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The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica serotype Typhi. Wild-type serotype Typhi induces less CXC chemokine production in tissue culture models than does an isogenic viaB mutant. Here we investigated the in vivo relevance of these observations by determining whether the presence of the viaB region prevents inflammation in two animal models of gastroenteritis. Unlike S. enterica serotype Typhimurium, serotype Typhi or a serotype Typhi viaB mutant did not elicit marked inflammatory changes in the streptomycin-pretreated mouse model. In contrast, infection of bovine ligated ileal loops with a serotype Typhi viaB mutant resulted in more fluid accumulation and higher expression of the chemokine growth-related oncogene alpha (GROα) and interleukin-17 (IL-17) than did infection with the serotype Typhi wild type. There was a marked upregulation of IL-17 expression in both the bovine ligated ileal loop model and the streptomycin-pretreated mouse model, suggesting that this cytokine is an important component of the inflammatory response to infection with Salmonella serotypes. Introduction of the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GROα and IL-17 expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in reducing intestinal inflammation in vivo.
Salmonella enterica serotype Typhi causes a severe systemic infection in humans known as typhoid fever. In contrast, nontyphoidal Salmonella serotypes, such as S. enterica serotype Typhimurium, cause a localized infection in humans manifesting as gastroenteritis (41, 64). The different clinical presentations of infections with serotype Typhi and serotype Typhimurium point to important differences during the interaction of these pathogens with their human host. One such difference is the host response elicited in the intestinal mucosa. Gastroenteritis is a typical diarrheal disease characterized by a massive neutrophil influx in the terminal ileum and colon and a predominance of neutrophils in the stool samples of patients (9, 17, 26). In contrast, typhoid fever is not a typical diarrheal disease, and the intestinal pathology is characterized by a predominantly mononuclear infiltrate (i.e., macrophages and dendritic cells) (17, 23, 29, 30, 46).
The nature of these differences is poorly understood, partly because the strict adaptation of serotype Typhi to its human host severely limits our ability to study host-pathogen interactions in vivo. While higher primates (i.e., chimpanzees) are susceptible to infections, nonprimate vertebrates and even lower primates (i.e., rhesus macaques) are resistant to serotype Typhi (11). As a result, differences between infections with serotypes Typhi and Typhimurium have mostly been explored with tissue culture models. Although the in vivo relevance of results from tissue culture assays remains to be established in many cases, these studies have revealed marked differences during the interactions of serotypes Typhi and Typhimurium with host cells. Microarray analysis shows that unlike serotype Typhimurium, serotype Typhi does not trigger a classical proinflammatory gene expression program in intestinal epithelial cell lines (62). Serotype Typhimurium can trigger the migration of neutrophils across monolayers of polarized colonic epithelial T84 cells, but serotype Typhi is not able to elicit this response (25). Furthermore, stimulation of macrophage-like THP-1 cells with serotype Typhi results in markedly reduced interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) expression compared to stimulation with serotype Typhimurium (19, 35).
Recent studies show that the viaB locus is required to prevent serotype Typhi from eliciting proinflammatory responses in tissue culture models. The viaB locus encodes the production of the Vi polysaccharide capsular antigen (3, 18, 22) and is located on a 134-kb serotype Typhi DNA region, termed SPI7, that is absent from the serotype Typhimurium chromosome (32). Deletion of the viaB locus from serotype Typhi results in increased expression of the CXC chemokine IL-8 after infection of human colonic epithelial cell lines (35, 43) and in increased IL-8 and TNF-α expression after infection of human macrophage-like cells (19, 35). These in vitro data suggest that the presence of the viaB locus may be in part responsible for the reduced propensity of serotype Typhi to rapidly elicit neutrophilic inflammation in the intestinal mucosa, which is characteristic of infections with serotype Typhimurium (36). However, the assumption that the viaB locus prevents intestinal inflammation in vivo has not been tested experimentally with an appropriate animal model.
