NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis.

Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1ß secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells.

The goblet cell-derived mediator RELM-ß drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.

Intestinal goblet cells are potentially key players in controlling susceptibility to ulcerative colitis (UC). Although impaired mucin (Muc2) production by goblet cells increases microbial stimulation of the colonic mucosa, goblet cells secrete other mediators that may influence or promote UC development. Correspondingly, Muc2-deficient ((-/-)) mice develop spontaneous colitis, concurrent with the dramatic upregulation of the goblet cell mediator, resistin-like molecule-beta (RELM-ß). Testing RELM-ß's role, we generated Muc2(-/-)/Retnlb(-/-) mice, finding that RELM-ß deficiency significantly attenuated colitis development and symptoms compared with Muc2(-/-) mice. RELM-ß expression in Muc2(-/-) mice strongly induced the production/secretion of the antimicrobial lectin RegIIIß, that exerted its microbicidal effect predominantly on Gram-positive Lactobacillus species. Compared with Muc2(-/-)/Retnlb(-/-) mice, this worsened intestinal microbial dysbiosis with a selective loss of colonic Lactobacilli spp. in Muc2(-/-) mice. Orally replenishing Muc2(-/-) mice with murine Lactobacillus spp., but not with a probiotic formulation containing several human Lactobacillus spp. (VSL#3), ameliorated their spontaneous colitis in concert with increased production of short-chain fatty acids. These studies demonstrate that the goblet cell mediator RELM-ß drives colitis in Muc2(-/-) mice by depleting protective commensal microbes. The ability of selective commensal microbial replacement to ameliorate colitis suggests that personalized bacterial therapy may prove beneficial for treatment of UC.

Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection.

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.

Bacterial stimulation of the TLR-MyD88 pathway modulates the homeostatic expression of ileal Paneth cell α-defensins.

Paneth cell α-defensins are antimicrobial peptides involved in the control of the intestinal microbiota and immunological homeostasis. In mice, they are encoded by multiple, highly homologous genes (Defa). The transcriptional activity of ileal Defa genes was studied in response to pharmacological and genetic perturbations of the intestinal environment of C57BL/6 mice. Defa gene transcription was sensitive to oral antibiotic administration suggesting that commensal microbes regulate Defa expression. Ileal microbiota analysis showed that decreased transcription of Defa genes correlated with depletion of Lactobacillus. Defa expression was partially restored in vivo by lactobacillus administration to antibiotic-treated mice. Defa transcripts were less abundant in ex vivo, microbiota-free intestinal explants but recovered after explant exposure to UV-killed bacteria, Toll-like receptor (TLR)-2 or TLR4 agonists. Genetic deficiency of several TLRs or MyD88 led to dramatic drops in Defa transcription in vivo. These results show that Paneth cell Defa genes are regulated by commensal bacteria through TLR-MyD88 signaling and provide a further understanding of the dysregulation of intestinal homeostasis that occurs as a result of imbalances in the populations of commensal bacteria.

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.

Antibiotic treatment alters the colonic mucus layer and predisposes the host to exacerbated Citrobacter rodentium-induced colitis.

Antibiotics are often used in the clinic to treat bacterial infections, but the effects of these drugs on microbiota composition and on intestinal immunity are poorly understood. Citrobacter rodentium was used as a model enteric pathogen to investigate the effect of microbial perturbation on intestinal barriers and susceptibility to colitis. Streptomycin and metronidazole were used to induce alterations in the composition of the microbiota prior to infection with C. rodentium. Metronidazole pretreatment increased susceptibility to C. rodentium-induced colitis over that of untreated and streptomycin-pretreated mice, 6 days postinfection. Both antibiotic treatments altered microbial composition, without affecting total numbers, but metronidazole treatment resulted in a more dramatic change, including a reduced population of Porphyromonadaceae and increased numbers of lactobacilli. Disruption of the microbiota with metronidazole, but not streptomycin treatment, resulted in an increased inflammatory tone of the intestine characterized by increased bacterial stimulation of the epithelium, altered goblet cell function, and thinning of the inner mucus layer, suggesting a weakened mucosal barrier. This reduction in mucus thickness correlates with increased attachment of C. rodentium to the intestinal epithelium, contributing to the exacerbated severity of C. rodentium-induced colitis in metronidazole-pretreated mice. These results suggest that antibiotic perturbation of the microbiota can disrupt intestinal homeostasis and the integrity of intestinal defenses, which protect against invading pathogens and intestinal inflammation.

