Role of Toll-Like Receptor 4 in the Proinflammatory Response to Vibrio cholerae O1 El Tor Strains Deficient in Production of Cholera Toxin a
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感染与免疫杂志 2005年第9期
Departments of Pathology
Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
ABSTRACT
Following intranasal inoculation, Vibrio cholerae KFV101 (ctxAB hapA hlyA rtxA) colonizes and stimulates tumor necrosis factor alpha and interleukin 1 (IL-1) in mice, similar to what occurs with isogenic strain P4 (ctxAB), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.
TEXT
Vibrio cholerae is a gram-negative pathogen that induces diarrhea dependent upon the action of the cyclic AMP-stimulating cholera toxin (CT) (11). During cholera disease, patients in the acute phase do not have extensive tissue damage such as might be observed with shigellosis (21, 22). However, low levels of infiltration of neutrophils, mast cells, and eosinophils in infection and the presence of mediators of inflammatory cell regulation on the second day of cholera disease indicate that V. cholerae stimulates a proinflammatory response early during infection (21). This inflammatory response possibly becomes excessive in the absence of genes for CT since volunteers given CT-deficient strains have significantly increased titers of lactoferrin and CXCL8, indicative of more severely inflammatory disease typified by symptoms that include vomiting, fever, mild diarrhea, and cramping (25). These symptoms are also typical of nonepidemic strains of V. cholerae that do not produce CT (4, 7, 10, 13, 16, 17). This increase in the inflammatory nature of V. cholerae infection could be due to the absence of the immunomodulatory activity of the B subunit of CT that blocks the secretion of proinflammatory cytokines by macrophages, dendritic cells, and epithelial cells in response to bacterial lipopolysaccharide (LPS) (24) due to downregulation of mitogen-activated protein kinase pathways (6).
A major problem with this residual disease of non-CT-producing V. cholerae is that these strains are frequently sufficiently virulent to make them unsafe for use as live attenuated vaccines (2, 14, 26, 27). It has been proposed that the actions of accessory toxins of V. cholerae are responsible for this increased inflammation and contribute to the reactogenicity of live attenuated vaccine strains (24). Recently, we developed a murine model that is sensitive to accessory toxins and can be used to investigate early events in the initiation of the host response to non-CT-producing V. cholerae. Although adult mice are resistant to intestinal colonization by V. cholerae (12), we found that acute inflammation develops in the lung tissue after intranasal inoculation with V. cholerae, providing a model for the assessment of the toxic action on inflammation of a mucosal epithelial layer (8). This model is based on experiments using Shigella flexneri dependent on the premise that the bronchial tree is composed of a mucosal lining with relevant characteristics similar to those of the intestine, including simple cubodial-to-columnar epithelial cells, lymphoid aggregates, bronchiole-associated lymphoid tissue similar to Peyer's patches, and the presence of several varieties of antigen-presenting cells (5, 19, 23, 28).
Using this new murine model, we previously found that wild-type CT-producing strains of V. cholerae induce a rapid cell death due to fluid accumulation in the lung but that a strain deleted of the genes for CT stimulates proinflammatory cytokine and chemokine production, leading to development of a lethal pneumonia characterized by infiltration of neutrophils, hemorrhages, and bacteremia (8). When mice were inoculated with a multitoxin mutant strain with deletions of genes for the accessory toxins hemagglutinin/protease (hapA), hemolysin (hlyA), and the actin cross-linking RTX toxin (rtxA), in addition to deletions of genes for CT (ctxAB), the mice did not develop severe pneumonia and cleared the infection. In this prior study, 100% of mice inoculated with this multitoxin mutant strain survived when inoculated at a dose that resulted in 80% lethality with the CT-deficient strain (8).
These results suggest that multitoxin mutant strains could be excellent candidates for safe, live attenuated vaccine. However, it is possible that in the absence of inflammation due to secreted toxins, a protective response will also not develop. We hypothesize that LPS and other bacterial factors will stimulate host immunity in the absence of accessory toxins. To demonstrate that V. cholerae stimulates the development of an innate immune response in response to LPS, we inoculated mice with a mutation in Toll-like receptor 4 (TLR4) and assessed if the absence of this signaling pathway would affect bacterial clearance and induction of proinflammatory mediators in both the presence and absence of accessory toxins. These data demonstrate that a multitoxin mutant of V. cholerae has inflammatory potential that is based in part on the activation of TLR4; yet, the strain is less virulent and is cleared more rapidly than a strain that produces the accessory toxins.
Intranasal inoculation of C3Heb/FeJ (tlr4+/+) and C3H/HeJ (tlr4–/–) mice. V. cholerae strains are derivatives of the streptomycin-resistant isolate of El Tor O1 P27459 originally isolated in Bangladesh (15). Strain P4 (also known as SM44) has a kanamycin cassette replacing the genes for ctxA and ctxB in P27459, creating the CT-deficient strain (9). Strain KFV101 is a derivative of P4 with unmarked deletions within the genes hapA, hlyA, and rtxA, creating a multitoxin mutant (8). Bacteria were grown on Luria agar supplemented with 50 μg/ml kanamycin and 100 μg/ml streptomycin. For inoculation to mice, bacterial cultures were grown at 30°C overnight in Luria broth supplemented with antibiotics. Bacteria were washed in phosphate-buffered saline, pH 7.4 (PBS), and then diluted to the desired number of CFU per milliliter for inoculation.