Two animal models used for the study of serotype Typhimurium-induced intestinal inflammation are bovine ligated ileal loops and streptomycin-pretreated mice. Mice are normally not well suited for the study of serotype Typhimurium-induced gastroenteritis, because these animals develop a systemic infection without diarrhea and neutrophils are scarce in intestinal infiltrates (44, 63). Pretreatment of mice with streptomycin drastically increases their susceptibility to oral infection with serotype Typhimurium (5) by promoting bacterial intestinal overgrowth (34), which triggers a neutrophil influx in the cecum (4). Streptomycin-pretreated mice can thus be used as a model for the study of serotype Typhimurium-induced neutrophil recruitment in the cecal mucosa (4, 7, 13, 14). Natural or experimental infection of calves with serotype Typhimurium results in an enteric disease with clinical and pathological features that parallel the disease in humans. Serotype Typhimurium causes a localized infection in calves, with the most severe pathological changes being restricted to the intestine (51, 59). Animals develop diarrhea and intestinal inflammation characterized by a severe diffuse inflammatory infiltrate composed predominantly of neutrophils (51, 59). Bovine ligated ileal loops have been used successfully for the study of fluid accumulation (a surrogate of diarrhea), neutrophil recruitment, and cytokine responses following serotype Typhimurium infection (10, 37-40, 42, 52, 53, 55, 56, 63, 65, 66). The goal of this study was to evaluate the role of the viaB locus in these two animal models of serotype Typhimurium-induced intestinal inflammation.
Serotype Typhimurium strain IR715 is a fully virulent, nalidixic acid-resistant derivative of isolate ATCC 14028 (48). Serotype Typhimurium strain ZA21 is a derivative of IR715 carrying mutations in sipA, sopA, sopB, sopD, and sopE2 (66). Strain EHW26 is a nonflagellated derivative of ATCC 14028 (fliC fljB mutant), which has been described previously (35). Serotype Typhi strain Ty2 was obtained from the American Type Culture Collection (ATCC 19430). Strain STY2, a derivative of Ty2 carrying a deletion of the viaB region (ΔtviABCDE vexABCDE::Km) (35). Plasmid pHP45Ω (33), carrying a streptomycin resistance gene, was introduced into serotype Typhi strains by electroporation prior to infection of streptomycin-pretreated mice. Cloning of the tviABCDE vexABCDE genes (viaB locus) was performed with Escherichia coli strain DH5α (12). The viaB region was amplified by PCR using the primers 5′-CGCAACACACGGAGTATCACC-3′ and 5′-TCGCCTACCAGCACAAAGCG-3′ for the upstream segment and the primers 5′-AAGTGCTGGAAGAACAGGTCG-3′ and 5′-ACTAGTGTGAATACTTAGGCTGGGGTG-3′ for the downstream segment. The resulting 7.255-kb and 7.113-kb PCR products were cloned into the vector pCR2.1 (Invitrogen), and amplification of the correct fragments was confirmed by sequence analysis. The upstream region was cloned into the SpeI and EcoRI sites of the low-copy-number vector pWSK29 (54) to give rise to the plasmid pDC3. The downstream fragment was then cloned into the EcoRI and KpnI sites of the plasmid pDC3 to give rise to the plasmid pDC5. The tviA gene and its 600-bp promoter region were amplified by PCR using the primers 5′-GGTACCCAGTATGACGTTCTG-3′ and 5′-CGAATTCTTGTCCGTGTTTTAC-3′ and cloned into the EcoRI and KpnI sites of the plasmid pWSK29 to give rise to the plasmid pTVIA1.
Strains were cultured aerobically at 37°C in Luria-Bertani (LB) broth supplemented with antibiotics, as appropriate, at the following concentrations: carbenicillin, 100 mg/liter (LB+Cb); chloramphenicol, 30 mg/liter (LB+Cm); tetracycline, 20 mg/liter (LB+Tc); kanamycin, 60 mg/liter (LB+Km); streptomycin, 100 mg/liter (LB+Str); nalidixic acid, 50 mg/liter (LB+Nal). For infection of bovine ligated ileal loops, each strain was grown overnight at 37°C in 4 ml of LB broth in a roller. A volume of 0.04 ml of this overnight culture was used for inoculation of 4 ml of LB broth, and bacteria were grown at 37°C for 3 h in a roller. Subsequently, this culture was used as the inoculum, and the numbers of CFU were determined by plating serial 10-fold dilutions on LB plates.
