Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 Prdx6 Deficiency Ameliorates DSS Colitis: Relevance of Compensatory Antioxidant Mechanisms Melhem, Hassan ; Spalinger, Marianne R ; Cosin-Roger, Jesus ; Atrott, Kirstin ; Lang, Silvia ; Wojtal, Kacper A ; Vavricka, Stephan R ; Rogler, Gerhard ; Frey-Wagner, Isabelle Abstract: Background and Aims An imbalance between cellular antioxidant defence system[s] and reactive oxygen species [ROS]-driven oxidative stress has been implicated in the pathogenesis of inflammatory bowel disease. Peroxiredoxin [PRDX] 6 contributes to an appropriate redox balance by clearing ROS and reducing peroxidized membrane phospholipids. We here studied the role of PRDX6 in acute and chronic dextran sodium sulphate [DSS]-induced colitis. Methods To investigate the impact of PRDX6 on intestinal inflammation, we used wild type [WT], Prdx6 knock-out mice [Prdx6-/-] and transgenic mice [Prdx6tg/tg], overexpressing Prdx6. Acute and chronic colitis was induced by DSS in WT, Prdx6-/- and Prdx6tg/tg mice. Colitis was evaluated by endoscopy, colon length, histopathological assessment and myeloperoxidase [MPO] activity. Changes in mRNA and protein expression of pro-inflammatory cytokines and antioxidant enzymes were evaluated by real-time quantitative polymerase chain reaction [RT-qPCR] and western blot. Total glutathione [GSH] levels in colon samples were determined. Results Prdx6-/- mice exposed to acute and chronic DSS showed a significant decrease in the clinical parameters and in colonic expression of pro-inflammatory cytokines compared with WT mice. mRNA expression of antioxidant enzymes in colon samples was significantly increased in Prdx6-/- compared with WT mice exposed to acute and chronic DSS. In addition, total GSH levels were increased in Prdx6-/- mice treated with DSS in comparison with WT. Overexpression of Prdx6 did not significantly influence acute and chronic colitis. Conclusions Our data indicate that a lack of the antioxidant enzyme PRDX6 protects against the development of acute and chronic experimental colitis and is associated with increased expression and function of other antioxidant enzymes, suggesting effective compensatory mechanisms. DOI: https://doi.org/10.1093/ecco-jcc/jjx016 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-145195 Journal Article Accepted Version Originally published at: Melhem, Hassan; Spalinger, Marianne R; Cosin-Roger, Jesus; Atrott, Kirstin; Lang, Silvia; Wojtal, Kacper A; Vavricka, Stephan R; Rogler, Gerhard; Frey-Wagner, Isabelle (2017). Prdx6 Deficiency Ame- liorates DSS Colitis: Relevance of Compensatory Antioxidant Mechanisms. Journal of Crohn’s Colitis, 11(7):871-884. DOI: https://doi.org/10.1093/ecco-jcc/jjx016 1 Prdx6 deficiency ameliorates DSS colitis: relevance of compensatory antioxidant 2 mechanisms 3 4 Hassan Melhem1, Marianne R. Spalinger1, Jesus Cosin-Roger1, Kirstin Atrott1, Silvia Lang1, 5 Kacper Wojtal1, Stephan Vavricka1,2,3, Gerhard Rogler1, 2 and Isabelle Frey-Wanger1, 2 6 7 1 Departement of Gastroenterology and Hepatology, University Hospital Zurich, University of 8 Zurich, Zurich, Switzerland 9 2Division of Gastroenterology and Hepatology, Hospital Triemli, Zurich, Switzerland 10 3 Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Zurich, 11 Switzerland 12 13 Short title: Lack of Prdx6 ameliorates DSS colitis 14 15 Correspondence: 16 Dr. Isabelle Frey-Wagner 17 Division of Gastroenterology and Hepatology 18 University Hospital of Zurich 19 Raemistrasse 100 20 CH-8091 Zurich 21 Switzerland 22 E-mail: [email protected] 23 24 Abbreviations 25 BCA, bicinchoninic acid assay; colonic lamina propria mononuclear cells (LPMC); CD, 26 Crohns’s disease; DSS, dextran sodium sulphate; GSH, reduced glutathione; IBD, 27 inflammatory bowel disease; intestinal epithelial cells, (IEC); MEICS, murine endoscopic 28 index of colitis severity; MPO, Myeloperoxidase; NO, Nitric oxide; ROS, reactive oxygen 29 species; Tg, transgenic; UC, ulcerative colitis; UV, ultraviolet 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Abstract 52 53 Background 54 An imbalance between cellular antioxidant defense system(s) and reactive oxygen species 55 (ROS)-driven oxidative stress has been implicated in the pathogenesis of inflammatory bowel 56 disease. Peroxiredoxin (PRDX) 6, contributes to an appropriate redox balance by clearing 57 ROS and reducing peroxidized membrane phospholipids. 58 59 Aims 60 We here studied the role of Prdx6 in acute and chronic DSS-induced colitis. 61 62 Design 63 To investigate the impact of PRDX6 on intestinal inflammation, we used wild type (WT), 64 Prdx6 knock-out mice (Prdx6-/-) and transgenic mice (Prdx6tg/tg), overexpressing Prdx6. 