Intestinal Expression of Fas and Fas Ligand Is Upregulated by Bacterial Signaling through TLR4 and TLR5, with Activation of Fas Modulating Intestinal TLR-Mediated This information is current as Inflammation of September 24, 2021. Philana Fernandes, Charlotte O'Donnell, Caitriona Lyons, Jonathan Keane, Tim Regan, Stephen O'Brien, Padraic Fallon, Elizabeth Brint and Aileen Houston J Immunol 2014; 193:6103-6113; Prepublished online 5 Downloaded from November 2014; doi: 10.4049/jimmunol.1303083 http://www.jimmunol.org/content/193/12/6103 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/11/05/jimmunol.130308 Material 3.DCSupplemental References This article cites 43 articles, 20 of which you can access for free at: http://www.jimmunol.org/content/193/12/6103.full#ref-list-1 by guest on September 24, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Intestinal Expression of Fas and Fas Ligand Is Upregulated by Bacterial Signaling through TLR4 and TLR5, with Activation of Fas Modulating Intestinal TLR-Mediated Inflammation Philana Fernandes,* Charlotte O’Donnell,* Caitriona Lyons,† Jonathan Keane,† Tim Regan,† Stephen O’Brien,† Padraic Fallon,‡ Elizabeth Brint,†,x,1 and Aileen Houston*,x,1 TLRs play an important role in mediating intestinal inflammation and homeostasis. Fas is best studied in terms of its function in apoptosis, but recent studies demonstrate that Fas signaling may mediate additional functions such as inflammation. The role of Fas, Downloaded from and the Fas ligand (FasL), in the intestine is poorly understood. The aim of this study was to evaluate potential cross-talk between TLRs and Fas/FasL system in intestinal epithelial cells (IECs). IECs were stimulated with TLR ligands, and expression of Fas and FasL was investigated. Treatment with TLR4 and TLR5 ligands, but not TLR2 and 9 ligands, increased expression of Fas and FasL in IECs in vitro. Consistent with this finding, expression of intestinal Fas and FasL was reduced in vivo in the epithelium of TLR4 knockout (KO), 5KO, and germ-free mice, but not in TLR2KO mice. Modulating Fas signaling using agonistic anti-Fas augmented TLR4- and TLR5-mediated TNF-a and IL-8 production by IECs. In addition, suppression of Fas in IECs reduced the ability of http://www.jimmunol.org/ TLR4 and TLR5 ligands and the intestinal pathogens Salmonella typhimurium and Listeria monocytogenes to induce the expres- sion of IL-8. In conclusion, this study demonstrates that extensive cross-talk in IECs occurs between the Fas and TLR signaling pathways, with the FasL/Fas system playing a role in TLR-mediated inflammatory responses in the intestine. The Journal of Immunology, 2014, 193: 6103–6113. he intestinal immune system consists of multiple cell TLRs are critical components of the innate immune response that types, including intestinal epithelial cells (IECs), which detect microorganisms through the recognition of conserved mo- constitute a single monolayer of cells found at the mucosal lecular motifs called pathogen-activated molecular patterns. Ex- T by guest on September 24, 2021 surface; specialized dendritic cells; and macrophages. In the pression of TLRs in the intestine has been well characterized. IECs gastrointestinal tract, IECs are in constant contact with luminal express several TLRs, including TLR2, TLR4, TLR5, and TLR9, bacteria, their metabolites, and their various inflammatory prod- with the location of these being restricted to either the apical or the ucts. The mucosal surface of the intestinal epithelium has evolved basolateral surface, or both (3). TLR5 is found exclusively on the to allow the correct balance of responsiveness, being broadly basolateral surface of IECs, thereby facilitating the detection of its unresponsive to the presence of the commensal bacteria in the gut ligand, flagellin (4). Conversely, TLR2, TLR4, and TLR9 have lumen while still being able to mount an immune response to the been found at both the apical and the basolateral surfaces (5, 6). presence of pathogenic bacteria (1). This colonic epithelial cell Studies performed to characterize signal transduction pathways of homeostasis is tightly regulated, as adverse effects can lead to TLRs in IECs have shown both inflammatory and homeostatic inflammatory conditions such as inflammatory bowel disease or to roles for these receptors