To determine the generation time (g), bacteria were cultured aerobically and the increase in bacterial numbers over time was monitored by measuring the optical density at 600 nm (OD600). The slope (m) of the logarithmic increase [plotted as the change in log2(OD600) over time] in bacterial numbers was calculated by linear regression by the program Excel (Microsoft). The number of bacteria (Nt) at a given time (t) is proportional to the number of bacteria at time zero (N0) and the number of doublings, which can be calculated from the generation time (g) by the formula 2t/g. After logarithmic conversion, this connection can be described as log2(Nt) = log2(N0) + t/g. The slope, m, of the logarithmic increase in bacterial numbers thus equals 1/g, which provides a simple means of calculating the generation time.
Flow cytometry to detect expression of the Vi capsule was performed as described previously (20, 35). DNA was labeled with propidium iodide, and Vi antigen was detected by labeling of cells with rabbit anti-Vi serum (1:250 dilution) (BD) and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugate (1:250 dilution) (Jackson ImmunoLabs). For each sample, the fluorescence of 10,000 particles (bacterial cells) was measured (FACSCalibur; Becton Dickinson). The gate for detection of Vi expression was set such that cells expressing the capsule (IR715[pDC5] or Ty2) were considered positive for expressing the Vi antigen when their FITC fluorescence intensity exceeded that of all but a small fraction (less than 2%) of the control population of an isogenic noncapsulated strain (IR715 or STY2, respectively).
To study inflammation in the cecum, 8-week-old streptomycin-pretreated C57BL/6 mice were orally infected with Salmonella serotypes as described previously (4). In brief, groups of four mice were inoculated intragastrically with streptomycin (0.1 ml of a 200 mg/ml solution in phosphate-buffered saline [PBS]). Mice were inoculated intragastrically 24 h later either with sterile LB broth or with bacteria (0.2 ml containing approximately 1 × 109 CFU/ml). At 24 h, 48 h, and 72 h after infection, groups of four mice were euthanized and samples of the cecum were collected for isolation of mRNA and histopathological analysis. Salmonella serotypes were enumerated in the cecal contents, the mesenteric lymph node, and the spleen by plating serial 10-fold dilutions of homogenates on selective agar plates (LB+Carb or LB+Nal).
Four male Holstein calves, 4 to 5 weeks of age, weighing 45 to 55 kg were used. They were fed milk replacer twice a day and water ad libitum. The calves were clinically healthy before the experiment and were culture negative for fecal excretion of Salmonella serotypes. Detection of Salmonella serotypes in fecal swabs was performed by enrichment in tetrathionate broth (Difco) followed streaking on brilliant green agar (BBL) and XLT4 (Difco).
Bovine ligated ileal loop surgery has been described previously (1, 39). In brief, the calves were fasted for 24 h prior to surgery. Anesthesia was induced with Propofol (Abbott Laboratories, Chicago, IL), followed by placement of an endotracheal tube and maintenance with isoflurane (Abbott Laboratories, Chicago, IL) for the duration of the experiment. A laparotomy was performed, the ileum was exposed, and loops with lengths ranging from 6 to 9 cm were ligated, leaving 1-cm loops between them. The loops were infected by intralumenal injection of 3 ml of either sterile LB broth or a suspension of bacterial strains in LB broth containing approximately 1 × 109 CFU. The loops were then replaced into the abdominal cavity until collection at the indicated time points. Each bacterial strain was tested in four different animals.
After surgical removal of loops, the fluid accumulated in the loops was measured and samples for bacteriological culture, histopathological analysis, and mRNA isolation were collected. Intestinal samples for bacteriological analysis were obtained with 3.5-mm biopsy punches and incubated in PBS containing 0.1 mg/liter gentamicin for 90 min. Tissue samples were then homogenized in PBS, serially diluted, and plated on LB agar plates containing antibiotics appropriate for determining CFU. Data on bacterial CFU were normalized to the length of the ligated loop and the CFU present in the inoculum prior to statistical analysis. Tissue collected for extraction of mRNA was snap frozen.
