Human epithelial-speci¢c response to pathogenic Campylobacter jejuni Erica S. Rinella1,2, Chevonne D. Eversley1,2, Ian M. Carroll1, Jason M. Andrus1,3, David W. Threadgill1,2,4 & Deborah S. Threadgill1 1
Department of Genetics, University of North Carolina, Chapel Hill, NC, USA; 2Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, USA; 3SPIRE Postdoctoral Fellowship Program, University of North Carolina, Chapel Hill, NC, USA; and 4Center for Gastrointestinal Biology and Disease, University of North Carolina, Chapel Hill, NC, USA
Correspondence: Deborah S. Threadgill, Department of Genetics, CB#7264 University of North Carolina, Chapel Hill NC 27599, USA. Tel.: 1919 843 4680; fax: 1919 966 3292; e-mail:
[email protected] Present address: Jason M. Andrus, Department of Biology, Augusta State University, Augusta, GA 30904, USA. Received 31 March 2006; revised 10 July 2006; accepted 10 July 2006. First published online 4 August 2006. DOI:10.1111/j.1574-6968.2006.00396.x Editor: Arnoud van Vliet Keywords Campylobacter jejuni ; Lactobacillus reuteri ; intestinal epithelial cells.
Abstract The gastrointestinal epithelia of mammals are tolerant of their resident gut microbiota but are usually highly responsive to entero-pathogens; the host-specific responses have not been well characterized. To this end, the transcriptional responses of cultured human (Caco-2) and murine (CT-26) colonic epithelial cells were compared after exposure with the microfloral bacterium Lactobacillus reuteri or the human gastrointestinal pathogen Campylobacter jejuni. When in bacterial broth, both species elicit a stronger differential gene expression response in human colonic cells compared with mouse colonic cells. However, when these data are adjusted to remove bacterial broth effects, only human colonic epithelia exposed to C. jejuni show altered gene expression, suggesting that the human pathogen C. jejuni induces a host-specific response. The genes with altered expression are involved in growth, transcription, and steroid biosynthesis. Interestingly, human genes involved in cell polarity and water transport were significantly changed in response to C. jejuni exposure, linking infection with gastrointestinal disease. This study demonstrates that mouse and human colonic epithelia remain relatively unresponsive to commensal bacterial challenge, while the human pathogen C. jejuni elicits a host-specific response.
Introduction The gastrointestinal (GI) tract of animals is host to a complex community of bacteria, archaea, and eukarya. The natural gut flora in the human colon can reach densities of up to 1011–1012 microbes mL 1 of luminal contents (Cummings et al., 1989) and contains greater than 500 species. With such a large and diverse gastrointestinal bacterial population, the human host has had to evolve mechanisms to tolerate and coexist with its normal flora. This has resulted in a symbiotic relationship as the nonpathogenic commensular bacteria, or microbiota, of the gastrointestinal tract provide a number of benefits to the host including immune stimulation (Haller et al., 2000), defense against pathogens (Bernet-Camard et al., 1997), and gastrointestinal mucosal barrier stability (Gotteland et al., 2001). Indeed, a deregulated mucosal immune response that is intolerant of the normal gut flora is a strong etiological factor for gastrointestinal diseases such as ulcerative colitis (UC) and Crohn’s disease (CD) (Yan & Polk, 2004). 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Because of the symbiotic relationship with the host, consumption of probiotics or ‘live microorganisms which when administered in adequate amounts confer a beneficial physiological effect on the host’ (Reid et al., 2003) is believed to exert beneficial effects either directly, by stimulating the host immune system (Tannock et al., 2000; Peran et al., 2005; Vinderola et al., 2005; O’Mahony et al., 2006), or indirectly, by altering the composition of the gut flora (Heilig et al., 2002; Garrido et al., 2005). The positive effects of the Lactobacillus sp., members of the lactic acid bacteria, have received particular attention as potential probiotics. Although these organisms are present at relatively low levels in the human colon, recent studies have demonstrated their ability to modulate inflammation in a murine colitis model [Lactobacillus reuteri (Madsen et al., 1999), a combination of Lactobacillus reuteri and Lactobacillus paracasei (Pena et al., 2005), Lactobacillus plantarum (Pavan et al., 2003) and Lactobacillus salavarius (O’Mahony et al., 2001; McCarthy et al., 2003)] and help prevent enteric infections [Lactobacillus casei (Alvarez et al., 2001)] when administered in FEMS Microbiol Lett 262 (2006) 236–243
