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• W2000707384 abstract "In response to enteric pathogens, the inflamed intestine produces antimicrobial proteins, a process mediated by the cytokines IL-17 and IL-22. Salmonella enterica serotype Typhimurium thrives in the inflamed intestinal environment, suggesting that the pathogen is resistant to antimicrobials it encounters in the intestinal lumen. However, the identity of these antimicrobials and corresponding bacterial resistance mechanisms remain unknown. Here, we report that enteric infection of rhesus macaques and mice with S. Typhimurium resulted in marked Il-17- and IL-22-dependent intestinal epithelial induction and luminal accumulation of lipocalin-2, an antimicrobial protein that prevents bacterial iron acquisition. Resistance to lipocalin-2, mediated by the iroBCDE iroN locus, conferred a competitive advantage to the bacterium in colonizing the inflamed intestine of wild-type but not of lipocalin-2-deficient mice. Thus, resistance to lipocalin-2 defines a specific adaptation of S. Typhimurium for growth in the inflamed intestine. In response to enteric pathogens, the inflamed intestine produces antimicrobial proteins, a process mediated by the cytokines IL-17 and IL-22. Salmonella enterica serotype Typhimurium thrives in the inflamed intestinal environment, suggesting that the pathogen is resistant to antimicrobials it encounters in the intestinal lumen. However, the identity of these antimicrobials and corresponding bacterial resistance mechanisms remain unknown. Here, we report that enteric infection of rhesus macaques and mice with S. Typhimurium resulted in marked Il-17- and IL-22-dependent intestinal epithelial induction and luminal accumulation of lipocalin-2, an antimicrobial protein that prevents bacterial iron acquisition. Resistance to lipocalin-2, mediated by the iroBCDE iroN locus, conferred a competitive advantage to the bacterium in colonizing the inflamed intestine of wild-type but not of lipocalin-2-deficient mice. Thus, resistance to lipocalin-2 defines a specific adaptation of S. Typhimurium for growth in the inflamed intestine. Salmonella enterica serotype Typhimurium (S. Typhimurium) causes a localized gastroenteritis in humans, characterized by acute intestinal inflammation and diarrhea. The pathogen encodes two type III secretion systems (T3SS-1 and T3SS-2) that are important for eliciting intestinal inflammation (Hapfelmeier et al., 2005Hapfelmeier S. Stecher B. Barthel M. Kremer M. Muller A.J. Heikenwalder M. Stallmach T. Hensel M. Pfeffer K. Akira S. et al.The Salmonella pathogenicity island (SPI)-2 and SPI-1 type III secretion systems allow Salmonella serovar typhimurium to trigger colitis via MyD88-dependent and MyD88-independent mechanisms.J. Immunol. 2005; 174: 1675-1685PubMed Google Scholar, Tsolis et al., 1999Tsolis R.M. Adams L.G. Ficht T.A. Bäumler A.J. Contribution of Salmonella typhimurium virulence factors to diarrheal disease in calves.Infect. Immun. 1999; 67: 4879-4885Crossref PubMed Google Scholar). The initiation of inflammatory responses in tissue requires direct contact between bacteria and host cells, including epithelial cells, macrophages, and dendritic cells. Macrophages and dendritic cells infected with S. Typhimurium are a source of cytokines, including interleukin (IL)-23 and IL-18, which help to amplify responses in tissue (Godinez et al., 2009Godinez I. Raffatellu M. Chu H. Paixão T.A. Haneda T. Santos R.L. Bevins C.L. Tsolis R.M. Bäumler A.J. IL-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine.Infect. Immun. 2009; 77: 387-398Crossref PubMed Scopus (115) Google Scholar, Srinivasan et al., 2007Srinivasan A. Salazar-Gonzalez R.M. Jarcho M. Sandau M.M. Lefrancois L. McSorley S.J. Innate immune activation of CD4 T cells in salmonella-infected mice is dependent on IL-18.J. Immunol. 2007; 178: 6342-6349PubMed Google Scholar). For example, IL-23 stimulates T cells in the intestinal mucosa to produce IL-17 and IL-22 (Godinez et al., 2009Godinez I. Raffatellu M. Chu H. Paixão T.A. Haneda T. Santos R.L. Bevins C.L. Tsolis R.M. Bäumler A.J. IL-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine.Infect. Immun. 