Sustained NF-kB Activation and Inhibition in b-Cells Have Minimal Effects on Function and Islet Transplant Outcomes Aileen J. F. King 1,2 , Yongjing Guo 3 , Dongsheng Cai 3 , Jennifer Hollister-Lock 2 , Brooke Morris 2 , Alison Salvatori 4 , John A. Corbett 5 , Susan Bonner-Weir 2 , Steven E. Shoelson 2 , Gordon C. Weir 2 * 1 Diabetes Research Group, King’s College London, London, United Kingdom, 2 Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Section on Pathophysiology and Molecular Pharmacology, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, United States of America, 4 Department of Pharmacology & Physiology, Saint Louis University, St. Louis, Missouri, United States of America, 5 Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America Abstract The activation of the transcription factor NF-kB leads to changes in expression of many genes in pancreatic b-cells. However, the role of NF-kB activation in islet transplantation has not been fully elucidated. The aim of the present study was to investigate whether the state of NF-kB activation would influence the outcome of islet transplantation. Transgenic mice expressing a dominant active IKKb (constitutively active) or a non-degradable form of IkBa (constitutive inhibition) under control of the rat insulin promoter were generated. Islets from these mice were transplanted into streptozotocin diabetic mice in suboptimal numbers. Further, the effects of salicylate (an inhibitor of NF-kB) treatment of normal islets prior to transplantation, and the effects of salicylate administration to mice prior to and after islet implantation were evaluated. Transplantation outcomes were not affected using islets expressing a non-degradable form of IkBa when compared to wild type controls. However, the transplantation outcomes using islets isolated from mice expressing a constitutively active mutant of NF-kB were marginally worse, although no aberrations of islet function in vitro could be detected. Salicylate treatment of normal islets or mice had no effect on transplantation outcome. The current study draws attention to the complexities of NF-kB in pancreatic beta cells by suggesting that they can adapt with normal or near normal function to both chronic activation and inhibition of this important transcription factor. Citation: King AJF, Guo Y, Cai D, Hollister-Lock J, Morris B, et al. (2013) Sustained NF-kB Activation and Inhibition in b-Cells Have Minimal Effects on Function and Islet Transplant Outcomes. PLoS ONE 8(10): e77452. doi:10.1371/journal.pone.0077452 Editor: Paolo Fiorina, Children’s Hospital Boston/Harvard Medical School, United States of America Received July 3, 2013; Accepted September 7, 2013; Published October 18, 2013 Copyright: ß 2013 King et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Juvenile Diabetes Research Foundation (GCW), grants from the National Institutes of Health: R01 DK 66056 and DK 93909 (SBW) and R01 DK 45943 and DK 51729 (SES), P30 DK36836 Joslin Diabetes Research Center (DRC) Advanced Microscopy Core, as well as the Diabetes Research and Wellness Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: gordon.weir@joslin.harvard.edu Introduction Inflammatory cytokines such as INF-c, TNF-a and IL-1 have been implicated in the autoimmune destruction of pancreatic b- cells in type 1 diabetes [1]. Since NF-kB is both activated by these cytokines, and drives their expression, considerable interest has been focused on NF-kB in b-cells [2,3]. But the situation is complex because NF-kB may increase the expression of both proapoptotic and antiapoptotic genes, and patterns of gene expression may vary depending on context and cell type. In b- cells, cytokine-induced activation of NF-kB has been associated with increased expression of inflammatory proteins such as iNOS and COX-2, and nitric oxide (the product of iNOS) has been implicated in IL-1b-induced b-cell death [4,5]. NF-kB activation has also been associated with the enhanced expression of proapoptotic and protective genes [6–8]. In vitro studies have shown that the inhibition of NF-kB can protect beta cells against cytokine-induced death [9–11]. However, others have suggested that NF-kB activation could play a protective role preventing TNF-induced b-cell apoptosis [12]. Indeed, it has been suggested that NF-kB may play a biphasic role in cytokine-induced b-cell death, by initially protecting the b-cells before leading to apoptosis [13]. It has also been recently suggested that NF-kB may act as an antiapoptotic factor in normoxic conditions but act as an apoptotic factor in hypoxic conditions [14]. Studies have shown that genetically modified mice with disrupted NF-kB may be resistant to b-cell toxins, such as multiple low-dose streptozotocin injections [15,16]. In transplantation settings it has been suggested that acute inhibition of NF-kB can improve islet transplantation outcome [14,17–21]. Transplantation of islets is an important breakthrough in the treatment of Type 1 diabetes [22]. It can reverse hyperglycaemia in humans [23], but long-term success is limited [24], indicating a failure to maintain islet mass. Because NF-kB is a potentially useful therapeutic target and seems to be involved in b-cell destruction in models of diabetes, we sought to determine if the state of NF-kB activation would influence the outcome of islet transplantation. The in vivo activity of NF-kB is tightly regulated by an inhibitory protein, IkBa [25] and an activating kinase, IKKb [26]. Once proinflammatory stimuli have activated IKKb, it phosphorylates IkBa, which is targeted for ubiquitination and proteasomal PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e77452