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sufficient quantities. Despite the wealth of research related to probiotic activity, the differential responses elicited specifically in the host epithelia by symbiotic or probiotic gut flora, like Lactobacillus sp., as opposed to that induced by entero-pathogens, like Campylobacter jejuni, remain unclear. Campylobacter jejuni is increasingly associated with episodes of gastrointestinal illness in developed countries. Although relatively little is known about the pathogenic events required for illness to occur, C. jejuni strains differ in their ability to adhere to and invade intestinal epithelial cells and it has been suggested that this is important to the pathogenic process (Harvey et al., 1999). While studies have recently been performed examining human cell responses to C. jejuni (Hu et al., 2006a, b), little is known about genomewide, host-specific responses, in particular human vs. animal models. The differential immune responses of the gut epithelium to both commensal and pathogenic bacteria have been described (Vinderola et al., 2005). However, to date, no direct comparisons have been reported describing genomewide responses of human epithelial cells to members of the indigenous gut flora and pathogenic C. jejuni. Moreover, little is known about the unique response of human colonic epithelium compared with other mammalian species, which are not susceptible to C. jejuni pathogenicity. The present study describes the differential responses of human and mouse colonic epithelial cells to the natural gut organism L. reuteri and the human pathogen C. jejuni, using DNA microarray analysis.
Methods Bacterial strains and mammalian cell lines Campylobacter jejuni NCTC11168 (isolate used is also known as C. jejuni 78-14 from the lab collection of Dr Martin Blaser, NYU) were grown in Mueller–Hinton (MH) broth (Difco Laboratories) under a hydrogen-containing microaerophilic atmosphere: 83% nitrogen, 8% oxygen, 4% hydrogen, and 5% carbon dioxide at 37 1C with shaking at 250 r.p.m. L. reuteri 4020 were grown shaking at 200 r.p.m. in MRS broth (Difco Laboratories) under anaerobic conditions at 37 1C. Human Caco-2 cells and mouse CT-26 cells (provided by Dr Robert Coffey, Vanderbilt University) were cultured in DMEM (Gibco-Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 1% L-glutamine (Gibco-Invitrogen), and 0.5% penicillin/ streptomycin (Gibco-Invitrogen), and grown at 37 1C in a 5% CO2 humidified incubator; no antibiotic was used during the exposure assay. FEMS Microbiol Lett 262 (2006) 236–243
Exposure assay Human and mouse cells were grown by seeding 106 cells onto 100 mm culture dishes 24 h before bacterial exposure. Each bacterial strain was grown to the mid-log phase (108 CFU mL 1), and 1 mL of culture was removed for addition to mammalian cells. Each cell line was exposed to nine different treatments: untreated, MH broth, MRS broth, C. jejuni NCTC 11168 in MH broth, L. reuteri 4020 in MRS broth, washed C. jejuni NCTC 11 168, washed L. reuteri 4020, spent MH, and spent MRS broth (Fig. 1). For the washed bacteria exposure, each bacterial sample was pelleted at 14 000 r.p.m. for 1 min before removing the supernatant. The pellet was resuspended in 1 mL of DMEM and pelleted again at 14 000 r.p.m. for 1 min before resuspending in 1 mL of DMEM for addition to the cells. The initial broth supernatant from the pelleted bacteria was used for the ‘spent’ broth treatment groups. Three replicates were performed for the untreated and washed bacteria treatment groups, and two replicates were performed for the broth, spent broth, and bacteria/broth treatment groups. After treatment, the cells were maintained at 37 1C in an aerobic atmosphere containing CO2 for 6 h.