Gut barrier disruption by an enteric bacterial pathogen accelerates insulitis in NOD mice.

AIMS/HYPOTHESIS: Increased exposure to enteric microbes as a result of intestinal barrier disruption is thought to contribute to the development of several intestinal inflammatory diseases; however, it less clear whether such exposure modulates the development of extra-intestinal inflammatory and autoimmune diseases. The goal of this study was to examine the potential role of pathogenic enteric microbes and intestinal barrier dysfunction in the pathogenesis of type 1 diabetes. METHODS: Using NOD mice, we assessed: (1) intrinsic barrier function in mice at different ages by measuring serum levels of FITC-labelled dextran; and (2) the impact on insulitis development of infection by strains of an enteric bacterial pathogen (Citrobacter rodentium) either capable (wild-type) or incapable (lacking Escherichia coli secreted protein F virulence factor owing to deletion of the gene [DeltaespF]) of causing intestinal epithelial barrier disruption. RESULTS: Here we demonstrate that prediabetic (12-week-old) NOD mice display increased intestinal permeability compared with non-obese diabetes-resistant and C57BL/6 mice. We also found that young (4-week-old) NOD mice infected with wild-type C. rodentium exhibited accelerated development of insulitis in concert with infection-induced barrier disruption. In contrast, insulitis development was not altered in NOD mice infected with the non-barrier-disrupting DeltaespF strain. Moreover, C. rodentium-infected NOD mice demonstrated increased activation and proliferation of pancreatic-draining lymph node T cells, including diabetogenic CD8(+) T cells, compared with uninfected NOD mice. CONCLUSIONS/INTERPRETATION: This is the first demonstration that a loss of intestinal barrier integrity caused by an enteric bacterial pathogen results in the activation of diabetogenic CD8(+) T cells and modulates insulitis.

Vasoactive intestinal peptide ameliorates intestinal barrier disruption associated with Citrobacter rodentium-induced colitis.

Attaching and effacing bacterial pathogens attach to the apical surface of epithelial cells and disrupt epithelial barrier function, increasing permeability and allowing luminal contents access to the underlying milieu. Previous in vitro studies demonstrated that the neuropeptide vasoactive intestinal peptide (VIP) regulates epithelial paracellular permeability, and the high concentrations and close proximity of VIP-containing nerve fibers to intestinal epithelial cells would support such a function in vivo. The aim of this study was to examine whether VIP treatment modulated Citrobacter rodentium-induced disruption of intestinal barrier integrity and to identify potential mechanisms of action. Administration of VIP had no effect on bacterial attachment although histopathological scoring demonstrated a VIP-induced amelioration of colitis-induced epithelial damage compared with controls. VIP treatment prevented the infection-induced increase in mannitol flux a measure of paracellular permeability, resulting in levels similar to control mice, and immunohistochemical studies demonstrated that VIP prevented the translocation of tight junction proteins: zonula occludens-1, occludin, and claudin-3. Enteropathogenic Escherichia coli (EPEC) infection of Caco-2 monolayers confirmed a protective role for VIP on epithelial barrier function. VIP prevented EPEC-induced increase in long myosin light chain kinase (MLCK) expression and myosin light chain phosphorylation (p-MLC). Furthermore, MLCK inhibition significantly attenuated bacterial-induced epithelial damage both in vivo and in vitro. In conclusion, our results indicate that VIP protects the colonic epithelial barrier by minimizing bacterial-induced redistribution of tight junction proteins in part through actions on MLCK and MLC phosphorylation.

Aggregation via the red, dry, and rough morphotype is not a virulence adaptation in Salmonella enterica serovar Typhimurium.

The Salmonella rdar (red, dry, and rough) morphotype is an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments. Growth of Salmonella enterica serovar Typhimurium was analyzed in a variety of nutrient-limiting conditions to determine whether aggregation would occur at low cell densities and whether the rdar morphotype was involved in this process. The resulting cultures consisted of two populations of cells, aggregated and nonaggregated, with the aggregated cells preferentially displaying rdar morphotype gene expression. The two groups of cells could be separated based on the principle that aggregated cells were producing greater amounts of thin aggregative fimbriae (Tafi or curli). In addition, the aggregated cells retained some physiological characteristics of the rdar morphotype, such as increased resistance to sodium hypochlorite. Competitive infection experiments in mice showed that nonaggregative DeltaagfA cells outcompeted rdar-positive wild-type cells in all tissues analyzed, indicating that aggregation via the rdar morphotype was not a virulence adaptation in Salmonella enterica serovar Typhimurium. Furthermore, in vivo imaging experiments showed that Tafi genes were not expressed during infection but were expressed once Salmonella was passed out of the mice into the feces. We hypothesize that the primary role of the rdar morphotype is to enhance Salmonella survival outside the host, thereby aiding in transmission.

