DUOX2 promotes the elimination of the Klebsiella pneumoniae strain K5 from T24 cells through the reactive oxygen species pathway

Lu,Huixia; Wu,Qi; Yang,Huijun

doi:10.3892/ijmm.2015.2234

DUOX2 promotes the elimination of the Klebsiella pneumoniae strain K5 from T24 cells through the reactive oxygen species pathway

Authors:
- Huixia Lu
- Qi Wu
- Huijun Yang
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Affiliations: Department of Anatomy, Basic Medical and Forensic Medical Institute, Sichuan University, Chengdu, Sichuan 610041, P.R. China, Innate Immunity Research Laboratory, Basic Medical and Forensic Medical Institute, Sichuan University, Chengdu, Sichuan 610041, P.R. China
Published online on: June 3, 2015 https://doi.org/10.3892/ijmm.2015.2234
Pages: 551-558

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Abstract

Dual oxidase 2 (DUOX2) plays a major role in host defense in intestinal and airway epithelial cells through the reactive oxygen species (ROS) pathway. Klebsiella pneumoniae is a uropathogen that causes urinary tract infections. It is not known whether DUOX2 plays a role in host defense in bladder cancer epithelial cells. It is also not known whether Klebsiella pneumoniae invades T24 human bladder carcinoma cells and whether DUOX2 plays a role in eliminating the Klebsiella pneumoniae strain K5 through the ROS pathway in T24 cells. Thus, in the present study, we aimed to investigate the infectious capability of the Klebsiella pneumoniae K5 strain and the immunity‑promoting capability of DUOX2 in T24 cells. We quantified the number of viable intracellular bacteria using the plate count method. DUOX2 expression was evaluated by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR) follwoing treatment with or without multiple cytokines, phorbol 12‑myristate 13‑acetate (PMA), muramyl dipeptide (MDP), N‑acetylmuramyl‑D‑alanyl‑D‑isoglutamine (MDP‑DD), H2O2 inhibitor, catalase (CAT), the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase inhibitor, diphenyleneiodonium (DPI), or siRNA targeting DUOX2 (siDUOX2). The levels of ROS in the T24 cells infected with the K5 strain were examined following treatment with DPI, CAT or siDUOX2. Our results revealed that DUOX2 expression increased and the number of viable intracellular bacteria decreased in the T24 cells following infection with the K4 bacteria. Treatment with the cytokines and MDP and PMA also induced DUOX2 expression and decreased the number of viable intracellular bacteria. The levels of ROS also increased following treatment with the cytokines and MDP and PMA. However, when the cells were treated with the inhibitors (DPI or CAT), these effects were all reversed. Our data demonstrated that DUOX2 played an important role in innate immunity against bacterial cytoinvasion through the ROS pathway in T24 cells. Our findings also provide insight into the protection of uroepithelial cells from Klebsiella pneumoniae K5 bacterial cytoinvasion, and thus lay the foundation for the development of novel therapies for urinary tract infections.

