Cytokine responses in primary chicken embryo intestinal cells infected with Campylobacter jejuni strains of human and chicken origin and the expression of bacterial virulence-associated genes
© Li et al; licensee BioMed Central Ltd. 2008
Received: 26 October 2007
Accepted: 27 June 2008
Published: 27 June 2008
Campylobacter jejuni is a major cause of inflammatory diarrhoea in humans and is considered a commensal of the gastroenteric tract of the avian host. However, little is known about the interaction between C. jejuni and the avian host including the cytokine responses and the expression of the bacterial genes. We have investigated the invasiveness of primary chicken embryo intestinal cells (CEICs) by C. jejuni strains of human and chicken origins and the production of pro-inflammatory cytokines as well as the expression of the bacterial virulence-associated genes during co-cultivation.
C. jejuni strains are capable of invading the CEICs and stimulate these cells in a pro-inflammatory manner and during this interaction the expression of the bacterial virulence-associated genes ciaB, dnaJ and racR is increased. Furthermore, incubation of bacteria with conditioned cell- and bacteria-free media from another co-cultivation experiment also increased the expression of the virulence-associated genes in the C. jejuni chicken isolate, indicating that the expression of bacterial genes is regulated by component(s) secreted upon co-cultivation of bacteria and CEICs.
We show that under in vitro culture condition C. jejuni strains of both human and chicken origins can invade avian host cells with a pro-inflammatory response and that the virulence-associated genes of C. jejuni may play a role in this process.
Campylobacter is a spiral Gram-negative, thermophilic, obligate microaerobic bacterial genus that is ubiquitous in temperate environments . Campylobacter jejuni is recognized as the leading cause of bacterial food-borne and water-borne enteric diarrhea in humans. In the United States and Great Britain, more than 1% of the population is infected with C. jejuni each year [2, 3], and the incidence of C. jejuni-induced disease may be even higher in developing countries, where infection often goes unreported. The clinical symptoms of Campylobacter infection include watery to bloody diarrhea, abdominal pain, fever, headache, nausea and vomiting in acute infection, even a severe inflammation of the intestinal mucosa with an influx of professional phagocytes . Mostly the infection of Campylobacter is self-limiting, but some infections are associated with more serious medical sequels such as Reiter's syndrome (reactive arthritis), Miller-Fisher syndrome (MFS) and Guillain-Barré syndrome (GBS) . The Campylobacter-associated illness has become an economic and health burden in the world [3, 6]. Although knowledge about the organism and host responses to infection has been growing rapidly in the past decades, the pathogenesis of C. jejuni is not yet well understood.
C. jejuni colonizes the intestinal mucosa of warm-blooded hosts, including farm animals and humans. Among these hosts the favored environment appears to be the intestines of avians, including chickens, that provide optimal temperature conditions for growth, e.g. 42°C. Unlike in the colonization of humans, C. jejuni colonizes the intestinal tracts of most mammals and birds at a high level with little or no pathology . The differences of pathology between human and chicken may relate partly to the host immune system. Cytokines are central to the development of effective immunity against microbial pathogens. Previous studies have shown that C. jejuni can invade human-derived epithelial cell cultures [8, 9] and induce the production of a number of cytokines and chemokines during the course of experimental infections [10–15], as well as in clinical Campylobacter infections . In general, cytokine responses have mainly been monitored on human-derived cell lines with only a few studies performed on chicken cells [15, 17] and none involving C. jejuni isolates of chicken origin. Furthermore, experimental colonization experiments have shown that passage through the chicken gut enhances the colonization potential and virulence of C. jejuni , suggesting that it is important to use C. jejuni of chicken origin in order to more closely mimic the interactions taking place in the avian gut. Therefore, we used primary chicken embryo intestinal cells (CEICs) as a model to compare the cytokine responses of CEICs elicited by C. jejuni strains of human and chicken origin, as well as the concomitant expression of the virulence-associated genes of C. jejuni.