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-alpha 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.

Saccharomyces boulardii ameliorates Citrobacter rodentium-induced colitis through actions on bacterial virulence factors.

Saccharomyces boulardii has received increasing attention as a probiotic effective in the prevention and treatment of infectious and inflammatory bowel diseases. The aim of this study was to examine the ameliorating effects of S. boulardii on Citrobacter rodentium colitis in vivo and identify potential mechanisms of action. C57BL/6 mice received 2.5 x 10(8) C. rodentium by gavage on day 0, followed by S. boulardii (25 mg; 5 x 10(8) live cells) gavaged twice daily from day 2 to day 9. Animal weights were monitored until death on day 10. Colons were removed and assessed for epithelial barrier function, histology, and myeloperoxidase activity. Bacterial epithelial attachment and type III secreted proteins translocated intimin receptor Tir (the receptor for bacterial intimin) and EspB (a translocation apparatus protein) required for bacterial virulence were assayed. In infected mice, S. boulardii treatment significantly attenuated weight loss, ameliorated crypt hyperplasia (234.7 +/- 7.2 vs. 297.8 +/- 17.6 microm) and histological damage score (0.67 +/- 0.67 vs. 4.75 +/- 0.75), reduced myeloperoxidase activity (2.1 +/- 0.4 vs. 4.7 +/- 0.9 U/mg), and attenuated increased mannitol flux (17.2 +/- 5.0 vs. 31.2 +/- 8.2 nm.cm(-2).h(-1)). The ameliorating effects of S. boulardii were associated with significantly reduced numbers of mucosal adherent C. rodentium, a marked reduction in Tir protein secretion and translocation into mouse colonocytes, and a striking reduction in EspB expression and secretion. We conclude that S. boulardii maintained colonic epithelial barrier integrity and ameliorated inflammatory sequelae associated with C. rodentium infection by attenuating C. rodentium adherence to host epithelial cells through putative actions on the type III secretion system.

Enteropathogenic Escherichia coli infection induces expression of the early growth response factor by activating mitogen-activated protein kinase cascades in epithelial cells.

Enteropathogenic Escherichia coli (EPEC) is an extracellular bacterial pathogen that infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. EPEC belongs to the group of attaching and effacing (A/E) pathogens. It uses a type III secretion system to deliver proteins into the host cell that mediate signal transduction events in host cells. We used gene array technology to study epithelial cell responses to EPEC infection at the level of gene expression. We found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide (LPS)-independent mechanism, including cytokines and early growth response factor 1 (Egr-1). The transcription factor Egr-1 is an immediate-early-induced gene that is activated in most cell types in response to stress. EPEC-induced upregulation of egr-1 is mediated by the activation of the MEK/extracellular signal-regulated kinase signal transduction pathway and is dependent on the type III secretion system. egr-1 is also induced during infection of mice by the A/E pathogen Citrobacter rodentium, suggesting that both Egr-1 and the activation of this mitogen-activated protein kinase signal transduction pathway may play a role in disease.

Locus of enterocyte effacement from Citrobacter rodentium: sequence analysis and evidence for horizontal transfer among attaching and effacing pathogens.