2009; 77: 387-398Crossref PubMed Scopus (115) Google Scholar), two cytokines whose expression is among the ones most prominently induced during S. Typhimurium infection (Godinez et al., 2008Godinez I. Haneda T. Raffatellu M. George M.D. Paixão T.A. Rolán H.G. Santos R.L. Dandekar S. Tsolis R.M. Bäumler A.J. T cells help to amplify inflammatory responses induced by Salmonella enterica serotype Typhimurium in the intestinal mucosa.Infect. Immun. 2008; 76: 2008-2017Crossref PubMed Scopus (116) Google Scholar, Raffatellu et al., 2008Raffatellu M. Santos R.L. Verhoeven D.E. George M.D. Wilson R.P. Winter S.E. Godinez I. Sankaran S. Paixao T.A. Gordon M.A. et al.Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.Nat. Med. 2008; 14: 421-428Crossref PubMed Scopus (434) Google Scholar). The cytokine storm ensuing from the amplification of inflammatory responses in tissue results in the activation of antimicrobial responses in the intestinal mucosa. One important component of the antimicrobial response induced by the IL-23/IL-17 axis is the recruitment of neutrophils (Godinez et al., 2008Godinez I. Haneda T. Raffatellu M. George M.D. Paixão T.A. Rolán H.G. Santos R.L. Dandekar S. Tsolis R.M. Bäumler A.J. T cells help to amplify inflammatory responses induced by Salmonella enterica serotype Typhimurium in the intestinal mucosa.Infect. Immun. 2008; 76: 2008-2017Crossref PubMed Scopus (116) Google Scholar, Godinez et al., 2009Godinez I. Raffatellu M. Chu H. Paixão T.A. Haneda T. Santos R.L. Bevins C.L. Tsolis R.M. Bäumler A.J. IL-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine.Infect. Immun. 2009; 77: 387-398Crossref PubMed Scopus (115) Google Scholar, Raffatellu et al., 2008Raffatellu M. Santos R.L. Verhoeven D.E. George M.D. Wilson R.P. Winter S.E. Godinez I. Sankaran S. Paixao T.A. Gordon M.A. et al.Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.Nat. Med. 2008; 14: 421-428Crossref PubMed Scopus (434) Google Scholar). The extravasation of neutrophils into the mucosa provides a formidable antibacterial defense to prevent S. Typhimurium dissemination. Support for this notion comes from clinical data, showing that neutropenia is a risk factor for bacteremia with nontyphoidal Salmonella serotypes (Noriega et al., 1994Noriega L.M. Van der Auwera P. Daneau D. Meunier F. Aoun M. Salmonella infections in a cancer center.Support. Care Cancer. 1994; 2: 116-122Crossref PubMed Scopus (24) Google Scholar). Thus, S. Typhimurium appears to be susceptible to this arm of the host defense. A second component of the antimicrobial response is the production in the intestinal mucosa of antimicrobial proteins (Godinez et al., 2009Godinez I. Raffatellu M. Chu H. Paixão T.A. Haneda T. Santos R.L. Bevins C.L. Tsolis R.M. Bäumler A.J. IL-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine.Infect. Immun. 2009; 77: 387-398Crossref PubMed Scopus (115) Google Scholar, Raffatellu et al., 2008Raffatellu M. Santos R.L. Verhoeven D.E. George M.D. Wilson R.P. Winter S.E. Godinez I. Sankaran S. Paixao T.A. Gordon M.A. et al.Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.Nat. Med. 2008; 14: 421-428Crossref PubMed Scopus (434) Google Scholar, Zheng et al., 2008Zheng Y. Valdez P.A. Danilenko D.M. Hu Y. Sa S.M. Gong Q. Abbas A.R. Modrusan Z. Ghilardi N. de Sauvage F.J. et al.Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens.Nat. Med. 2008; 14: 282-289Crossref PubMed Scopus (1321) Google Scholar), whose release into the intestinal lumen may be responsible for the dramatic changes in the microbiota observed during inflammation (Lupp et al., 2007Lupp C. Robertson M.L. Wickham M.E. Sekirov I. Champion O.L. Gaynor E.C. Finlay B.B. Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae.Cell Host Microbe. 2007; 2: 119-129Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, Stecher et al., 2007Stecher B. Robbiani R. Walker A.W. Westendorf A.M. Barthel M. Kremer M. Chaffron S. Macpherson A.J. Buer J. Parkhill J. et al.Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota.PLoS Biol. 2007; 5: 2177-2189Crossref PubMed Scopus (686) Google Scholar). The cytokines IL-17 and IL-22 are required for the production in the inflamed intestine of antimicrobials (Godinez et al., 2009Godinez I. Raffatellu M. Chu H. Paixão T.A. Haneda T. Santos R.L. Bevins C.L. Tsolis R.M. Bäumler A.J. IL-23 orchestrates mucosal responses to Salmonella enterica serotype Typhimurium in the intestine.Infect. Immun. 2009; 77: 387-398Crossref PubMed Scopus (115) Google Scholar, Raffatellu et al., 2008Raffatellu M. Santos R.L. Verhoeven D.E. George M.D. Wilson R.P. Winter S.E. Godinez I. Sankaran S. Paixao T.A. Gordon M.A. et al.Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.Nat. Med. 2008; 14: 421-428Crossref PubMed Scopus (434) Google Scholar), including lipocalin-2, a protein that inhibits bacterial growth by interfering with the acquisition of an essential nutrient, iron (Berger et al., 2006Berger T. Togawa A. Duncan G.S. Elia A.J. You-Ten A. Wakeham A. Fong H.E. Cheung C.C. Mak T.W. Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury.Proc. Natl. Acad. Sci. USA. 2006; 103: 1834-1839Crossref PubMed Scopus (333) Google Scholar, Flo et al., 2004Flo T.H. Smith K.D. Sato S. Rodriguez D.J. Holmes M.A. Strong R.K. Akira S. Aderem A. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.Nature. 2004; 432: 917-921Crossref PubMed Scopus (1214) Google Scholar, Goetz et al., 2002Goetz D.H. Holmes M.A. Borregaard N. Bluhm M.E. Raymond K.N. Strong R.K. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.Mol. Cell. 2002; 10: 1033-1043Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). However, the cells targeted by IL-17 and IL-22 in vivo to induce production of these antimicrobials have not been identified. S. Typhimurium appears to be resistant against this arm of the host defense, because its numbers in the intestinal lumen increase dramatically during inflammation, resulting in increased fecal oral transmission (Barman et al., 2008Barman M. Unold D. Shifley K. Amir E. Hung K. Bos N. Salzman N. Enteric salmonellosis disrupts the microbial ecology of the murine gastrointestinal tract.Infect. Immun. 2008; 76: 907-915Crossref PubMed Scopus (291) Google Scholar, Lawley et al., 2008Lawley T.D. Bouley D.M. Hoy Y.E. Gerke C. Relman D.A. Monack D.M. Host transmission of Salmonella enterica serovar Typhimurium is controlled by virulence factors and indigenous intestinal microbiota.Infect. Immun. 2008; 76: 403-416Crossref PubMed Scopus (191) Google Scholar, Stecher et al., 2007Stecher B. Robbiani R. Walker A.W. Westendorf A.M. Barthel M. Kremer M. Chaffron S. Macpherson A.J. Buer J. Parkhill J. et al.Salmonella enterica serovar typhimurium exploits inflammation to compete with the intestinal microbiota.PLoS Biol. 2007; 5: 2177-2189Crossref PubMed Scopus (686) Google Scholar). We thus hypothesized that, in addition to virulence mechanisms to induce intestinal inflammation, S. Typhimurium should possess virulence mechanisms to survive the ensuing antimicrobial responses in the intestinal lumen. While the virulence factors involved in triggering intestinal inflammation, such as T3SS-1 and T3SS-2, are well characterized, the identity of the antimicrobial responses potentially encountered in the lumen of the inflamed intestine and the identity of the corresponding bacterial resistance genes are currently unknown. To gain further insights into this “inflammation-adapted” pathogenic lifestyle of S. Typhimurium, we set out to identify antimicrobials released by intestinal epithelial cells into the intestinal lumen during inflammation. After identifying one such antimicrobial, we determined whether the corresponding S. Typhimurium resistance genes conferred an advantage during bacterial growth in the inflamed intestine. To identify antimicrobial responses elicited by IL-17 and IL-22 in the intestinal epithelium, human colonic cancer epithelial (T84) cells were polarized and basolaterally stimulated with IL-17 or IL-22. Expression of CCL20, a chemokine gene known to be induced by IL-17 in lung epithelial cells (Kao et al., 2005Kao C.Y. Huang F. Chen Y. Thai P. Wachi S. Kim C. Tam L. Wu R. Up-regulation of CC chemokine ligand 20 expression in human airway epithelium by IL-17 through a JAK-independent but MEK/NF-kappaB-dependent signaling pathway.J. Immunol. 