Tissue samples were fixed in formalin, processed according to standard procedures for paraffin embedding, sectioned at 5 μm, and stained with hematoxylin and eosin. Inflammatory changes were scored from 0 to 3 according to the following criteria: 0, no inflammation; 1, mild inflammatory changes, characterized by multifocal intravascular margination and mild perivascular infiltration of neutrophils in the lamina propria and submucosa; 2, moderate inflammatory changes, characterized by moderate multifocal to coalescent or diffuse infiltration of neutrophils in the lamina propria and submucosa, associated with mild to moderate edema; 3, severe inflammatory changes, characterized by diffuse severe infiltration of neutrophils in the lamina propria and submucosa, associated with moderate to severe edema and/or multifocal hemorrhage and epithelial loss. Although the same scoring system was adopted for both calves and mice, lesions scored as 2 and 3 tended to be more severe in calves, in which necrosis and erosion/ulceration of the mucosa were often observed. Therefore, this scoring system is intended for comparisons between treatments within a host species but not for comparisons between host species. Scores of 0, 1, 2, and 3 corresponded to averages of 3.3, 16.9, 46.7, and 144,9 neutrophils per high-magnification microscopic field (10 fields per animal were counted and then averaged within each score). Such quantification of neutrophils in calves was not performed due to the higher numbers of neutrophils and more-severe tissue damage.
J774.A1 cells (ATCC TIB-67) were grown in Dulbecco's modified Eagle medium with glucose (4.5 g/liter) supplemented with 10% calf serum (Gibco). Cells were seeded on a 24-well plate at a density of 2.5 × 105 cells/well overnight. The next day, cells were infected with bacteria at a multiplicity of infection (MOI) of 10 for gene expression analysis and at an MOI of 0.1 for detection of protein levels in the supernatant. After 1 h, the cells were washed and incubated with 0.1 mg/ml gentamicin for 1 h. Subsequently, the medium was replace to contain 0.025 mg/ml gentamicin for the remaining time of the experiment. The level of TNF-α in the supernatant was then quantified at 24 h postinfection by enzyme-linked immunosorbent assay (ELISA) (eBioscience) according to the instructions provided by the manufacturer. RNA extraction and real-time PCR analysis were performed 6 h after infection, as described below.
For analysis of changes in gene expression after Salmonella infection, tissue samples of murine cecum and bovine ileum were collected, immediately snap-frozen in liquid nitrogen at the site of surgery, and stored at −80 °C until processing. RNA was then extracted from snap-frozen tissue with TriReagent (Molecular Research Center) according to the instructions of the manufacturer. Next, 1,000 ng of RNA from each sample was retrotranscribed in a 0.05-ml volume (Taqman reverse transcription reagent; Applied Biosystems), and 0.004 ml of cDNA was used for each real-time reaction. Real-time PCR was performed with SYBR green (Applied Biosystems) and the 7900HT Fast Real-Time PCR System. The data were analyzed by the comparative CT method (Applied Biosystems). Increases in cytokine expression in infected mice were calculated relative to the average level of the respective cytokine in four control animals from the corresponding time point after inoculation with sterile LB broth. Increases in cytokine expression in calves were calculated for each infected loop relative to a loop (collected at the same time point from the same animal) that was inoculated with sterile LB broth. A list of genes analyzed in this study with the respective primers is provided in Table Table11.
For statistical analysis of ratios (i.e., increases in cytokine expression or data expressed as percentages), data were transformed logarithmically prior to performance of a statistical analysis. A parametric test (paired Student's t test for ligated loop samples and Student's t test for murine samples) was used to calculate whether differences in increases or percentages between treatment groups were statistically significant. Significance levels for Pearson's correlation analyses were determined with Instat software (GraphPad, San Diego, CA).