The family of attaching and effacing (A/E) bacterial pathogens, which includes diarrheagenic enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), remains a significant threat to human and animal health. These bacteria intimately attach to host intestinal cells, causing the effacement of brush border microvilli. The genes responsible for this phenotype are encoded in a pathogenicity island called the locus of enterocyte effacement (LEE). Citrobacter rodentium is the only known murine A/E pathogen and serves as a small animal model for EPEC and EHEC infections. Here we report the full DNA sequence of C. rodentium LEE and provide a comparative analysis with the published LEEs from EPEC, EHEC, and the rabbit diarrheagenic E. coli strain RDEC-1. Although C. rodentium LEE shows high similarities throughout the entire sequence and shares all 41 open reading frames with the LEE from EPEC, EHEC, and RDEC-1, it is unique in its location of the rorf1 and rorf2/espG genes and the presence of several insertion sequences (IS) and IS remnants. The LEE of EPEC and EHEC is inserted into the selC tRNA gene. In contrast, the Citrobacter LEE is flanked on one side by an operon encoding an ABC transport system, and an IS element and sequences homologous to Shigella plasmid R100 and EHEC pO157 flank the other. The presence of plasmid sequences next to C. rodentium LEE suggests that the prototype LEE resided on a horizontally transferable plasmid. Additional sequence analysis reveals that the 3-kb plasmid in C. rodentium is nearly identical to p9705 in EHEC O157:H7, suggesting that horizontal plasmid transfer among A/E pathogens has occurred. Our results indicate that the LEE has been acquired by C. rodentium and A/E E. coli strains independently during evolution.

IL-12 gene transfer alters gut physiology and host immunity in nematode-infected mice.

Immune responses elicited by nematode parasite infections are characterized by T helper 2 (Th2) cell induction. The immunologic basis for changes in intestinal physiology accompanying nematode infection is poorly understood. This study examined whether worm expulsion and associated goblet cell hyperplasia and muscle contractility share a similar immune basis by shifting the response from Th2 to Th1 using interleukin-12 (IL-12) overexpression. We used a single administration of recombinant adenovirus vector expressing IL-12 (Ad5IL-12) in Trichinella spiralis-infected mice. Ad5IL-12 administered 1 day after infection prolonged worm survival and inhibited infection-induced muscle hypercontractility and goblet cell hyperplasia. This was correlated with upregulated interferon-gamma (IFN-gamma) expression and downregulated IL-13 expression in the muscularis externa layer. We also observed increased IFN-gamma production and decreased IL-4 and IL-13 production from in vitro stimulated spleen and mesenteric lymph node cells of infected Ad5IL-12-treated mice. These results indicate that transfer and overexpression of the IL-12 gene during Th2-based nematode infection shifts the immune response toward Th1 and delays worm expulsion. Moreover, the immune response shift abrogated the physiological responses to infection, attenuating both muscle hypercontractility and goblet cell hyperplasia. These findings strongly indicate that worm expulsion, muscle hypercontractility, and goblet cell hyperplasia share a common immunologic basis and may be causally linked.

Neural change in Trichinella-infected mice is MHC II independent and involves M-CSF-derived macrophages.

Intestinal inflammation due to nematode infection impairs enteric cholinergic nerve function and induces hypercontractility of intestinal muscle. Macrophages have been implicated in the neural changes, but the subpopulation and mechanism involved are unknown. We examined whether macrophages alter nerves by virtue of their ability to activate lymphocytes via major histocompatibility complex (MHC) II-restricted antigen presentation. We also attempted to evaluate the role of macrophage subsets using op/op mice deficient in macrophage colony-stimulating factor (M-CSF). ACh release from the myenteric plexus was measured in MHC II- and M-CSF-deficient (op/op) mice infected with Trichinella spiralis. F4/80-positive macrophages and interleukin-1 beta were constitutively present in op/op and op/? mice but increased only in op/? mice postinfection. After infection, a marked suppression of ACh release occurred only in infected MHC II-deficient and op/? mice. Muscle hypercontractility remained evident in infected op/? mice. Treatment with M-CSF restored macrophage number, and this was accompanied by suppression of cholinergic nerve function during infection. Thus M-CSF plays a critical role in this model by recruiting a subset of macrophages that selectively suppresses enteric neural function.

Mast cell-independent impairment of host defense and muscle contraction in T. spiralis-infected W/W(V) mice.

In response to nematode infection, the host presumably attempts to create an unfavorable environment to prevent larval penetration of the host and to expedite parasite expulsion from the gut. In this study, we have used W/W(V) mice with or without mast cells after bone marrow reconstitution (BMR-W/W(V)) to examine the role of mast cells in the host response. W/W(V), BMR-W/W(V), and wild-type (+/+) mice were infected with Trichinella spiralis. Infected W/W(V) mice exhibited less tissue damage and experienced a delay in worm expulsion and a greater degree of larval penetration of the gut leading to encystment in skeletal muscle. Tissue injury was greater and worm expulsion was normalized in BMR-W/W(V) mice, but larval penetration remained unchanged. Spontaneous contractile activity of jejunal muscle was disrupted in W/W(V) mice, as was the contractile response to carbachol. These abnormalities were also present in BMR-W/W(V) mice. These results indicate that mast cells mediate tissue damage and contribute to the timely expulsion of nematodes from the gut during primary infection.

