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.     

Role of inflammasomes in host defense against Citrobacter rodentium infection

Citrobacter rodentium is an enteric bacterial pathogen of the mouse intestinal tract that triggers inflammatory responses resembling those of humans infected with enteropathogenic and enterohemorrhagic Escherichia coli. Inflammasome signaling is emerging as a central regulator of inflammatory and host responses to several pathogens, but the in vivo role of inflammasome signaling in host defense against C. rodentium has not been characterized. Here, we show that mice lacking the inflammasome components Nlrp3, Nlrc4, and caspase-1 were hypersusceptible to C. rodentium-induced gastrointestinal inflammation. This was due to defective interleukin (IL)-1β and IL-18 production given that il-1β(-/-) and il-18(-/-) mice also suffered from increased bacterial burdens and exacerbated histopathology. C. rodentium specifically activated the Nlrp3 inflammasome in in vitro-infected macrophages independently of a functional bacterial type III secretion system. Thus, production of IL-1β and IL-18 downstream of the Nlrp3 and Nlrc4 inflammasomes plays a critical role in host defense against enteric infections caused by C. rodentium.     

Toll-like receptor 2 plays a critical role in maintaining mucosal integrity during Citrobacter rodentium-induced colitis

Inflammatory bowel diseases and infectious gastroenteritis likely occur when the integrity of intestinal barriers is disrupted allowing luminal bacterial products to cross into the intestinal mucosa, stimulating immune cells and triggering inflammation. While specific Toll-like receptors (TLR) are involved in the generation of inflammatory responses against enteric bacteria, their contributions to the maintenance of intestinal mucosal integrity are less clear. These studies investigated the role of TLR2 in a model of murine colitis induced by the bacterial pathogen Citrobacter rodentium . C. rodentium supernatants specifically activated TLR2 in vitro while infected TLR2–/– mice suffered a lethal colitis coincident with colonic mucosal ulcerations, bleeding and increased cell death but not increased pathogen burden. TLR2–/– mice suffered impaired epithelial barrier function mediated via zonula occludens (ZO)-1 in naïve mice and claudin-3 in infected mice, suggesting this could underlie their susceptibility. TLR2 deficiency was also associated with impaired production of IL-6 by bone marrow-derived macrophages and infected colons cultured ex vivo . As IL-6 has antiapoptotic and epithelial repair capabilities, its reduced expression could contribute to the impaired mucosal integrity. These studies report for the first time that TLR2 plays a critical role in maintaining intestinal mucosal integrity during infection by a bacterial pathogen.     

Autophagy in the intestinal epithelium regulates Citrobacter rodentium infection

Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been fully elucidated. In this study, we showed that the essential autophagy protein Atg7 is required for resistance to Citrobacter rodentium infection in the intestinal epithelium. Infected mice in which Atg7 had been conditionally deleted from the intestinal epithelium exhibited greater clinical evidence of disease and higher expression levels of pro-inflammatory cytokine mRNA in the large intestine. Moreover, C. rodentium clearance was reduced in the Atg7 conditional knockout mice. These results demonstrate that autophagy in intestinal epithelial cells plays an important role in host defense against C. rodentium infection and the regulation of C. rodentium infectious colitis.     

Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae

While the normal microbiota has been implicated as a critical defense against invading pathogens, the impact of enteropathogenic infection and host inflammation on intestinal microbial communities has not been elucidated. Using mouse models of Citrobacter rodentium, which closely mimics human diarrheal pathogens inducing host intestinal inflammation, and Campylobacter jejuni infection, as well as chemically and genetically induced models of intestinal inflammation, we demonstrate that host-mediated inflammation in response to an infecting agent, a chemical trigger, or genetic predisposition markedly alters the colonic microbial community. While eliminating a subset of indigenous microbiota, host-mediated inflammation supported the growth of either the resident or introduced aerobic bacteria, particularly of the Enterobacteriaceae family. Further, assault by an enteropathogen and host-mediated inflammation combined to significantly reduce the total numbers of resident colonic bacteria. These findings underscore the importance of intestinal microbial ecosystems in infectious colitis and noninfectious intestinal inflammatory conditions, such as inflammatory bowel disease.     

Host-mediated inflammation disrupts the intestinal microbiota and promotes the overgrowth of Enterobacteriaceae

While the normal microbiota has been implicated as a critical defense against invading pathogens, the impact of enteropathogenic infection and host inflammation on intestinal microbial communities has not been elucidated. Using mouse models of Citrobacter rodentium, which closely mimics human diarrheal pathogens inducing host intestinal inflammation, and Campylobacter jejuni infection, as well as chemically and genetically induced models of intestinal inflammation, we demonstrate that host-mediated inflammation in response to an infecting agent, a chemical trigger, or genetic predisposition markedly alters the colonic microbial community. While eliminating a subset of indigenous microbiota, host-mediated inflammation supported the growth of either the resident or introduced aerobic bacteria, particularly of the Enterobacteriaceae family. Further, assault by an enteropathogen and host-mediated inflammation combined to significantly reduce the total numbers of resident colonic bacteria. These findings underscore the importance of intestinal microbial ecosystems in infectious colitis and noninfectious intestinal inflammatory conditions,such as inflammatory bowel disease.     

