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

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.     

Development of fatal colitis in FVB mice infected with Citrobacter rodentium

Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia. The disease is characterized by severe but temporary epithelial hyperplasia with limited inflammation in the descending colon of adult mice on a variety of genetic backgrounds. The natural history of infection with this murine pathogen has been characterized in outbred Swiss Webster (SW) mice but not in the cognate inbred FVB strain. In contrast to subclinical infection in SW mice, 12-week-old FVB mice developed overt disease with significant weight loss and mortality beginning by 9 days postinoculation (dpi). By 21 dpi, more than 75% of infected FVB mice died or had to be euthanized, whereas no mortality developed in SW mice. Mortality in FVB mice was fully prevented by fluid therapy. Fecal shedding of bacteria was similar in both groups through 9 dpi; however, a slight but significant delay in bacterial clearance was observed in FVB mice by 12 to 18 dpi. SW mice developed hyperplasia with minimal inflammation in the descending colon. FVB mice developed epithelial cell hyperproliferation, severe inflammation with erosions and ulcers, and epithelial atypia by 6 dpi in the descending colon. In the majority of surviving FVB mice, colonic lesions, including epithelial atypia, were reversible, although a small percentage (5 to 7%) exhibited chronic colitis through 7 months postinoculation. The existence of susceptible and resistant lines of mice with similar genetic backgrounds will facilitate the identification of host factors responsible for the outcome of infection and may lead to the development of novel strategies for preventing and treating infectious colitis.     

Immunization of mice with Lactobacillus casei expressing a beta-intimin fragment reduces intestinal colonization by Citrobacter rodentium

Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in children from developing countries. Intimate adhesion of the bacteria to intestinal cells occurs via binding of the adhesin intimin to the TIR receptor exposed on cell surfaces. Here, Lactobacillus casei expressing a fragment of β-intimin (L. casei-Int(cv)) was tested as mucosal vaccines in mice against intestinal colonization with the murine pathogen Citrobacter rodentium. Oral or sublingual immunization of C57BL/6 mice with L. casei-Int(cv) induced anti-Int(cv) IgA in feces but no IgG in sera. Conversely, anti-Int(cv) IgG was induced in the sera of mice after sublingual immunization with purified Int(cv). All vaccines were able to decrease C. rodentium recovery from feces. However, this reduction was more evident and sustained over time in mice immunized with L. casei-Int(cv) by the sublingual route. These mice also displayed an increase in interleukin 6 (IL-6) and gamma interferon (IFN-γ) secretion by spleen cells 10 days after infection. Additionally, oral or sublingual immunization of C3H/HePas mice, which are highly susceptible to C. rodentium infection, with L. casei-Int(cv) induced anti-Int(cv) antibodies and significantly increased survival after challenge. Immunohistological analysis of colon sections revealed that C. rodentium was located in deep fractions of the tissue from C3H/HePas mice immunized with L. casei whereas superficial staining was observed in colon sections from mice immunized with L. casei-Int(cv.) The results indicate that vaccines composed of L. casei expressing intimin may represent a promising approach and that the C3H/HePas infection model with C. rodentium can be used to evaluate potential vaccines against EPEC.     

Tight junctional disruption and apoptosis in an in vitro model of Citrobacter rodentium infection

The murine model of Citrobacter rodentium infection has been used to complement in vitro studies of enteropathogenic Escherichia coli (EPEC) infections of human intestinal epithelial cells (IECs). However, the differences in epithelial cell responses between these two models are not fully understood. We used an in vitro model of C. rodentium infection to examine important, yet incompletely understood, cellular responses of murine IECs to this pathogen. C. rodentium attached to CMT-93 cells and disrupted their tight junctional expression of claudins-4 and -5. This was associated with a loss of barrier function that required live bacteria and was partially prevented by the inhibition of Rho kinase. Furthermore, C. rodentium caused an upregulation of IEC apoptosis that was associated with the cytoplasmic accumulation of apoptosis-inducing factor, but not with the activation of caspase-3. These studies demonstrate for the first time that C. rodentium affects murine IECs in ways that may be similar, but distinct, to the effects of EPEC on human IECs.     

