexpression through adventitial application of recombinant CTGF. Injury induced medial expression of Smad3; overexpression of Smad3 caused neointimal thickening and luminal expansion, suggesting adaptive remodelling. Smad3 overexpression, though exclusively medial, caused adventitial changes: myofibroblast transformation, proliferation, and collagen production, all of which are associated with adaptive remodelling. Supporting the hypothesis that Smad3 initiated remodelling and these adventitial changes via a secreted product of medial smooth muscle cells (SMCs), we found that media conditioned by Smad3-expressing recombinant adenoviral vector (AdSmad3)-infected SMCs stimulated adventitial fibroblast transformation, proliferation, and collagen production in vitro. This effect was attenuated by pre-treatment of SMCs with siRNA specific for CTGF, abundantly produced by AdSmad3-infected SMCs, and significantly up-regulated in Smad3-overexpressing arteries. Moreover, periadventitial administration of CTGF replicated the effect of medial Smad3 overexpression on adaptive remodelling and neointimal hyperplasia.ConclusionMedial gene transfer of Smad3 promotes adaptive remodelling by indirectly influencing the behaviour of adventitial fibroblasts. This arterial cell-cell communication is likely to be mediated by Smad3-dependent production of CTGF.

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The role of apoptosis in immune hyporesponsiveness following AAV8 liver gene transfer.

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