Enteric infection with Citrobacter rodentium induces coagulative liver necrosis and hepatic inflammation prior to peak infection and colonic disease

Acute and chronic forms of inflammation are known to affect liver responses and susceptibility to disease and injury. Furthermore, intestinal microbiota has been shown critical in mediating inflammatory host responses in various animal models. Using C. rodentium, a known enteric bacterial pathogen, we examined liver responses to gastrointestinal infection at various stages of disease pathogenesis. For the first time, to our knowledge, we show distinct liver pathology associated with enteric infection with C. rodentium in C57BL/6 mice, characterized by increased inflammation and hepatitis index scores as well as prominent periportal hepatocellular coagulative necrosis indicative of thrombotic ischemic injury in a subset of animals during the early course of C. rodentium pathogenesis. Histologic changes in the liver correlated with serum elevation of liver transaminases, systemic and liver resident cytokines, as well as signal transduction changes prior to peak bacterial colonization and colonic disease. C. rodentium infection in C57BL/6 mice provides a potentially useful model to study acute liver injury and inflammatory stress under conditions of gastrointestinal infection analogous to enteropathogenic E. coli infection in humans.     

Cathelicidin mediates innate intestinal defense against colonization with epithelial adherent bacterial pathogens

Cathelicidin-related antimicrobial peptide (mCRAMP), the sole murine cathelicidin, is encoded by the gene Cnlp. We show that mCRAMP expression in the intestinal tract is largely restricted to surface epithelial cells in the colon. Synthetic mCRAMP had antimicrobial activity against the murine enteric pathogen Citrobacter rodentium, which like the related clinically important human pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli, adheres to the apical membrane of intestinal epithelial cells. Colon epithelial cell extracts from Cnlp+/+ mice had significantly greater antimicrobial activity against C. rodentium than those of mutant Cnlp-/- mice that lack mCRAMP. Cnlp-/- mice developed significantly greater colon surface and crypt epithelial cell colonization, surface epithelial cell damage, and systemic dissemination of infection than Cnlp+/+ mice after oral infection with C. rodentium. Moreover, Cnlp+/+ mice were protected from oral infections with C. rodentium inocula that infected the majority of Cnlp-/- mice. These results establish cathelicidin as an important component of innate antimicrobial defense in the colon.     

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.     

The intestinal microbiota plays a role in Salmonella-induced colitis independent of pathogen colonization

The intestinal microbiota is composed of hundreds of species of bacteria, fungi and protozoa and is critical for numerous biological processes, such as nutrient acquisition, vitamin production, and colonization resistance against bacterial pathogens. We studied the role of the intestinal microbiota on host resistance to Salmonella enterica serovar Typhimurium-induced colitis. Using multiple antibiotic treatments in 129S1/SvImJ mice, we showed that disruption of the intestinal microbiota alters host susceptibility to infection. Although all antibiotic treatments caused similar increases in pathogen colonization, the development of enterocolitis was seen only when streptomycin or vancomycin was used; no significant pathology was observed with the use of metronidazole. Interestingly, metronidazole-treated and infected C57BL/6 mice developed severe pathology. We hypothesized that the intestinal microbiota confers resistance to infectious colitis without affecting the ability of S. Typhimurium to colonize the intestine. Indeed, different antibiotic treatments caused distinct shifts in the intestinal microbiota prior to infection. Through fluorescence in situ hybridization, terminal restriction fragment length polymorphism, and real-time PCR, we showed that there is a strong correlation between the intestinal microbiota composition before infection and susceptibility to Salmonella-induced colitis. Members of the Bacteroidetes phylum were present at significantly higher levels in mice resistant to colitis. Further analysis revealed that Porphyromonadaceae levels were also increased in these mice. Conversely, there was a positive correlation between the abundance of Lactobacillus sp. and predisposition to colitis. Our data suggests that different members of the microbiota might be associated with S. Typhimurium colonization and colitis. Dissecting the mechanisms involved in resistance to infection and inflammation will be critical for the development of therapeutic and preventative measures against enteric pathogens.     

