title: Low-dose rapamycin unmasks the protective potential of targeting intragraft NF-κB for islet transplants.
abstract: Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 β-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.
authors.affiliation: Gene Therapy and Autoimmunity Group, Immunology Program, Garvan Institute, Darlinghurst, NSW, Australia.
authors.forename: Nathan W
authors.initials: NW
authors.lastname: Zammit
authors.forename: Bernice M
authors.initials: BM
authors.lastname: Tan
authors.forename: Stacey N
authors.initials: SN
authors.lastname: Walters
authors.forename: David
authors.initials: D
authors.lastname: Liuwantara
authors.forename: Jeanette E
authors.initials: JE
authors.lastname: Villanueva
authors.forename: Elisabeth K
authors.initials: EK
authors.lastname: Malle
authors.forename: Shane T
authors.initials: ST
authors.lastname: Grey
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D003921:Diabetes Mellitus, Experimental / Q000401:mortality / Q000473:pathology / Q000601:surgery*; D004357:Drug Synergism; D006085:Graft Survival / Q000187:drug effects*; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology*; D013552:Swine; D014184:Transplantation, Homologous; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D018925:Chemokines; C090509:Icam1 protein, mouse; D007166:Immunosuppressive Agents; D015850:Interleukin-6; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D051550:I-kappa B Kinase; D020123:Sirolimus