Eosinophil-Mediated Signalling Attenuates Inflammatory Responses
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Gut Online First, published on September 10, 2014 as 10.1136/gutjnl-2014-306998 Inflammatory bowel disease ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2014-306998 on 10 September 2014. Downloaded from Eosinophil-mediated signalling attenuates inflammatory responses in experimental colitis Joanne C Masterson,1,2,3 Eóin N McNamee,2,3,4 Sophie A Fillon,1,2,3 Lindsay Hosford,1,2,3 Rachel Harris,1,2,3 Shahan D Fernando,1,2,3 Paul Jedlicka,3,5 Ryo Iwamoto,6 Elizabeth Jacobsen,7,8 Cheryl Protheroe,7,8 Holger K Eltzschig,2,3,4 Sean P Colgan,2,3,9 Makoto Arita,6,7 James J Lee,8 Glenn T Furuta1,2,3 ▸ Additional material is ABSTRACT published online only. To view Objective Eosinophils reside in the colonic mucosa Significance of this study please visit the journal online fi (http://dx.doi.org/10.1136/ and increase signi cantly during disease. Although a gutjnl-2014-306998). number of studies have suggested that eosinophils contribute to the pathogenesis of GI inflammation, the For numbered affiliations see What is already known on this subject? end of article. expanding scope of eosinophil-mediated activities ▸ IBD is a disease characterised by increased indicate that they also regulate local immune responses numbers of eosinophils. Correspondence to and modulate tissue inflammation. We sought to define ▸ The exact role of eosinophils during the onset Dr Glenn T Furuta, Section of the impact of eosinophils that respond to acute phases of inflammation and in chronic disease remains Pediatric Gastroenterology, Hepatology and Nutrition, of colitis in mice. unclear. Design Acute colitis was induced in mice by Department of Pediatrics, fi University of Colorado School administration of dextran sulfate sodium, 2,4,6- What are the new ndings? of Medicine, 13123 East trinitrobenzenesulfonic acid or oxazolone to C57BL/6J ▸ In the absence of eosinophils, mice succumb to 16th Ave, B290, Aurora, (control) or eosinophil deficient (PHIL) mice. Eosinophils a greater colonic inflammation during acute CO 80045, USA; were also depleted from mice using antibodies against experimental models of colitis. glenn.furuta@ ▸ fi childrenscolorado.org interleukin (IL)-5 or by grafting bone marrow from PHIL Eosinophil-de cient mice develop an exuberant mice into control mice. Colon tissues were collected and and compensatory neutrophilic inflammation of Received 11 February 2014 analysed by immunohistochemistry, flow cytometry and the colon, accompanied by greater increases in Revised 18 August 2014 levels of proinflammatory molecules. Accepted 19 August 2014 reverse transcription PCR; lipids were analysed by mass spectroscopy. ▸ Eosinophil-deficient mice have diminished Results Eosinophil-deficient mice developed levels of arachidonate-15-lipoxygenase, the significantly more severe colitis, and their colon tissues lipid biosynthetic enzyme, and the proresolving http://gut.bmj.com/ contained a greater number of neutrophils, than lipid mediator protectin D1 during acute colonic controls. This compensatory increase in neutrophils was inflammation, and administration of protectin accompanied by increased levels of the chemokines D1-isomer to eosinophil-deficient mice CXCL1 and CXCL2, which attract neutrophils. Lipidomic attenuates inflammatory indices of colitis, analyses of colonic tissue from eosinophil-deficient mice including neutrophilic infiltration. identified a deficiency in the docosahexaenoic acid- fl How might it impact on clinical practice in on October 6, 2021 by guest. Protected copyright. derived anti-in ammatory mediator 10, 17- the foreseeable future? dihydroxydocosahexaenoic acid (diHDoHE), namely ▸ Since eosinophils are normal residents of the protectin D1 (PD1). Administration of an exogenous colonic mucosa, further studies are necessary PD1-isomer (10S, 17S-DiHDoHE) reduced the severity of ’ fi to determine eosinophil s role in health and colitis in eosinophil-de cient mice. The PD1-isomer also disease. Protective factors released or regulated attenuated neutrophil infiltration and reduced levels of fi α β by eosinophils may be bene cial to colonic tumour necrosis factor- , IL-1 , IL-6 and inducible NO- health. synthase in colons of mice. Finally, in vitro assays identified a direct inhibitory effect of PD1-isomer on neutrophil transepithelial migration. – Conclusions Eosinophils exert a protective effect in role in a number of diseases such as asthma3 5 and fl – acute mouse colitis, via production of anti-in ammatory eosinophilic GI diseases.6 8 In these diseases, eosi- lipid mediators. nophils increase within the mucosal surfaces where they may participate in processes that often contrib- ute to tissue damage910and/or remodelling,11 either way leading to eventual organ dysfunction. INTRODUCTION