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1	Meca G, Sospedra I, Valero MA, Mañes J, Font G and Ruiz MJ: Antibacterial activity of the enniatin B, produced by Fusarium tricinctum in liquid culture, and cytotoxic effects on Caco-2 cells. Toxicol Mech Methods. 21:503–512. 2011. View Article : Google Scholar : PubMed/NCBI
2	Rhim AD, Stoykova L, Glick MC and Scanlin TF: Terminal glycosylation in cystic fibrosis (CF): Aa review emphasizing the airway epithelial cell. Glycoconj J. 18:649–659. 2001. View Article : Google Scholar
3	Swords WE, Chance DL, Cohn LA, Shao J, Apicella MA and Smith AL: Acylation of the lipooligosaccharide of Haemophilus influenzae and colonization: an htrB mutation diminishes the colonization of human airway epithelial cells. Infect Immun. 70:4661–4668. 2002. View Article : Google Scholar : PubMed/NCBI
4	Loose M, Hudel M, Zimmer KP, Garcia E, Hammerschmidt S, Lucas R, Chakraborty T and Pillich H: Pneumococcal hydrogen peroxide-induced stress signaling regulates inflammatory genes. J Infect Dis. 211:306–316. 2014. View Article : Google Scholar : PubMed/NCBI
5	Cortés G, Alvarez D, Saus C and Albertí S: Role of lung epithelial cells in defense against Klebsiella pneumoniae pneumonia. Infect Immun. 70:1075–1080. 2002. View Article : Google Scholar : PubMed/NCBI
6	Rosen DA, Pinkner JS, Walker JN, Elam JS, Jones JM and Hultgren SJ: Molecular variations in Klebsiella pneumoniae and Escherichia coli FimH affect function and pathogenesis in the urinary tract. Infect Immun. 76:3346–3356. 2008. View Article : Google Scholar : PubMed/NCBI
7	Zgair AK and Al-Adressi AM: Stenotrophomonas maltophilia fimbrin stimulates mouse bladder innate immune response. Eur J Clin Microbiol Infect Dis. 32:139–146. 2013. View Article : Google Scholar
8	Michelet C, Avril JL, Cartier F and Berche P: Inhibition of intracellular growth of Listeria monocytogenes by antibiotics. Antimicrob Agents Chemother. 38:438–446. 1994. View Article : Google Scholar : PubMed/NCBI
9	Nakagawa I, Amano A, Mizushima N, Yamamoto A, Yamaguchi H, Kamimoto T, Nara A, Funao J, Nakata M, Tsuda K, et al: Autophagy defends cells against invading group A Streptococcus. Science. 306:1037–1040. 2004. View Article : Google Scholar : PubMed/NCBI
10	Grasberger H, El-Zaatari M, Dang DT and Merchant JL: Dual oxidases control release of hydrogen peroxide by the gastric epithelium to prevent Helicobacter felis infection and inflammation in mice. Gastroenterology. 145:1045–1054. 2013. View Article : Google Scholar : PubMed/NCBI
11	Rada B and Leto TL: Oxidative innate immune defenses by Nox/Duox family NADPH oxidases. Contrib Microbiol. 15:164–187. 2008. View Article : Google Scholar : PubMed/NCBI
12	Enyedi B and Niethammer P: H₂O₂: a chemoattractant? Methods Enzymol. 528:237–255. 2013. View Article : Google Scholar
13	Geiszt M and Leto TL: The Nox family of NAD(P)H oxidases: host defense and beyond. J Biol Chem. 279:51715–51718. 2004. View Article : Google Scholar : PubMed/NCBI
14	Lees MS, H Nagaraj S, Piedrafita DM, Kotze AC and Ingham AB: Molecular cloning and characterisation of ovine dual oxidase 2. Gene. 500:40–46. 2012. View Article : Google Scholar : PubMed/NCBI
15	Rigutto S, Hoste C, Grasberger H, Milenkovic M, Communi D, Dumont JE, Corvilain B, Miot F and De Deken X: Activation of dual oxidases Duox1 and Duox2: differential regulation mediated by camp-dependent protein kinase and protein kinase C-dependent phosphorylation. J Biol Chem. 284:6725–6734. 2009. View Article : Google Scholar : PubMed/NCBI
16	Kim HJ, Kim CH, Ryu JH, Kim MJ, Park CY, Lee JM, Holtzman MJ and Yoon JH: Reactive oxygen species induce antiviral innate immune response through IFN-λ regulation in human nasal epithelial cells. Am J Respir Cell Mol Biol. 49:855–865. 2013. View Article : Google Scholar : PubMed/NCBI
17	Fink K, Martin L, Mukawera E, Chartier S, De Deken X, Brochiero E, Miot F and Grandvaux N: IFNβ/TNFα synergism induces a non-canonical STAT2/IRF9-dependent pathway triggering a novel DUOX2 NADPH oxidase-mediated airway antiviral response. Cell Res. 23:673–690. 2013. View Article : Google Scholar : PubMed/NCBI
18	Strengert M, Jennings R, Davanture S, Hayes P, Gabriel G and Knaus UG: Mucosal reactive oxygen species are required for antiviral response: Rrole of Duox in influenza a virus infection. Antioxid Redox Signal. 20:2695–2709. 2014. View Article : Google Scholar
19	Lipinski S, Till A, Sina C, Arlt A, Grasberger H, Schreiber S and Rosenstiel P: DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses. J Cell Sci. 122:3522–3530. 2009. View Article : Google Scholar : PubMed/NCBI
20	Ronald A and Ludwig E: Urinary tract infections in adults with diabetes. Int J Antimicrob Agents. 17:287–292. 2001. View Article : Google Scholar : PubMed/NCBI
21	Hansen DS, Gottschau A and Kolmos HJ: Epidemiology of Klebsiella bacteraemia: Aa case control study using Escherichia coli bacteraemia as control. J Hosp Infect. 38:119–132. 1998. View Article : Google Scholar : PubMed/NCBI