To investigate whether the deletion of the viaB locus would result in increased intestinal inflammation during serotype Typhi infection in vivo, we infected streptomycin-pretreated mice with serotype Typhi (Ty2), a serotype Typhi viaB mutant (STY2), or serotype Typhimurium (IR715), a pathogen that has been shown previously to cause inflammation in this model (4). Each strain was used to inoculate groups of mice (n = 12 [per group]) that had been pretreated with streptomycin 24 h earlier. As a negative control, streptomycin-pretreated mice were inoculated with sterile LB broth (n = 12). For each treatment, a subgroup of four mice was euthanized at 24, 48, and 72 h after infection.
Serotype Typhimurium was retrieved from the mesenteric lymph node at significantly (P < 0.05) higher numbers than either the serotype Typhi wild type or viaB mutant at 48 and 72 h after infection. The serotype Typhi wild type (Ty2) was recovered on average in significantly (P < 0.05) higher numbers than was the viaB mutant (STY2) from the mesenteric lymph nodes at 72 h after infection. Serotype Typhimurium was isolated from the spleen starting at 48 h after infection, and a high bacterial load, indicative of a severe systemic infection, was observed in this organ by 72 h after infection. In contrast, serotype Typhi was not recovered in large numbers from the spleen at any time point.
Gross pathological changes were not observed at any time point in ceca from mice infected with serotype Typhi strains or with sterile LB broth. In contrast, at 48 and 72 h after inoculation the ceca from mice infected with serotype Typhimurium were reduced in size, were devoid of contents, and had a thickened wall with whitish discoloration and a gelatinous appearance indicative of severe edema. Histopathological analysis of hematoxylin- and eosin-stained sections from the ceca revealed that at 48 and 72 h after infection, only serotype Typhimurium induced a strong inflammatory response (Fig. (Fig.1A),1A), which was characterized by moderate to severe diffuse neutrophil infiltrate in the lamina propria and submucosa, associated with severe edema, particularly in the submucosa, and multifocal epithelial detachment of the luminal epithelium.
To further characterize the inflammatory response in the cecum, we analyzed the expression of cytokines in cecal tissue by real-time PCR. Expression of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2), two neutrophil chemoattractants related to the human GRO proteins (6, 28, 31, 45, 50, 58), were strongly upregulated 48 and 72 h after infection with serotype Typhimurium but not with serotype Typhi (data not shown). Expression of IL-17 (also known as IL-17A), a cytokine contributing to neutrophil recruitment in response to bacterial infection in the mouse lung (15, 16, 24, 60, 61), was increased in all infected mice by 24 h after inoculation. However, at later time points (48 h and 72 h postinfection), IL-17 expression was considerably higher in the ceca of mice infected with serotype Typhimurium (Fig. (Fig.1B).1B). TNF-α expression was significantly (P = 0.014) higher in mice 72 h after infection with the serotype Typhi viaB mutant than in animals infected with the serotype Typhi wild type (Fig. (Fig.1C1C).
The only significant difference in the host response of streptomycin-pretreated mice associated with the viaB locus was an induction of TNF-α expression observed 72 h after infection with the viaB mutant but not with the serotype Typhi wild type. To model expression of TNF-α in vitro, a murine macrophage-like cell line (J774.A1 cells) was infected with serotype Typhi strains and cytokine production was monitored by real-time PCR and ELISA. The serotype Typhi viaB mutant elicited significantly higher TNF-α expression in J774.A1 cells than did the serotype Typhi wild type (Fig. (Fig.2).2). We constructed low-copy-number plasmids carrying the whole viaB region (plasmid pDC5) or the cloned tviA regulatory gene (pTVIA1) and introduced each into the serotype Typhi viaB mutant by electroporation. Introduction of the cloned viaB locus, but not of the cloned tviA regulatory gene, complemented the phenotype of the viaB mutant. These data suggested that reduced TNF-α expression in murine macrophages depends on the presence of capsule biosynthesis genes. TviA, the positive regulator encoded within the viaB region, was not sufficient to complement this phenotype. Deletion of the serotype Typhi flagellin gene (fliC) did not significantly reduce TNF-α expression elicited by the viaB mutant. These data suggested that pathogen-associated molecular patterns, in addition to flagellin, contributed to TNF-α expression elicited by the serotype Typhi viaB mutant in murine J774.A1 macrophages.