following binding of their cognate ligands neoplastic conditions such as colon cancer (2). (7). TLR signaling in the intestine is tightly controlled, as aberrant signaling can give rise to uncontrolled inflammation or impaired *Department of Medicine, University College Cork, Cork, Ireland; †Department of defense against pathogens. Indeed, aberrant TLR signaling underlies Pathology, University College Cork, Cork, Ireland; ‡Trinity Biomedical Sciences x numerous pathologic conditions of the colon (5). Institute, Trinity College Dublin, Dublin 2, Ireland; and Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland Several studies have also investigated the role of the death re- 1E.B. and A.H. contributed equally to this work. ceptor Fas and the Fas ligand (FasL) in IECs (8–10). Fas has been Received for publication November 19, 2013. Accepted for publication October 5, shown to be constitutively expressed in the normal intestinal ep- 2014. ithelium, whereas the expression of FasL is more restricted, being This work was supported by Science Foundation Ireland Grants 10/RFP/CAN2894 most pronounced in the hematopoietic cells. Although best studied and 10/RFP/BIC2737. in terms of its role in apoptosis, recent studies have identified Address correspondence and reprint requests to Dr. Elizabeth Brint, Department of several nonapoptotic processes for Fas, such as proliferation, Pathology, Cork University Hospital, University College Cork, Wilton, Cork, Ireland. E-mail address: [email protected] migration, invasion and inflammation and tissue regeneration The online version of this article contains supplemental material. (11). In IECs, ligation of Fas has been shown to induce proin- Abbreviations used in this article: DSS, dextran sodium sulfite; FADD, Fas- flammatory cytokines in vitro (12), while also having a prosurvival associated protein with death domain; FasL, Fas ligand; FU, fluorescence unit; function in dextran sodium sulfite (DSS)–induced colitis (13). IEC, intestinal epithelial cell; KO, knockout; PGN, peptidoglycan; shRNA, short Several studies have shown that a level of cross-talk exists hairpin RNA. between the Fas and TLR signaling pathways. TLR4 and IL-1R1 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 signaling is reduced in peritoneal macrophages from Fas-deficient www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303083 6104 CROSS-TALK BETWEEN Fas/FasL AND TLRs IN IECs (lpr) mice, whereas blocking FasL/Fas interactions in macrophages Ab (ab15285) was obtained from Abcam (Cambridge, U.K.). Anti–b-actin suppresses LPS-induced (TLR4 agonist) and IL-1R1–induced in- Ab, TPCA-1, and LY294002 were obtained from Sigma-Aldrich (St Louis, flammatory cytokine production (14). Furthermore, the Fas adaptor MO). Anti-SIGIRR Ab was obtained from Pro-Sci (Poway, CA), anti– IRAK-m from AbboMax (San Jose, CA), anti-TLR2, and anti-TLR9 Ab molecule, Fas-associated protein with death domain (FADD), has from Novus (Littleton, CO) been shown to negatively regulate TLR signaling (15). Although no Cell lines and tissue study has directly investigated the cross-talk between these path- ways in IECs, a recent investigation into Fas signaling in alveolar HT29, HCT116, SW480, and CT26 colon epithelial cells and Jurkat T cells were epithelial cells indicated that Fas-induced inflammation occurred in obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in DMEM containing 10% FCS and penicillin–streptomycin. a MyD88-dependent manner in these cells (16). Cells were seeded at 1 3 105 cells per milliliter unless otherwise stated, cul- In the current study, we sought to determine whether Fas plays tured overnight, and then treated as specified in the figure legends. a role in the induction of TLR-induced inflammation in IECs. We Tissue from Swiss Webster wild-type and germ-free mice and from found that cross-talk exists between these receptors in IECs, in C57BL/6 TLR2, TLR4, and TLR5 knockout (KO) and wild-type mice was terms of both the expression level of Fas and FasL, and the in- kindly provided by the Alimentary Pharmabiotic Centre, University College Cork. Germ-free mice were colonized for 49 d, and tissue was obtained. duction of an inflammatory response. Collectively, these results Tissue from C57BL/6 MyD88 and TRIF KO mice was obtained from Prof. indicate a new role