Inoculations were then performed according to protocols approved by the Northwestern University IACUC. Five- to 6-week-old C3Heb/FeJ (tlr4+/+) and C3H/HeJ (tlr4–/–) female mice (Jackson Labs, Bar Harbor, ME) were inoculated intranasally with 20 μl of a V. cholerae suspension while under anesthesia with 60 to 75 mg/kg ketamine and 12 to 15 mg/kg xylazine, as previously detailed (8). At 12 or 20 h after inoculation, mice were exsanguinated by cardiac puncture and euthanized. The caudate lobe of the lung, the spleen, and the liver were weighed and then homogenized in 5 ml LB medium, and dilutions were plated for recovered CFU. Total numbers of CFU per organ were calculated by multiplying the number of CFU/ml by 5 ml. The number of CFU in the lung was then multiplied by 2 to adjust for homogenization of only one lobe. One milliliter of undiluted lung homogenate was preserved at –80°C. At a later time, titers of tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), IL-6, and IL-12 p70 in the clarified lung homogenates were determined by enzyme-linked immunosorbent assay (ELISA) using Quantikine kits (R & D Systems, Minneapolis, MN) according to manufacturer's protocol. Values reported as nanograms per lung were calculated as nanograms per milliliter and multiplied by 5 ml homogenate and by 2 to adjust for homogenization of only one lobe. Values were not calculated per gram of lung since lung weight changed up to 300% due to fluid accumulation in some groups, while lung weight among mock-inoculated mice varied by less than 20%.
The remaining lung lobes were perfused with 4% paraformaldehyde for preservation and later were mounted in paraffin, sectioned, and stained with hematoxylin and eosin (H/E) at the Robert Lurie Cancer Center Core Facility for Histopathology. Slides were coded and then scored by a single pathologist (G.K.H.) for the degrees of bronchiole per peribronchiole inflammation, alveolar inflammation, and respiratory epithelial damage (on a scale of 0 to 4). Neutrophilic inflammation was quantified by counting the number of neutrophils in four randomly selected high-power fields, using an Olympus BX40 microscope with an Olympus DP12 digital camera.
Experiments were done with four to five animals per group. Data from duplicate or triplicate experiments were pooled and analyzed using Microsoft Excel X for Macintosh computers and a two-sample t test assuming unequal variances. P values were considered significant at a P of <0.05 for quantitative data (numbers of CFU, lung weight, and ELISA results) and a P of <0.005 for semiquantitative data (health score and histopathology).
Clearance of infection and reduced morbidity at 20 h depend upon TLR4 and production of accessory toxins. C3H/HeJ mice have a point mutation in the tlr4 gene that leads to a defective response to bacterial endotoxin, rendering the animals resistant to LPS-induced shock (20). Previously, we demonstrated that BALB/c mice succumbed to infection with the ctxAB mutant P4 between 24 and 72 h when inoculated with 107 CFU/ml (8). At this same dosage, three of four P4-inoculated tlr4+/+ mice and all P4-inoculated tlr4–/– mice died within 18 h. The lung, spleen, and liver of the sole surviving P4-inoculated tlr4+/+ mouse were colonized; the mouse was in moderate health, with fluid in the lungs, and had a high titer of TNF- and dense neutrophil infiltration in its lung tissue (data not shown). Data from this pilot study suggest that C3H inbred mice may be more susceptible to this bacterial infection than the previously used BALB/c mice. Therefore, mice for these series of experiments were inoculated with a dosage of 1 x 106 CFU for 20-h experiments and 3 x 106 to 7 x 106 CFU for 12-h experiments to ensure sufficient survivors for analysis.
For experiments terminated at 20 h, two experiments with four to nine mice per group were performed. At a dosage of 106 CFU, all nine P4-inoculated tlr4+/+ mice survived and were colonized with an average of 3,800 CFU in their lungs, with dissemination to the liver occurring in only four of nine mice and to the spleen in none of the mice (Table 1). By comparison, 4 of 13 P4-inoculated tlr4–/– mice died in less than 18 h, while the remaining 9 survivors were heavily colonized with an average of 3.3 x 106 CFU, a 3-fold increase over the inoculum and a 100-fold increase over the level in tlr4+/+ mice. These nine surviving mice also had dissemination of the infection to their livers and spleens (Table 1). In addition to a difference in colonization levels, tlr4–/– mice appeared to be in worse health, as shown by the decrease in the health rating from near normal to poor or near death. These data demonstrate the importance of the TLR4 signaling pathway to containment of infection to the point that the infection clears in the tlr4+/+ mice by 20 h postinoculation (p.i.) but expands in the tlr4–/– mice.
In contrast to the mice inoculated with the ctxAB strain P4, mice inoculated with KFV101, a derivative of P4 with additional deletions in the accessory toxin genes hapA, hlyA, and rtxA, cleared the infection with no deaths and appeared to be in normal health after 20 h (Table 1). Mice in this group had neutrophils present in their lungs, indicating that the infection was recently cleared, rather than that KFV101 initially failed to colonize (Table 1). The infection was less efficiently cleared in KFV101-inoculated tlr4–/– mice, whose lungs were still colonized at 103 to 105 CFU, with only one of nine mice clearing the infection entirely (Table 1). However, the infection was less severe than in the P4-inoculated mice as shown by the 2-log-unit decrease in colonization of the lung, the failure of cells to disseminate to the spleen and liver, and the improved health rating to normal. In all, these data show that TLR4 is essential for early containment of the infection, both in the presence and absence of accessory toxins, and that accessory toxins affect the severity of the disease.
The production of accessory toxins is also important for increased lung weight. All mice inoculated with the ctxAB strain P4 showed increases in weight of the caudate lobe of the lung: tlr4+/+ mice showed a 1.9-fold increase in lung weight above that of PBS-inoculated control groups, while the tlr4–/– mice had a significantly greater 2.8-fold increase in lung weight. By contrast, KFV101-inoculated mice showed only a 10 to 30% increase compared to the lung weight of control mice, and lung weight was significantly reduced compared to that of P4-inoculated mice (Table 1). These data comparing P4- and KFV101-inoculated mice suggest that increased lung weight is probably due to toxin-specific damage to capillary endothelial cells, resulting in fluid leakage into the lung tissue.
