Marie LeVine5* Abstract IL-36a Exerts Pro-Inflammatory Effects in the Lungs of Mice Ravisankar A. Ramadas1,2*, Susan L. Ewart3, Yoichiro Iwakura4, Benjamin D. Medoff1,2, Ann 1 Center for Immunology and Inflammatory Diseases, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 2 Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, Massachusetts, United States of America, 3 Department of Large Animal Clinical Sciences, Michigan State University, East Lansing, Michigan, United States of America, 4 Center for Experimental Medicine and Systems Biology, University of Tokyo, Tokyo, Japan, 5 Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, United States of America Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36a (IL-1F6) and test the hypothesis that IL-36a acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36a induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1ab2/2 mice in vivo. IL-36a induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1ab2/2 mice in vivo. In addition, intratracheal instillation of IL- 36a enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1ab2/2 mice in vivo. Furthermore, in vitro incubation of CD11c+ cells with IL-36a resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFa. IL-36a increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c+ cells to induce CD4+ T cell proliferation in vitro. Furthermore, stimulation with IL-36a activated NF-kB in a mouse macrophage cell line. These results demonstrate that IL-36a acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1a and IL-1b. The current study describes the pro-inflammatory effects of IL-36a in the lung, demonstrates the functional redundancy of IL-36a with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases. Citation: Ramadas RA, Ewart SL, Iwakura Y, Medoff BD, LeVine AM (2012) IL-36a Exerts Pro-Inflammatory Effects in the Lungs of Mice. PLoS ONE 7(9): e45784. doi:10.1371/journal.pone.0045784 Editor: Bernhard Ryffel, French National Centre for Scientific Research, France Received May 17, 2012; Accepted August 22, 2012; Published September 20, 2012 Copyright: 2012 Ramadas 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 research work was supported by Grants from the National Institute of Allergy and Infectious Diseases 1R03AI101366 (R.A.R) and the National Heart, Lung & Blood Institute 5T32HL007874 (B.D.M). 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: rramadas@partners.org (RAR); anlevine@med.umich.edu (AML) Introduction The Interleukin 1 (IL-1) cytokine cluster consists of a network of agonist, antagonist and receptor molecules that are expressed in a variety of immune and structural cells [1]. Since its initial identification as the human leukocytic pyrogen that causes fever [2], IL-1 has emerged as highly organized facilitator of early immune events in a plethora of inflammatory diseases [3]. The role of IL-1 cytokines in the development of acute and chronic inflammatory disorders of the lung has been extensively docu- mented and mechanistically characterized in human patients and in animal models of human lung disorders. For example, increased levels of IL-1 protein or mRNA, reflective of increased IL-1 cytokine activity, has consistently been observed in tissues from human patients with inflammatory lung disorders such as asthma [4–7], acute lung injury [8–10] and pulmonary fibrosis [11]. Similarly, studies from animal models have demonstrated that IL- 1 cytokines such as IL-1a and IL-1b play critical roles in the pathogenesis of asthma [12–14], chronic obstructive pulmonary disease [15,16], acute lung injury [17] and pulmonary fibrosis [18– 20]. In pulmonary infection models, IL-1 promoted or dampened bacterial clearance from the lungs depending on the bacterial pathogen [21–23]. While IL-1 induced the recruitment of neutrophils and pulmonary inflammation during the early stages of influenza infection in mice [24], it was critical in conferring protective immunity and enhancing survival of mice during the later stages of the disease [24–26]. In the last decade, genes encoding a novel cytokine cluster with structural and functional similarities to interleukin 1 (IL-1) were discovered and mapped to chromosome 2 in humans and mice [27–31]. These cytokines, initially termed IL-1 family members 5– 10 (IL1F5– IL1F10) have been recently reclassified according to an updated cytokine nomenclature scheme [32]. This novel IL-1 like cytokine cluster comprise a collection of agonists IL-36a (IL- 1F6), IL-36b (IL-1F8), IL-36c (IL-1F9), an antagonist IL-36Ra (IL-36Ra), a nuclear factor IL-37 (IL-1F7) and a functional heterodimeric receptor complex consisting of a novel receptor IL- 36R (IL-36R) and the adapter molecule IL-1 receptor accessory protein (IL-1RAcP) [33]. In addition, IL-38 (IL-1F10) has recently been reported to act as a partial antagonist for IL-36R [34]. The organization and function of this novel IL-1 like cytokine cluster is strikingly similar to that of the classical IL-1 cytokine cluster, PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45784