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.     

Subcellular expression pattern and role of IL-15 in pneumococci induced lung epithelial apoptosis

Streptococcus pneumoniae is the leading causative agent of community-acquired pneumonia. Induction of apoptosis in pulmonary epithelial cells by bacteria during pneumonia might be harmful to the host. Interleukin-15 (IL-15) has been demonstrated as an effective inhibitor of apoptosis and is expressed in lung epithelium on the mRNA and protein level. Therefore, we characterized the sub-cellular expression pattern of the short and long IL-15 isoforms in lung epithelial cells in vitro as well as its role in pneumococci-related lung epithelial cell apoptosis. We found an expression pattern for both IL-15 signal peptides in the pulmonary epithelial cell lines A549 and Beas-2B. Moreover, a strong co-localization of IL-15 and IL-15Ralpha was detected on cell surfaces. Compared to pro-inflammatory cytokine stimulation, neither IL-15 nor its trimeric receptor complex was up-regulated after pneumococcal infection. However, overexpression of IL-15 isoforms revealed IL-15LSP and IL-15Vkl as inhibitors of pneumococci induced apoptosis in pulmonary epithelial cells. Thus, IL-15 may act as an anti-apoptotic molecule in pneumococci infection, thereby suggesting IL-15 as a benefical cytokine in pulmonary host defense against infection.     

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.     

Citrobacter rodentium infection causes both mitochondrial dysfunction and intestinal epithelial barrier disruption in vivo: role of mitochondrial associated protein (Map)

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli are non-invasive attaching/effacing (A/E) bacterial pathogens that infect their host's intestinal epithelium, causing severe diarrhoeal disease. These bacteria utilize a type III secretion apparatus to deliver effector molecules into host cells, subverting cellular function. Mitochondrial associated protein (Map) is a multifunctional effector protein that targets host cell mitochondria and contributes to infection-induced epithelial barrier dysfunction in vitro. Unfortunately, the relevance of these actions to the pathogenesis of EPEC-induced disease is uncertain. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that Map colocalized with host cell mitochondria, and that in vivo infection led to a disruption of mitochondrial morphology in infected colonocytes as assessed by electron microscopy. Histochemical staining for the mitochondrial enzyme succinate dehydrogenase also revealed a significant loss of mitochondrial respiratory function in the infected intestinal epithelium; however, both pathologies were attenuated in mice infected with a Deltamap strain. C. rodentium Map was also implicated in the disruption of epithelial barrier function both in vitro and in vivo. These studies thus advance our understanding of how A/E pathogens subvert host cell functions and cause disease, demonstrating that Map contributes to the functional disruption of the intestinal epithelium during enteric infection by C. rodentium.     

Citrobacter rodentium infection causes iNOS-independent intestinal epithelial dysfunction in mice

Attaching-effacing bacteria are major causes of infectious diarrhea in humans worldwide. Citrobacter rodentium is an attaching-effacing enteric pathogen that causes transmissible murine colonic mucosal hyperplasia. We characterized colonic inflammation and ion transport at 3, 7, 10, 30, and 60 d after infection of C57Bl/6 mice with C. rodentium. Macroscopic damage score was significantly increased 7 and 10 d after infection. Colonic wall thickness was increased at 7, 10, 30, and 60 d. Myeloperoxidase (MPO) activity was significantly increased at 3, 7, and 10 d and returned to control levels by days 30 and 60. The expressions of inducible nitric oxide synthase and cyclooxygenase-2 were increased by C. rodentium infection. Significant reductions in the epithelial secretory response to carbachol, but not to electrical field stimulation or forskolin, were observed at 3 and 10 d of infection. Translocation of enteric bacteria into the mesenteric lymph nodes was observed 10 d following infection. There was no difference in response to infection between animals deficient in inducible nitric oxide synthase and wild-type controls. The COX-2 inhibitor rofecoxib caused decreased wall thickness and MPO activity at day 10. However, COX-2 inhibition did not alter infection-induced changes in ion transport. Citrobacter rodentium infection causes colonic inflammation, mucosal hyperplasia, and nitric-oxide-independent epithelial dysfunction in association with increased permeability to luminal bacteria.     

Haemophilus influenzae stimulates ICAM-1 expression on respiratory epithelial cells

Epithelial cells interact directly with bacteria in the environment and play a critical role in airway defense against microbial pathogens. In this study, we examined the response of respiratory epithelial cells to infection with nontypable Haemophilus influenzae. Using an in vitro cell culture model, we found that epithelial cell monolayers released significant quantities of IL-8 and expressed increased levels of ICAM-1 mRNA and surface protein in response to H. influenzae. In contrast, levels of IL-1beta, TNF-alpha, and MHC class I were not significantly affected, suggesting preferential activation of a specific subset of epithelial genes directed toward defense against bacteria. Induction of ICAM-1 required direct bacterial interaction with the epithelial cell surface and was not reproduced by purified H. influenzae lipooligosaccharide. Consistent with a functional role for this response, induction of ICAM-1 by H. influenzae mediated increased neutrophil adherence to the epithelial cell surface. Furthermore, in an in vivo murine model of airway infection with H. influenzae, increased epithelial cell ICAM-1 expression coincided with increased chemokine levels and neutrophil recruitment in the airway. These results indicate that ICAM-1 expression on human respiratory epithelial cells is induced by epithelial cell interaction with H. influenzae and suggest that an ICAM-1-dependent mechanism can mediate neutrophil adherence to these cells independent of inflammatory mediator release by other cell types. Direct induction of specific epithelial cell genes (such as ICAM-1 and IL-8) by bacterial infection may allow for rapid and efficient innate defense in the airway.     