Microarray experiments Total RNA was isolated from the exposed cells using Trizol (Invitrogen) according to the manufacturer’s protocol. Isolated RNA was quantified using OD260 nm measurements in a DU 800 spectrophotometer (Beckman Coulter). RNA sample integrity and concentration was verified using the RNA Nano 6000 Chip Assay on a BioAnalyzer 2100 (Agilent). RNA labeling was carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent). Two hundred and fifty nanograms of RNA isolated from cell lines or 250 ng reference RNA was incubated with 0.7 mL T7 promoter primer at 65 1C for 10 min. For the human arrays, a Human Universal Reference RNA (Stratagene) was used, and for the mouse arrays, a total RNA reference pool isolated from C57BL/6J embryos was used. The reactions were incubated on ice for 5 min. cDNA was synthesized using 5 First Strand Buffer, 0.1 M dithiothreitol, dNTP mix, RNase OUT, and MMLV-RT at 40 1C for 120 min and the reaction stopped at 65 1C for 15 min. The reactions were placed on ice for 5 min, tap spun, and 1mM of cyanine 3-CTP or cyanine 5-CTP (Perkin Elmer) was added to the reactions for the reference and cell lines, respectively. Fluorescent cRNA was synthesized using 4 transcription buffer, 0.1 M dithiothreitol, NTP mix, 50% PEG, RNase OUT, inorganic pyrophosphatase, and T7 RNA polymerase at 40 1C for 120 min. The labeled cRNA was purified using an RNeasy Kit (Qiagen). cRNA was quantified using a DU 800 spectrophotometer (Beckman-Coulter), and 1 mg of labeled reference and 1 mg 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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MH or MRS Broth
Untreated
Spent MH or Spent MRS
C. jejuni or L. reuteri
C. jejuni /MH or L. reuteri /MRS
Mammalian cell lines Caco-2 (Human) or CT-26 (Mouse)
Data normalization and filtering
SAM analysis Multiclass
Caco-2 + C. jejuni treatments
Multiclass
Caco-2 + L. reuteri treatments
CT-26 + C. jejuni treatments
CT-26 + L.reuteri treatments
Two-class, unpaired
Control
Treatment
Untreated MH or MRS Spent MH or Spent MRS
C. jejuni or L. reuteri C. jejuni/ MH or L.reuteri / MRS
of labeled sample were combined with 30 mL control target solution, 9 mL 25 fragmentation buffer, and 225 mL 2 hybridization buffer before loading onto hybridization chambers containing the appropriate microarray. The slides were incubated in a Rotisserie hybridization oven at 60 1C for 17 h before the microarrays were washed and scanned using a microarray scanner (Agilent). Human G4110B and mouse G4121A Arrays (Agilent) were used for the Caco-2 and CT-26 cells, respectively. Both array platforms contain 22 K 60-mer probes, representing genes with well documented annotations.
Data analysis Data from the microarrays were extracted using Feature Extraction Software (Agilent) and uploaded into GeneSpring (Agilent). The data were normalized first by Lowess and then centered around the ‘untreated’ samples before filtering using the default minimum expression level of 0.1. Filtered gene lists were exported for statistical analysis using Significance Analysis of Microarrays (SAM) software (Stan2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Fig. 1. Flow chart representing bacterial exposure and microarray analyses.
ford). Lists of differentially expressed genes for each exposure were generated using a multiclass comparison at a false discovery rate (FDR) of 10%. Bacteria-specific responses were also determined in SAM using a two-class unpaired analysis, dividing the five C. jejuni treatments and five L. reuteri treatments for each host into two groups: ‘control’ and ‘treatment’ (Fig. 1). Data from the exposure of the human cell line (Caco-2) to the C. jejuni treatments were further evaluated at an FDR of 5%. Gene Ontology Tree Machine (GOTM) software (http://genereg.ornl.gov/gotm) was used to group the human genes into ontological categories determined to be statistically significant when compared with the entire Agilent 4110B microarray gene list (P o 0.01).
Results Host-specific response to bacterial exposure Exposing mammalian colonic cells to bacteria resulted in a host-specific response as measured by changes in gene FEMS Microbiol Lett 262 (2006) 236–243
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expression. Four separate multiclass analyses were initially performed using SAM at 10% FDR (Fig. 1). When exposed to either pathogenic or probiotic bacteria and/or bacterial broth, human epithelial cells had an overall stronger response compared with that of mouse epithelium (Table 1). Orthology tables, built in GeneSpring, revealed that 13 of 18 mouse genes whose expression was altered in response to C. jejuni, had recognizable human orthologs. Venn diagrams showing overlap of human and mouse genes with altered expression in response to C. jejuni revealed that three genes, out of the 13 mouse genes with human homologs, were common to both hosts; the three were upregulated in both host cell lines and included GDF15 (growth differentiation factor 15), TRIB3 (Tribbles homolog), and DDIT4 (DNAdamage-inducible transcript 4). GDF15 and DDIT4 have roles in cell growth, maintenance, and communication, with GDF15 being part of the TGF-b signaling pathway. DDIT4 and TRIB3 have roles in transcription; TRIB3 is also a negative regulator of NF-kB and can induce apoptosis. No overlap was observed between the two cell lines in response to L. reuteri-related treatments.