22	Donkó A, Ruisanchez E, Orient A, Enyedi B, Kapui R, Péterfi Z, de Deken X, Benyó Z and Geiszt M: Urothelial cells produce hydrogen peroxide through the activation of Duox1. Free Radic Biol Med. 49:2040–2048. 2010. View Article : Google Scholar : PubMed/NCBI
23	El Hassani RA, Benfares N, Caillou B, Talbot M, Sabourin JC, Belotte V, Morand S, Gnidehou S, Agnandji D, Ohayon R, et al: Dual oxidase2 is expressed all along the digestive tract. Am J Physiol Gastrointest Liver Physiol. 288:G933–G942. 2005. View Article : Google Scholar
24	Ha EM, Oh CT, Bae YS and Lee WJ: A direct role for dual oxidase in Drosophila gut immunity. Science. 310:847–850. 2005. View Article : Google Scholar : PubMed/NCBI
25	Flores MV, Crawford KC, Pullin LM, Hall CJ, Crosier KE and Crosier PS: Dual oxidase in the intestinal epithelium of zebrafish larvae has anti-bacterial properties. Biochem Biophys Res Commun. 400:164–168. 2010. View Article : Google Scholar : PubMed/NCBI
26	Harper RW, Xu C, Eiserich JP, Chen Y, Kao CY, Thai P, Setiadi H and Wu R: Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium. FEBS Lett. 579:4911–4917. 2005. View Article : Google Scholar : PubMed/NCBI
27	Geiszt M, Lekstrom K, Brenner S, Hewitt SM, Dana R, Malech HL and Leto TL: NAD(P)H oxidase 1, a product of differentiated colon epithelial cells, can partially replace glycoprotein 91phox in the regulated production of superoxide by phagocytes. J Immunol. 171:299–306. 2003. View Article : Google Scholar : PubMed/NCBI
28	Geiszt M, Lekstrom K, Witta J and Leto TL: Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells. J Biol Chem. 278:20006–20012. 2003. View Article : Google Scholar : PubMed/NCBI
29	O’Quinn DB, Palmer MT, Lee YK and Weaver CT: Emergence of the Th17 pathway and its role in host defense. Adv Immunol. 99:115–163. 2008. View Article : Google Scholar
30	Miljkovic D and Trajkovic V: Inducible nitric oxide synthase activation by interleukin-17. Cytokine Growth Factor Rev. 15:21–32. 2004. View Article : Google Scholar : PubMed/NCBI
31	Trajkovic V, Stosic-Grujicic S, Samardzic T, Markovic M, Miljkovic D, Ramic Z and Mostarica Stojkovic M: Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes. J Neuroimmunol. 119:183–191. 2001. View Article : Google Scholar : PubMed/NCBI
32	Willems MM, Zom GG, Meeuwenoord N, Ossendorp FA, Overkleeft HS, van der Marel GA, Codée JD and Filippov DV: Design, automated synthesis and immunological evaluation of NOD2-ligand-antigen conjugates. Beilstein J Org Chem. 10:1445–1453. 2014. View Article : Google Scholar : PubMed/NCBI
33	Kim HJ, Kim CH, Ryu JH, Joo JH, Lee SN, Kim MJ, Lee JG, Bae YS and Yoon JH: Crosstalk between platelet-derived growth factor-induced Nox4 activation and MUC8 gene overexpression in human airway epithelial cells. Free Radic Biol Med. 50:1039–1052. 2011. View Article : Google Scholar : PubMed/NCBI
34	van der Vliet A: NADPH oxidases in lung biology and pathology: hHost defense enzymes, and more. Free Radic Biol Med. 44:938–955. 2008. View Article : Google Scholar : PubMed/NCBI
35	Arnoult D, Carneiro L, Tattoli I and Girardin SE: The role of mitochondria in cellular defense against microbial infection. Semin Immunol. 21:223–232. 2009. View Article : Google Scholar : PubMed/NCBI
36	Gao XP, Standiford TJ, Rahman A, Newstead M, Holland SM, Dinauer MC, Liu QH and Malik AB: Role of NADPH oxidase in the mechanism of lung neutrophil sequestration and microvessel injury induced by Gram-negative sepsis: Studies in p47^phox−/− and gp91^phox−/− mice. J Immunol. 168:3974–3982. 2002. View Article : Google Scholar : PubMed/NCBI
37	Koarai A, Sugiura H, Yanagisawa S, Ichikawa T, Minakata Y, Matsunaga K, Hirano T, Akamatsu K and Ichinose M: Oxidative stress enhances toll-like receptor 3 response to double-stranded RNA in airway epithelial cells. Am J Respir Cell Mol Biol. 42:651–660. 2010. View Article : Google Scholar :
38	Zmijewski JW, Lorne E, Zhao X, Tsuruta Y, Sha Y, Liu G and Abraham E: Antiinflammatory effects of hydrogen peroxide in neutrophil activation and acute lung injury. Am J Respir Crit Care Med. 179:694–704. 2009. View Article : Google Scholar : PubMed/NCBI
39	Hou Y, An J, Hu XR, Sun BB, Lin J, Xu D, Wang T and Wen FQ: Ghrelin inhibits interleukin-8 production induced by hydrogen peroxide in A549 cells via NF-kappaB pathway. Int Immunopharmacol. 9:120–126. 2009. View Article : Google Scholar
40	Kim JY, Omori E, Matsumoto K, Núñez G and Ninomiya-Tsuji J: TAK1 is a central mediator of NOD2 signaling in epidermal cells. J Biol Chem. 283:137–144. 2008. View Article : Google Scholar
41	Forteza R, Salathe M, Miot F, Forteza R and Conner GE: Regulated hydrogen peroxide production by Duox in human airway epithelial cells. Am J Respir Cell Mol Biol. 32:462–469. 2005. View Article : Google Scholar : PubMed/NCBI
42	Gattas MV, Forteza R, Fragoso MA, Fregien N, Salas P, Salathe M and Conner GE: Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli. Free Radic Biol Med. 47:1450–1458. 2009. View Article : Google Scholar : PubMed/NCBI
43	Geiszt M, Witta J, Baffi J, Lekstrom K and Leto TL: Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J. 17:1502–1504. 2003.PubMed/NCBI