2005; 175: 6676-6685PubMed Google Scholar), was found to be induced optimally 4 hr after stimulation with IL-17 or IL-22 (data not shown). To determine the complete molecular profile of epithelial responses, RNA from four replicates was isolated 4 hr after stimulation of T84 cells with IL-22 or IL-17, and global gene expression profiles were elucidated by microarray analysis. In general, IL-22 produced more robust responses in T84 cells than IL-17 (Figure S1). For example, the number of genes upregulated 2-fold or more was 849 for IL-22 treatment compared to only 62 for IL-17 treatment. A table containing a complete listing of genes with significantly (p < 0.05) altered expression in T84 epithelial cells after stimulation with IL-17 or IL-22 can be accessed at the Gene Expression Omnibus database (GSE11345). To identify those antimicrobial responses induced in vitro in T84 cells that are also observed in the inflamed intestine in vivo, we performed meta analysis of the overlap in upregulated gene transcription (>2-fold) between T84 cells stimulated with IL-22 and the ileal mucosa of rhesus macaques infected with S. Typhimurium (Figure 1). The in vivo gene expression profile analyzed in this study had previously been generated using an adult healthy rhesus macaque that had undergone ligated ileal loop surgery (Raffatellu et al., 2008Raffatellu M. Santos R.L. Verhoeven D.E. George M.D. Wilson R.P. Winter S.E. Godinez I. Sankaran S. Paixao T.A. Gordon M.A. et al.Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.Nat. Med. 2008; 14: 421-428Crossref PubMed Scopus (434) Google Scholar). Two ligated ileal loops had been inoculated with either sterile culture medium (mock infection) or with S. Typhimurium, and 5 hr later, both loops had been surgically removed to isolate RNA for gene expression profiling (Raffatellu et al., 2008Raffatellu M. Santos R.L. Verhoeven D.E. George M.D. Wilson R.P. Winter S.E. Godinez I. Sankaran S. Paixao T.A. Gordon M.A. et al.Simian immunodeficiency virus-induced mucosal interleukin-17 deficiency promotes Salmonella dissemination from the gut.Nat. Med. 2008; 14: 421-428Crossref PubMed Scopus (434) Google Scholar). Meta analysis revealed a substantial overlap between transcripts whose levels were increased in the ileal mucosa of a rhesus macaque during S. Typhimurium infection and those whose expression was induced in human T84 epithelial cells upon stimulation with IL-22 (302 transcripts) (Figure 1A). A functional category that was statistically overrepresented included genes involved in defense responses, encoding antimicrobials (LCN2, NOS2, and MUC4) and cytokines (CCL20) whose expression was increased most dramatically both in vitro in IL-22-treated T84 cells and during S. Typhimurium infection in vivo (Figure 1A). These data supported the hypothesis that IL-22 induced expression of antimicrobials in epithelial cells during S. Typhimurium infection in the intestinal mucosa. Transcripts encoding the antimicrobial lipocalin-2 were among the ones most prominently increased both in vitro after IL-22 stimulation and in vivo after S. Typhimurium infection (Figure 1A). Thus, after validating results from gene expression profiling for selected genes by real-time PCR (Figure S2), we sought to further investigate whether intestinal epithelial cells may be a source of lipocalin-2 during S. Typhimurium infection. First, we verified that lipocalin-2 is an antimicrobial produced by intestinal epithelial cells. Quantification of changes in gene expression by real-time PCR confirmed that stimulation of T84 cells with IL-22 resulted in an induction of LCN2 expression (7.9-fold) (Figure 1B). Stimulation with IL-17 alone did not alter LCN2 transcription (1.3-fold change), but further increased LCN2 transcription when added in combination with IL-22 (17-fold). We next determined protein secretion by ELISA. Nonstimulated T84 cells secreted a small amount of lipocalin-2 apically (approximately 2 ng/ml), while the amount