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Infection and Immunity, May 2006, p. 2522-2536, Vol. 74, No. 5
0019-9567/06/$08.00+0 doi:10.1128/IAI.74.5.2522-2536.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Toll-Like Receptor 4 Contributes to Colitis Development but Not to Host Defense during Citrobacter rodentium Infection in Mice
Mohammed A. Khan,1,
Caixia Ma,1,
Leigh A. Knodler,2,
Yanet Valdez,1,2
Carrie M. Rosenberger,3
Wanyin Deng,2
B. Brett Finlay,2 and
Bruce A. Vallance1*
Division of Gastroenterology, BC's Children's Hospital,1 Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada,2 Institute for Systems Biology, Seattle, Washington3
Received 1 August 2005/ Returned for modification 28 October 2005/ Accepted 20 January 2006
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Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are noninvasive bacterial pathogens that infect their hosts' intestinal epithelium, causing severe diarrheal disease. These infections also cause intestinal inflammation, although the mechanisms underlying the inflammatory response, as well as its potential role in host defense, are unclear. Since these bacteria are gram-negative, Toll-like receptor 4 (TLR4), the innate receptor for bacterial lipopolysaccharide may contribute to the host response; however, the role of TLR4 in the gastrointestinal tract is poorly understood, and its impact has yet to be tested against this family of enteric bacterial pathogens. Since EPEC and EHEC are human specific, we infected mice with Citrobacter rodentium, a mouse-adapted attaching and effacing (A/E) bacterium that infects colonic epithelial cells, causing colitis and epithelial hyperplasia, using a similar array of virulence proteins as EPEC and EHEC. We demonstrated that C. rodentium activates TLR4 and rapidly induced NF-
B nuclear translocation in host cells in a partially TLR4-dependent manner. Infection of TLR4-deficient mice revealed that TLR4-dependent responses mediate much of the inflammation and tissue pathology seen during infection, including the induction of the chemokines MIP-2 and MCP-1, as well as the recruitment of macrophages and neutrophils into the infected intestine. Surprisingly, spread of C. rodentium through the colon was delayed in TLR4-deficient mice, whereas the duration of the infection was unaffected, indicating that TLR4-mediated responses against this A/E pathogen are not host protective and are ultimately maladaptive to the host, contributing to both the morbidity and the pathology seen during infection. |
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Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are among the most important bacterial causes of diarrheal disease worldwide (15, 35). EPEC infection leads to the deaths of hundreds of thousands of children each year in developing countries (35, 56), while EHEC, the causal agent of "hamburger disease," is a growing health threat in North America and Europe, causing sporadic diarrheal outbreaks and deaths (21, 29). Belonging to a family of related gram-negative pathogenic bacteria that includes the mouse pathogen Citrobacter rodentium (30), these bacteria are noninvasive, infecting their hosts by attaching to the surface of intestinal epithelial cells, effacing the epithelial microvilli and producing pedestal-like structures (3, 4, 12, 30). The formation of attaching and effacing (A/E) lesions correlates with the ability of these microbes to cause diarrheal disease (1, 14); thus, much work has been done to characterize the virulence mechanisms used by A/E bacteria. These studies have revealed a series of elegant strategies used by A/E bacteria to infect their hosts (16, 21, 56). Despite these advances, it is now becoming clear that the host also plays an important role in causing the tissue pathology and diarrheal disease seen during infection by these intestinal lumen-dwelling pathogens (10).
Recent reports indicate that EPEC and EHEC infections induce significant intestinal inflammation, with fecal leukocytes more frequently seen in patients infected with EHEC than in those infected with other enteric pathogens, including Salmonella and Shigella spp. (28, 35, 56). Similarly, studies examining colonic biopsies from patients infected with A/E pathogens reveal inflammatory cell infiltration into the intestinal lamina propria and crypts of infected tissues (19, 27, 28). Although the mechanisms involved in triggering this inflammatory response are unclear, they likely require the activation of the host's innate immune system. Innate immunity in multicellular organisms involves a group of pattern recognition receptors that detect and respond to conserved molecules expressed by pathogenic microorganisms (17). Toll-like receptors (TLRs) are a family of these innate receptors (34, 51), the best described of which is TLR4, which in association with the LBP (lipopolysaccharide [LPS]-binding protein), CD14, and MD-2 proteins, specifically triggers an inflammatory response to LPSderived from gram-negative bacteria (34). We and others have demonstrated that TLR4-dependent responses play a critical role in host defense against Salmonella enterica serovar Typhimurium, a gram-negative bacterium that causes a systemic disease in mice. TLR4 expression limited the spread of this microbe through the host's spleen, liver, and other internal organs (36, 57) and, similarly, TLR4 has proven important in providing protection against a variety of other gram-negative bacterial pathogens, including uropathogenic E. coli (47), as well as Bordetella and Brucella spp. (8, 32).
In contrast to the actions of TLR4 during systemic bacterial infections, the role of TLR4 in recognizing and responding to gram-negative bacterial infections of the gastrointestinal (GI) tract has not been examined. In part this reflects the lack of suitable models, since most clinically important enteric bacterial pathogens, including EPEC and EHEC, do not cause a relevant gastroenteritis in laboratory animal species (30). As a result, it is unclear what aspects of innate immunity are activated during A/E bacterial infections of the GI tract or what role they play in intestinal host defense. In fact, although innate responses usually trigger an inflammatory response that is toxic to pathogens, inflammation can also disrupt mucosal integrity (45), weakening physical barriers that protect the intestine from enteric pathogens. Several in vitro and in vivo studies have indicated that innate inflammatory responses may actually aid bacterial pathogens such as Shigella flexneri colonize their host's intestines by disrupting the intestinal epithelium that normally prevents these microbes from invading their hosts (37, 44, 45). Since EPEC and EHEC utilize infectious strategies different from Shigella, the impact of innate immunity on intestinal host defense against A/E bacterial pathogens remains an unresolved, yet important issue.
To investigate the role of TLR4 in an A/E bacterial infection, we chose the C. rodentium mouse model. An excellent paradigm for EPEC and EHEC infections, C. rodentium infects the colonic epithelium of mice, triggering colitis and mucosal hyperplasia (3, 4, 12, 30). C. rodentium colonizes epithelial cells using a homologous array of virulence proteins to those expressed by EPEC and EHEC and was recently used to identify new translocated effectors shared by these pathogens (13). We and others have also begun to assess what roles T and B lymphocytes (7, 48, 55), as well as the cytokines interleukin-12 (IL-12), gamma interferon (49), and tumor necrosis factor alpha (23), play in host defense and the inflammatory response to C. rodentium infection. Thus far, however, innate receptors have not been assessed in this model. In the present study, we compared the susceptibility of wild-type (WT) mice and TLR4-deficient mice to C. rodentium, studying their inflammatory responses, as well as the disease symptoms and pathology they suffered during infection. We found that TLR4 deficiency significantly reduced tissue pathology and inflammatory cell recruitment into the infected gut and, surprisingly, the absence of TLR4 also delayed the spread of C. rodentium through the mouse colon, suggesting that the innate inflammatory response may facilitate the ability of C. rodentium to colonize the distal colon. These studies thus introduce the novel concept that TLR4-dependent innate responses in the GI tract are not host protective but instead may prove maladaptive during infection by A/E bacteria, contributing to the resulting disease and facilitating the ability of these pathogens to infect and spread through their hosts' GI tracts.