Bacteria-specific response by hosts In order to measure the bacteria-specific responses of the two mammalian cell lines, two-class unpaired comparisons were generated in SAM between control (combining the ‘untreated’, ‘broth’, and ‘spent broth’ groups) and bacteria treatment (combining the ‘bacteria’ and ‘bacteria/broth’ groups). The resulting list contained only those genes that were modified in response to the bacteria and not in response to the bacterial broth. Only exposure of the human colonic cells to pathogenic C. jejuni resulted in changes in gene expression; C. jejuni elicited expression changes in 290 human genes.
Human-specific response to pathogenic C. jejuni Exposure of human colonic cells to C. jejuni resulted in 290 differentially expressed genes at a 10% FDR. The 290 genes were grouped by function into 32 categories determined to be statistically significant when compared with the entire Agilent 4110B microarray gene content (P o 0.01). Categories were considered ‘over-represented’ if the gene list of interest contained significantly more genes within particular categories than what would be expected based on a random Table 1. Number of differentially expressed genes at 10% FDR
Human Mouse
L. reuteri treatments
C. jejuni treatments
125 4
1591 18
L. reuteri, Lactobacillus reuteri; C. jejuni, Campylobacter jejuni.
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sample from the reference gene list. Conversely, categories were considered ‘under-represented’ if fewer genes than expected were on the gene list of interest compared with the reference list. The major categories over-represented in the list of differentially expressed genes were cell motility, adhesion, growth, inflammatory response, response to various external stimuli, cytokine activity, and protein binding. No categories were under-represented. A more stringent analysis of the human colonic cell response to pathogenic C. jejuni, by reducing the FDR to 5%, resulted in the detection of 102 differentially expressed genes (Fig. 2). The majority of these genes were upregulated in the treatment group (C. jejuni and C. jejuni/MH) compared with the control group (untreated, MH alone, and spent MH); only six of the 102 genes were downregulated in the treatment group. GOTM was used to organize the 102 genes into 21 gene ontology categories that were significantly over-represented (Fig. 3). The major categories over-represented were cell growth, transcription, and steroid biosynthesis. No categories were significantly under-represented. One biological response that was of particular interest is immune activity as some natural gut flora, as well as pathogenic bacteria, have been shown to elicit responses from the immune system (O’Mahony et al., 2006). Several interleukin and interleukin receptor genes were modified in the human cell response to bacteria and/or broth including IL1Ra, IL3, NFIL3, IL8, IL13, IL17RB, IL17RD, IL18R1, and IL28RA. Three interleukins were upregulated specifically in response to C. jejuni, NFIL3, IL11, and IL17B at 10% FDR (only NFIL3 was on the 5% FDR gene list).
Discussion The use of microarray analysis has revealed a host-specific, differential response of human colonic epithelial cells to challenge by C. jejuni NCTC11168. Although C. jejuni and/ or its broth elicited a much greater response in human cells, both human and mouse cells showed a stronger response to the pathogen than to exposure to microfloral bacteria, possibly suggesting an evolutionarily adapted response of the host to tolerate indigenous bacteria. Importantly, only C. jejuni exposure to human cells elicited a bacteria-specific response, independent of broth treatments; the mouse cells were generally unresponsive to either pathogenic or nonpathogenic bacteria. The lack of response to C. jejuni by mouse colonic cells is of particular interest as there is also a lack of pathogenic response in normal mice to C. jejuni in vivo. Indeed, an adequate mouse model that mimics human C. jejuni infection has yet to be established (Vuckovic et al., 1998); C. jejuni colonizes the mouse gastrointestinal tract but does not cause subsequent gastroenteritis, suggesting a species-specific adaptation of pathogenic response. 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Fig. 2. Human genes differentially expressed at 5% FDR in response to Campylobacter jejuni. The means of each treatment group are shown, with heat map representing genes that are upregulated (red) or downregulated (blue) relative to untreated cells. The genes are ranked based upon their response to C. jejuni alone.