Innate host responses to enteric bacterial pathogens: a balancing act between resistance and tolerance

Infection by enteric bacterial pathogens activates pathogen recognition receptors, leading to innate responses that promote host defence. While responses that promote host 'resistance' to infection, through the release of antimicrobial mediators, or the recruitment of inflammatory cells aimed at clearing the infection are best known, recent studies have begun to identify additional innate driven responses that instead promote intestinal tissue repair and host survival. Described as infection 'tolerance' responses, we and others have primarily studied these responses in the Citrobacter rodentium infection model. In this review we discuss the impact of innate resistance mechanisms on host defence, and describe how 'tolerance' responses act primarily on the intestinal epithelium, triggering epithelial cell proliferation, repair or promoting barrier function. Resistance and tolerance responses appear to work together, with tolerance repairing the tissue injury caused by resistance driven inflammation. Tolerance responses fit a pattern where innate immunity and inflammation are tightly regulated in the gastrointestinal tract. Moreover, tolerance may have developed due to the successful subversion and avoidance of host resistance by enteric bacterial pathogens. Further studies are needed to clarify the contribution of different pathogen recognition receptors to tolerance and resistance responses against bacterial pathogens, in the gut or in other host tissues.     

Citrobacter rodentium of mice and man

The major classes of enteric bacteria harbour a conserved core genomic structure, common to both commensal and pathogenic strains, that is most likely optimized to a life style involving colonization of the host intestine and transmission via the environment. In pathogenic bacteria this core genome framework is decorated with novel genetic islands that are often associated with adaptive phenotypes such as virulence. This classical genome organization is well illustrated by a group of extracellular enteric pathogens, which includes enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium, all of which use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. Both EHEC and EPEC are poorly pathogenic in mice but infect humans and domestic animals. In contrast, C. rodentium is a natural mouse pathogen that is related to E. coli, hence providing an excellent in vivo model for A/E lesion forming pathogens. C. rodentium also provides a model of infections that are mainly restricted to the lumen of the intestine. The mechanism's by which the immune system deals with such infections has become a topic of great interest in recent years. Here we review the literature of C. rodentium from its emergence in the mid-1960s to the most contemporary reports of colonization, pathogenesis, transmission and immunity.     

Colonic microbiota alters host susceptibility to infectious colitis by modulating inflammation, redox status, and ion transporter gene expression

Individuals vary in their resistance to enteric infections. The role of the intestinal microbiota in altering susceptibility to enteric infection is relatively unknown. Previous studies have identified that C3H/HeOuJ mice suffer 100% mortality during Citrobacter rodentium-induced colitis, whereas C57BL/6 mice recover from infection. The basis for their differences in susceptibility is unclear and has been mainly attributed to differences in host genetics. This study investigated the role of the intestinal microbiota in altering susceptibility to C. rodentium-induced colitis. When the feces of C57BL/6 mice were gavaged into antibiotic treated C3H/HeOuJ mice, the C57BL/6 microflora led to a complete reversal in mortality patterns where 100% of the C3H/HeOuJ mice survived infection. This protection corresponded with reduced colonic pathology and less systemic pathogen load and was associated with increased inflammatory and redox responses with reduced epithelial cell death. C3H/HeOuJ mice are normally susceptible to infection-induced dehydration due to defective expression of colonic ion transporters such as Dra, CA IV, and CA I; expression of these genes was normalized when C3H/HeOuJ mice were colonized with the C57BL/6 microflora. Together, these data reveal that the colonic microbiota play a critical role in protecting against intestinal infection by inducing proinflammatory and prooxidant responses that control pathogen load as well as ion transporter gene expression previously shown to prevent fatal dehydration. Protection of mice from lethal colitis was associated with higher levels of bacteria from Bacteroidetes. This study reveals that the microbiota is sufficient to overcome inherent genetic susceptibility patterns in C3H/HeOuJ mice that cause mortality during C. rodentium infection.     