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Mice. Six- to eight-week-old wild-type (WT), TLR4-expressing mice (C57BL/10J and C3H/HeOuJ) were obtained from Jackson Laboratories (Bar Harbor, Maine). Several TLR4-deficient strains were used in the present study, including the C3H/HeJ and C57BL/10ScNJ strains that were also obtained from Jackson, while C57BL/10ScNCr mice were purchased from the National Cancer Institute (Frederick, MD). Mice were kept in sterilized, filter-topped cages; handled in tissue culture hoods; and fed autoclaved food and water under specific-pathogen-free conditions at our animal facilities. Sentinel animals were routinely tested for common pathogens. The protocols used were approved by the University of British Columbia's Animal Care Committee and in direct accordance with guidelines drafted by the Canadian Council on the Use of Laboratory Animals.
Bacterial strains and infection of mice. Mice were orally inoculated with wild-type C. rodentium (formerly C. freundii biotype 4280), strain DBS100 (46). For inoculations, bacteria were grown overnight in Luria broth. Mice were infected by oral gavage using 0.1 ml of Luria broth containing ca. 2.5 x 108 CFU of C. rodentium. To minimize differences between infections, both WT and TLR4-deficient mouse strains were infected with the same bacterial preparation. Mice were euthanized at various time points postinfection (p.i.), and tissues were prepared for histological analysis, viable bacterial counts, or RNA isolation as described below.
Survival and body weight measurement. To assess the systemic effects of C. rodentium infection on the host, the survival of infected mice and changes in their body weight were measured over the course of infection. Mice were monitored throughout the infection, and any that showed extreme distress or became moribund were euthanized. Mice were also weighed just prior to infection and at specified intervals until day 24 p.i. The survival and body weight data presented are from a representative experiment (n = 3). Body weight data are presented as the mean percentage of the starting weight of 10 mice at each time point, whereas survival data are presented as the percentage of the initial 10 mice still surviving at each time point.
Tissue and stool collection and colon weight measurement. Over the course of the infection, mice were euthanized and, after careful dissection, the first 4 cm of each colon, beginning at the anal verge, was collected. Fecal pellets were removed before the colonic tissue was weighed. Tissues were then placed in 10% neutral buffered formalin (Fisher) for histological analysis. For viable bacterial count studies, the colons and pellets were collected in phosphate-buffered saline (PBS; pH 7.4) and kept on ice before being processed for viable bacterial counts. For studies on stool water content, the pellets were weighed immediately (wet weight) and then allowed to dry overnight in a 37°C dry oven. The next day the dried stool was weighed and, by subtracting the dry stool weight from the wet stool weight and dividing the result by the original wet stool weight, we determined the original water content of the stool.
Bacterial counts. Colonic tissues (plus any fecal stool pellets) or mesenteric lymph nodes (MLNs) were homogenized at low speed using a Kinematica tissue homogenizer (Brinkmann). Homogenates were then serially diluted and plated onto MacConkey agar plates. Bacterial colonies were enumerated the following day. C. rodentium colonies were easily distinguished from commensal flora by their size and appearance (pink center with white rim), as previously described (46). The validity of this approach was verified by PCR analysis for LEE genes.
Crypt heights and histopathological analysis. Full thickness colonic tissues were fixed in 10% neutral buffered formalin. Sections (3 µm) were cut and stained with hematoxylin and eosin. Photomicrographs were taken using a Nikon Eclipse E400 microscope. Crypt heights were measured by micrometry by an observer blinded to the experimental condition, with 10 measurements taken in the distal colon for each mouse. Only well-oriented crypts were measured. Similarly, colon sections were assessed for quantitative analysis of intestinal inflammation by a blinded observer, using a previously described scoring system (5). In brief, the system assessed submucosal edema, polymorphonuclear infiltration into the lamina propria, goblet cell depletion, and epithelial integrity. The combined pathological score ranged from 0 to 13 arbitrary units and covers the following levels of inflammation: 0 = intestine intact with no sign of inflammation; 1 to 2 = minimal signs of inflammation; 3 to 4 = slight inflammation; 5 to 8 = moderate inflammation; and 9 to 13 = profound inflammation.
TLR4 activation.
Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) were cultured at 37°C in a 5% CO2 incubator in Dulbecco modified Eagle medium containing 10% cosmic calf serum (HyClone, Logan, UT). The day prior to transfection, HEK293 cells were plated in a 96-well dish at 5 x 104 per well. Transient transfection was performed by using Polyfect reagent (QIAGEN, Chatsworth, CA). Cells were transfected with 150 ng of NF-B reporter (endothelial leukocyte molecule-1 [ELAM-1] firefly luciferase construct), 15 ng of thymidine kinase Renilla luciferase vector (pRL-TK; Promega, Madison, WI) to control for transfection efficiency, 20 ng of EF6 hTLR4, 100 ng of hMD-2, and 10 ng of hCD14, or a hTLR8 vector. The empty vector pEF6 was used as a control and to normalize the DNA concentrations for all transfections to 250 ng per well. At 20 h after transfection, the cells were stimulated with 10% supernatant from an overnight culture of C. rodentium in LB broth, 10 ng of Ultrapure LPS (Salmonella enterica serovar Minnesota R595; List Biological Laboratories, Campbell, CA)/ml, or LB alone. Cells were lysed after 4 h by using passive lysis buffer, and the luciferase activity was quantified by using the dual-luciferase reporter assay system (Promega). Firefly luciferase units were divided by Renilla units to normalize for transfection efficiency between wells.
Generation and infection of bone marrow-derived macrophages and CMT-93 epithelial cells: immunofluorescence studies.
Bone marrow was isolated from the femurs of C57BL/10ScNJ (TLR4-deficient) or C57BL/10J (WT) mice, cultured for 7 days in DMEM (HyClone) supplemented with 20% fetal bovine serum (Invitrogen)-2 mM L-glutamine-1 mM sodium pyruvate-30% L cell-conditioned medium at 37°C in 5% CO2. The bone marrow-derived macrophages (BMDM) were seeded in 24-well plates (105 cells per well). The BMDM, as well as CMT-93 murine colonic epithelial cells (106 cells per well), were infected with 10 µl of C. rodentium grown overnight in LB. At 1 to 4 h p.i., cells were fixed in 2.5% paraformaldehyde at 37°C for 10 min, followed by extensive washing in PBS. Fixed cells were then permeabilized in PBS containing 10% (vol/vol) goat serum and 0.3% Triton X-100 for 30 min at room temperature. Primary rat antibodies to TLR4/MD2 (MTS510 kindly provided by Kensuke Miyake) and a mouse monoclonal directed to the p65 subunit of NF-B (Santa Cruz Biotechnology), as well as Alexa-conjugated secondary antibodies (all at a 1:300 dilution) were applied in PBS—0.3% Triton X-100—5% goat serum for 1 h at room temperature. DAPI (4',6'-diamidino-2-phenylindole; Sigma) was used at 1 µg/ml to stain both host cell and bacterial DNA. Coverslips were mounted in Mowiol mounting medium (Aldrich) and viewed at 350, 488, and 594 nm with a Nikon microscope.
Immunofluorescence staining of colon tissues. Immunofluorescence staining of control and infected tissues was performed in a fashion similar to that described previously (14, 55). In brief, tissues were rinsed in ice-cold PBS, embedded in optimal cutting template compound (OCT; Sakura, Finetech), frozen with isopentane (Sigma) and liquid N2, and stored at –70°C. Serial sections were cut at a thickness of 6 µm and fixed in ice-cold acetone for 10 min. Tissue sections were directly blocked with 1% bovine serum albumin, followed by the addition of polyclonal rabbit antisera (dilution 1:1,000) generated against LPS (E. coli Poly 8 [Biotec Laboratories, England]), rat antibodies to the macrophage marker F4/80 (Serotec; dilution 1:300), or rat antibodies to the neutrophil marker Gr1 (Serotec; dilution 1:100). After extensive washing with Tris-buffered saline, Alexa 488-conjugated goat anti-rabbit immunoglobulin G (IgG) antibodies or Alexa 568-conjugated goat anti-rat IgG antibodies (all at a dilution of 1:300) were added. Lastly, DAPI was used to stain the host cell DNA and visualized as described above.