Overall, the lack of an acute inflammatory reaction in response to infection of streptomycin-pretreated mice with serotype Typhi wild type and the viaB mutant suggested that this animal model may not be well suited for the study of serotype Typhi-specific virulence factors. To study the role of the viaB locus during infection of cattle, we introduced the cloned viaB region (pDC5) into serotype Typhimurium (IR715) by electroporation. Expression of the Vi capsular antigen in serotype Typhimurium strain IR715(pDC5) was detected by slide agglutination (data not shown). Flow cytometry with anti-Vi antiserum revealed that serotype Typhimurium carrying pDC5 expressed the Vi antigen on its surface at levels similar to those detected in the serotype Typhi wild type (Fig. 3A and B). Introduction of pCD5 into S. Typhimurium did not reduce its generation time during growth in LB broth (Fig. (Fig.3C3C).
Bovine ligated ileal loops were infected with the serotype Typhimurium wild-type strain (IR715) and its capsulated derivative [IR715(pDC5)]. Recovery of bacteria from the ileal mucosa at 2 h and 8 h after infection showed that plasmid pDC5 was maintained by serotype Typhimurium for the duration of the experiment (data not shown). To evaluate the magnitude of the inflammatory response, expression of the CXC chemokine GROα and the cytokine IL-17 were detected in the ileal mucosa by real-time PCR at 2 h after infection. GROα expression was induced approximately sevenfold in loops infected with the serotype Typhimurium wild type (IR715) compared to mock-infected loops. Strain IR715(pDC5) elicited only a 3.6-fold upregulation of GROα expression, suggesting that the presence of the Vi capsule attenuated the inflammatory response to serotype Typhimurium (Fig. (Fig.4A4A).
By 2 h after infection of loops with serotype Typhimurium (IR715), IL-17 expression was increased, on average, 56-fold compared to loops inoculated with sterile LB broth. Similarly, strain IR715(pDC5) (serotype Typhimurium expressing the Vi capsule) elicited approximately 14-fold upregulation of IL-17 expression compared to control loops inoculated with sterile LB broth (Fig. (Fig.4B4B).
At 8 h after infection, fluid accumulation elicited by each strain was measured. Capsulated bacteria [IR715(pDC5)] triggered reduced fluid accumulation compared to the serotype Typhimurium wild-type strain (IR715) (Fig. (Fig.4C).4C). However, these differences were not statistically significant. A milder inflammatory response and reduction of neutrophil influx was observed in the histopathological analysis of sections from the ileal mucosa infected with IR715(pDC5) compared to the serotype Typhimurium wild-type strain (Fig. (Fig.55).
Previous studies have implicated flagellin as a major contributor to serotype Typhimurium-induced inflammation in bovine ligated ileal loops (42). To investigate whether the anti-inflammatory effect of the viaB locus was solely dependent on flagella, plasmid pDC5 was introduced into a serotype Typhimurium fliC fljB mutant (EHW26). Compared to infection with the fliC fljB mutant (EHW26), infection with the fliC fljB mutant carrying pDC5 elicited significantly (P < 0.05) less GROα expression (Fig. (Fig.6).6). The fliC fljB mutant carrying pDC5 also elicited less IL-17 expression, less fluid accumulation, and a milder inflammatory response; however, these differences were not statistically significant. Although these data do not rule out the possibility that the viaB locus reduces inflammatory responses triggered by flagellin, the fact that the viaB locus significantly reduced GROα expression in a fliC fljB mutant demonstrated that the anti-inflammatory effect mediated by this DNA region cannot be exclusively attributed to an inhibition of flagellin-mediated responses.