65 Acute and chronic colitis was induced by dextran sulfate sodium (DSS) in WT, Prdx6-/-, and 66 Prdx6tg/tg mice. Colitis was evaluated by endoscopy, colon length, histopathological 67 assessment, and myeloperoxidase (MPO) activity. Changes in mRNA and protein expression 68 of pro-inflammatory cytokines and antioxidant enzymes were evaluated by RT-qPCR and 69 western blot. Total GSH levels in colon samples were determined. 70 71 Results 72 Prdx6-/- mice exposed to acute and chronic DSS showed a significant decrease in the clinical 73 parameters and in colonic expression of pro-inflammatory cytokines compared to WT mice. 74 mRNA expression of antioxidant enzymes in colon samples was significantly increased in 75 Prdx6-/- compared to WT mice exposed to acute and chronic DSS. In addition, total GSH 76 levels were increased in Prdx6-/- mice treated with DSS in comparison to WT. Overexpression 77 of Prdx6 did not significantly influence acute and chronic colitis. 78 79 Conclusion 80 Our data indicate that a lack of the antioxidant enzyme PRDX6 protects against the 81 development of acute and chronic experimental colitis and is associated with increased 82 expression and function of other antioxidant enzymes suggesting effective compensatory 83 mechanisms. 84 85 Keywords 86 IBD, colitis, ROS, Prdx6, antioxidant enzymes 87 88 Introduction 89 The major forms of inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative 90 colitis (UC), are chronic, relapsing inflammatory disorders of the gastrointestinal (GI) tract 91 causing a life-long burden on affected patients (1). Both disorders have a multifactorial 92 pathogenesis with three main factors involved in their pathogenesis: environmental exposure 93 (e.g. to gut microbiota and microbial pathogens), genetic susceptibility (e.g. predisposing 94 factors affecting the gut barrier function) and consecutive dysregulation of the immune 95 system (on the innate and adaptive level) (2-3). 96 The mechanisms by which imbalances of the immune system initiate and perpetuate intestinal 97 damage and inflammation are not entirely clear. However, inflammatory conditions lead to an 98 increased oxidative burden due to the release of reactive oxygen species (ROS) by activated 99 macrophages and neutrophils as a part of the body’s defense mechanisms (4). Low levels of 100 ROS have a protective effect by activating protective signaling pathways against 101 inflammation and by modulating the immune-mediated attack against extrinsic pathogens. 102 Conversely, high levels of ROS create oxidative stress within the cells and, in case of chronic 103 exposure, changes in the genome, such as single strand DNA breaks, sugar moiety 104 modifications, and deamination and adduction of bases (5), leading to a loss of homeostasis 105 (6). 106 Several studies have established a direct relation between oxidative stress and IBD 107 pathogenesis in both, human subjects and animals subjected to experimental colitis. Clinical 108 studies showed an increase of ROS as well as biomarkers of oxidative injury, including lipid 109 peroxidation products and protein modifications, in the intestinal mucosa of patients with 110 either UC or CD (7-8). These highly cytotoxic molecules contribute to tissue damage in IBD 111 (9). In line with this, Barbosa et al., (10) showed a correlation between the activity of free 112 radicals in the intestinal mucosa of IBD patients, and the clinical disease activity. To maintain 113 adequate ROS levels, a balance between oxidant and antioxidant mechanisms is necessary. A 114 depletion of antioxidant levels in the intestinal mucosa and in peripheral red blood cells has 115 been observed in IBD patients (11-13). Genetic polymorphisms in antioxidant enzymes are 116 associated with altered enzyme activity and a higher risk of developing IBD (14). In animal 117 models of IBD, multiple studies demonstrated an increased formation of ROS in the colonic 118 mucosa (15-16). In line with this, experimental colitis is associated with a decrease in the 119 levels of endogenous antioxidants in colonic tissue (17). Genetic deletion of ROS-detoxifying 120 enzymes increases the susceptibility towards tissue destruction (18), whereas an 121 overexpression of antioxidant enzymes resulted in attenuation of colitis in mice (19-20). 122 Among the most important antioxidant proteins is the peroxiredoxin (PRDX) family. PRDX6, 123 a bifunctional 25-kDa protein, is the only member of PRDXs that confers both GSH 124 peroxidase and phospholipase A2 activities (21). Despite intensive investigations, the role of 125 PRDX6 in inflammation is still controversial and both protective (experimental autoimmune 126