Mast cells limit systemic bacterial dissemination but not colitis in response to Citrobacter rodentium

Enteropathogenic Escherichia coli and enterohemorrhagic E. coli cause an inflammatory colitis in human patients characterized by neutrophil infiltration, proinflammatory cytokine expression, and crypt hyperplasia. Citrobacter rodentium causes a similar colitis in mice and serves as a model for enteropathogenic E. coli infection in humans. C. rodentium induces systemic T-cell-dependent antibody production that facilitates clearance of the bacteria and protects the host from reinfection. The role of innate immune cells in infectious colitis, however, is less well understood. In this study, we have determined the role of mast cells in the inflammatory response and disease induced by C. rodentium. Mice deficient in mast cells exhibit more severe colonic histopathology and have a higher mortality rate following infection with C. rodentium than do wild-type animals. Despite unimpaired neutrophil recruitment and lymphocyte activation, mast cell-deficient mice have a disseminated infection evident in crucial organ systems that contributes to sepsis. Importantly, mast cells also have the capacity to directly kill C. rodentium. Together, these results suggest that mast cells protect the host from systemic infection by reducing the bacterial load and preventing dissemination of the bacterium from the colon.     

Antibiotic-induced perturbations of the intestinal microbiota alter host susceptibility to enteric infection

Intestinal microbiota comprises microbial communities that reside in the gastrointestinal tract and are critical to normal host physiology. Understanding the microbiota's role in host response to invading pathogens will further advance our knowledge of host-microbe interactions. Salmonella enterica serovar Typhimurium was used as a model enteric pathogen to investigate the effect of intestinal microbiota perturbation on host susceptibility to infection. Antibiotics were used to perturb the intestinal microbiota. C57BL/6 mice were treated with clinically relevant doses of streptomycin and vancomycin in drinking water for 2 days, followed by oral infection with Salmonella enterica serovar Typhimurium. Alterations in microbiota composition and numbers were evaluated by fluorescent in situ hybridization, differential plating, and Sybr green staining. Antibiotics had a dose-dependent effect on intestinal microbiota composition. The chosen antibiotic regimen did not significantly alter the total numbers of intestinal bacteria but altered the microbiota composition. Greater preinfection perturbations in the microbiota resulted in increased mouse susceptibility to Salmonella serovar Typhimurium intestinal colonization, greater postinfection alterations in the microbiota, and more severe intestinal pathology. These results suggest that antibiotic treatment alters the balance of the microbial community, which predisposes the host to Salmonella serovar Typhimurium infection, demonstrating the importance of a healthy microbiota in host response to enteric pathogens.     

The pathogenicity of an enteric Citrobacter rodentium Infection is enhanced by deficiencies in the antioxidants selenium and vitamin E

The pathogenesis of a Citrobacter rodentium infection was evaluated in mice fed diets with a single deficiency in either selenium or vitamin E or with a double deficiency in both selenium and vitamin E compared to mice on nutritionally adequate diets. Mice fed the selenium- and vitamin E-deficient diet for 6 weeks had increased loads of C. rodentium in the colon and spleen, which were not observed in mice fed either of the singly deficient diets or the adequate diet. Infected mice fed the doubly deficient diet had increased colon crypt hyperplasia and an influx of infiltrating cells along with gross changes to crypt architecture, including ulceration and denuding of the epithelial layer. Cytokine and chemokine mRNA levels in the colon were measured by real-time PCR. Expression of proinflammatory cytokines and chemokines was upregulated on day 12 after infection with C. rodentium in mice fed the doubly deficient diet compared to mice fed the control diet. Heme oxygenase 1, an enzyme upregulated by oxidative stress, also was more highly induced in infected mice fed the doubly deficient diet. Production of C. rodentium antigen-specific IgM and IgG antibodies was not affected by feeding the doubly deficient diet. The results indicated that selenium and vitamin E play an important role in host resistance and in the pathology induced by C. rodentium, an infection that mimics disease caused by common food-borne bacterial pathogens in humans.     