Serotype Typhi does not elicit marked inflammation in bovine ligated ileal loops (37). To investigate whether the absence of the viaB locus would increase the response to serotype Typhi infection in calves, loops were infected with the serotype Typhi wild type (Ty2) and a viaB mutant (STY2). The serotype Typhi wild type (Ty2) elicited significantly less GROα expression (P = 0.018) and IL-17 expression (P = 0.045) than did the serotype Typhimurium wild type (Fig. (Fig.4).4). The serotype Typhi viaB mutant elicited significantly more GROα expression (P = 0.02) and IL-17 expression (P = 0.049) than did the wild type (Ty2) in ligated ileal loops. Interestingly, at 2 h after infection, the serotype Typhi viaB mutant (STY2) elicited IL-17 expression (approximately 38-fold upregulation compared to mock-infected loops) at levels similar to those elicited by the serotype Typhimurium wild type (P = 0.1). Loops infected with the serotype Typhi viaB mutant (STY2) contained amounts of fluid at 8 h after infection similar to those infected with the serotype Typhimurium wild-type strain (P = 0.09). The amount of fluid elicited by the serotype Typhi viaB mutant (STY2) was significantly higher than that elicited by the serotype Typhi wild type (Ty2) (P = 0.005). Furthermore, the serotype Typhi viaB mutant triggered more inflammation than did its capsulated parent (Ty2) (Fig. (Fig.55).
In summary, data from the bovine ligated ileal loop model show that the presence of the viaB region significantly reduces the inflammatory responses to serotype Typhi or serotype Typhimurium infection in vivo.
Previous studies of inflammation in the respiratory tract suggest that IL-17 contributes to neutrophil recruitment by stimulating other cells to produce CXC chemokines (15, 16, 24, 60, 61). The assumption that IL-17 significantly contributes to neutrophil recruitment in the intestine would predict that there should be a positive correlation between the expression levels of IL-17 and the expression levels of neutrophil chemoattractants (CXC chemokines) in infected tissue. To test this prediction, we compared the expression levels of IL-17 and of CXC chemokines in the bovine ileal mucosa. The expression levels of IL-17 and GROα determined for individual loops collected at 2 h after infection of the bovine ileal mucosa with serotype Typhi or serotype Typhimurium strains were compared (Fig. (Fig.7A).7A). This analysis revealed a positive correlation between expression of IL-17 and GROα (R2, 0.66; P, 0.001). The β-defensins are a group of inducible antimicrobial peptides that contribute to host defense at several mucosal surfaces, including the enteric mucosa. IL-17 has been shown to induce expression of β-defensin in human bronchial epithelial cells (21). Comparison of the expression levels of IL-17 and bovine enteric β-defensin (Fig. (Fig.7B)7B) revealed a significant positive correlation (R2, 0.60; P, <0.0001).
Humans infected with serotype Typhimurium develop a massive neutrophil influx in the intestine, but this host response does not develop in patients infected with serotype Typhi. It has been proposed that serotype Typhi does not elicit neutrophil influx in the human intestine because it possesses the capsule-encoding viaB locus, a DNA region that is absent from the serotype Typhimurium genome (35, 36). This hypothesis is based on in vitro data showing that deletion of the viaB locus from serotype Typhi causes an upregulation of IL-8 expression during infection of human cell lines (T84, THP-1) and human colonic tissue explants (35, 43). To investigate the in vivo relevance of these findings, we investigated whether the presence of the viaB locus influences the intestinal inflammatory response in two animal models of serotype Typhimurium-induced neutrophil influx.
Bovine ligated ileal loops are well suited for the study of cytokine expression, neutrophil influx, and fluid accumulation in response to serotype Typhimurium infection (10, 37, 39, 40, 52, 53, 55-57, 63, 65, 66). However, infection of bovine ligated ileal loops with serotype Typhi strain Ty2 does not result in profound inflammatory changes or fluid accumulation (37). We show that introduction of the capsule-encoding viaB locus into serotype Typhimurium resulted in reduced inflammatory cytokine production, reduced severity of histopathological changes, and reduced fluid accumulation in the bovine ligated ileal loop model. Remarkably, deletion of the viaB locus from serotype Typhi strain Ty2 resulted in increased inflammation in the calf intestine and in fluid secretion at levels that were similar to those elicited by serotype Typhimurium (35).