detected in the basolateral compartment was negligible. Stimulation of T84 cells with IL-22 but not with IL-17 resulted in apical and basolateral secretion of lipocalin-2 at levels above those detected with nonstimulated controls. Stimulation with both IL-17 and IL-22 resulted in a significant (p < 0.05) further increase of lipocalin-2 secretion both apically and basolaterally, reaching substantial concentrations (approximately 20 ng/ml) in both compartments (Figure 1C). In contrast, CCL20 was secreted exclusively into the basolateral compartment after cytokine stimulation of T84 cells (Figure S2). Although IL-17 by itself did not markedly alter LCN2 transcription, our data suggested that this cytokine synergized with IL-22 in controlling production of lipocalin-2 in the intestinal epithelium. Because lipocalin-2 was highly induced during S. Typhimurium infection in vivo, we next investigated whether expression of LCN2 was induced by cytokines (i.e., IL-17 and IL-22) present in the inflamed intestine, by direct contact of bacteria with epithelial cells, or by a combination of both mechanisms. Basolateral stimulation of T84 cells with purified flagellin or infection of T84 cells with the S. Typhimurium wild-type strain, an invasion-deficient strain (invA mutant), or a nonflagellated strain (fliC fljB mutant) did not result in marked induction in LCN2 expression (Figure 1D). Treatment of T84 cells with IFNγ did not increase LCN2 transcription. The marked increase in LCN2 transcription observed after basolateral stimulation of T84 cells with IL-17 and IL-22 was modestly increased by basolateral stimulation with purified flagellin (p = 0.03) or by S. Typhimurium infection (p = 0.004). These data suggested that induction of LCN2 transcription was mediated largely through stimulation with IL-17 and IL-22. Although direct interaction between bacteria and host cells was not sufficient to induce LCN2 transcription, these interactions could further increase LCN2 mRNA levels induced by treatment with IL-17 and IL-22. To investigate the biological significance of the IL-17/IL-22-mediated lipocalin-2 production by intestinal epithelial cells, we determined whether the protein was produced at levels that exhibited an antimicrobial activity. Lipocalin-2 specifically binds enterochelin, a small molecular weight iron chelator (siderophore) produced by many members of the Enterobacteriaceae. As a result, lipocalin-2 exhibits a bacteriostatic effect on bacteria that depend exclusively on enterochelin to acquire iron, an essential trace element, during growth in the host (Berger et al., 2006Berger T. Togawa A. Duncan G.S. Elia A.J. You-Ten A. Wakeham A. Fong H.E. Cheung C.C. Mak T.W. Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemia-reperfusion injury.Proc. Natl. Acad. Sci. USA. 2006; 103: 1834-1839Crossref PubMed Scopus (333) Google Scholar, Flo et al., 2004Flo T.H. Smith K.D. Sato S. Rodriguez D.J. Holmes M.A. Strong R.K. Akira S. Aderem A. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.Nature. 2004; 432: 917-921Crossref PubMed Scopus (1214) Google Scholar, Goetz et al., 2002Goetz D.H. Holmes M.A. Borregaard N. Bluhm M.E. Raymond K.N. Strong R.K. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.Mol. Cell. 2002; 10: 1033-1043Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar). The iroBCDE iroN gene cluster of S. Typhimurium encodes proteins involved in the biosynthesis and uptake of salmochelin, a glycosylated derivative of enterochelin (Bäumler et al., 1996Bäumler A.J. Tsolis R.M. van der Velden A.W. Stojiljkovic I. Anic S. Heffron F. Identification of a new iron regulated locus of Salmonella typhi.Gene. 1996; 183: 207-213Crossref PubMed Scopus (133) Google Scholar, Bäumler et al., 1998Bäumler A.J. Norris T.L. Lasco T. Voight W. Reissbrodt R. Rabsch W. Heffron F. IroN, a novel outer membrane siderophore receptor characteristic of Salmonella enterica.J. Bacteriol. 1998; 180: 1446-1453PubMed Google Scholar, Bister et al., 2004Bister B. Bischoff D. Nicholson G.J. Valdebenito M. Schneider K. Winkelmann G. Hantke K. Sussmuth R.D. The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica.Biometals. 