Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021 is online at: average * Drive Diverse Biological The Journal of Immunology , 27 of which you can access for free at: 2012; 188:781-792; Prepublished online 19 from submission to initial decision 4 weeks from acceptance to publication Benjamin L. Duell, Alison J. Carey, Chee K. Tan, Xiangqin Cui, Richard I. Webb, Makrina Totsika, Mark A. Schembri, Petra Derrington, Helen Irving-Rodgers, Andrew J. Brooks, Allan W. Cripps, Michael Crowley and Glen C. Ulett December 2011; doi: 10.4049/jimmunol.1101231 http://www.jimmunol.org/content/188/2/781 Innate Transcriptional Networks Activated in Bladder in Response to Uropathogenic Escherichia coli Pathways and Rapid Synthesis of IL-10 for Defense against Bacterial Urinary Tract Infection J Immunol cites 82 articles Submit online. 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The Journal of Immunology Innate Transcriptional Networks Activated in Bladder in Response to Uropathogenic Escherichia coli Drive Diverse Biological Pathways and Rapid Synthesis of IL-10 for Defense against Bacterial Urinary Tract Infection Benjamin L. Duell,*,1 Alison J. Carey,*,1 Chee K. Tan,* Xiangqin Cui,†,‡ Richard I. Webb,x Makrina Totsika,{ Mark A. Schembri,{ Petra Derrington,‖ Helen Irving-Rodgers,# Andrew J. Brooks,** Allan W. Cripps,* Michael Crowley,†† and Glen C. Ulett* Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC coloni- Downloaded from zation of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regu- lated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems- level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple http://www.jimmunol.org/ distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alter- ations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed signif- icantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI. The Journal of Immu- nology, 2012, 188: 781–792. by guest on October 1, 2021 rinary tract infections (UTI) are among the most common not well defined. Studies have shown that UPEC triggers inflam- infectious diseases of humans. Up to 40% of healthy mation that consists of immune mediators, including ILs (9–12), U adult women experience at least one UTI episode in their although some of these responses, including, for example, IL-1a, lifetime (1). Escherichia coli is the most common cause of UTI, occur in a pathogen-specific manner (13). Roles for TNF, NO (14), and the spectrum of UTI caused by uropathogenic E. coli (UPEC) CXCR 2 (15), Tamm-Horsfall protein (16, 17), and CD44 (18) includes asymptomatic bacteriuria, cystitis, pyelonephritis, and have been shown in models of human UPEC UTI, although, in urosepsis. UPEC expresses multiple adhesins and virulence fac- most cases, the functions of these host factors at the cellular and tors that provoke inflammation and enable bacterial colonization molecular levels remain unclear. UPEC induces host cell apoptosis of the bladder as the first step in UTI pathogenesis (2–5). UPEC in cystitis patients (19–21) and macrophage inflammatory peptide may also suppress innate immune responses (6–8). 2 that is induced in response to neutrophils attracted to the bladder The immediate steps that form the host response to UPEC in the to counteract colonization by the bacterium (6). Specific UPEC bladder and mediate the pathogenesis of UPEC UTI in patients are adhesins, including type 1 and P fimbriae, trigger some of these *School of Medical Sciences, Centre for Medicine and Oral Health, Griffith Univer- The sequences presented in this article have been submitted to the Gene Expression sity Gold Coast Campus, Queensland 4222, Australia; †Department of Biostatistics, Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE26509 University of Alabama at Birmingham, Birmingham, AL 35294; ‡Department of and GSE33210. Genetics, University of Alabama at Birmingham, Birmingham, AL 35294; xCentre Address correspondence and reprint requests to Dr. Glen C. Ulett, School of Medical for Microscopy and Microanalysis, University of Queensland, Brisbane, Queensland { Sciences, Centre for Medicine and Oral Health, Griffith University, Gold Coast 4072, Australia; School of Chemistry and Molecular Biosciences, University of ‖ Campus, QLD 4222, Australia. E-mail address: g.ulett@griffith.edu.au Queensland, Brisbane, Queensland 4072, Australia; Pathology Queensland, Gold Coast Hospital, Southport, Queensland 4215, Australia; #School of Obstetrics and The online version of this article contains supplemental material. Gynaecology, University of Adelaide, Adelaide, South Australia 5055, Australia; Abbreviations used in this article: CT, cycle threshold; GO, gene ontology; IHC, **Institute for Molecular Biosciences, University of Queensland, Brisbane, Queens- immunohistochemistry; IPA, ingenuity pathway analysis; KEGG, Kyoto Encyclope- land 4072, Australia; and ††Heflin Center for Human Genetics, University of Ala- dia of Genes and Genomes; qRT-PCR, quantitative RT-PCR; RT, room temperature; bama at Birmingham, Birmingham, AL 35294 SEM, scanning electron microscope; UPEC, uropathogenic Escherichia coli; UTI, 1B.L.D. and A.J.C. contributed equally to this work. urinary tract infection; WT, wild-type. Received for publication May 2, 2011. Accepted for publication November 15, 2011. Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 This work was supported by National Health and Medical Research Council Project Grant 569674, a Griffith University Research Infrastructure Fund grant, and a Gold Coast Hospital Collaborative grant. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101231 782 EARLY BLADDER TRANSCRIPTOME OF UPEC UTI AND IL-10 processes (22, 23). Host responses that arbitrate these very early a T7 promoter sequence. Gene 1.0 ST Mouse Microarrays (Affymetrix) steps in the pathogenesis of UPEC UTI are largely unknown, and were performed, according to the manufacturer’s instructions, in quintu- the overall contributions of innate immune elements beyond pre- plicate for each treatment group using one microarray per bladder. viously identified mechanisms of attachment are not well defined. Quantitative RT-PCR To better understand the nature of innate immunity to UPEC in Quantitative RT-PCR (qRT-PCR) was carried out for groups of genes the bladder, we performed detailed genome-wide gene expression identified as differentially regulated by microarray analysis. Amplification microarray profiling using the murine model of UPEC UTI to of cDNA was performed using a GeneAmp 7700 System (Applied Bio- establish the global host transcriptome of mouse bladder to UPEC systems). Target genes were amplified using thermal cycling conditions cystitis. The overall results of the transcriptome were interrogated previously described (13, 31). Primer sequences are listed in Supplemental Table I. GAPDH and b-actin were used as reference genes. Separate using several cutting-edge bioinformatics platforms and reveal reactions were carried out to ensure that the efficiency of amplification of collections of innate transcriptional networks not previously rec- the reference gene was approximately equal to that of the target gene. ognized in bacterial UTI. Systems-level bioinformatic analyses