Alternatively, an observed statistically significant reduction in neutrophil infiltration could account for the decreased lung weight in the KFV101-inoculated mice (Table 1). In addition, the presence of neutrophils is dependent on TLR4 since neutrophils are slightly reduced in the tlr4–/– mice inoculated with either P4 or KFV101. Still, all V. cholerae-inoculated groups showed both focal and diffuse inflammation. Indeed, some inflammation was observed in all mice even though TNF- titers in P4-inoculated tlr4–/– mice were sixfold reduced compared to those of P4-inoculated tlr4+/+ mice and below detectable limits in KFV101-inoculated tlr4–/– mice (Table 1). These data indicate that both TLR4 and accessory toxins contribute to neutrophil infiltration by 20 h p.i., although it is possible that effects on inflammation could be due to down-regulation of cytokine expression and clearance of neutrophils by this time point.
Progression of disease depends on both TLR4 and accessory toxins as early as 12 h after inoculation. To further investigate which host mediators and bacterial factors are essential for stimulation of the host innate immune system, tlr4+/+ and tlr4–/– mice were inoculated with 3 x 106 to 7 x 106 CFU of ctxAB strain P4 or the multitoxin mutant KFV101 and the mice were analyzed at 12 h for colonization, dissemination, and expression of proinflammatory cytokines. Lungs were also fixed in paraformaldehyde and stained for gross pathology changes. This experiment allows for comparison at the early stage of infection under a greater bacterial load, eliminating comparison to mice that have completely cleared the infection, as occurred in the 20-h experiments. Experiments terminated at 12 h were performed in triplicate with four to five mice per group, and data were pooled for analysis. No deaths were recorded prior to 12 h.
P4-inoculated tlr4+/+ mice and all KFV101-inoculated mice were colonized to similar levels 12 h p.i. (Fig. 1). P4-inoculated tlr4–/– mice were colonized more heavily, showing a threefold expansion from the inoculum and a 1-log-unit increase in lung colonization compared to levels in other inoculation groups (Fig. 1). These data demonstrate that the loss of TLR4 does impact early containment of the infection such that tlr4+/+ mice are colonized to a lesser extent at 20 h than tlr4–/– mice (Table 1).
Even though all KFV101-inoculated mice are colonized at 12 h, there is a significant difference in the progressions of disease between P4- and KFV101-inoculated mice at this early time point, suggesting that accessory toxins are important early in the infectious process. This difference was apparent in the improvement in KFV101-inoculated mice compared to P4-inoculated mice in their health scores, lung weights, and failure of the infection to disseminate to the spleen and liver (Table 2 and Fig. 1).
Accessory toxins, but not TLR4, are associated with epithelial damage. All of these differences between P4- and KFV101-inoculated mice could be related to a decrease in epithelial damage, as previously observed in BALB/c mice (8). As shown in Fig. 2, extensive epithelial damage is observed in the P4-inoculated mice, including patchy denudation of the bronchiolar respiratory epithelium in both tlr4+/+ and tlr4–/– mice. Remaining epithelial cells are cuboidal, with gaps between the normally columnar cells. Higher magnification revealed loss of cilia from cells (data not shown). These changes are less dramatic in the KFV101-inoculated mice, in which the epithelial cells remained tightly packed and morphologically unaltered. Under higher magnification, neutrophils were observed on the apically exposed surface despite an intact epithelial barrier, demonstrating that the damage observed in P4-inoculated mice was not caused by movement of neutrophils to the bronchiolar spaces (data not shown). No quantifiable difference in epithelial damage was noted to be dependent upon the expression of TLR4 (Table 2).
The production of the proinflammatory cytokines TNF- and IL-1 depends on TLR4. The differences in epithelial damage as well as the detection of LPS could lead to the development of inflammation in the lung through induction of proinflammatory cytokines. The homogenized caudate lobe of the lung was used to measure the presence of the locally released cytokines TNF-, IL-1, and IL-12 in response to inoculation with V. cholerae. IL-12 p70 was not present in any of the preserved lung homogenates (data not shown). However, all mice inoculated with V. cholerae had increased titers of TNF- and IL-1 over control mice, with differences in titer being dependent upon whether TLR4 was present. As shown in Fig. 3, both P4 and KFV101 stimulated the production of TNF- in tlr4+/+ mice, demonstrating that accessory toxins are not involved in the stimulation of TNF-, consistent with previous results with BALB/c mice (8). The titer of IL-1 also did not vary depending upon accessory toxins (Fig. 3). Unlike results from the 20-h experiments with C3H mice (Table 1), there was no statistically significant difference in the titers of TNF- between P4- and KFV101-inoculated mice, indicating that the observed fivefold difference at 20 h likely results from a down-regulation of synthesis once bacteria are cleared.
In all, these data demonstrate that signaling through TLR4 is important for an increased production of TNF- and IL-1. However, even in the absence of TLR4, both cytokines are expressed above control levels, indicating that detection of LPS and signaling through TLR4 are not the sole pathways for the stimulation of inflammation in response to V. cholerae.
Lack of a detectable difference in inflammation among groups of mice. Even though there were differences in levels of production of proinflammatory cytokines among the groups, no observable or quantifiable differences in inflammation were observed in the lungs of mice at 12 h. Bronchiole/peribronchiole and alveolar inflammation tended to be patchy, resulting in large standard deviations when the numbers of neutrophils per x100 field were counted in either contiguous (data not shown) or randomly selected fields (Table 2). Semiquantitative assessment of inflammation (on a scale of 0 to 4) did not reveal any obvious changes in the distribution of inflammation (Table 2 and Fig. 4). Even though levels of neutrophil infiltration and inflammation are similar between all groups, a distinct increase in lung weight is observed in the P4-inoculated mice compared to that in the KFV101-inoculated mice. Thus, it is clear that fluid accumulation due to damaged endothelial cells, not differences in neutrophil infiltration, as considered in the 20-h experiment, is the likely cause of the observed increases in lung weight in P4-inoculated mice.