RNA extraction, semiquantitative, and real time-PCR. Immediately after dissection, colonic tissues were transferred to TRIzol reagent (Gibco-BRL), frozen in liquid N2, and stored at –70°C until required. RNA was purified according to the manufacturer's instructions. Total RNA was treated with DNase I (Ambion) to remove any contaminating DNA. DNase I was removed with DNase inactivation reagent (Ambion) according to the manufacturer's instructions. cDNA was synthesized with Superscript II reverse transcriptase (Gibco-BRL) using 3 µg of DNase-treated RNA. An aliquot (1/40) of the cDNA reaction was subject to PCR analysis according to the manufacturer's instructions for AmpliTaq Gold DNA polymerase (Perkin-Elmer). For TLR4 gene transcription, the oligonucleotide primers mTLR4+ (5'-GGG TCA AGG AAC AGA AGC AG-3') and mTLR4– (5'-AGC CTT CCT GGA TGA TGT TG-3') were used with cycling conditions of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for a total of 28 cycles. For semiquantitative chemokine analysis, the primers mMIP-2F (5'-TCC TCG GGC ACT CCA GAC-3') and mMIP-2R (5'-GCC TTG CCT TTG TTC AGT AT-3') or mMCP-1F (5'-CTT CCT CCA CCA CCA TGC AG-3') and mMCP-1R (5'-AAA ATG GAT CCA CAC CTT GC-3') were used with cycling conditions of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for a total of 35 cycles. As a control for a constitutively expressed gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified with the oligonucleotides CR127 (5'-ACA TCA TCC CTG CAT CC-3') and CR128 (5'-GGA TGG AAA TTG TGA GG-3') with cycling conditions as follows: 95°C for 1 min, 55°C for 1 min, and 72°C for 1 min for a total of 22 cycles. PCR products were separated by agarose gel electrophoresis.
Quantitative real-time PCR was performed by using the Bio-Rad Laboratories MJ mini Opticon Real-Time PCR System. For quantitative chemokine analysis, the primers MIP-2F (5'-GCC AAG GGT TGA CTT CA-3') and MIP-2R (5'-TGT CTG GGC GCA GTG) or MCP-1F (5'-TAC TCA TTA ACC AGC AAG AT-3') and MCP-1R (5'-TTG AGG TGG TGG TGG AA-3') were used, while PCR conditions were as previously described (20, 33). Expression of GAPDH was used as a reference, using the GAPDH primers mentioned above for semiquantitative analysis. PCR was conducted with IQ SYBR Green Supermix (Bio-Rad), using cycling conditions of 95°C for 5 min and 35 cycles of 95°C for 30 s, followed by 55°C for 30s and 72°C for 30s. To calculate the relative gene expression, the Pfaffl method was used, as outlined in the Gene Ex Macro software by Bio-Rad.
Data presentation and statistical analysis. All of the results are expressed as the mean value ± the standard error of the mean (SEM). The results presented are from one representative infection out of three. Statistical significance was calculated by using the nonparametric Mann-Whitney test. Multiple comparisons were performed by using the Neuman-Keuls multiple comparison test. A P value of <0.05 was considered significant.
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Lower infectious doses reveal a maladaptive role for TLR4 during C. rodentium infection. In a previous study, it was noted that the TLR4-deficient mouse strain, C3H/HeJ, and the closely related, but TLR4-expressing C3H/HeOuJ mice were both highly susceptible to infection by C. rodentium, carrying bacterial burdens several logs higher than the less susceptible C57BL/6 and BALB/c mouse strains (54). Since both C3H/HeJ and C3H/HeOuJ mice ultimately succumbed to infection, we determined that TLR4 played no significant role in the host response to C. rodentium infection (52). However, considering the speed with which these mice succumbed, it was possible that the susceptibility of these strains and the large oral dose (2.5 x 108 CFU) initially used had obscured any potential role for TLR4. To test this hypothesis, we infected both strains of susceptible mice with the original dose, as well as lower doses, of C. rodentium (2.5 x 106 CFU and 2.5 x 104 CFU). Although the C3H/HeJ and C3H/HeOuJ strains succumbed at similar times (7.5 ± 1.2 days and 8.1 ± 1.9 days p.i., respectively) after the high infectious dose, we observed a distinct delay in the time of death of the TLR4-deficient C3H/HeJ mice (see Fig. 1 for representative infections) compared to their WT C3H/HeOuJ controls at the lower doses. The delay was ca. 3 days (12.6 days ± 2.5 versus 9.7 ± 2.1 days p.i.) at the 2.5 x 106 CFU dose and was even more pronounced at the lower dose of 2.5 x 104 CFU, with a significant delay of almost 6 days (17.3 ± 3.3 days versus 10.8 ± 2.6 days p.i. [P < 0.05]). These data suggested that mice expressing a functional TLR4 gene might suffer a more severe disease during C. rodentium infection than TLR4-deficient mice. Unfortunately, because of the low infectious doses required to observe this effect in C3H/HeJ mice, the resulting variability of disease onset made the C3H/HeJ mouse less than ideal for exploring this newly hypothesized role for TLR4 during C. rodentium infection. Therefore, these studies were continued with TLR4-deficient C57BL/10ScNJ mice (39) and their WT (C57BL/10J) TLR4-expressing mice that are on a genetic background that is relatively resistant to C. rodentium infection.
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FIG. 1. Lower infectious doses of C. rodentium reveal prolonged survival of TLR4-deficient compared to TLR4-expressing mice. TLR4-deficient C3H/HeJ mice (A) and TLR4-expressing C3H/HeOuJ mice (B) mice were inoculated with 2.5 x 108 (circles), 2.5 x 106 (squares), or 2.5 x 104 CFU (diamonds), and the survival of the C3H/HeJ mice (open markers) and the C3H/HeOuJ mice (filled markers) was monitored over the first 24 days of infection. In this experiment, the reduction in infectious doses dramatically prolonged the survival of the C3H/HeJ mice, whereas it had only a modest impact on the survival of the C3H/HeOuJ mice. Each datum point represents the percentage of surviving mice from an initial population of five mice. Survival data from one experiment representative of three is shown.
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FIG. 2. Immunolocalization of C. rodentium LPS in the infected colon. Immunofluorescence staining of an uninfected colon (A), as well as a C. rodentium-infected colon from C57BL/10J mice at day 10 p.i. (B and C). Panel A shows a low-magnification (x40) image, with the structures of the colonic mucosa and muscle layers highlighted by phalloidin staining of the actin cytoskeleton (red) and DAPI staining of host cell nuclei (blue). Note that there is no staining with the anti-C. rodentium LPS antisera (green). In panel B, a low-magnification image of an infected colon shows that C. rodentium LPS (green) is predominantly located in the lumen and on the colonic epithelial surface. Panel C shows a higher-magnification (x1,000) image, using the same staining as in panel B, demonstrating intact C. rodentium on the apical surface of colonic epithelial cells at the base of a colonic crypt. Note the spread of intact bacteria (arrows), and shed LPS across the epithelial barrier and into the underlying lamina propria.