Epidermal growth factor receptor acts as a negative regulator for bacterium nontypeable Haemophilus influenzae-induced Toll-like receptor 2 expression via an Src-dependent p38 mitogen-activated protein kinase signaling pathway

Epidermal growth factor receptor (EGFR) has been shown to play important roles in regulating diverse biological processes, including cell growth, differentiation, apoptosis, adhesion, and migration. Its role in regulating human Toll-like receptors (TLRs), key host defense receptors that recognize invading bacterial pathogens, however, remains unknown. Here we show for the first time that EGFR acts as a negative regulator for TLR2 induction by the bacterium nontypeable Haemophilus influenzae (NTHi) in vitro and in vivo. The negative regulation of TLR2 induction by EGFR is mediated via an Src-MKK3/6-p38 alpha/beta MAP kinase-dependent mechanism. Moreover, direct activation of EGFR signaling by the bacterium NTHi-derived EGF-like factor appears to be responsible for triggering the downstream Src-MKK3/6-p38 MAPK signaling, which in turn leads to the negative regulation of TLR2 induction. Finally, exogenous EGF increases NTHi invasion of host epithelial cells, thereby demonstrating the biological significance of TLR2 regulation by EGFR signaling. The evidence we provided in the present study may suggest a novel strategy utilized by bacteria to attenuate host defensive and immune response by negatively regulating the expression of host defense receptor TLR2. These studies may bring new insight for fully understanding the important role of EGFR signaling in regulating host defense and immune response by tightly controlling TLR2 induction during bacterial infections.     

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.     

肠粘膜上皮细胞在天然免疫中的作用

粘膜免疫是机体防御系统的主要成分。致病性细菌侵入机体后,首先遭遇到天然免疫的抵抗,随后产生获得性免疫,两共同执行机体的防御功能,消灭入侵细菌。最近的研究表明上皮细胞对细菌感染有重要的免疫调节作用,在天然免疫与获得性免疫防御机制中起重要作用。本重点介绍肠上皮细胞在天然免疫中的作用。     

TLR2 signaling contributes to rapid inflammasome activation during F. novicida infection

BACKGROUND: Early detection of microorganisms by the innate immune system is provided by surface-expressed and endosomal pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). Detection of microbial components by TLRs initiates a signaling cascade leading to the expression of proinflammatory cytokines including IL-6 and IL-1β. Some intracellular bacteria subvert the TLR response by rapidly escaping the phagosome and entering the cytosol. However, these bacteria may be recognized by the inflammasome, a multi-protein complex comprised of a sensor protein, ASC and the cysteine protease caspase-1. Inflammasome activation leads to release of the proinflammatory cytokines IL-1β and IL-18 and death of the infected cell, an important host defense that eliminates the pathogen's replicative niche. While TLRs and inflammasomes are critical for controlling bacterial infections, it is unknown whether these distinct host pathways cooperate to activate defenses against intracellular bacteria. METHODOLOGY/SIGNIFICANT FINDINGS: Using the intracellular bacterium Francisella novicida as a model, we show that TLR2(-/-) macrophages exhibited delayed inflammasome activation compared to wild-type macrophages as measured by inflammasome assembly, caspase-1 activation, cell death and IL-18 release. TLR2 also contributed to inflammasome activation in response to infection by the cytosolic bacterium Listeria monocytogenes. Components of the TLR2 signaling pathway, MyD88 and NF-κB, were required for rapid inflammasome activation. Furthermore, TLR2(-/-) mice exhibited lower levels of cell death, caspase-1 activation, and IL-18 production than wild-type mice upon F. novicida infection. CONCLUSIONS/SIGNIFICANCE: These results show that TLR2 is required for rapid inflammasome activation in response to infection by cytosolic bacterial pathogens. In addition to further characterizing the role of TLR2 in host defense, these findings broaden our understanding of how the host integrates signals from spatiotemporally separated PRRs to coordinate an innate response against intracellular bacteria.     

Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C. rodentium in situ and in real time. The bioluminescent C. rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo. We have demonstrated that primary colonization of the mouse by C. rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch. Following colonization of the caecum C. rodentium established a colonic infection. Clearance of C. rodentium ICC180 parallels the colonization dynamics, i.e. the caecum was first to be cleared followed by the colon. A bioluminescent eae (encoding the outer membrane adhesin intimin) C. rodentium mutant failed to establish long-term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.