In all, these data indicate that either other cytokines and/or chemokines are key to the recruitment of neutrophils or the low levels detected in the tlr4–/– mice are sufficient to induce optimal neutrophil recruitment.
IL-6 production in lung and serum depends on accessory toxins, not TLR4. To further assess what might signal inflammation, even in the absence of TLR4, another proinflammatory cytokine, IL-6, was measured. In contrast to results with TNF- and IL-1, IL-6 levels were elevated in the lung tissues of P4-inoculated mice, with no difference being dependent upon TLR4 (Fig. 3). In addition, IL-6 titers were 10- and 16-fold reduced in the KFV101-inoculated tlr4+/+ and tlr4–/– mice, respectively. These data argue that expression of IL-6 is only marginally dependent upon signaling through TLR4 and is instead correlated with the presence of toxins. In addition, detection of IL-6 in serum was dependent upon the presence of toxins, as previously determined (8; data not shown). The accessory-toxin-dependent release of chemokine MIP-2 to serum (8) also did not vary in titer, depending upon TLR4, even though TNF- and IL-1 act on endothelial cells to stimulate the release of this neutrophil-recruiting chemokine, further indicating that other signaling pathways stimulate neutrophil recruitment (data not shown).
These data indicate that IL-6, a proinflammatory cytokine, is produced independently of TLR4 and the detection of LPS, even though IL-6 is known to be induced by LPS (6). Therefore, it seems reasonable that this cytokine is induced by tissue damage and the subsequent presence of bacteria within the bloodstream. Alternatively, it is possible that one of the accessory toxins is directly responsible for the stimulation of the production of IL-6.
Discussion. In this study, we have shown in agreement with our previous study that accessory toxins lead to the development of epithelial tissue damage and subsequent dissemination to the spleen and liver. In addition, the toxins apparently enhance a fluid leakage from blood vessels and capillaries that is not correlated with increased neutrophil infiltration. By all measurements, KFV101 is less virulent in this model, and in the presence of an intact immune system, the infection is cleared with no observable detriment to animal health at either 12 or 20 h.
However, measurements at 12 h reveal that KFV101 stimulated key innate immune effectors similar to what occurred with the accessory-toxin-producing strain and inflammation developed. This engagement of the immune system resulted in the eventual clearance of the bacterial infection by 20 h. Thus, KFV101 does stimulate innate immunity resulting in bacterial clearance without causing the severe pathology associated with the accessory toxins.
With some variables measured, TLR4 plays an important role in the stimulation of innate immunity. Most obviously, in the absence of TLR4, the bacterial infection was not contained as well; thus, bacteria were recovered from the lung in 10- to 100-fold-greater numbers of CFU. These data are similar to those of a study that found that tlr4–/– mice are more heavily colonized than tlr4+/+ mice after intranasal inoculation with the gram-negative pathogen Klebsiella pneumoniae and the gram-positive pathogen Streptococcus pneumoniae (3). In our V. cholerae infections, a similar failure to contain the infection occurred despite no visually observable difference in the levels of recruitment of neutrophils dependent upon the absence of TLR4. Furthermore, recruitment of macrophages is not apparent in the histological examination, eliminating the potential hypothesis that TLR4-dependent activation of macrophages is a critical factor. Rather, it seems likely that, in the absence of TLR4, inflammation is signaled by other stimulatory pathways, especially the potential activation of TLR5 by V. cholerae flagella and detection of tissue damage. These pathways could lead to the stimulation of IL-6 and MIP-2 and residual titers of TNF- or IL-1 in the tlr4–/– mice. However, the rates of neutrophil recruitment prior to 12 h could differ, accounting for the failure of the tlr4–/– mice to control the infection when inoculated with the ctxAB strain P4.
This inability to control the infection in P4-inoculated tlr4–/– mice is likely due to the significant decrease in the production of TNF- or IL-1, even though these cytokines were still produced in the absence of TLR4, albeit at much lower titers. These observations are distinct from those reported for intranasal infection with K. pneumoniae. C3H/HeJ mice inoculated with this gram-negative pathogen showed no observable difference in TNF- production but did have a 65% increase in IL-6 compared to the level in wild-type C3H/HeN mice (3). Thus, both V. cholerae and K. pneumoniae stimulate proinflammatory responses by multiple pathways, but the levels of importance of TLR4 in signaling are distinct between the two pathogens. Taken together, these data suggest that each gram-negative bacterium may engage the innate immune system slightly differently, even within the now well-characterized paradigm of TLR4 detection of LPS.
This study supports our hypothesis that a multitoxin mutant of V. cholerae could be developed as a safe live attenuated vaccine. The multitoxin mutant KFV101 does not cause extensive damage to epithelial layers but does stimulate the innate immune system in a classical fashion, through multiple pathways both dependent on and independent of TLR4.
Although these results were obtained through lung infection, they are likely to apply to the infection of a gut mucosal layer as well. Studies of infant mice revealed preferential colonization of the distal small intestine by V. cholerae (1), and similar results have been shown for oral infection of rabbits (K.J.F.S, unpublished results). Within the distal small intestine, TLR4 has been detected by various methods, including reverse transcription-PCR, Northern blotting, Western blotting, and immunohistochemistry to be apically expressed by intestinal epithelial cells, particularly in cells that line the crypts (18). Further investigation of whether KFV101 or similar strains could be used as a vaccine during a gut infection will require additional analysis using oral inoculation in either the rabbit oral inoculation or human volunteer models. However, preliminary assessment using the lung model is apparently useful to validate strains prior to initiation of more-complex trials.
ACKNOWLEDGMENTS
We thank Yanping Tan and Brian Meehan for technical assistance. Stephen Miller and Neil Clipstone are thanked for their scientific input.
This work was supported by a Biomedical Research Support Program Award from the Howard Hughes Medical Institute to K.J.F.S. and Public Health Services grant AI051490 from the National Institute for Allergy and Infectious Diseases.