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FIG. 3. TLR4 is expressed in the mouse colon and C. rodentium supernatants activate TLR4. (A) Colonic tissues taken from wild-type (TLR4+) and TLR4-deficient (TLR4–) mice were assessed for the expression of TLR4 mRNA under uninfected conditions and at days 6 and 10 of C. rodentium infection. reverse transcription-PCR for TLR4 showed expression under control conditions (lane 4), with only slight increases in TLR4 expression at days 6 (lane 5) and 10 p.i. (lane 6). In contrast, colonic tissues from TLR4 deficient mice lacked any TLR4 gene expression under uninfected or infected conditions (lanes 1 to 3), in keeping with their spontaneous loss of the TLR4 gene. GAPDH was used as the housekeeping gene control. (B) Luria broth (LB) ( ) and the supernatants from C. rodentium cultures grown overnight in LB ( ) were used to stimulate HEK 293 cells transiently transfected with an NF-B reporter construct along with either an empty vector, vectors encoding TLR4 (as well as MD-2 and CD14), or a vector encoding TLR8. The NF-B activation (as assessed by firefly luciferase units) resulting from this stimulation was normalized for transfection efficiency and compared to the response with the empty vector. The asterisk indicates that the level of NF-B activation by the C. rodentium supernatant is significantly greater ( , P < 0.01) in cells transfected with the TLR4 vector than the activation caused by LB alone, whereas the supernatant caused no significant NF-B activation of cells transfected with the TLR8 vector.
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B activity and cotransfected with TLR4, MD-2, and CD14. We assessed NF-B activity by measuring luminescence of transfected cells after exposure to either purified LPS or to the supernatant taken from overnight cultures of C. rodentium. Ultrapure Salmonella enterica serovar Minnesota LPS (10 ng/ml) activated NF-B in TLR4-transfected cells by two- to fourfold compared to unstimulated cells or cells transfected with empty vector. Similarly, C. rodentium supernatant activated the TLR4 reporter by 13- to 14-fold (see Fig. 3B) relative to TLR4 transfectants treated with an equivalent volume of LB, confirming that C. rodentium can activate cells through TLR4. This activation was specific, since C. rodentium supernatant did not activate NF-B in HEK293 cells transfected with TLR8, whereas stimulation with the TLR8 agonist R848 led to a five- to eightfold increase in NF-B-dependent luciferase activity. No significant NF-B activity was measured in cells transfected with empty vector and stimulated under the same conditions.
Exposure to C. rodentium causes NF-B nuclear translocation: partial TLR4 dependence.
Once we had confirmed that C. rodentium activated TLR4, we assessed the impact of TLR4 on the inflammatory response of host cells. A/E pathogens most closely interact with intestinal epithelial cells; however, several studies have demonstrated that colonic epithelial cells are normally hyporesponsive to LPS (17, 22, 25, 38). Although we did find that the CMT-93 murine colonic epithelial cell line expresses TLR4 mRNA (not shown), we were unable to immunofluorescently detect TLR4 protein expression by these cells (Fig. 4A and B) using MTS510, an antibody that recognizes the complex formed by TLR4 and its coreceptor MD-2 (2). Moreover, we were unable to detect overt NF-B activation in the CMT93 cells following S. enterica serovar Minnesota LPS stimulation (not shown), confirming the hyporesponsiveness of these epithelial cells to LPS. In contrast, professional phagocytes such as macrophages and dendritic cells are known to express functional levels of this innate receptor and respond to purified LPS (42). Since previous immunostaining studies of uninfected mouse colons had identified numerous resident macrophages, but few resident dendritic cells (C. Ma and B. A. Vallance, unpublished data), we examined TLR4 protein expression and its role in the inflammatory response to C. rodentium in macrophages. For these studies, viable C. rodentium from overnight cultures were added to macrophages derived from the bone marrow of WT mice or from TLR4-deficient mice. After 1 to 4 h of exposure, cells were fixed and processed for immunofluorescence. The localization of TLR4/MD-2 was determined by using the MTS510 antibody (2). As assessed by immunofluorescence staining, C. rodentium were phagocytosed by both types of macrophages, while bacterial counts revealed similar numbers of C. rodentium were phagocytosed by the two macrophage populations (not shown). TLR4/MD-2 staining (red) was readily seen in WT-derived macrophages (Fig. 4C) but not in macrophages derived from TLR4-deficient mice (Fig. 4D). Much of the TLR4/MD-2 staining appeared to localize to the perinuclear region, presumably within the Golgi apparatus, as previously described (34). The specificity of the TLR4 antibody was confirmed when control slides receiving irrelevant isotype-matched antibodies, or the secondary antibody alone, showed no staining (not shown).
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FIG. 4. Immunostaining of TLR4/MD-2 and NF-B in epithelial cells and macrophages exposed to C. rodentium. (A and B) Immunofluorescence staining demonstrating the lack of TLR4/MD-2 immunoreactivity (red) in uninfected murine CMT93 colonic epithelial cells (A), as well as in CMT93 cells exposed to C. rodentium (B). The cell nuclei are DAPI stained (blue). (C) In contrast, to the lack of TLR4/MD2 staining in colonic epithelial cells, there is considerable expression of TLR4/MD-2 in WT BMDM exposed to C. rodentium. Note the immunolocalization of TLR4/MD2 (in red, see arrow) within the cytoplasm, and the internalized bacteria (an arrowhead indicates the DAPI-stained bacteria in blue). (D) In contrast, macrophages derived from TLR4-deficient mice showed no evidence of immunoreactive TLR4, despite the presence of phagocytosed C. rodentium (arrowhead). (E) Localization of NF-B was predominantly cytoplasmic in naive BMDM derived from WT mice, with little if any staining in the nucleus (arrow). (F) A similar cytoplasmic localization of NF-B was seen in naive BMDM derived from TLR4-deficient mice (see arrow for nucleus). (G) Within 2 h of exposure to C. rodentium, there was a dramatic recruitment of NF-B to the nucleus (arrow) of WT derived cells. (H) Under the same conditions, TLR4-deficient BMDM showed modest but significantly attenuated recruitment of NF-B. All images were taken at x800 magnification.
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B. Macrophages were exposed to C. rodentium for the times described above; the cells were then fixed and immunostained for NF-B. As shown in Fig. 4E and F, uninfected WT and TLR4-deficient macrophages both showed punctate NF-B staining throughout the cytoplasm, with little nuclear staining. Within 2 h of exposure to C. rodentium, there was a dramatic recruitment of NF-B to the nucleus of WT macrophages (Fig. 4G), and this was maintained until at least 4 h postexposure (not shown). In contrast, NF-B translocation to the nuclei of macrophages derived from TLR4-deficient mice was attenuated at 2 h (Fig. 4H) compared to WT-derived cells. Although the extent of nuclear translocation did increase over time (not shown), it always remained less than that seen in WT macrophages. These results demonstrate that exposure to C. rodentium triggers NF-B nuclear translocation in macrophages in a partially TLR4-dependent manner. Early inflammatory cell recruitment during C. rodentium infection is TLR4 dependent. We next assessed the impact of TLR4 expression on the host inflammatory response to C. rodentium infection. In a recent study by Wiles et al. (58), it was demonstrated that after oral inoculation, C. rodentium initially colonize the cecum and, after bacterial growth at that site, the infection progresses down the length of the colon. Therefore, we examined cecal tissues at day 6 p.i., the earliest time point when we identified the cecum to be consistently and heavily colonized. Infected tissues were immunostained and then compared to control tissues for the presence of macrophages and neutrophils by using antibodies recognizing F4/80 and Gr1 as the respective markers. Under control conditions, occasional resident F4/80-positive macrophages were identified at the base of cecal crypts and in subepithelial locations in both WT and TLR4-deficient mice (Fig. 5A and C). Similarly, we saw few other inflammatory cells, such as Gr1-positive neutrophils, in control cecal tissues (Fig. 5E and G).
