Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection

et al. (2013) Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection. PLoS ONE 8(4): e61394. doi:10.1371/journal.pone.0061394 Ecrg4 Attenuates the Inflammatory Proliferative Response of Mucosal Epithelial Cells to Infection Arwa Kurabi 0 Kwang Pak 0 Xitong Dang 0 Raul Coimbra 0 Brian P. Eliceiri 0 Allen F. Ryan 0 Andrew Baird 0 Johanna M. Brandner, University Hospital Hamburg-Eppendorf, Germany 0 1 Department of Surgery, University of California, San Diego School of Medicine, La Jolla, California, United States of America, 2 Veterans Administration Medical Center , San Diego, California , United States of America We report an inverse relationship between expression of the orphan candidate tumor suppressor gene esophageal cancer related gene 4 (Ecrg4), and the mucosal epithelial cell response to infection in the middle ear (ME). First, we found constitutive Ecrg4 mRNA expression in normal, quiescent ME mucosa that was confirmed by immunostainning of mucosal epithelial cells and immunoblotting of tissue lysates for the 14 kDa Ecrg4 protein. Upon experimental ME infection, Ecrg4 gene expression rapidly decreased by over 80%, between 3 to 48 hrs, post infection. When explants of this infected mucosa were placed in culture and transduced with an adenovirus (AD) encoding Ecrg4 gene (ADEcrg4), the proliferative and migratory responses of mucosal cells were significantly inhibited. ADEcrg4 transduction of control explants from uninfected MEs had no effect on basal growth and migration. Over-expression of Ecrg4 in vivo, by pre-injecting MEs with ADEcrg4 48 hrs prior to infection, prevented the natural down-regulation of Ecrg4, reduced mucosal proliferation and prevented inflammatory cell infiltration normally observed after infection. Taken together, these data support a hypothesis that Ecrg4 plays a role in coordinating the inflammatory and proliferative response to infection of mucosal epithelium suggesting a possible mechanism for its putative anti-tumor activity. - Funding: Research was supported by the National Institutes of Health grants P20-GM78421 (AB) and RO1-DC000129 (AFR), and by the VA Research Service (AFR). 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. Mucosal surfaces represent a major barrier lining and protecting the ducts of the eye, ear, exocrine glands and the aero-digestive and uro-genital tracts [1]. In particular, mucosal epithelia serve critical homeostatic functions as biological, physical and mechanical barriers that regulate innate and adaptive immunities and the tissue response to infection and injury [2,3]. It is generally accepted that the expression of cytokines, chemokines and antimicrobial factors mediate much of the mucosal response to injury [4,5,6,7]. However, the mechanisms that regulate local homeostasis in normal mucosal epithelium and how these mechanisms may participate in response to injury are less understood. In recent years, the identification of paracrine, juxtacrine and autocrine factors that control the inflammatory response has led to significant refinements in our understanding of tissue homeostasis. Local factors that are constitutively produced in tissues respond to changes in the local milieu to play critical roles in defining ultimate biological responses. For example, constitutively expressed defensins [8,9] and pathogen receptors [1,4,6,10] encode classes of molecules poised to defend tissues from infection. In a similar manner, the induction of alarmin genes [11,12] after inflammation is a response to the detection of biological, chemical and physical threats that disrupt tissue homeostasis. We recently identified a candidate gene called Esophageal cancer related gene-4 (Ecrg4) that we proposed plays a sentinel function to monitor set points of homeostasis [13,14,15,16]. Constitutively expressed by numerous cell types, localized in many normal tissues, and found in selected biological fluids, Ecgr4 is a member of both the secretome [17,18] and neuropeptidome [13,14,19,20] that is tethered to the epithelial cell surface [13,14,15,16]. In cancer, its expression is epigenetically regulated by DNA methylation of .16 CpG sites in its promoter region [21,22,23] and as such, it is highly down-regulated in epithelial cancers via hypermethylation [21,22,23,24,25,26]. At 148 residues in length, the Ecrg4 protein is slightly basic (pI 8) and, depending on its posttranslational processing, can generate several potential ligands of 2 to 14 kDa [14]. Because previous studies demonstrated that Ecrg4 gene expression is associated with epithelial cells, cancers and barriers [16,25,27,28], we tested the possibility that Ecrg4 could regulate the inflammatory response to infection. In the studies presented here, we exploited the capacity of ME mucosal epithelia to respond to infection with both proliferation and inflammation. We showed that while Ecrg4 is constitutively expressed in normal epithelial mucosa, it is rapidly down-regulated during bacterially mediated otitis media (OM) and that with its inappropriate expression during infection, it can modulate the natural course of the inflammatory response both in vitro and in vivo. Materials and Methods Hyperplastic epithelial middle ear mucosa in vitro model All animal studies were performed in strict accordance to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH). All animal studies performed were carried out in strict accordance with an approved UCSD Institutional Animal Care and Use Committee (IACUC) protocol (Protocol no. S08281) specifically approved for this study. All surgeries were performed under anesthesia, and all efforts were made to minimize suffering. The bullae of ,300 g male Sprague-Dawley rats were bilaterally injected with ,50 mL saline (control) or saline containing 105 cells/mL Haemophillus influenzae strain 3655 (nontypeable Hi/ biotype II). Following the inoculation, the tympanic membrane was visually confirmed to be intact. At ,48 hrs post surgery, the animals were sacrificed and the ME mucosa were surgically removed and divided into 0.5 mm2 square explants and individually seeded into a 24 well culture dish in media (75% DMEM, 25% HEMs-F12 supplemented with 5% bovine serum, and containing the following additives: 100 IU/mL penicillin, 100 mg/mL streptomycin, 0.4 mg/mL hydrocortisone and 1026 M isoproterenol), as described in Palacios et al [29]. The explants were incubated in a 5% CO2 humidified atmosphere at 37uC. Culture media was replaced every 3 days. Photographs of each explant were taken daily with a RT-SPOT color digital camera to document the extent of primary culture growth. The diameter of explant outgrowth, which was approximately circular, was measured and its area was cal (...truncated)