Intracellular bacterial counts
Production of cytokines by CEICs
Induction of iNOS and production of NO by CEICs
Expression of the virulence-associated genes of C. jejuni
In this study, primary chicken embryo intestinal cells (CEICs) were used to study the commensal interaction between chicken intestinal cells and C. jejuni of human and chicken origin. The results clearly show that both C. jejuni strains of chicken (SC11) and clinical (HM5040) origin can invade CEICs to a level approximate to that seen with human-derived cell lines . However, HM5040 demonstrated a higher percentage of internalized bacteria than SC11 at early stage after inoculation (1 and 2 h p.i., Figure 1). The internalized bacteria did not persist, and declined to a low level at 24 h p.i.. These results are consistent with previous studies, in which only a few viable bacteria remained intracellularly in human dendritic cells  and human peripheral monocytes/macrophages  after a prolonged infection period (24 or 48 h) with C. jejuni. In contrast, other studies have demonstrated that C. jejuni may survive intracellular for relatively long periods of time in both phagocytes and intestinal epithelial cells [24, 25], and the phagocytosis may even promote the survival of C. jejuni , which has lead to its suggested classification as a facultative intracellular pathogen. The intracellular survival may enhance the ability of C. jejuni to evade the host immune system, possibly favoring its long-term persistent infections . However, to survive, the intracellular bacteria also have to overcome certain challenges, such as hydrogen peroxide  and ferrous iron . For C. jejuni and some other enteric pathogens, it is however still largely unclear as to which primary factor(s) determines intracellular bacterial survival.
We further studied the interaction of C. jejuni-avian host in the aspect of the cytokine responses to C. jejuni. The results showed that both C. jejuni strains investigated were capable of inducing the expression of the pro-inflammatory cytokines IL-1β and IL-6 and the pro-inflammatory chemokines CXCLi1 and CXCLi2 in CEICs. Furthermore, we measured the production of iNOS and NO from CEICs. Inducible NOS was increased transcriptionally in CEICs at 4 h p.i. (Figure 2), and an increased NO level was also detected in the co-cultivated medium at 4 and 24 h p.i. (Figure 3). Inducible NOS, IL-1β, IL-6, and CXCLi2 are major markers of inflammatory disease. Thus, it appears that C. jejuni can stimulate inflammatory responses in CEICs. Our results are consistent with previous reports using avian cells, in which Campylobacter induced pro-inflammatory cytokines and chemokines in avian primary chick kidney cells and the avian macrophage cell line HD11 , as well as in primary intestinal chick cells . Interleukin-8 is a pro-inflammatory cytokine, a potent chemotactic factor for many immune effector cells, and a mediator of localized inflammatory responses . Campylobacter strains have exhibited variability in the induction of CXCL8, and those strains with a high ability to invade intestinal epithelial cells frequently induce the high levels of CXCL8 . Although HM5040 showed higher invasion efficiency than SC11 shortly after inoculation, we did not observe any convincing correlation linking the invasion efficiency to the induction of cytokines and chemokines. In addition, a previous study has demonstrated that the induction of CXCL8 from INT407 cells may be regulated by two independent mechanisms, one of which requires adherence and/or invasion and the other requires cytolethal distending toxin (CDT) . The functional role of the avian CXCLi2 measured in this study awaits further investigations.
Potential virulence properties of Campylobacter include motility, chemotaxis, colonization ability, adhesion to intestinal cells, invasion and epithelial translocation, intracellular survival, and formation of toxins and a number of putative virulence genes have been reported among the Campylobacter isolates . CiaB gene and racR gene are highly prevalent in Campylobacter isolates from various sources , and conserved across the species . DnaJ is a functional homologue of the dnaJ gene from E. coli and plays a role in C. jejuni thermotolerance and colonization of chickens [33, 34]. The RacR-RacS (reduced ability to colonize) system is a two-component regulatory system and is involved in the ability to colonize the chicken intestinal tract . It is important to study how these genes react to the co-cultivation as they could provide more information about Campylobacter-host interaction and the pathogenesis of Campylobacter. We thus measured the expression of these virulence-associated genes at the transcriptional level. All the three virulence-associated genes were up-regulated in invaded/adhered bacteria. The level of the expression in the suspended bacteria in contact with CEICs was generally lower than that in the internalized/adhered bacteria, which is likely a consequence of less direct contact than experienced by the internalized/adhered bacteria. Expression of ciaB gene of the internalized/adhered bacteria was induced to a lower extent as compared to that of dnaJ and racR (Figure 4). Previously, in in vitro studies have shown that ciaB is involved in the internalization of C. jejuni into host cells and required for the protein secretion process . The up-regulation of the virulence-associated genes during the co-cultivation with and/or invasion into CEICs may suggest that these genes play a role in the invasion process of C. jejuni. Although few statistical differences were observed, SC11 showed in general a higher level of expression of these virulence-associated genes than observed in HM5040 except with a few differences (Figure 4). Shortly after inoculation (1 and 2 h p.i., Figure 1), the invasion efficiency of SC11 was significantly lower than that of HM5040. It is likely that the virulence-associated genes in SC11 are more inducible by co-cultivation with CEICs and this induction may decrease the invasion efficiency of SC11.
