In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-kBdependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-kB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-kB target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-kB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1b and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG -/recipients. Thus, human islets can provide a reservoir of NF-kB-dependent inflammatory factors that have the potential to contribute to the antiislet-graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-kB, and transplanted them into allogenic recipients. These NF-kB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-kB-activated islets were rejected with a faster tempo as compared to non-NF-kB-activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention.
Malle et al. identify a role for nuclear factor inducing κB (NIK) in pancreatic β cell failure. NIK activation disrupts glucose homeostasis in zebrafish in vivo and impairs glucose-stimulated insulin secretion in mouse and human islets in vitro. NIK activation also perturbs β cell insulin secretion in a diet-induced obesity mouse model. These studies reveal that NIK contributes a central mechanism for β cell failure in obesity.
The CRISPR-Cas9 and related systems offer a unique genome editing tool allowing facile and efficient introduction of heritable and locus-specific sequence modifications in the genome.Despite its molecular precision, temporal and spatial control of gene editing with CRISPR-Cas9 system is very limited. We developed a light-sensitive liposome delivery system that offers a high degree of spatial and temporal control of gene editing with CRISPR/Cas9 system. We demonstrated its high transfection efficiency, by assessing the targeted knockout of eGFP gene in human HEK293 cells (52.8% knockout). We further validated our results at a single-cell resolution using an in vivo eGFP reporter system in zebrafish (77% knockout). To the best of our knowledge we reported the first proof-of-concept of spatio-temporal control of CRISPR/Cas9 by using lighttriggered liposomes in both in vitro and in vivo environment.
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