Critical role for signal transducer and activator of transcription factor 6 in mediating intestinal muscle hypercontractility and worm expulsion in Trichinella spiralis-infected mice.

Intestinal nematode infections in rats or mice are accompanied by intestinal muscle hyper contractility that may contribute to parasite expulsion from the gut. Previous studies demonstrated that both the expulsion of nematode parasites and the associated muscle hyper contractility are dependent on CD4(+) T helper cells. Nevertheless, the precise immunological mechanism underlying changes in intestinal muscle function remains to be determined. In this study, we investigated the role of interleukin 4 (IL-4) and signal transducer and activator of transcription factor 6 (STAT6) in the development of intestinal muscle hypercontractility and worm expulsion by infecting IL-4 and STAT6-deficient mice with Trichinella spiralis. Worm expulsion was almost normal in IL-4-deficient mice but substantially delayed in STAT6-deficient mice. Consistent with delayed worm expulsion, we also observed a marked attenuation of carbachol-induced muscle contraction in STAT6-deficient mice but only a moderate decrease in muscle hypercontractility in IL-4-deficient mice. In addition, we also observed severe impairment of T helper type 2 cytokine responses and intestinal mucosal mastocytosis in STAT6-deficient mice, although some degree of intestinal tissue eosinophilia was evident in these animals. These results are consistent with the hypothesis that STAT6-dependent changes in intestinal muscle function contribute to host protection in nematode infection.

Interleukin-5 deficient mice exhibit impaired host defence against challenge Trichinella spiralis infections.

Enteric nematode infections are characterized by both peripheral and tissue eosinophilia. The cytokine interleukin (IL)-5 is considered a critical factor in the proliferation and recruitment of eosinophils, however, studies suggest it plays little role in host defence, at least during primary Trichinella spiralis infections. Less is known concerning its role in host defence or in the inflammatory response that develops against challenge infections with the same parasite. We examined these questions by infecting IL-5 deficient and wild-type mice, with T. spiralis parasites. Both strains expelled the primary infection by day 21. Forty days after the primary infection, we challenged the mice with a second T. spiralis infection and counted tissue eosinophils and worms in the intestine. While wild-type mice developed a large tissue eosinophilia, IL-5 deficient mice showed little increase in eosinophil numbers within the intestine. Throughout the challenge infection, significantly larger worm burdens were recovered from IL-5 deficient mice, and worm expulsion was also significantly slower (day 21) compared to wild-type mice (day 14). Thus, unlike in a primary infection, IL-5 is not only essential for the onset of intestinal eosinophilia, but also makes a significant contribution to enteric host defence during challenge T. spiralis infections.

Exploitation of host cells by enteropathogenic Escherichia coli.

Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells. One such subversive microbe is enteropathogenic Escherichia coli (EPEC). A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions. Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir). Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells. The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria. This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions. Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

Inflammation-induced impairment of enteric nerve function in nematode-infected mice is macrophage dependent.

Trichinella spiralis infection in rodents is associated with suppression of ACh release from myenteric plexus that can be mimicked by macrophage-derived cytokines. We verified the presence of a macrophage infiltrate in the intestine during T. spiralis infection and determined the extent to which this cell type is responsible for the neural changes. C57BL/6 mice were infected with 375 T. spiralis larvae by gavage, and the presence of macrophages (F4/80 positive) in the jejunum was determined immunohistochemically. In another experiment, infected mice were treated intravenously with liposomes containing dichloromethylene diphosphonate (clodronate, Cl(2)MDP), which causes apoptosis of macrophages, and killed at postinfection day 6, and jejunal tissues were evaluated for the presence of F4/80-positive cells and for [(3)H]ACh release from the myenteric plexus. Infection caused an infiltration of F4/80-positive cells into the intestinal mucosa, muscle layers, and myenteric plexus region and a significant suppression of ACh release (50%). Depletion of F4/80-positive macrophages using Cl(2)MDP-containing liposomes prevented the suppression in [(3)H]ACh release, identifying macrophages as the cell type involved in the functional impairment of enteric cholinergic nerves.