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FIG. 5. Macrophage and neutrophil recruitment during C. rodentium infection is TLR4 dependent. Cecal tissues from uninfected WT (A and E) and TLR4-deficient (C and G) mice, as well as day 6 post-C. rodentium-infected WT (B and F) and TLR4-deficient (D and H) mice, were stained for the presence of F4/80-positive macrophages (A to D) and Gr1-positive neutrophils (E to H). Inflammatory cells are shown in red, and DAPI-stained host cell nuclei are in blue. A resident network of macrophages is seen in both uninfected WT (A) and TLR4-deficient (C) mice, as shown in panel B, numerous infiltrating F4/80-positive macrophages were seen in the cecal submucosa and mucosa of C. rodentium-infected WT mice. (D) However, no infiltrating macrophages were observed in the TLR4-deficient mice. The few F4/80-positive macrophages detected in the mucosa of infected TLR4-deficient mice (B) were similar in location and number to the network of resident macrophages seen in uninfected mice (C). Few if any neutrophils were seen in uninfected WT (E) and TLR4-deficient (G) mice, whereas large numbers of infiltrating Gr1-positive neutrophils were also detected in the cecal tissues of infected WT mice (F). (H) This recruitment was also significantly attenuated in the TLR4-deficient mice. All images were taken at x200 magnification and are representative of 10 sections assessed for each group.
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TLR4-dependent changes in chemokine gene expression in the infected cecum. To explore the basis for the impaired inflammatory cell recruitment seen in the TLR4-deficient mice, we examined the gene expression of the neutrophil chemoattractant MIP-2 (11), as well as the monocyte/macrophage chemokine MCP-1 (50) through both semiquantitiative PCR (Fig. 6A) and quantitative real-time PCR (Fig. 6B). We chose these two chemokines as representative of the chemokine response to C. rodentium infection, since we had previously determined their expression to be upregulated in colonic tissues during infection in WT mice (C. Ma and B. A. Vallance, unpublished data). As shown in Fig. 6, MCP-1 was expressed at similarly low levels in cecal tissues taken from uninfected WT (lane 1) and TLR4-deficient (lane 3) mice, whereas MIP-2 gene expression was very low in WT mice, and even lower in TLR4-deficient mice. Infection led to a significant (88-fold) increase in MIP-2 expression in WT mice (lane 2), whereas only a twofold increase was seen in tissues taken from infected TLR4-deficient mice (Fig. 6A, lane 4, and B). Similarly, MCP-1 expression was upregulated by sixfold in tissues from infected WT mice, but no change in expression was detected in TLR4-deficient mice. Based on these findings, a lack of chemokine induction after infection may be the basis for the impaired recruitment of inflammatory cells into the ceca of infected TLR4-deficient mice. Corresponding to the observed infiltration of more inflammatory cells at later time points in infected TLR4-deficient mice, we also detected a modest increase in these chemokines at later time points during the infection (not shown).
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FIG. 6. Increased chemokine expression during C. rodentium infection is TLR4 dependent. (A) Cecal tissues from WT (TLR4+) and TLR4-deficient (TLR4–) mice were assessed for the expression of the chemokine genes, MIP-2 and MCP-1. Tissues were collected from uninfected mice and at day 6 p.i. (A) Both mouse strains expressed detectable but low mRNA levels of the chemokine MCP-1 but no expression of MIP-2 (lanes 1 and 3) under uninfected conditions. By day 6 p.i., the expression of both chemokine genes (lane 2) strongly increased in WT mice but not in TLR4-deficient cecal tissues (lane 4). GAPDH was used as the housekeeping gene control. (B) The same samples (from uninfected and day 6 p.i. WT and TLR4 knockout mice) were subjected to real-time PCR analysis, and the expression of MCP-1 () and MIP-2 () were quantified relative to the mRNA levels in uninfected WT samples. The asterisks indicate that the expression levels of both chemokines were significantly increased in WT day 6 p.i. tissues over that of uninfected control tissues (, P < 0.01). In contrast, the modest increase in the expression of these genes in infected TLR4 knockout mice did not reach significance.
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FIG. 7. Infection induced weight loss and mortality are attenuated in TLR4-deficient mice. (A) Mice were inoculated with 2.5 x 108 CFU, and the survival of WT (•) and TLR4 deficient mice ( ) was monitored over the first 24 days of infection. In this experiment, 30% of the WT mice in this particular experiment required euthanization between days 10 and 14 p.i.; however, no TLR4-deficient mice required euthanization over the 24 days. Each datum point represents the percentage of surviving mice from an initial population of ten mice. Survival data from one experiment representative of three is shown. (B) The body weights of WT (•) and TLR4-deficient mice () were followed over the first 24 days of a C. rodentium infection. Each datum point represents average weight data pooled from ten mice and is expressed as the percentage of the initial body weight. Although both mouse strains lost weight during the course of infection, the body weight loss was dramatically greater for WT mice. The asterisks indicate that the body weight of the WT mice was significantly less than that found for TLR4-deficient mice at days 2, 10, and 14 p.i. (, P < 0.05).
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103-fold fewer bacteria recovered from the distal colon at day 6 p.i. [(7.3 ± 1.2) x 105 CFU; P < 0.001].
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FIG. 8. C. rodentium spread to the colon is delayed in TLR4-deficient mice. WT and TLR4-deficient mice were inoculated with 2.5 x 108 CFU, and the number of C. rodentium CFU recovered from the ceca of individual infected WT mice (•) and TLR4-deficient mice () are presented on days 6 and 10 p.i. (A), while panel B indicates the number of C. rodentium CFU recovered from the colons of these mouse strains on days 6, 10, 14, 18, and 21 p.i.. The mean CFU values are indicated by the horizontal solid lines for WT mice and horizontal dashed lines for TLR- deficient mice. Asterisks denote the recovery of significantly more bacteria (mean value) from the colons of WT mice compared to TLR4-deficient mice at days 6 and 10 p.i. and significantly more bacteria recovered from the TLR4-deficient mice at day 18 p.i. (, P < 0.01). Each group contained five mice, and the data shown are from one experiment representative of three.
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Considering that TLR4 frequently limits bacterial growth during systemic gram-negative bacterial infections, the discovery that C. rodentium colonization of the distal GI tract was delayed by 4 to 8 days in the TLR4-deficient mice was a surprising result. To evaluate whether this was a common feature in murine hosts lacking TLR4 function, we assessed the time course of C. rodentium infection in the other two spontaneously TLR4-deficient mouse strains: the C57BL/10ScNCr strain and the C3H/HeJ mouse strain. Using the same oral dose (2.5 x 108 CFU), C. rodentium colonization of the C57BL/10ScNCr mice also showed a significant delay, carrying 10- to 100-fold fewer bacteria than their WT controls at day 6 p.i., and 2- to 5-fold fewer bacteria at day 10 p.i. (not shown). Because the TLR4-deficient C3H/HeJ strain is highly susceptible to infection, we used a smaller infectious dose of C. rodentium in order to visualize any potential differences in colonization rates. After an oral dose of 104 CFU, heavy colonization of the WT C3H/HeOuJ strain occurred at day 7 p.i.. With the same dose, heavy colonization of the C3H/HeJ strain was delayed and did not occur until 4 to 6 days later (not shown). These results confirm that the loss of TLR4 function in the host delays C. rodentium colonization and spread to the murine colon.