Identification and characterization of Cri1, a locus controlling mortality during Citrobacter rodentium infection in mice

Infection of inbred mouse strains with Citrobacter rodentium represents an ideal model to reveal the genetic factors controlling host resistance to noninvasive enteric bacterial pathogens. We have chosen a positional cloning approach to identify putative gene(s) that control the known difference in survival between resistant C57BL/6J and susceptible C3H/HeJ and C3H/HeOuJ mice. Our work has identified one major locus within proximal chromosome 15 that is responsible for the marked susceptibility of both C3H strains, and we formally exclude Tlr4 from control of survival to this pathogen. We have named this new host resistance locus Cri1 (Citrobacter rodentium infection 1). The Cri1 genetic interval currently spans ～16 Mb and it confers survival to the infection in a recessive manner. Transfer of the Cri1 locus from the surviving B6 mice into a congenic mouse with a C3Ou genetic background confirms its overall chromosomal localization and its highly significant effect on host survival. The C3Ou.B6-Cri1 mice thus produced have also enabled us to dissociate the control of mouse survival from the control of bacterial load early in the infection as well as from control of colonic hyperplasia.     

Neutrophils play an important role in host resistance to respiratory infection with Acinetobacter baumannii in mice

Acinetobacter baumannii has emerged as a major cause of both community-associated and nosocomial pneumonia, but little is known about the cellular and molecular mechanisms of host defense against respiratory infection with this bacterial pathogen. In this study, we examined the role of neutrophils in host resistance to pulmonary A. baumannii infection in a mouse model of intranasal (i.n.) infection. We found that neutrophils were rapidly recruited to the lungs following i.n. inoculation of the pathogen and declined to baseline level upon clearance of the infection. Depletion of neutrophils using monoclonal antibody RB6-8C5 prior to infection resulted in an acute lethal infection that was associated with enhanced bacterial burdens in the lung (P < 0.05) and extrapulmonary dissemination to the spleen. The increased susceptibility to A. baumannii in neutropenic mice was associated with a delay in the mRNA expression and production of early proinflammatory cytokines such as tumor necrosis factor alpha, interleukin-6, keratinocyte chemoattractant protein, monocyte chemoattractant protein 1, and macrophage inflammatory protein 2 (MIP-2) in the lungs and development of severe bronchopneumonia and lymphoid tissue destruction in the spleen. Moreover, i.n. administration of the neutrophil-inducing chemokine MIP-2 to normal mice induced a pulmonary influx of neutrophils and significantly enhanced the clearance of A. baumannii from the lungs (P < 0.01). These results imply that neutrophils play a critical role in host resistance to respiratory A. baumannii infection.     

Treatment with anti-interleukin-10 monoclonal antibody enhances early resistance to but impairs complete clearance of Listeria monocytogenes infection in mice.

Mice that received an anti-interleukin-10 (anti-IL-10) neutralizing monoclonal antibody (MAb) (SXC-1) prior to infection with Listeria monocytogenes initially demonstrated resistance to the infection, as indicated by reduced recovery of L. monocytogenes from their spleens and livers during the first 5 days after challenge. Anti-IL-10 MAb-treated mice then demonstrated reduced resistance during the later stage of infection, as indicated by persistent infection with L. monocytogenes in their livers 11 days after challenge. Aspartate aminotransferase (AST) levels (a measure of liver damage) in the sera of control mice increased between 1 and 5 days after challenge, while anti-IL-10 MAb-treated mice maintained lower AST levels. At 7 days after challenge, AST levels in the sera of control mice decreased as the numbers of organisms declined. In contrast, AST levels increased as the infections persisted in anti-IL-10 MAb-treated mice. The AST levels in serum reflected liver histopathology as anti-IL-10 MAb-treated mice exhibited fewer granulomatous lesions and less necrosis of liver tissue than the control mice during the first 5 days after challenge. Anti-IL-10 MAb treatment altered the expression of inflammatory cytokine mRNAs during L. monocytogenes infection. Control MAb-treated mice exhibited increased expression of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNA in their lives during L. monocytogenes infection, but this increase did not occur in anti-IL-10 MAb-treated mice. Gamma interferon mRNA expression in the livers of the control MAb-treated mice was increased between 1 and 5 days after L. monocytogenes challenge and then decreased at 7 days after challenge. In contrast, gamma interferon mRNA expression in the livers of anti-IL-10 MAb-treated mice was not decreased until 7 days after challenge. These results indicate that endogenous IL-10 has both beneficial and detrimental effects on the host response to L. monocytogenes infection in mice.     