To study whether the induction of the virulence-associated genes is contact dependent, the bacteria were incubated with the bacteria- and cell-free supernatant from another co-cultivation experiments and with the medium of non-inoculated CEICs. The bacteria-and cell-free supernatant induced the expression of the genes tested to a greater extent in SC11 (p ≤ 0.170) than in HM5040 (p ≥ 0.30). The medium of non-inoculated CEICs suppressed the expression of the virulence-associated genes in chicken strain SC11, especially for genes ciaB and dnaJ (p ≤ 0.081), while slightly increased in human strain HM5040 (Figure 5). Previous studies have showed that both host cell components and a cell-free serum-supplemented tissue culture medium can induce Cia proteins secretion, and the secretion of C. jejuni Cia proteins is contact dependent [37, 38]. In our study, the results from SC11 may indicate that a physically contact of bacteria and CEICs is not necessary for the transcriptional up-regulation of the genes tested, since the induction of the genes occurred in those bacteria inoculated only with bacteria- and cell-free media prepared from an another co-cultivation experiments. These results may indicate that CEICs and/or bacteria secrete components into the environment after physical contact event(s). The secreted component(s) may subsequently initiate the expression of the virulence-associated genes in other bacterium that had never been physically contacted with either CEICs or invaded/adhered bacteria. Thus, the up-regulation of these virulence-associated genes may not be bacteria-cell contact dependent, but the secretion of the active component(s) is bacteria-cell contact dependent.
In summary, we have demonstrated that both C. jejuni strains of human and chicken origin can invade CEICs and stimulate CEICs to undergo an inflammatory response in a similar manner. The expression of virulence-associated genes of C. jejuni is increased during the course of invasion, and may play a role in the invasion process.
Two C. jejuni strains SC11, a Danish chicken isolate, and HM5040, a human isolate, were used in this study. These two strains were the most common serotype (Penner serotype 2) and flaA type (flaA type 1) among isolates from broilers and human cases in Denmark . Both strains were taken from -80°C stock and streaked on modified CCDA (mCCDA) (blood-free agar base with cefoperazone [32 mg/liter] and amphotericin B [10 mg/liter]) (CM739 plus SR155; Oxoid, Basingstoke, UK) agar plates and incubated under microaerobic condition (10% CO2, 2–4% H2 and 86%–88% N2) at 42°C for 48 h. The bacteria were then sub-cultured on a new mCCDA plate for another 24 h prior to experimentation. The bacteria were harvested and well suspended in room temperature PBS (10 mM) to an OD595 = 1.0 (approximately 109 bacteria/ml). The bacteria were then diluted in pre-warmed Dulbecco's modified Eagle's growth medium (DMEM) (Invitrogen, UK) without antibiotics to the desired density for inoculation of cell cultures at a multiplicity of infection (MOI) of 100 bacteria per cell.