C. rodentium dissemination to the MLNs is TLR4 independent. Although C. rodentium are primarily found in the colonic lumen of their hosts, some bacteria do translocate across the intestinal epithelial barrier, reaching the lamina propria (Fig. 2C), and the MLNs of infected mice (54). Since we and others have shown that serovar Typhimurium and other invasive gram-negative bacteria rapidly proliferate and spread through the internal organs of TLR4-deficient mice, we examined whether TLR4 expression also impacted on C. rodentium translocation and systemic survival in their murine hosts. The C. rodentium populations found in the MLN of WT (C57BL/10J) and TLR4-deficient (C57BL/10ScNJ) mice were enumerated during the peak phase of the infection, days 10 to 14 p.i., when both strains carried large C. rodentium burdens in their colons. As shown in Table 1, the numbers of C. rodentium recovered from the MLN of infected WT mice were 5.9 ± 1.7 x 106 on day 10 p.i., with the number falling to 4.5 ± 1.5 x 105 CFU on day 14 p.i. Similar, but consistently lower numbers [(8.4 ± 3.3) x 105 and (1.1 ± 0.6) x 105 CFU] were recovered from the TLR4-deficient mice. Interestingly, the MLN numbers in both mouse strains correlated well with the load of C. rodentium found in their colons, reflecting ca. 1.6% of the colonic C. rodentium population at day 10 p.i. and 0.1% at day 14 p.i.. These results indicate that the loss of TLR4 expression did not increase C. rodentium translocation to, or growth in, the MLNs but instead that the C. rodentium population in the MLN was directly related to the load of this pathogen in the colon.
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TABLE 1. C. rodentium dissemination to the MLN is TLR4 independenta
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(i) Colon weights. After C. rodentium infection, the colons of infected WT mice rapidly increased in weight, as shown in Fig. 9A, demonstrating a significant increase (P < 0.05) over the weight of uninfected colons from days 10 to 21 p.i. Colon weights increased from a mean of 105 ± 8 mg in uninfected mice to 118 ± 12 mg at day 6 p.i. and 185 ± 16 mg at day 10 p.i. The colon weights continued to rise to 235 ± 18 mg at day 14 p.i. but peaked at day 18 p.i., reaching weights of 275 ± 8 mg, after which they fell to 220 ± 15 mg at day 21 p.i. Although the TLR4-deficient mice also exhibited a time-dependent increase over control values, their increases were only significant over their uninfected values between days 14 and 21 p.i. As a result of this attenuated response, the mean colon weights of the TLR4-deficient mice were significantly lower than that seen in WT mice at all time points during infection.
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FIG.9. Infection-induced colitis and pathology are attenuated in TLR4-deficient mice. WT and TLR4-deficient mice were inoculated with 2.5 x 108 CFU and assessed for colitis and intestinal pathology for 21 days. (A) Both WT () and TLR4-deficient mice () developed significant increases in colon weights during the course of C. rodentium infection (P < 0.05). The WT tissues showed a significant increase over uninfected controls from days 10 to 21 p.i., whereas the TLR4-deficient mice showed significant increases from days 14 to 21 p.i. However, at all time points between days 6 to 21 p.i. inclusive, the mean colon weights (in milligrams) were significantly greater in the WT mice than in the TLR4-deficient mice (indicated by asterisks). Error bars represent standard errors, and each group contained five mice. , P < 0.05. (B) WT mice () show significantly increased colonic crypt heights compared to uninfected controls at all time points tested during the course of C. rodentium infection, whereas TLR4-deficient mice () only showed significant increases from days 14 to 21 p.i. When the two strains are compared, the mean crypt heights (in microns) in the colons of WT mice were significantly greater than those from TLR4-deficient mice at days 6 and 10 p.i. (, P < 0.01). (C) Both WT () and TLR4 deficient mice () show evidence of inflammation and tissue damage in the distal colon during C. rodentium infection, but the response was increased and more prolonged in the WT mice. Inflammation scores of WT tissues were significantly greater than those from TLR4-deficient mice at days 6, 10, and 18 p.i. (, P < 0.05). For each panel, error bars represent standard errors. Each group contained five mice, and the data shown are from one representative experiment out of three. (D and E) Low-magnification (x40) histological images of colons taken from WT (D) and TLR4-deficient (E) mice at day 10 p.i. Significant inflammation, crypt hyperplasia, superficial epithelial damage, and goblet cell depletion are all evident in the tissues from WT mice. In contrast, only a limited inflammatory infiltrate is seen in the TLR4-deficient mouse tissues, with few signs of mucosal hyperplasia or other overt signs of pathology. The epithelium appears relatively undamaged and numerous goblet cells are evident in the TLR4-deficient tissue.
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(iii) Colitis/inflammation score. Although the TLR4-dependent attenuation of the initial inflammatory response in the cecum was dramatic, since the infection progressed into the distal colon, inflammation, and tissue damage were observed in both WT and TLR4-deficient mice, but to a lesser degree in the TLR4-deficient mice. As shown in Fig. 9C, colitis in WT mice was evident by day 6 p.i. (score, 2.4 ± 0.4 U), with the score primarily due to polymorphonuclear infiltration and epithelial sloughing. By days 10 to 14 p.i., neutrophil infiltration was less prominent in WT mice, but submucosal edema, epithelial damage, and goblet cell depletion (Fig. 9D) were prominent aspects of the colonic pathology, increasing the inflammatory score to over 6.0 U. The colitis score was maintained in WT mice at day 18 p.i. (score, 6.3 ± 0.5 U), but by day 21 p.i., as the infection began to clear, the inflammation score in the WT mice decreased to 1.8 ± 0.2 U. In contrast, the inflammatory response seen in the TLR4-deficient mice was reduced and more transient, showing little inflammation except at day 14 p.i. As a result, the inflammation scores in the TLR4-deficient mice were significantly attenuated compared to WT mice (Fig. 8E) at all time points studied, except days 14 and 21 p.i. Even on day 14 p.i., when the inflammation peaked in the TLR4-deficient mice, the score was primarily due to epithelial disruption and polymorphonuclear cell infiltration, while other pathological features such as edema and the degree of goblet cell depletion remained attenuated compared to WT mice.
Infection-induced diarrhea is less severe in TLR4- deficient mice. Both EPEC and EHEC induce profuse watery diarrhea in infected human hosts (29, 35); however, C. rodentium infection of mice causes a less severe form of intestinal dysfunction, resulting in soft and watery stools (30). At the most severe stage of C. rodentium infection, the colons of infected mice contain no formed stool but rather a mixture of fecal material, mucus, and fluids. Since diarrhea is such an important component of A/E bacterial infections, we studied what role TLR4 played in these pathophysiological changes using a scoring system assessing the water content of the stool. The stools from uninfected mice were well formed and relatively dry (water content 41%), but by day 6 p.i. the C. rodentium infection had already altered the stool found in WT mice, with fewer stools present and the average water content increasing from control values to 82% (Fig. 10). By days 10 and 14 p.i., this response had worsened (water content of 85 to 90%), with most animals carrying only unformed watery fecal material. Although TLR4-deficient mice also developed watery stool during infection, this response was significantly attenuated at day 6 and 10 p.i., with stool water content determined to average 56 and 72%, respectively. At day 14 p.i., the water content in the stool from TLR4-deficient mice reached more than 75% and was no longer significantly different from WT mice. After day 14 p.i., the stool water content in both mouse strains began to return to control levels, falling at day 18 p.i. to 55% in the WT mice and to 45% at 21 p.i. A similar trend was seen in the TLR4-deficient mice and, although the water content was consistently higher in the stool from WT mice, the difference between strains was not significant at either day 18 or 21 p.i.