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Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois
ABSTRACT
Following intranasal inoculation, Vibrio cholerae KFV101 (ctxAB hapA hlyA rtxA) colonizes and stimulates tumor necrosis factor alpha and interleukin 1 (IL-1) in mice, similar to what occurs with isogenic strain P4 (ctxAB), but is less virulent and stimulates reduced levels of IL-6, demonstrating a role for accessory toxins in pathogenesis. Morbidity is enhanced in C3H/HeJ mice, indicating that Toll-like receptor 4 is important for infection containment.
TEXT
Vibrio cholerae is a gram-negative pathogen that induces diarrhea dependent upon the action of the cyclic AMP-stimulating cholera toxin (CT) (11). During cholera disease, patients in the acute phase do not have extensive tissue damage such as might be observed with shigellosis (21, 22). However, low levels of infiltration of neutrophils, mast cells, and eosinophils in infection and the presence of mediators of inflammatory cell regulation on the second day of cholera disease indicate that V. cholerae stimulates a proinflammatory response early during infection (21). This inflammatory response possibly becomes excessive in the absence of genes for CT since volunteers given CT-deficient strains have significantly increased titers of lactoferrin and CXCL8, indicative of more severely inflammatory disease typified by symptoms that include vomiting, fever, mild diarrhea, and cramping (25). These symptoms are also typical of nonepidemic strains of V. cholerae that do not produce CT (4, 7, 10, 13, 16, 17). This increase in the inflammatory nature of V. cholerae infection could be due to the absence of the immunomodulatory activity of the B subunit of CT that blocks the secretion of proinflammatory cytokines by macrophages, dendritic cells, and epithelial cells in response to bacterial lipopolysaccharide (LPS) (24) due to downregulation of mitogen-activated protein kinase pathways (6).
A major problem with this residual disease of non-CT-producing V. cholerae is that these strains are frequently sufficiently virulent to make them unsafe for use as live attenuated vaccines (2, 14, 26, 27). It has been proposed that the actions of accessory toxins of V. cholerae are responsible for this increased inflammation and contribute to the reactogenicity of live attenuated vaccine strains (24). Recently, we developed a murine model that is sensitive to accessory toxins and can be used to investigate early events in the initiation of the host response to non-CT-producing V. cholerae. Although adult mice are resistant to intestinal colonization by V. cholerae (12), we found that acute inflammation develops in the lung tissue after intranasal inoculation with V. cholerae, providing a model for the assessment of the toxic action on inflammation of a mucosal epithelial layer (8). This model is based on experiments using Shigella flexneri dependent on the premise that the bronchial tree is composed of a mucosal lining with relevant characteristics similar to those of the intestine, including simple cubodial-to-columnar epithelial cells, lymphoid aggregates, bronchiole-associated lymphoid tissue similar to Peyer's patches, and the presence of several varieties of antigen-presenting cells (5, 19, 23, 28).
Using this new murine model, we previously found that wild-type CT-producing strains of V. cholerae induce a rapid cell death due to fluid accumulation in the lung but that a strain deleted of the genes for CT stimulates proinflammatory cytokine and chemokine production, leading to development of a lethal pneumonia characterized by infiltration of neutrophils, hemorrhages, and bacteremia (8). When mice were inoculated with a multitoxin mutant strain with deletions of genes for the accessory toxins hemagglutinin/protease (hapA), hemolysin (hlyA), and the actin cross-linking RTX toxin (rtxA), in addition to deletions of genes for CT (ctxAB), the mice did not develop severe pneumonia and cleared the infection. In this prior study, 100% of mice inoculated with this multitoxin mutant strain survived when inoculated at a dose that resulted in 80% lethality with the CT-deficient strain (8).
These results suggest that multitoxin mutant strains could be excellent candidates for safe, live attenuated vaccine. However, it is possible that in the absence of inflammation due to secreted toxins, a protective response will also not develop. We hypothesize that LPS and other bacterial factors will stimulate host immunity in the absence of accessory toxins. To demonstrate that V. cholerae stimulates the development of an innate immune response in response to LPS, we inoculated mice with a mutation in Toll-like receptor 4 (TLR4) and assessed if the absence of this signaling pathway would affect bacterial clearance and induction of proinflammatory mediators in both the presence and absence of accessory toxins. These data demonstrate that a multitoxin mutant of V. cholerae has inflammatory potential that is based in part on the activation of TLR4; yet, the strain is less virulent and is cleared more rapidly than a strain that produces the accessory toxins.
Intranasal inoculation of C3Heb/FeJ (tlr4+/+) and C3H/HeJ (tlr4–/–) mice. V. cholerae strains are derivatives of the streptomycin-resistant isolate of El Tor O1 P27459 originally isolated in Bangladesh (15). Strain P4 (also known as SM44) has a kanamycin cassette replacing the genes for ctxA and ctxB in P27459, creating the CT-deficient strain (9). Strain KFV101 is a derivative of P4 with unmarked deletions within the genes hapA, hlyA, and rtxA, creating a multitoxin mutant (8). Bacteria were grown on Luria agar supplemented with 50 μg/ml kanamycin and 100 μg/ml streptomycin. For inoculation to mice, bacterial cultures were grown at 30°C overnight in Luria broth supplemented with antibiotics. Bacteria were washed in phosphate-buffered saline, pH 7.4 (PBS), and then diluted to the desired number of CFU per milliliter for inoculation.