Primary chicken embryo intestinal cells (CEICs) were prepared from 19-day-old specific pathogen free (SPF) chicken embryos (Lohmann Tierzucht GmbH, Cuxhaven, Germany). Briefly, SPF chicken embryos were dissected, and intestines were separated and cut into small pieces in PBS prior to being digested with 0.25% trypsin-EDTA (Invitrogen, UK) for 3 times, 5 min for each. The cells were then collected by centrifugation at 500 × g for 10 min at room temperature (~25°C). The cells were seeded in 12-well plates at a density of 3.5 × 105 cells/ml (1 ml per well) in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 0.3% (w/v) tryptose phosphate broth (containing 0.13% pancreatic digest of casein, 0.07% proteose peptone No. 3, 0.02% dextrose, 0.05% sodium chloride, 0.026% disodium phosphate), and 0.02% penicillin and streptomycin each (Invitrogen). The cells were incubated in a humidified environment at 37°C with 5% CO2 throughout the experiment. At 24 h the medium was replaced with fresh growth medium, the cells were incubated for anther 72 h prior to bacterial inoculation experiments. The cells were washed three times with pre-warmed PBS, freshly prepared 300 μl of antibiotic-free medium containing the desired number of bacteria was added and co-cultivated with cells to various time points. All inoculations were carried out at a MOI of 100:1, unless stated otherwise. Controls consisted of mock infections using antibiotic-free medium alone or positive controls of lipopolysaccharide (LPS, 5 μg/ml) (Sigma, Schnelldorf, Germany).
To prepare bacteria- and cell-free cultured medium, cells were inoculated with the desired number of bacteria for 4 h (MOI = 100), the medium was collected, the bacteria was removed by centrifugation three times at 10,000 × g, 3 min for each, or filtration through a 0.22 μm filter. Three hundred μl of this medium was added to freshly harvested bacteria of ~1.7 × 107 and incubated for 4 h. As controls, the bacteria incubated with fresh and CEICs-cultured medium were included in parallel. The incubated bacteria were collected by centrifugation and subject to bacterial total RNA isolation as described below.
Intracellular bacterial counts
The number of intracellular bacteria per eukaryotic cell culture was assessed by the gentamicin protection assay . At 1, 2, 3, 4 and 24 h post inoculation (p.i.), the culture medium was removed, Hank's Buffered Salt Solution (HBSS, Invitrogen) containing gentamicin (200 μg/ml) was added, and incubated at 37°C, 5% CO2 for 2 h. The cells were then washed three times with pre-warmed PBS and lysed with 500 μl of 0.2% (v/v) Triton X-100 at room temperature for 15 min. The bacteria counts were numerated by plates counting and were present as a percentage of invaded bacteria to total inoculated bacteria.
RNA extraction, reverse transcription, and PCR conditions
Genes and primers used in this study.
Primer sequences (5'-3')
Annealing temperatures (°C)
Correlation coefficient (r2)
GenBank access no.
β-actin (Gallus gallus/chicken)
IL-6 (Gallus gallus/chicken)
CXCLi1 (K60) (Gallus gallus/chicken)
CXCLi2 (CAF/IL-8) (Gallus gallus/chicken)
TGF-β4 (Gallus gallus/chicken)
iNOS (Gallus gallus/chicken)
16S (C. jejuni) rRNA (C. jejuni)
ciaB (C. jejuni)
dnaJ (C. jejuni)
racR (C. jejuni)
Quantification of cytokines and bacterial virulence-associated genes
The transcripts of cytokine interleukin 1β (IL-1β), IL-6, CXCLi1, CXCLi2, tumor growth factor β4 (TGF-β4) and the inducible nitric oxide synthese (iNOS) were measured in inoculated and mock-inoculated cells using qPCR relative to β-actin. The transcripts of bacterial virulence-associated genes ciaB, dnaJ and racR in invaded/adhered, suspended and those bacteria treated with various media were quantitatively determined by qPCR relative to 16S rRNA, by use of the 2-ΔΔCt method previously described . PCR was performed in duplicate including no-template controls.
Measurement of NO production
Nitric oxide production was determined by measuring nitrite in cell culture media using the Griess Reagent System (Promega) assay according to the manufacture's instruction. The absorbance was read at 550 nm, using a Multiskan EX microtiter plate reader (Thermo Labsystem, Multiskan EX, Denmark).
The values were expressed as the average ± standard deviation (SD). The data were analyzed for statistical significance using one-way ANOVA (ANalysis Of VAriance, Microsoft Excel). A p-value ≤ 0.05 was considered to be statistically significant.
We thank Jonas Hoeberg for his assistance in RNA isolation and real time RT-PCR experiments. This work was supported by the Danish Research Council.
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