Toll-like receptor 4 contributes to colitis development but not to host defense during Citrobacter rodentium infection in mice - PubMed (original) (raw)

Toll-like receptor 4 contributes to colitis development but not to host defense during Citrobacter rodentium infection in mice

Mohammed A Khan et al. Infect Immun. 2006 May.

Abstract

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-kappaB 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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Figures

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 × 108 (circles), 2.5 × 106 (squares), or 2.5 × 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.

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 (×40) 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 (×1,000) 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.

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-κB 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-κB 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-κB 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-κB activation of cells transfected with the TLR8 vector.

FIG. 4.

Immunostaining of TLR4/MD-2 and NF-κB 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-κB 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-κB 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-κB to the nucleus (arrow) of WT derived cells. (H) Under the same conditions, TLR4-deficient BMDM showed modest but significantly attenuated recruitment of NF-κB. All images were taken at ×800 magnification.

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 ×200 magnification and are representative of 10 sections assessed for each group.

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.

FIG. 7.

Infection induced weight loss and mortality are attenuated in TLR4-deficient mice. (A) Mice were inoculated with 2.5 × 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).

FIG. 8.

C. rodentium spread to the colon is delayed in TLR4-deficient mice. WT and TLR4-deficient mice were inoculated with 2.5 × 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.

FIG.9.

Infection-induced colitis and pathology are attenuated in TLR4-deficient mice. WT and TLR4-deficient mice were inoculated with 2.5 × 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 (×40) 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.

FIG.9.

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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Toll-like receptor 4 contributes to colitis development but not to host defense during Citrobacter rodentium infection in mice - PubMed (original) (raw)