Inoculations were then performed according to protocols approved by the Northwestern University IACUC. Five- to 6-week-old C3Heb/FeJ (tlr4+/+) and C3H/HeJ (tlr4–/–) female mice (Jackson Labs, Bar Harbor, ME) were inoculated intranasally with 20 μl of a V. cholerae suspension while under anesthesia with 60 to 75 mg/kg ketamine and 12 to 15 mg/kg xylazine, as previously detailed (8). At 12 or 20 h after inoculation, mice were exsanguinated by cardiac puncture and euthanized. The caudate lobe of the lung, the spleen, and the liver were weighed and then homogenized in 5 ml LB medium, and dilutions were plated for recovered CFU. Total numbers of CFU per organ were calculated by multiplying the number of CFU/ml by 5 ml. The number of CFU in the lung was then multiplied by 2 to adjust for homogenization of only one lobe. One milliliter of undiluted lung homogenate was preserved at –80°C. At a later time, titers of tumor necrosis factor alpha (TNF-), interleukin 1 (IL-1), IL-6, and IL-12 p70 in the clarified lung homogenates were determined by enzyme-linked immunosorbent assay (ELISA) using Quantikine kits (R & D Systems, Minneapolis, MN) according to manufacturer's protocol. Values reported as nanograms per lung were calculated as nanograms per milliliter and multiplied by 5 ml homogenate and by 2 to adjust for homogenization of only one lobe. Values were not calculated per gram of lung since lung weight changed up to 300% due to fluid accumulation in some groups, while lung weight among mock-inoculated mice varied by less than 20%.
The remaining lung lobes were perfused with 4% paraformaldehyde for preservation and later were mounted in paraffin, sectioned, and stained with hematoxylin and eosin (H/E) at the Robert Lurie Cancer Center Core Facility for Histopathology. Slides were coded and then scored by a single pathologist (G.K.H.) for the degrees of bronchiole per peribronchiole inflammation, alveolar inflammation, and respiratory epithelial damage (on a scale of 0 to 4). Neutrophilic inflammation was quantified by counting the number of neutrophils in four randomly selected high-power fields, using an Olympus BX40 microscope with an Olympus DP12 digital camera.
Experiments were done with four to five animals per group. Data from duplicate or triplicate experiments were pooled and analyzed using Microsoft Excel X for Macintosh computers and a two-sample t test assuming unequal variances. P values were considered significant at a P of <0.05 for quantitative data (numbers of CFU, lung weight, and ELISA results) and a P of <0.005 for semiquantitative data (health score and histopathology).
Clearance of infection and reduced morbidity at 20 h depend upon TLR4 and production of accessory toxins. C3H/HeJ mice have a point mutation in the tlr4 gene that leads to a defective response to bacterial endotoxin, rendering the animals resistant to LPS-induced shock (20). Previously, we demonstrated that BALB/c mice succumbed to infection with the ctxAB mutant P4 between 24 and 72 h when inoculated with 107 CFU/ml (8). At this same dosage, three of four P4-inoculated tlr4+/+ mice and all P4-inoculated tlr4–/– mice died within 18 h. The lung, spleen, and liver of the sole surviving P4-inoculated tlr4+/+ mouse were colonized; the mouse was in moderate health, with fluid in the lungs, and had a high titer of TNF- and dense neutrophil infiltration in its lung tissue (data not shown). Data from this pilot study suggest that C3H inbred mice may be more susceptible to this bacterial infection than the previously used BALB/c mice. Therefore, mice for these series of experiments were inoculated with a dosage of 1 x 106 CFU for 20-h experiments and 3 x 106 to 7 x 106 CFU for 12-h experiments to ensure sufficient survivors for analysis.
For experiments terminated at 20 h, two experiments with four to nine mice per group were performed. At a dosage of 106 CFU, all nine P4-inoculated tlr4+/+ mice survived and were colonized with an average of 3,800 CFU in their lungs, with dissemination to the liver occurring in only four of nine mice and to the spleen in none of the mice (Table 1). By comparison, 4 of 13 P4-inoculated tlr4–/– mice died in less than 18 h, while the remaining 9 survivors were heavily colonized with an average of 3.3 x 106 CFU, a 3-fold increase over the inoculum and a 100-fold increase over the level in tlr4+/+ mice. These nine surviving mice also had dissemination of the infection to their livers and spleens (Table 1). In addition to a difference in colonization levels, tlr4–/– mice appeared to be in worse health, as shown by the decrease in the health rating from near normal to poor or near death. These data demonstrate the importance of the TLR4 signaling pathway to containment of infection to the point that the infection clears in the tlr4+/+ mice by 20 h postinoculation (p.i.) but expands in the tlr4–/– mice.
In contrast to the mice inoculated with the ctxAB strain P4, mice inoculated with KFV101, a derivative of P4 with additional deletions in the accessory toxin genes hapA, hlyA, and rtxA, cleared the infection with no deaths and appeared to be in normal health after 20 h (Table 1). Mice in this group had neutrophils present in their lungs, indicating that the infection was recently cleared, rather than that KFV101 initially failed to colonize (Table 1). The infection was less efficiently cleared in KFV101-inoculated tlr4–/– mice, whose lungs were still colonized at 103 to 105 CFU, with only one of nine mice clearing the infection entirely (Table 1). However, the infection was less severe than in the P4-inoculated mice as shown by the 2-log-unit decrease in colonization of the lung, the failure of cells to disseminate to the spleen and liver, and the improved health rating to normal. In all, these data show that TLR4 is essential for early containment of the infection, both in the presence and absence of accessory toxins, and that accessory toxins affect the severity of the disease.
The production of accessory toxins is also important for increased lung weight. All mice inoculated with the ctxAB strain P4 showed increases in weight of the caudate lobe of the lung: tlr4+/+ mice showed a 1.9-fold increase in lung weight above that of PBS-inoculated control groups, while the tlr4–/– mice had a significantly greater 2.8-fold increase in lung weight. By contrast, KFV101-inoculated mice showed only a 10 to 30% increase compared to the lung weight of control mice, and lung weight was significantly reduced compared to that of P4-inoculated mice (Table 1). These data comparing P4- and KFV101-inoculated mice suggest that increased lung weight is probably due to toxin-specific damage to capillary endothelial cells, resulting in fluid leakage into the lung tissue.