2004; 17: 471-481Crossref PubMed Scopus (125) Google Scholar, Hantke et al., 2003Hantke K. Nicholson G. Rabsch W. Winkelmann G. Salmochelins, siderophores of Salmonella enterica and uropathogenic Escherichia coli strains, are recognized by the outer membrane receptor IroN.Proc. Natl. Acad. Sci. USA. 2003; 100: 3677-3682Crossref PubMed Scopus (253) Google Scholar, Rabsch et al., 1999Rabsch W. Voigt W. Reissbrodt R. Tsolis R.M. Bäumler A.J. Salmonella typhimurium IroN and FepA proteins mediate uptake of enterobactin but differ in their specificity for other siderophores.J. Bacteriol. 1999; 181: 3610-3612PubMed Google Scholar, Zhu et al., 2005Zhu M. Valdebenito M. Winkelmann G. Hantke K. Functions of the siderophore esterases IroD and IroE in iron-salmochelin utilization.Microbiology. 2005; 151: 2363-2372Crossref PubMed Scopus (77) Google Scholar). Salmochelin is not bound by lipocalin-2, and its production therefore renders S. Typhimurium lipocalin-2 resistant. However, in the absence of a functional iroBCDE iroN gene cluster, S. Typhimurium produces enterochelin as its sole siderophore and is lipocalin-2 sensitive (Crouch et al., 2008Crouch M.L. Castor M. Karlinsey J.E. Kalhorn T. Fang F.C. Biosynthesis and IroC-dependent export of the siderophore salmochelin are essential for virulence of Salmonella enterica serovar Typhimurium.Mol. Microbiol. 2008; 67: 971-983Crossref PubMed Scopus (127) Google Scholar, Fischbach et al., 2006Fischbach M.A. Lin H. Zhou L. Yu Y. Abergel R.J. Liu D.R. Raymond K.N. Wanner B.L. Strong R.K. Walsh C.T. et al.The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2.Proc. Natl. Acad. Sci. USA. 2006; 103: 16502-16507Crossref PubMed Scopus (215) Google Scholar). Growth of the S. Typhimurium wild-type (lipocalin-2 resistant) and a S. Typhimurium iroBC mutant (lipocalin-2 sensitive) were compared in medium collected either from nonstimulated T84 cells (control) or from T84 cells after stimulation with IL-17 and IL-22 (Figure 2). No differences between the S. Typhimurium wild-type and the iroBC mutant were observed in control media; however, growth of the iroBC mutant was significantly (p < 0.05) reduced in media in which lipocalin-2 production had been elicited by stimulation with IL-17 and IL-22 (Figure 2A). These data suggested that the amount of lipocalin-2 produced by intestinal epithelial cells upon IL-17/IL-22 stimulation was sufficient to reduce growth of the iroBC mutant in vitro. A mutation in iroBC did not impair bacterial growth in rich media (Luria-Bertani broth) (Figure 2B). Tissue culture medium was spiked with lipocalin-2 to confirm that the presence of this antimicrobial was responsible for the growth defect observed with the iroBC mutant. The presence of lipocalin-2 significantly (p < 0.05) reduced growth of the iroBC mutant, while growth of the S. Typhimurium wild-type was not affected (Figure 2C). Complementation of the iroBC mutant with the cloned iroBCDE iroN gene cluster restored resistance to lipocalin-2, as indicated by equal growth in tissue culture medium in the presence or absence of lipocalin-2 (Figure 2C). The lipocalin-2-mediated growth inhibition of the iroBC mutant could be prevented when iron was supplied in the form of ferrioxamine B (iron-desferal), a siderophore that can be internalized by the FoxA outer membrane receptor of S. Typhimurium (Figure 2C). Collectively, these data suggested lipocalin-2 inhibited growth of the iroBC mutant through iron sequestration. To investigate the in vivo relevance of lipocalin-2 production by T84 cells, we examined the contribution of epithelial cells expressing this antimicrobial protein during S. Typhimurium infection in a relevant host. To this end, we first localized the LCN2 transcripts in the ileal mucosa of rhesus macaques using in situ hybridization (Figure 3). LCN2 transcripts localized to epithelial cells lining the villi and crypts, with intense staining in tissue from S. Typhimurium-infected ileal loops with a LCN2-specific cDNA probe (Figure 3A). These data suggested that LCN2 was mainly expressed by intestinal epithelial