Mechanisms controlling pathogen colonization of the gut

The intestinal microbiota can protect efficiently against colonization by many enteric pathogens ('colonization resistance', CR). This phenomenon has been known for decades, but the mechanistic basis of CR is incompletely defined. At least three mechanisms seem to contribute, that is direct inhibition of pathogen growth by microbiota-derived substances, nutrient depletion by microbiota growth and microbiota-induced stimulation of innate and adaptive immune responses. In spite of CR, intestinal infections are well known to occur. In these cases, the multi-faceted interactions between the microbiota, the host and the pathogen are shifted in favor of the pathogen. We are discussing recent progress in deciphering the underlying molecular mechanisms in health and disease.     

The role of microbiota in infectious disease

The intestine harbors an ecosystem composed of the intestinal mucosa and the commensal microbiota. The microbiota fosters development, aids digestion and protects host cells from pathogens - a function referred to as colonization resistance. Little is known about the molecular basis of colonization resistance and how it can be overcome by enteropathogenic bacteria. Recently, studies on inflammatory bowel diseases and on animal models for enteric infection have provided new insights into colonization resistance. Gut inflammation changes microbiota composition, disrupts colonization resistance and enhances pathogen growth. Thus, some pathogens can benefit from inflammatory defenses. This new paradigm will enable the study of host factors enhancing or inhibiting bacterial growth in health and disease.     

Mining zebrafish microbiota reveals key community-level resistance against fish pathogen infection

The long-known resistance to pathogens provided by host-associated microbiota fostered the notion that adding protective bacteria could prevent or attenuate infection. However, the identification of endogenous or exogenous bacteria conferring such protection is often hindered by the complexity of host microbial communities. Here, we used zebrafish and the fish pathogen Flavobacterium columnare as a model system to study the determinants of microbiota-associated colonization resistance. We compared infection susceptibility in germ-free, conventional and reconventionalized larvae and showed that a consortium of 10 culturable bacterial species are sufficient to protect zebrafish. Whereas survival to F. columnare infection does not rely on host innate immunity, we used antibiotic dysbiosis to alter zebrafish microbiota composition, leading to the identification of two different protection strategies. We first identified that the bacterium Chryseobacterium massiliae individually protects both larvae and adult zebrafish. We also showed that an assembly of 9 endogenous zebrafish species that do not otherwise protect individually confer a community-level resistance to infection. Our study therefore provides a rational approach to identify key endogenous protecting bacteria and promising candidates to engineer resilient microbial communities. It also shows how direct experimental analysis of colonization resistance in low-complexity in vivo models can reveal unsuspected ecological strategies at play in microbiota-based protection against pathogens.Subject terms: Microbiome, Microbial ecology     

Alternatively activated macrophages in intestinal helminth infection: effects on concurrent bacterial colitis

The distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, including enteric bacterial infections. To dissect the mechanisms by which helminths modulate the host's response to enteric bacteria and bacteria-mediated intestinal inflammation, we have recently established a coinfection model and shown that coinfection with the helminth Heligmosomoides polygyrus exacerbates colitis induced by infection with the gram-negative bacterial pathogen Citrobacter rodentium. The disease severity of the coinfected mice was correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments, delayed bacterial clearance, and a significantly enhanced colonic TNF-alpha response. In the present study, using our in vivo coinfection model as well as in vitro approaches, we test the hypothesis that the phenotypic and functional alterations in macrophages induced by the helminth-driven T cell response may contribute to the observed alterations in the response to C. rodentium. We show that via a STAT6-dependent mechanism H. polygyrus coinfection results in a marked infiltration into the colonic lamina propria of F4/80+ cells that have the phenotype of alternatively activated macrophages. Functional analysis of these macrophages further shows that they are impaired in their killing of internalized bacteria. Yet, these cells produce an enhanced amount of TNF-alpha in response to C. rodentium infection. These results demonstrate that helminth infection can impair host protection against concurrent enteric bacterial infection and promote bacteria-induced intestinal injury through a mechanism that involves the induction of alternatively activated macrophages.     

Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens

Infections by attaching and effacing (A/E) bacterial pathogens, such as Escherichia coli O157:H7, pose a serious threat to public health. Using a mouse A/E pathogen, Citrobacter rodentium, we show that interleukin-22 (IL-22) has a crucial role in the early phase of host defense against C. rodentium. Infection of IL-22 knockout mice results in increased intestinal epithelial damage, systemic bacterial burden and mortality. We also find that IL-23 is required for the early induction of IL-22 during C. rodentium infection, and adaptive immunity is not essential for the protective role of IL-22 in this model. Instead, IL-22 is required for the direct induction of the Reg family of antimicrobial proteins, including RegIIIbeta and RegIIIgamma, in colonic epithelial cells. Exogenous mouse or human RegIIIgamma substantially improves survival of IL-22 knockout mice after C. rodentium infection. Together, our data identify a new innate immune function for IL-22 in regulating early defense mechanisms against A/E bacterial pathogens.     

MyD88 signalling plays a critical role in host defence by controlling pathogen burden and promoting epithelial cell homeostasis during Citrobacter rodentium-induced colitis

Myeloid differentiation factor (MyD)88, an adaptor protein shared by the Toll-interleukin 1 receptor superfamily, plays a critical role in host defence during many systemic bacterial infections by inducing protective inflammatory responses that limit bacterial growth. However, the role of innate responses during gastrointestinal (GI) infections is less clear, in part because the GI tract is tolerant to commensal antigens. The current study investigated the role of MyD88 following infection by the murine bacterial pathogen, Citrobacter rodentium . MyD88-deficient mice suffered a lethal colitis coincident with colonic mucosal ulcerations and bleeding. Their susceptibility was associated with an overwhelming bacterial burden and selectively impaired immune responses in colonic tissues, which included delayed inflammatory cell recruitment, reduced iNOS and abrogated production of TNF-α and IL-6 from MyD88-deficient macrophages and colons cultured ex vivo . Immunostaining for Ki67 and BrDU revealed that MyD88 signalling mediated epithelial hyper-proliferation in response to C. rodentium infection. Thus, MyD88-deficient mice could not promote epithelial cell turnover and repair, leading to deep bacterial invasion of colonic crypts, intestinal barrier dysfunction and, ultimately, widespread mucosal ulcerations. In conclusion, MyD88 signalling within the GI tract plays a critical role in mediating host defence against an enteric bacterial pathogen, by controlling bacterial numbers and promoting intestinal epithelial homeostasis.     

The contributing role of the intestinal microbiota in stressor-induced increases in susceptibility to enteric infection and systemic immunomodulation

This article is part of a Special Issue "Neuroendocrine-Immune Axis in Health and Disease." The body is colonized by highly complex and genetically diverse communities of microbes, the majority of which reside within the intestines in largely stable but dynamically interactive climax communities. These microbes, referred to as the microbiota, have many functions that enhance the health of the host, and it is now recognized that the microbiota influence both mucosal and systemic immunity. The studies outlined in this review demonstrate that the microbiota are also involved in stressor-induced immunomodulation. Exposure to different types of stressors, including both physical and psychological stressors, changes the composition of the intestinal microbiota. The altered profile increases susceptibility to an enteric pathogen, i.e., Citrobacter rodentium, upon oral challenge, but is also associated with stressor-induced increases in innate immune activity. Studies using germfree mice, as well as antibiotic-treated mice, provide further evidence that the microbiota contribute to stressor-induced immunomodulation; stressor-induced increases in splenic macrophage microbicidal activity fail to occur in mice with no, or reduced, intestinal microbiota. While the mechanisms by which microbiota can impact mucosal immunity have been studied, how the microbiota impact systemic immune responses is not clear. A mechanism is proposed in which stressor-induced degranulation of mucosal mast cells increases the permeability of the intestines. This increased permeability would allow intact bacteria and/or bacterial products (like peptidoglycan) to translocate from the lumen of the intestines to the interior of the body, where they directly, or indirectly, prime the innate immune system for enhanced reactivity to antigenic stimulation.     