Alternatively, an observed statistically significant reduction in neutrophil infiltration could account for the decreased lung weight in the KFV101-inoculated mice (Table 1). In addition, the presence of neutrophils is dependent on TLR4 since neutrophils are slightly reduced in the tlr4–/– mice inoculated with either P4 or KFV101. Still, all V. cholerae-inoculated groups showed both focal and diffuse inflammation. Indeed, some inflammation was observed in all mice even though TNF- titers in P4-inoculated tlr4–/– mice were sixfold reduced compared to those of P4-inoculated tlr4+/+ mice and below detectable limits in KFV101-inoculated tlr4–/– mice (Table 1). These data indicate that both TLR4 and accessory toxins contribute to neutrophil infiltration by 20 h p.i., although it is possible that effects on inflammation could be due to down-regulation of cytokine expression and clearance of neutrophils by this time point.
Progression of disease depends on both TLR4 and accessory toxins as early as 12 h after inoculation. To further investigate which host mediators and bacterial factors are essential for stimulation of the host innate immune system, tlr4+/+ and tlr4–/– mice were inoculated with 3 x 106 to 7 x 106 CFU of ctxAB strain P4 or the multitoxin mutant KFV101 and the mice were analyzed at 12 h for colonization, dissemination, and expression of proinflammatory cytokines. Lungs were also fixed in paraformaldehyde and stained for gross pathology changes. This experiment allows for comparison at the early stage of infection under a greater bacterial load, eliminating comparison to mice that have completely cleared the infection, as occurred in the 20-h experiments. Experiments terminated at 12 h were performed in triplicate with four to five mice per group, and data were pooled for analysis. No deaths were recorded prior to 12 h.
P4-inoculated tlr4+/+ mice and all KFV101-inoculated mice were colonized to similar levels 12 h p.i. (Fig. 1). P4-inoculated tlr4–/– mice were colonized more heavily, showing a threefold expansion from the inoculum and a 1-log-unit increase in lung colonization compared to levels in other inoculation groups (Fig. 1). These data demonstrate that the loss of TLR4 does impact early containment of the infection such that tlr4+/+ mice are colonized to a lesser extent at 20 h than tlr4–/– mice (Table 1).
Even though all KFV101-inoculated mice are colonized at 12 h, there is a significant difference in the progressions of disease between P4- and KFV101-inoculated mice at this early time point, suggesting that accessory toxins are important early in the infectious process. This difference was apparent in the improvement in KFV101-inoculated mice compared to P4-inoculated mice in their health scores, lung weights, and failure of the infection to disseminate to the spleen and liver (Table 2 and Fig. 1).
Accessory toxins, but not TLR4, are associated with epithelial damage. All of these differences between P4- and KFV101-inoculated mice could be related to a decrease in epithelial damage, as previously observed in BALB/c mice (8). As shown in Fig. 2, extensive epithelial damage is observed in the P4-inoculated mice, including patchy denudation of the bronchiolar respiratory epithelium in both tlr4+/+ and tlr4–/– mice. Remaining epithelial cells are cuboidal, with gaps between the normally columnar cells. Higher magnification revealed loss of cilia from cells (data not shown). These changes are less dramatic in the KFV101-inoculated mice, in which the epithelial cells remained tightly packed and morphologically unaltered. Under higher magnification, neutrophils were observed on the apically exposed surface despite an intact epithelial barrier, demonstrating that the damage observed in P4-inoculated mice was not caused by movement of neutrophils to the bronchiolar spaces (data not shown). No quantifiable difference in epithelial damage was noted to be dependent upon the expression of TLR4 (Table 2).
The production of the proinflammatory cytokines TNF- and IL-1 depends on TLR4. The differences in epithelial damage as well as the detection of LPS could lead to the development of inflammation in the lung through induction of proinflammatory cytokines. The homogenized caudate lobe of the lung was used to measure the presence of the locally released cytokines TNF-, IL-1, and IL-12 in response to inoculation with V. cholerae. IL-12 p70 was not present in any of the preserved lung homogenates (data not shown). However, all mice inoculated with V. cholerae had increased titers of TNF- and IL-1 over control mice, with differences in titer being dependent upon whether TLR4 was present. As shown in Fig. 3, both P4 and KFV101 stimulated the production of TNF- in tlr4+/+ mice, demonstrating that accessory toxins are not involved in the stimulation of TNF-, consistent with previous results with BALB/c mice (8). The titer of IL-1 also did not vary depending upon accessory toxins (Fig. 3). Unlike results from the 20-h experiments with C3H mice (Table 1), there was no statistically significant difference in the titers of TNF- between P4- and KFV101-inoculated mice, indicating that the observed fivefold difference at 20 h likely results from a down-regulation of synthesis once bacteria are cleared.
In all, these data demonstrate that signaling through TLR4 is important for an increased production of TNF- and IL-1. However, even in the absence of TLR4, both cytokines are expressed above control levels, indicating that detection of LPS and signaling through TLR4 are not the sole pathways for the stimulation of inflammation in response to V. cholerae.
Lack of a detectable difference in inflammation among groups of mice. Even though there were differences in levels of production of proinflammatory cytokines among the groups, no observable or quantifiable differences in inflammation were observed in the lungs of mice at 12 h. Bronchiole/peribronchiole and alveolar inflammation tended to be patchy, resulting in large standard deviations when the numbers of neutrophils per x100 field were counted in either contiguous (data not shown) or randomly selected fields (Table 2). Semiquantitative assessment of inflammation (on a scale of 0 to 4) did not reveal any obvious changes in the distribution of inflammation (Table 2 and Fig. 4). Even though levels of neutrophil infiltration and inflammation are similar between all groups, a distinct increase in lung weight is observed in the P4-inoculated mice compared to that in the KFV101-inoculated mice. Thus, it is clear that fluid accumulation due to damaged endothelial cells, not differences in neutrophil infiltration, as considered in the 20-h experiment, is the likely cause of the observed increases in lung weight in P4-inoculated mice.