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FIG. 10. Infection-induced diarrhea is less severe in TLR4-deficient mice. The percentage of water in the colonic stool contents of WT mice infected by C. rodentium increased in a time-dependent manner, with the water content significantly elevated over uninfected control levels at days 6, 10, and 14 p.i. (all P < 0.01). While the percent water content of the stool also increased in TLR4-deficient mice, it was only significantly elevated over control levels at day 10 and 14 p.i. (P < 0.05). When the two strains are directly compared, the water content in the stool of WT mice was significantly greater than in TLR4-deficient mice at days 6 and 10 p.i. (, P < 0.05). Each group contained five mice, and the data shown are from one representative experiment out of three.
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There is a growing recognition that the immune response triggered during enteric bacterial infections not only protects the host (10) but also inadvertently causes much of the associated disease (43, 55). Therefore, our laboratory and others have begun to explore how the hosts' immune system recognizes and combats A/E bacterial infections in the GI tract (48, 49, 53). However, our knowledge of how the innate immune system responds to these enteric pathogens is limited. Among the key issues that remain unresolved are the identity of the innate receptors that are expressed and function in the GI tract and whether these receptors respond to the presence of A/E bacteria. Moreover, we have yet to distinguish whether the innate responses that are induced actually protect the host or, alternatively, merely cause inflammation and tissue damage without providing significant protection against infection. We began our investigations by testing the role of the innate LPS receptor, TLR4, in the host response against the model pathogen, C. rodentium. Surprisingly, despite the prominent health threat posed by A/E bacteria, this is the first study to specifically assess the role of an innate receptor in the host response to these pathogens.
Our initial examination of infected mice found the vast majority of C. rodentium residing on the lumenal surface of the gut epithelium. However, these microbes also translocated out of infected colonic crypts and into the lamina propria, exposing not just the epithelium but resident macrophages and other professional immune cells to C. rodentium LPS during infection. The basis for this translocation is unclear, but EPEC infection has been repeatedly shown to disrupt intestinal epithelial barrier function in tissue culture cells (26). Similarly, we recently found that C. rodentium infection causes increased permeability in gut segments (unpublished data); therefore, the passage of C. rodentium and its products across the epithelium likely reflects passive movement across a weakened intestinal barrier rather than an active invasive process.
Our examination of TLR4 expression found TLR4 mRNA to be constitutively expressed in the mouse colon, with little change in expression during the course of C. rodentium infection. While immunostaining for TLR4/MD-2 showed diffuse staining in a limited number of cells within the colonic mucosa (not shown), we were unable to clearly identify which cell types expressed these proteins. However, most previous studies on this subject suggest that resident macrophages and other inflammatory cells are the primary source of TLR4/MD-2 expression in the colon (25), with colonic epithelial cells expressing little if any TLR4 protein, in keeping with their relative nonresponsiveness to LPS (17, 40). Our results support these studies, since although we did detect TLR4 mRNA in the CMT-93 murine colonic epithelial cell line, we were unable to detect immunoreactive TLR4/MD-2 in these cells, whereas it was readily detectable in murine BMDM. Future studies will be needed to examine whether the colonic epithelium of a C. rodentium-infected mouse becomes more responsive to LPS during the course of infection, potentially in response to the inflammatory mediators released during infection. Interestingly, while previous studies have shown that EPEC possesses strong antiphagocytic abilities (9), C. rodentium were rapidly phagocytosed by BMDM. This may reflect differences between EPEC and C. rodentium in their pathogenic strategies or, alternatively, it may simply reflect that the infectivity of C. rodentium as well as the secretion of their type III effectors is attenuated in tissue culture medium compared to EPEC (14).
Using nuclear translocation of NF-B as a measure of inflammatory activation (38), we found that exposure to C. rodentium caused a rapid activation of NF-B in BMDM in a partially TLR4-dependent manner. The limited TLR4-independent response likely reflects the activation of other innate receptors in the BMDM, since A/E bacteria are sources of a number of other proinflammatory molecules aside from LPS, such as flagellin (6) and bacterial DNA. Since NF-B activation is a central player in inflammation, our in vitro assays led us to test what role TLR4 plays in the inflammatory response during C. rodentium infection in mice.
C. rodentium infection in vivo triggers the recruitment of large numbers of macrophages and neutrophils to the infected intestine (30), and these inflammatory cells undoubtedly contribute to the tissue damage and disease suffered during infection. Prior to infection, we found few neutrophils in the cecal mucosa; however, immunostaining did reveal a broad network of resident macrophages within these tissues in both mouse strains. A similar network of macrophages has been previously identified in other regions of the mammalian GI tract (18) and may function to provide early recognition of translocating intestinal pathogens. We observed the rapid and dramatic recruitment of macrophages, as well as neutrophils, into the cecal mucosa and submucosa of WT mice during C. rodentium infection; however, few of these inflammatory cells were in direct proximity to the infected epithelium and the luminal C. rodentium population. Despite similarly large numbers of C. rodentium recovered from the cecal tissues of TLR4 deficient mice, we found few signs of macrophage or neutrophil recruitment in these mice, demonstrating a clear TLR4 dependence for this early inflammatory response. Similarly, we found a clear TLR4 dependence in the induction of MIP-2 and MCP-1 mRNA expression within the infected ceca. These two chemokines potently recruit neutrophils and macrophages, respectively (11, 50). These findings are similar to an earlier characterization of the role of TLR4 during hepatic serovar Typhimurium infection in mice (57). In that study, serovar Typhimurium infection led to the upregulated expression of MIP-2 and other chemokines in the infected livers of WT mice. In contrast, these chemokine responses were attenuated in serovar Typhimurium-infected TLR4-deficient mice. Furthermore, the impaired inflammatory response ultimately proved fatal to TLR4 deficient mice as the serovar Typhimurium infection spread through their internal organs, leading to sepsis (36, 57).
Our present findings indicate that TLR4 plays a more complex role during C. rodentium infection, with infected WT mice suffering more severe morbidity and greater mortality rates than TLR4-deficient mice. While these results contrast with an earlier study that found no difference in susceptibility to C. rodentium infection between TLR4 deficient C3H/HeJ mice and their TLR4 expressing controls (54), we hypothesized that the heightened susceptibility of the C3H mice as well as the large oral bacterial inocula given in that earlier study masked any role for TLR4. In the present study, gavaging these mice with lower infectious doses revealed a previously unrecognized delay in disease onset in the C3H/HeJ mice compared to their WT controls, confirming that TLR4 expression worsens the disease outcome in this infection model.
Understanding how A/E bacterial pathogens colonize their hosts is obviously of great importance in combating these infections, and a recent study found that after oral inoculation of mice, C. rodentium initially colonizes the epithelium that overlies lymphoid tissue in the cecum (known as the cecal patch) (58). From that site, the bacteria replicate within the cecum and then pass from that site to colonize the distal colon of infected mice. The mechanisms that underlie the spread from the cecum to the distal colon are unclear. Our data indicate that C. rodentium was able to colonize the ceca of both WT and TLR4-deficient mice to roughly equal levels but the spread to and colonization of the distal colon by C. rodentium was significantly delayed in TLR4 deficient mice, while TLR4 ultimately had little impact on pathogen clearance from the gut. Similarly, TLR4 expression did not directly affect the population of C. rodentium found in the MLN during infection; therefore, we conclude that TLR4 does not contribute to host defense against C. rodentium in the GI tract or its associated lymphoid tissues or to bacterial clearance from infected mice.