Acute inflammatory/immune reparative responses To cite: Masterson JC, Eosinophils synthesise and release a broad range of are necessary self-limited processes that participate McNamee EN, Fillon SA, et al. Gut Published Online biologically active mediators, including antimicro- in maintaining host health and tissue function. First: [please include Day bial, proremodelling or anti-inflammatory asso- Mucosal surfaces use and amplify acute restorative Month Year] doi:10.1136/ ciated factors (reviewed in12). An increasing body anti-inflammatory activities through a number of gutjnl-2014-306998 of evidence suggests that eosinophils play a primary mechanisms including anti-inflammatory lipid Copyright Article author (or their employer)Masterson JC, et 2014. al. Gut 2014; Produced0:1–12. doi:10.1136/gutjnl-2014-306998 by BMJ Publishing Group Ltd (& BSG) under licence.1 Inflammatory bowel disease mediator synthesis.12 13 In this regard, omega-3 polyunsaturated PD1-isomer) (0.05 mg/kg) (Cayman Chemical, Ann Arbor, Gut: first published as 10.1136/gutjnl-2014-306998 on 10 September 2014. Downloaded from fatty acid (PUFA)-derived resolvins and protectins are potent Michigan, USA) was administered to mice. anti-inflammatory lipid mediators. For example, mice ingesting chow rich in endogenous omega-3 fatty acids have less severe Generation of bone marrow chimeric mice 14 colitis. By contrast, increased consumption of dietary proin- Bone marrow chimeric mice were generated by transferring iso- flammatory omega-6 PUFA correlates with an increase in IBD lated bone marrow from PHIL (PHIL->WT) or WT 15 16 incidence. Thus, dietary omega-3 fatty acid-derived anti- (WT->WT) donor mice into the retro-orbital plexus of subleth- inflammatory molecules have emerged as potent options for ally irradiated WT recipients, as described previously.30 – therapeutic intervention.17 20 Docosahexaenoic acid (DHA), an omega-3 PUFA abundant in fish oils, is converted to hydroxydo- Tissue processing and histological analysis cosahexaenoic acids (HDoHE), by enzymatic oxidation. Whole length colons were removed, processed and a global Protectin D1 (PD1) (10,17- dihydroxydocosahexaenoic acid inflammatory score was developed from H&E stained sections (DiHDoHE)) is biosynthesised by 15-lipoxygenase (LOX) in modified to account for area of tissue affected from that humans and 12/15-LOX in mice, and present in inflamed 28 – described previously. tissues.21 24 Recent reports show that both human and mouse eosinophils express high levels of leucocyte-type 15-LOX and Immunohistochemical assessment of eosinophil, 12/15-LOX, respectively, and are capable of producing PD1 fl 24 25 myeloperoxidase or F4/80 cellular in ammation when activated in vitro. Other cells with the potential to Eosinophils were localised in mouse and human tissues express 12/15-LOX include dendritic cells, tissue-resident 21 (Institutional Review Board approved) by eosinophil major basic macrophage, mast cells and epithelial cells. While the presence protein-1 (MBP-1) and eosinophil peroxidase immunohisto- fi of the enzyme is necessary, it is not always suf cient for PD1 chemistry as previously described.11 31 Neutrophils were loca- biosynthesis, as expression may be regulated by environmental lised in mouse tissues by myeloperoxidase (MPO) factors, such as cytokines or cell differentiation/activation 21 immunohistochemistry. Macrophage and other F4/80+ cells states. Administration of exogenous PD1 to mouse models of were localised in mouse tissues by F4/80 immunohistochemistry. inflammation protects tissues from inflammatory damage, expediting phagocytic resolution of acute peritonitis and retinal In situ hybridisation assessment of CXCL1 production injury models, attenuating inflammation and airway hyper- Formalin-fixed paraffin-embedded sections were subjected to responsiveness in mouse asthma models, while mouse eosino- in situ hybridisation with probes targeted to the CXCL1 mRNA phils contribute to the resolution of acute peritonitis in a – as per manufacturer’s recommendations (Advanced Cell 12/15-LOX-dependent manner.12 22 24 26 To date, the receptor Diagnostics, Hayward, California, USA). Slight modifications and, therefore, cellular target for PD1 has not been identified. are detailed in online supplementary methods. Probes directed Expression of these lipid mediators, association with colonic towards Ubc and DapB were used as positive and negative con- eosinophils and their role in resolution responses during colonic trols, respectively. inflammation is incompletely understood. The aim of our study was to determine the role(s) of eosinophils in mouse models of Colonic lamina propria and bone marrow leucocyte isolation http://gut.bmj.com/ acute colitis. Here we provide evidence for a novel protective fl role of eosinophils and concurrently show that this protection