In all, these data indicate that either other cytokines and/or chemokines are key to the recruitment of neutrophils or the low levels detected in the tlr4–/– mice are sufficient to induce optimal neutrophil recruitment.
IL-6 production in lung and serum depends on accessory toxins, not TLR4. To further assess what might signal inflammation, even in the absence of TLR4, another proinflammatory cytokine, IL-6, was measured. In contrast to results with TNF- and IL-1, IL-6 levels were elevated in the lung tissues of P4-inoculated mice, with no difference being dependent upon TLR4 (Fig. 3). In addition, IL-6 titers were 10- and 16-fold reduced in the KFV101-inoculated tlr4+/+ and tlr4–/– mice, respectively. These data argue that expression of IL-6 is only marginally dependent upon signaling through TLR4 and is instead correlated with the presence of toxins. In addition, detection of IL-6 in serum was dependent upon the presence of toxins, as previously determined (8; data not shown). The accessory-toxin-dependent release of chemokine MIP-2 to serum (8) also did not vary in titer, depending upon TLR4, even though TNF- and IL-1 act on endothelial cells to stimulate the release of this neutrophil-recruiting chemokine, further indicating that other signaling pathways stimulate neutrophil recruitment (data not shown).
These data indicate that IL-6, a proinflammatory cytokine, is produced independently of TLR4 and the detection of LPS, even though IL-6 is known to be induced by LPS (6). Therefore, it seems reasonable that this cytokine is induced by tissue damage and the subsequent presence of bacteria within the bloodstream. Alternatively, it is possible that one of the accessory toxins is directly responsible for the stimulation of the production of IL-6.
Discussion. In this study, we have shown in agreement with our previous study that accessory toxins lead to the development of epithelial tissue damage and subsequent dissemination to the spleen and liver. In addition, the toxins apparently enhance a fluid leakage from blood vessels and capillaries that is not correlated with increased neutrophil infiltration. By all measurements, KFV101 is less virulent in this model, and in the presence of an intact immune system, the infection is cleared with no observable detriment to animal health at either 12 or 20 h.
However, measurements at 12 h reveal that KFV101 stimulated key innate immune effectors similar to what occurred with the accessory-toxin-producing strain and inflammation developed. This engagement of the immune system resulted in the eventual clearance of the bacterial infection by 20 h. Thus, KFV101 does stimulate innate immunity resulting in bacterial clearance without causing the severe pathology associated with the accessory toxins.
With some variables measured, TLR4 plays an important role in the stimulation of innate immunity. Most obviously, in the absence of TLR4, the bacterial infection was not contained as well; thus, bacteria were recovered from the lung in 10- to 100-fold-greater numbers of CFU. These data are similar to those of a study that found that tlr4–/– mice are more heavily colonized than tlr4+/+ mice after intranasal inoculation with the gram-negative pathogen Klebsiella pneumoniae and the gram-positive pathogen Streptococcus pneumoniae (3). In our V. cholerae infections, a similar failure to contain the infection occurred despite no visually observable difference in the levels of recruitment of neutrophils dependent upon the absence of TLR4. Furthermore, recruitment of macrophages is not apparent in the histological examination, eliminating the potential hypothesis that TLR4-dependent activation of macrophages is a critical factor. Rather, it seems likely that, in the absence of TLR4, inflammation is signaled by other stimulatory pathways, especially the potential activation of TLR5 by V. cholerae flagella and detection of tissue damage. These pathways could lead to the stimulation of IL-6 and MIP-2 and residual titers of TNF- or IL-1 in the tlr4–/– mice. However, the rates of neutrophil recruitment prior to 12 h could differ, accounting for the failure of the tlr4–/– mice to control the infection when inoculated with the ctxAB strain P4.
This inability to control the infection in P4-inoculated tlr4–/– mice is likely due to the significant decrease in the production of TNF- or IL-1, even though these cytokines were still produced in the absence of TLR4, albeit at much lower titers. These observations are distinct from those reported for intranasal infection with K. pneumoniae. C3H/HeJ mice inoculated with this gram-negative pathogen showed no observable difference in TNF- production but did have a 65% increase in IL-6 compared to the level in wild-type C3H/HeN mice (3). Thus, both V. cholerae and K. pneumoniae stimulate proinflammatory responses by multiple pathways, but the levels of importance of TLR4 in signaling are distinct between the two pathogens. Taken together, these data suggest that each gram-negative bacterium may engage the innate immune system slightly differently, even within the now well-characterized paradigm of TLR4 detection of LPS.
This study supports our hypothesis that a multitoxin mutant of V. cholerae could be developed as a safe live attenuated vaccine. The multitoxin mutant KFV101 does not cause extensive damage to epithelial layers but does stimulate the innate immune system in a classical fashion, through multiple pathways both dependent on and independent of TLR4.
Although these results were obtained through lung infection, they are likely to apply to the infection of a gut mucosal layer as well. Studies of infant mice revealed preferential colonization of the distal small intestine by V. cholerae (1), and similar results have been shown for oral infection of rabbits (K.J.F.S, unpublished results). Within the distal small intestine, TLR4 has been detected by various methods, including reverse transcription-PCR, Northern blotting, Western blotting, and immunohistochemistry to be apically expressed by intestinal epithelial cells, particularly in cells that line the crypts (18). Further investigation of whether KFV101 or similar strains could be used as a vaccine during a gut infection will require additional analysis using oral inoculation in either the rabbit oral inoculation or human volunteer models. However, preliminary assessment using the lung model is apparently useful to validate strains prior to initiation of more-complex trials.
ACKNOWLEDGMENTS
We thank Yanping Tan and Brian Meehan for technical assistance. Stephen Miller and Neil Clipstone are thanked for their scientific input.
This work was supported by a Biomedical Research Support Program Award from the Howard Hughes Medical Institute to K.J.F.S. and Public Health Services grant AI051490 from the National Institute for Allergy and Infectious Diseases.
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