To explore possible reasons for the delayed colonization of the colons in the TLR4 deficient mice, we assessed pathological factors characteristic of C. rodentium infection, including increased colon weights and crypt heights, as well as intestinal inflammation (30) and water content in the stools of infected mice. Although C. rodentium does not cause overt diarrhea in infected mice, infected mice do exhibit watery stool. There was evidence that compensatory inflammatory pathways were activated in the absence of TLR4; however, all of the pathological features that we assessed were attenuated and/or delayed in TLR4-deficient mice, suggesting that TLR4-mediated inflammatory changes may facilitate the ability of C. rodentium to colonize the distal colon. Even after bacterial burdens had reached similar levels in the two mouse strains, the degree of inflammation and pathology was still significantly greater and longer lasting in the WT mice, indicating that differences in bacterial burdens are unable to explain the dramatic TLR4 dependence of the inflammation and tissue damage seen in this model.
Our results with C. rodentium-induced colitis agree with the contention that TLR4 contributes to intestinal inflammation and pathology, as proposed in two recent studies testing the role of TLR4 in the dextran sodium sulfate (DSS)-induced colitis model (22, 41). In both studies, TLR4-deficient mice suffered less inflammation than WT mice, with Fukata et al. linking this attenuated response to reduced MIP-2 production by TLR4-deficient macrophages. Curiously, despite the reduced inflammation, both groups found TLR4-deficient mice suffered greater body weight loss, intestinal bleeding, and mortality than WT mice during DSS-induced colitis. This heightened susceptibility was linked to impaired epithelial proliferation and increased commensal bacterial translocation to the MLN (22). Epithelial cell hyperplasia was also impaired in our C. rodentium-infected TLR4-deficient mice; in contrast, we saw less morbidity and mortality in these mice, and no significant increase in bacterial translocation. This difference in pathological outcomes may reflect the nature of the injurious agent and the severity of the resulting colitis. DSS is a sulfated polysaccharide known to be directly toxic to colonic epithelium, resulting in rapid and significant epithelial cell injury and apoptosis irrespective of the inflammatory response (22). In contrast, the colitis that develops during C. rodentium infection occurs more slowly, causes only limited apoptosis (54), and appears to be predominantly mediated by the host immune response (10, 55). We therefore hypothesize that differences in the mode and degree of epithelial damage differentiate the impact of TLR4 deficiency in these two models of colitis.
Overall, our results were unexpected, since in most previously published reports, the loss of TLR4 signaling leads to increased susceptibility to gram-negative bacterial infections. We hypothesize that the lack of a beneficial effect of TLR4 against C. rodentium infection may reflect the environmental niche occupied by this pathogen and the cell types it colonizes. In previous studies, TLR4 expression has been shown to protect mice against infection by Salmonella and uropathogenic E. coli, as well as other gram-negative bacterial pathogens (47, 57). In these models, salmonellae preferentially infect macrophages, whereas uropathogenic E. coli infects bladder epithelial cells. Both cell types express functional TLR4 (47, 57), and several studies have shown that infection of these cells by gram-negative bacteria induces a rapid inflammatory response. In contrast, there is little evidence that under basal conditions, the colonocytes preferentially infected by C. rodentium express TLR4 or its required cofactors at the protein level. While C. rodentium can induce an inflammatory response in macrophages, very few of the C. rodentium found in the gut lumen would directly contact these cells in vivo. Instead intestinal macrophages might be activated by translocating bacteria or by LPS, passively entering the colonic mucosa. The resulting innate response, as we have shown with TLR4-deficient mice, contributes to both the tissue damage and the disease symptoms caused by this infection; however, C. rodentium and perhaps other luminal pathogens may be sufficiently distant from these cells to be protected from their responses. Our findings therefore may prove relevant to the wide variety of gram-negative bacterial pathogens that preferentially infect the intestinal epithelium. Interestingly, our results bear some similarity to work showing that broadly innate immune-impaired MyD88-deficient mice suffer less severe gastroenteritis than WT mice after oral infection with serovar Typhimurium (24).
While it was surprising that TLR4 deficient mice would be less susceptible to infection than WT mice, the ability of bacterial pathogens to exploit inflammation in order to colonize the colon is not unprecedented. Shigella spp. are known to trigger an inflammatory response in order to invade and spread through the host's colonic epithelium (37, 44, 45). Although A/E pathogens do not invade the epithelium like Shigella organisms do, several of the translocated virulence proteins used by A/E pathogens are homologous to Shigella effector proteins (52), suggesting these pathogens may share common virulence strategies. While more studies are needed to determine whether C. rodentium is truly exploiting the TLR4-induced host response, there are many reasons why colonization of the colon might be facilitated by a strong innate host response. As we demonstrated, TLR4 responses aid in the depletion of goblet cells, potentially weakening luminal protection against infection. Also, the aggravated diarrheal response seen in infected WT mice, along with the sloughing of colonized enterocytes, would increase fecal shedding and transmission of the organism down the GI tract and to other potential hosts. Similarly, the rapid epithelial cell proliferation and mucosal thickening seen during C. rodentium infection would provide a rapidly renewing supply of uninfected cells, as well as a larger and potentially less protected epithelial surface area for bacterial colonization. Another possibility is that TLR4-dependent responses may alter the population of commensal microflora found in the GI tract, providing C. rodentium with a selective advantage in colonizing this tissue.
In summary, our studies indicate that TLR4 expression mediates much of the colitis and tissue pathology seen during C. rodentium infection; however, in contrast to the host protective role observed in other types of gram-negative infections, TLR4 expression demonstrated no obvious benefit to the host during infection by this A/E bacterium. The putative maladaptive role played by TLR4 in this model may reflect the unique environment of the colon and the luminal niche occupied by A/E bacteria. While translocating C. rodentium and shed LPS appear to activate TLR4-dependent inflammation, the vast majority of these bacteria reside within the colonic lumen, a location that is relatively safe from the resulting inflammatory response. It should be noted, however, that other, more broadly acting, host immune factors do contribute to host defense in this model, since mice deficient in downstream inflammatory mediators such as IL-12, gamma interferon (49), and NF-B (B. A. Vallance, unpublished data) suffer increased susceptibility to C. rodentium infection. Therefore, although our findings indicate that TLR4 activation is not effective in promoting host defense against C. rodentium, other inflammatory pathways may prove beneficial, presumably differing from TLR4-dependent responses in their timing, location, or the cell types involved. Although more studies are needed to establish how inflammation may facilitate C. rodentium colonization of the host gut, it is clear that the role of innate immunity in the lower GI tract cannot simply be extrapolated from tissue culture or systemic infection studies. Considering the importance of this site, and the vast number of pathogens that target the GI tract, additional studies examining the actions of the innate immune system in relevant enteric infection models should be a high priority for the field, to aid in the rational design of vaccines and therapies.
We thank P. Beck for advice concerning the Gr1 staining.
This study was supported by operating grants from the Canadian Institutes for Health Research to B.A.V. and B.B.F. and by an establishment award from the University of British Columbia to B.A.V. C.M.R. is a Broad Medical Research Program Fellow of the Life Sciences Research Foundation. B.B.F. is a Howard Hughes International Research Scholar, a CIHR Distinguished Investigator, and the UBC Peter Wall Distinguished Professor. B.A.V. is the Children with Intestinal and Liver Disorders (CHILD) Foundation Research Scholar, a Michael Smith Foundation for Health Research Scholar, and the Canada Research Chair in Pediatric Gastroenterology.
* Corresponding author. Mailing address: ACB, Rm. K4-188, 4480 Oak Street, BC's Children's Hospital, Vancouver, British Columbia V6H 3V4, Canada. Phone: (604) 875-2345, x5118. Fax: (604) 875-3244. E-mail: bvallance{at}cw.bc.ca.
M.A.K. and C.M. contributed equally to this study.
Present address: Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana.
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Infection and Immunity, May 2006, p. 2522-2536, Vol. 74, No. 5
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