The ability to invade the intestinal epithelium with the invasion-associated type III secretion system (T3SS-1) is critically important for the induction of inflammation and fluid accumulation by serotype Typhimurium in vivo (38, 66). Expression of the Vi antigen reduces the invasiveness of serotype Typhi for epithelial cells in vitro (2, 27). These data suggest that one possible mechanism by which the viaB locus may prevent inflammatory responses in vivo is by reducing bacterial invasion. Although this possibility cannot be ruled out, we did not find a significant correlation between the numbers of capsulated or noncapsulated bacteria recovered from gentamicin-treated tissue and the magnitude of proinflammatory cytokine expression (R2, 0.18; P, 0.12) (Fig. (Fig.7C).7C). Furthermore, deletion of the viaB locus results in increased IL-8 expression during serotype Typhi infection of colonic epithelial T84 cells regardless of whether T3SS-1 is functional or inactivated by a mutation in invA (35). These data suggest that the viaB locus can reduce inflammatory responses by a T3SS-1-independent mechanism. A T3SS-1-independent mechanism by which the Vi antigen may inhibit inflammatory responses in vivo is by its interference with Toll-like receptor (TLR) recognition (19, 35). Human epithelial kidney 293 (HEK293) cells produce IL-8 in response to infection with a serotype Typhi viaB mutant only when they are transfected with TLR5 or TLR4/MD2/CD14. IL-8 production by TLR5 or TLR4/MD2/CD14-transfected HEK293 cells is significantly reduced during infection with serotype Typhi wild type (35), supporting the idea that the viaB region may interfere with bacterial recognition by TLRs expressed on host cells.
Oral infection of streptomycin-pretreated mice with serotype Typhimurium triggers the development of a neutrophil influx in the cecum (typhlitis) (4). Host responses to infection with serotype Typhimurium in the cecum of streptomycin-pretreated mice are similar to those elicited in germfree mice (47). Oral infection of germfree mice with serotype Typhi strain Ty2 results in substantial bacteria growth in the cecum, but bacteria are not recovered from the liver or spleen (8). Similarly, we recovered serotype Typhi strain Ty2 in large numbers from the cecum of streptomycin-pretreated mice, while animals were able to contain growth at systemic sites of infection (data not shown). Furthermore, in contrast to serotype Typhimurium, serotype Typhi caused very few inflammatory changes in the cecum of streptomycin-pretreated mice. These results are similar to those obtained by infecting streptomycin-pretreated mice with another strictly human -adapted pathogen, S. enterica serotype Paratyphi A, which colonizes the cecum in large numbers but does not trigger pronounced inflammation in the cecum (49).
The only significant difference between inflammatory responses elicited by capsulated and noncapsulated serotype Typhi strains was a 10-fold upregulation in TNF-α expression in ceca from mice infected with the viaB mutant at 72 h after infection. A viaB-mediated reduction in TNF-α expression was also observed during serotype Typhi infection of murine macrophage-like cells (J774). Deletion of the viaB region from serotype Typhi increases TNF-α expression during infection of a human macrophage-like cell line (differentiated THP-1 cells) with serotype Typhi (19). In this model, TNF-α expression depends on the presence of the TLR4 adaptor protein CD14 (19).
In conclusion, this report describes the first evidence for a role of the capsule-encoding viaB operon in downregulating intestinal inflammation in vivo. In addition, our data implicate a new cytokine, IL-17, as a contributor to gastroenteritis elicited by serotype Typhimurium.
We thank Josely F. Figueiredo, Sangeeta Khare, and Tamara Gull for assistance with calf surgeries.
These studies were supported by USDA/NRICGP grant 2002-35204-12247 (L.G.A.) and by Public Health Service grants AI060933 (S.D.L.), AI040124 (A.J.B.), AI044170 (A.J.B.), AI065534 (A.J.B.), AI032738 (C.L.B.), and AI050843 (C.L.B.).
Editor: J. L. Flynn
Published ahead of print on 25 June 2007.