cells during S. Typhimurium infection. Next, we determined the absolute number of LCN2 transcripts using quantitative real-time PCR. For each rhesus macaque (n = 4), a tissue sample from a mock-infected loop and from a S. Typhimurium-infected loop were processed for mRNA isolation. Transcripts encoding β-actin (ACT1) were detected at similar levels in both treatment groups, averaging approximately 20,000 copies/ng RNA in samples from mock-infected and S. Typhimurium-infected loops (Figure 3B). Based on theoretical assumptions about the RNA content of 106 cells (5–8 μg) (Ausubel et al., 1994Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current protocols in molecular biology. John Wiley & Sons, Media, PA1994Google Scholar), these data would predict that ACT1 was present on average in 100–160 copies/cell. Our data were thus within the range of previous measurements by real-time PCR, indicating that human leukocytes contain between 84 and 491 copies of ACT1 per cell (Lupberger et al., 2002Lupberger J. Kreuzer K.A. Baskaynak G. Peters U.R. le Coutre P. Schmidt C.A. Quantitative analysis of beta-actin, beta-2-microglobulin and porphobilinogen deaminase mRNA and their comparison as control transcripts for RT-PCR.Mol. Cell. Probes. 2002; 16: 25-30Crossref PubMed Scopus (100) Google Scholar). LCN2 transcripts averaged 1400 copies/ng RNA in samples from mock-infected loops, but the copy number was significantly (p = 0.03) increased after S. Typhimurium infection, averaging 48,000 copies/ng RNA. Collectively, these data suggested that during S. Typhimurium infection, the LCN2 gene was expressed at a very high level in the epithelium of the ileal mucosa. We next studied the production of lipocalin-2 in situ using immunohistochemistry (Figures 3C–3E). Rabbit polyclonal antibody derived from recombinant rhesus lipocalin-2 was generated. Immunostaining of formalin-fixed tissue revealed that after S. Typhimurium infection, lipocalin-2 was abundant in intestinal epithelial cells and in the underlying lamina propria. These data suggested that lipocalin-2 protein is produced and secreted in tissue during the course of an infection with S. Typhimurium. Next, we wanted to detect the amount of lipocalin-2 secreted into the intestinal lumen in vivo. An ELISA was developed to detect the amount of lipocalin-2 secreted into the luminal fluid previously collected from ligated ileal loops of rhesus macaques 8 hr after inoculation with S. Typhimurium (n = 4) or sterile culture medium (n = 4). Luminal contents of S. Typhimurium-infected loops contained on average 419 ng of lipocalin-2, which was significantly higher (p = 0.007) than the 64 ng lipocalin-2 detected in luminal contents of mock-infected loops (Figure 3F). Collectively, these data identified lipocalin-2 production as an antimicrobial response encountered in the lumen of the inflamed intestine. After identifying lipocalin-2 as an antimicrobial response encountered in the intestinal lumen, we wanted to determine whether the corresponding S. Typhimurium resistance genes would confer an advantage during bacterial growth in the inflamed intestine. To establish cause and effect using lipocalin-2-deficient animals, we used the streptomycin-pretreated mouse model for these studies. S. Typhimurium infection of streptomycin-pretreated mice results in acute inflammation of the cecal mucosa (Barthel et al., 2003Barthel M. Hapfelmeier S. Quintanilla-Martinez L. Kremer M. Rohde M. Hogardt M. Pfeffer K. Russmann H. Hardt W.D. Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.Infect. Immun. 2003; 71: 2839-2858Crossref PubMed Scopus (633) Google Scholar), which is accompanied by overgrowth of S. Typhimurium in the lumen of the large intestine (Que et al., 1986Que J.U. Casey S.W. Hentges D.J. Factors responsible for increased susceptibility of mice to intestinal colonization after treatment with streptomycin.Infect. Immun. 1986; 53: 116-123PubMed Google Scholar, Stecher et al., 2007Stecher B. Robbiani R. Walker A.W. Westendorf A.M. Barthel M. Kremer M. Chaffron S. Macp" @default.
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