ARTICLE Pharmacological application of carbon monoxide ameliorates islet-directed autoimmunity in mice via anti-inflammatory and anti-apoptotic effects Ivana Nikolic & Tamara Saksida & Katia Mangano & Milica Vujicic & Ivana Stojanovic & Ferdinando Nicoletti & Stanislava Stosic-Grujicic Received: 19 July 2013 /Accepted: 18 December 2013 /Published online: 2 February 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Aims/hypothesis Recent studies have identified carbon mon- oxide (CO) as a potential therapeutic molecule for the treat- ment of autoimmune diseases owing to its anti-inflammatory and anti-apoptotic properties. We explored the efficacy and the mechanisms of action of the CO-releasing molecule (CORM)-A1 in preclinical models of type 1 diabetes. Methods The impact of CORM-A1 on diabetes development was evaluated in models of spontaneous diabetes in NOD mice and in diabetes induced in C57BL/6 mice by multiple low-dose streptozotocin (MLDS). Ex vivo analysis was per- formed to determine the impact of CORM-A1 both on T helper (Th) cell and macrophage differentiation and on their production of soluble mediators in peripheral tissues and in infiltrates of pancreatic islets. The potential effect of CORM- A1 on cytokine-induced apoptosis in pancreatic islets or beta cells was evaluated in vitro. Results CORM-A1 conferred protection from diabetes in MLDS-induced mice and reduced diabetes incidence in NOD mice as confirmed by preserved insulin secretion and improved histological signs of the disease. In MLDS- challenged mice, CORM-A1 attenuated Th1, Th17, and M1 macrophage response and facilitated Th2 cell differentiation. In addition, CORM-A1 treatment in NOD mice upregulated the regulatory arm of the immune response (M2 macrophages and FoxP3 + regulatory T cells). Importantly, CORM-A1 in- terfered with in vitro cytokine-induced beta cell apoptosis through the reduction of cytochrome c and caspase 3 levels. Conclusions/interpretation The ability of CORM-A1 to pro- tect mice from developing type 1 diabetes provides a valuable proof of concept for the potential exploitation of controlled CO delivery in clinical settings for the treatment of autoim- mune diabetes. Keywords Beta cell apoptosis . Carbon monoxide-releasing molecule-A1 . Cytokines . Type 1 diabetes Abbreviations CO Carbon monoxide ConA Concanavalin A CORM-A1 Carbon monoxide-releasing molecule-A1 ERK Extracellular signal-regulated kinases HO Haem oxygenase iCORM-A1 Inactive carbon monoxide-releasing molecule-A1 JNK Jun N-terminal kinase MLDS Multiple low-dose streptozotocin MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide NBT Nitroblue tetrazolium NFκB Nuclear factor κB NO Nitric oxide PC Peritoneal cell PLNC Pancreatic lymph node cell PMC Pancreatic-infiltrating mononuclear cell RORγt Retinoic acid-related orphan receptor-γt SC Splenocyte Electronic supplementary material The online version of this article (doi:10.1007/s00125-014-3170-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users. I. Nikolic : T. Saksida : M. Vujicic : I. Stojanovic : S. Stosic-Grujicic (*) Department of Immunology, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Bul. Despota Stefana 142, 11060 Belgrade, Serbia e-mail: duta@eunet.rs K. Mangano : F. Nicoletti (*) Department of Biomedical Sciences, School of Medicine, University of Catania, Via Androne 83, 95124 Catania, Italy e-mail: ferdinic@unict.it Diabetologia (2014) 57:980–990 DOI 10.1007/s00125-014-3170-7