Androstenediol stimulates myelopoiesis and enhances resistance to infection in gamma-irradiated mice

The ionizing radiation-induced hemopoietic syndrome is characterized by defects in immune function and increased mortality due to infections and hemorrhage. Since the steroid 5-androstene-3beta, 17beta-diol (5-androstenediol, AED) modulates cytokine expression and increases resistance to bacterial and viral infections in rodents, we tested its ability to promote survival after whole-body ionizing radiation in mice. In unirradiated female B6D2F1 mice, sc AED elevated numbers of circulating neutrophils and platelets and induced proliferation of neutrophil progenitors in bone marrow. In mice exposed to whole-body (60)Co gamma-radiation (3 Gy), AED injected 1 h later ameliorated radiation-induced decreases in circulating neutrophils and platelets and marrow granulocyte-macrophage colony-forming cells, but had no effect on total numbers of circulating lymphocytes or erythrocytes. In mice irradiated (0, 1 or 3 Gy) and inoculated four days later with Klebsiella pneumoniae, AED injected 2 h after irradiation enhanced 30-d survival. Injecting AED 24 h before irradiation or 2 h after irradiation increased survival to approximately the same extent. In K. pneumoniae-inoculated mice (irradiated at 3-7 Gy) and uninoculated mice (irradiated at 8-12 Gy), AED (160 mg/kg) injected 24 h before irradiation significantly promoted survival with dose reduction factors (DRFs) of 1.18 and 1.26, respectively. 5-Androstene-3beta-ol-17-one (dehydroepiandrosterone, DHEA) was markedly less efficacious than AED in augmenting survival, indicating specificity. These results demonstrate for the first time that a DHEA-related steroid stimulates myelopoiesis, and ameliorates neutropenia and thrombocytopenia and enhances resistance to infection after exposure of animals to ionizing radiation.     

Immunity to enteric infection in mice

Specific pathogen-free CD-1 mice infected orally with sublethal doses (10⁴ to 10⁶ viable organisms) of Salmonella enteritidis rapidly developed extensive bacterial populations in the liver, spleen, and mesenteric lymph nodes. Although the pathogen did not multiply extensively in the gut, the infection persisted in the intestine at between 10⁴ and 10⁵ viable organisms throughout the experiment. S. gallinarum was less invasive than S. enteritidis when given by mouth; S. pullorum failed to survive in the intestine or to invade the tissues of orally infected mice. Vaccination with a sublethal dose of living S. enteritidis, either orally or intravenously, completely prevented the establishment of liver and spleen populations of a drug-resistant, virulent strain of S. enteritidis. Vaccination with an ethyl alcohol-killed vaccine given by various routes delayed the spread of the orally introduced challenge population to the liver and spleen by 1 to 2 days but was unable to prevent the subsequent growth of the pathogen in vivo, although the vaccinated mice survived the infection. The importance of these findings in relation to vaccination against typhoid fever in man is discussed.     

Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection

The bacterial pathogen Citrobacter rodentium belongs to a family of gastrointestinal pathogens that includes enteropathogenic and enterohemorrhagic Escherichia coli and is the causative agent of transmissible colonic hyperplasia in mice. The molecular mechanisms used by these pathogens to colonize host epithelial surfaces and form attaching and effacing (A/E) lesions have undergone intense study. In contrast, little is known about the host's immune response to these infections and its importance in tissue pathology and bacterial clearance. To address these issues, wild-type mice and mice lacking T and B lymphocytes (RAG1 knockout [KO]) were infected with C. rodentium. By day 10 postinfection (p.i.), both wild-type and RAG1 KO mice developed colitis and crypt hyperplasia, and these responses became more exaggerated in wild-type mice over the next 2 weeks, as they cleared the infection. By day 24 p.i., bacterial clearance was complete, and the colitis had subsided; however, crypt heights remained increased. In contrast, inflammatory and crypt hyperplastic responses in the RAG1 KO mice were transient, subsiding after 2 weeks. By day 24 p.i., RAG1 KO mice showed no signs of bacterial clearance and infection was often fatal. Surprisingly, despite remaining heavily infected, tissues from RAG1 KO mice surviving the acute colitis showed few signs of disease. These results thus emphasize the important contribution of the host immune response during infection by A/E bacterial pathogens. While T and/or B lymphocytes are essential for host defense against C. rodentium, they also mediate much of the tissue pathology and disease symptoms that occur during infection.     