Campylobacter jejuni NCTC11168 is routinely used in invasion studies (Konkel et al., 1992; Carrillo et al., 2004). The advantage of this strain is that its entire genome has been sequenced. Although NCTC11168 is not as virulent as some other C. jejuni strains, it was originally isolated from a patient with gastroenteritis (Skirrow, 1977; Ahmed et al., 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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2002) and this study demonstrates that, compared with the commensal strain L. reuteri, C. jejuni NCTC11168 elicits a highly significant response in human epithelial cells. It has also been demonstrated that invasion is not necessary for such responses as cytokine activation, as both 81-176, a more virulent strain of C. jejuni, and NCTC11168 induce FEMS Microbiol Lett 262 (2006) 236–243
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Fig. 3. Gene ontology categories significantly over-represented in the list of human genes with altered expression in response to Campylobacter jejuni exposure.
Human response to Campylobacter jejuni
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production of IL-1b, IL-6, IL-8, IL-10, IL-12, IFN-g, and TNF-a after a 24 h exposure (Hu et al., 2006a). Although the previous study used dendritic cells and a longer exposure, in the current study IFNG and IL8 are activated (FDR 10%), but the activation of IL-8 is not bacteria-specific. In addition, invasion of human intestinal cells with C. jejuni 81-176 involves protein phosphorylation and activation of G proteins, PI3-K, and MAPK (Wooldridge et al., 1996; Watson & Galan, 2005; Hu et al., 2006b). In the current study, expression of genes involved with protein binding, including Gprotein-coupled receptor binding, is increased when human intestinal cells are exposed to C. jejuni (FDR 10%). PIK3C2B and MAP3K8 are also upregulated, in addition to genes coding for other kinases (IKBKB and PFKFB4). Interestingly, analysis of the microarray data reveals that the majority of the differentially expressed genes are upregulated in response to treatment. As the average half-life of mRNA in human cultured cells is about 10 h, the exposure of cells to bacteria for 6 h may not have been sufficient to observe downregulation of specific genes. The decision to expose the cells to bacteria for 6 h was based on previous studies in our lab and others indicating that C. jejuni invades human epithelial cells within 2 h (Konkel et al., 1992; Carrillo et al., 2004). We were also concerned that longer exposures may be detrimental to the survival of the cells or may cause consequential changes in genes expression that were not directly effected by bacterial exposure. Groups of genes that were upregulated in the human colonic epithelial cells in response to C. jejuni exposure included those over-represented in the biological functions’ cell growth/maintenance, transcription, steroid synthesis, and inflammation. The human genes showing some of the greatest changes in expression after exposure to C. jejuni provide insight into host-specific responses that are associated with C. jejuni-induced gastrointestinal disease. Specifically, genes involved in cell polarity (EFNA1 and PECAM1), water movement (AQP4), and solute transport (SLCO2A1), thus potentially altering water absorption in the gut, showed altered expression. In addition, CDH1, PCDH12, and PCDHB8, involved in cell–cell adhesion, and FOS, an early response gene to various stimuli, were upregulated in response to C. jejuni exposure. Regulation of tissue maintenance (GDF15) and inflammatory response (MAP3K8 and NFKBIZ) were also upregulated in response to C. jejuni. Unexpectedly, NFKBIZ, an inhibitor of NFKB, was upregulated contrary to previous reports indicating that NFKB is activated in response to C. jejuni (Mellits et al., 2002; Hu et al., 2006a). Our finding potentially results from differences in experimental design, in particular the choice of bacterial strains, cell lines, and the fact that we controlled for broth effects. Only three of the genes that were altered in human epithelial cells were also altered in the mouse cells in response to C. jejuni-related exposure, but this response was 2006 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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not bacteria-specific, also occurring in broth-treated cells. Neither mammalian cell line had a significant L. reuterispecific response, exclusive of broth treatment. The results presented here provide evidence that differences between host responses to specific commensal and pathogenic bacteria exist. Furthermore, the human genes that are altered in response to C. jejuni suggest why humans and not mice develop gastroenteritis upon colonization.
Acknowledgements This work was supported by an NSF POWRE award (9973861) and an NIH Grant subcontract (U01CA084239) to D.S.T. E.R. was supported by an NIH Predoctoral Fellowship (F31AT002835). J.M.A. was supported by an SPIRE Postdoctoral Fellowship (K12GM000678). The NIDDKsupported CGIBD provided important infrastructure (P30DK034987).
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