Disentangling the effect of host genetics and gut microbiota on resistance to an intestinal parasite

Resistance to infection is a multifactorial trait, and recent work has suggested that the gut microbiota can also contribute to resistance. Here, we performed a fecal microbiota transplant to disentangle the contribution of the gut microbiota and host genetics as drivers of resistance to the intestinal nematode Heligmosomoides polygyrus. We transplanted the microbiota of a strain of mice (SJL), resistant to H. polygyrus, into a susceptible strain (CBA) and vice-versa. We predicted that if the microbiota shapes resistance to H. polygyrus, the FMT should reverse the pattern of resistance between the two host strains. The two host strains had different microbiota diversities and compositions before the start of the experiment, and the FMT altered the microbiota of recipient mice. One mouse strain (SJL) was more resistant to colonization by the heterologous microbiota, and it maintained its resistance profile to H. polygyrus (lower parasite burden) independently of the FMT. On the contrary, CBA mice harbored parasites with lower fecundity during the early stage of the infection, and had an up-regulated expression of the cytokine IL-4 (a marker of H. polygyrus resistance) after receiving the heterologous microbiota. Therefore, while host genetics remains the main factor shaping the pattern of resistance to H. polygyrus, the composition of the gut microbiota also seems to play a strain-specific role.     

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Compete with the Intestinal Microbiota

Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10^−/−, VILLIN-HA^CL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen.     

Helminth-primed dendritic cells alter the host response to enteric bacterial infection

To examine whether intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established using the intestinal helminth Heligomosomoides polygyrus and a murine pathogen Citrobacter rodentium, which causes infectious colitis. Using this model we recently have shown that coinfection with the Th2-inducing H. polygyrus and C. rodentium promotes bacterial-associated disease and colitis. In this study, we expand our previous observations and examine the hypothesis that dendritic cells (DC) stimulated by helminth infection may play an important role in the regulation of the intestinal immune response to concurrent C. rodentium infection as well as in the modulation of the bacterial pathogenesis. We show that H. polygyrus infection induces DC activation and IL-10 expression, and that adoptive transfer of parasite-primed DC significantly impairs host protection to C. rodentium infection, resulting in an enhanced bacterial infection and in the development of a more severe colonic injury. Furthermore, we demonstrate that adoptive transfer of parasite-primed IL-10-deficient DCs fails to result in the development of a significantly enhanced C. rodentium-mediated colitis. Similarly, when the DC IL-10 response was neutralized by anti-IL-10 mAb treatment in mice that received parasite-primed DC, no deleterious effect of the parasite-primed DC on the host intestinal response to C. rodentium was detected. Thus, our results provide evidence to indicate that the H. polygyrus-dependent modulation of the host response to concurrent C. rodentium infection involves IL-10-producing DCs.     

TLR signaling is required for Salmonella typhimurium virulence

Toll-like receptors (TLRs) contribute to host resistance to microbial pathogens and can drive the evolution of virulence mechanisms. We have examined the relationship between host resistance and pathogen virulence using mice with a functional allele of the nramp-1 gene and lacking combinations of TLRs. Mice deficient in both TLR2 and TLR4 were highly susceptible to the intracellular bacterial pathogen Salmonella typhimurium, consistent with reduced innate immune function. However, mice lacking additional TLRs involved in S. typhimurium recognition were less susceptible to infection. In these TLR-deficient cells, bacteria failed to upregulate Salmonella pathogenicity island 2 (SPI-2) genes and did not form a replicative compartment. We demonstrate that TLR signaling enhances the rate of acidification of the Salmonella-containing phagosome, and inhibition of this acidification prevents SPI-2 induction. Our results indicate that S. typhimurium requires cues from the innate immune system to regulate virulence genes necessary for intracellular survival, growth, and systemic infection.