Induction of anxiety-like behavior in mice during the initial stages of infection with the agent of murine colonic hyperplasia Citrobacter rodentium

Symptoms of anxiety frequently occur concomitant to the development and persistence of inflammatory bowel disease (IBD) in patients. In the present study, we utilized an animal model of IBD, infection with Citrobacter rodentium, to determine whether the infection per se can drive anxiety-like behavior. Nine-week-old CF-1 male mice were challenged orally with either saline or C. rodentium. Early in the infective process (7-8 h later), mice were tested on a hole-board open field apparatus for anxiety-like behavior measurement. Immediately following behavioral testing, plasma samples were obtained for immune cytokine analysis and colons were excised for histological analysis. In additional animals, vagal ganglia were removed and processed for c-Fos protein detection. Challenge with C. rodentium significantly increased anxiety-like behavior as evidenced by avoidance of the center area and increased risk assessment behavior. Plasma levels of the cytokines IFN-gamma, TNF-alpha and IL-12 were not different. However vagal sensory ganglia from C. rodentium-treated animals evinced significantly more c-Fos protein-positive neurons, consistent with vagal afferent transmission of C. rodentium-related signals from gut to brain. Histological examination of the colon indicated a lack of overt inflammation at the 8 h post-challenge time point, indicating that the differences in behavior were unlikely to follow from inflammation-related stress. The results of the present study demonstrate that infection with C. rodentium can induce anxiety-like symptoms that are likely mediated via vagal sensory neurons.     

Osteopontin participates in Th1-mediated host resistance against nonlethal malaria parasite Plasmodium chabaudi chabaudi infection in mice

Osteopontin (OPN) knockout mice (OPN-KO mice) died of Plasmodium chabaudi chabaudi infection, although wild-type (WT) mice had self-limiting infections. OPN was detected in the WT mice at 2 days postinfection. OPN-KO mice produced significantly smaller amounts of interleukin-12 and gamma interferon than WT mice produced. These results suggested that OPN is involved in Th1-mediated immunity against malaria infection.     

A critical role for stem cell factor and c-kit in host protective immunity to an intestinal helminth

In common with many intestinal nematode infections, Trichinellaspiralls infections in mice are associated with a pronouncedintestinal mast cell hyperplasia. The expulsion of the parasitefrom the gut is temporally associated with intestinal mastocytosisand mast cell function reflected by the secretion of mast cellprotease into tissue and serum. In vivo, mucosal mast cell productionis highly dependent upon T cell-derived cytokines includingIL-3 and lL-4. We present data here to show that intestinalmast cell hyperplasia induced by helminth infection is alsodependent upon the production of stem cell factor (SCF). Neutralizationof SCF by anti-SCF or anti-SCF receptor mAb completely abrogatedthe mast cell hyperplasia generated by T spiralls infection.Moreover, worm expulsion was dramatically delayed in treatedmice and a reduced intestinal eoslnophilla was observed. Theseeffects did not appear to be mediated through alteration ofT^h cell responses and the parasite-specific serum antibody responsewas not affected. The reduction in the mast cell response andworm expulsion observed after SCF neutralization were reversiblefollowing cessation of monoclonal treatment. The data presentedhere clearly demonstrate a major role for SCF in the generationof intestinal mastocytosis and the host protective immune responsefollowing parasitic infection.     

Sex differences in host resistance to Mycobacterium marinum infection in mice.

Sex differences were observed in host resistance to Mycobacterium marinum infection in mice. Males were found to be more susceptible than females in terms of mortality, incidence of gross skin lesions, and bacterial load in the lungs and spleen. The degree of sex differences varied from strain to strain of test mice, in the order C3H/He, A/J, and BALB/c greater than DBA/2, B10.A, and C57BL/6, on the basis of survival time and multiplication of organisms in the visceral organs. Although this ordering corresponded to the susceptibilities of both male and female mice to the organisms, much greater strain dependency was seen in males than in females. Castration caused an increase in the host resistance of males, but this effect was substantially reversed by continuous testosterone treatment. Testosterone also increased the susceptibility of female mice to this infection. These findings imply that the male sex hormone is involved in the lowered anti-M. marinum resistance of males. Although athymic mice were more susceptible than euthymic mice, a substantial degree of sex difference was also observed in the T-cell-depleted animals, indicating that natural host resistance to this infection is sex dependent. Indeed, more efficient macrophage mobilization at the site of infection was seen in females than in males. Although female T-cell transplantation improved the lowered resistance of athymic mice, there was a sex difference in bacterial growth in the lungs. This implies that sex hormones affect T-cell functions.