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Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits.

ABSTRACT: Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent. In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC). Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for the induction of proinflammatory cytokines in enterocytes. The role of lymphocyte-dependent immunity was determined through the expression of proinflammatory cytokines by lymphocytes from Peyer's patches (PP) and the spleen. EspA and intimin mutants were used to explore the role of A/E lesions in the expression of these cytokines. REPEC-infected rabbit enterocytes showed increased interleukin 1beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) mRNA expression, but that of anti-inflammatory IL-10 was increased only slightly. In contrast, intimin mutant-infected rabbits were unable to produce this proinflammatory cytokine profile but did produce a remarkable increase in IL-10 expression. Bacteria lacking EspA increased the expression of IL-8 and TNF-alpha, but that of IL-10 was increased only slightly. PP lymphocytes also produced proinflammatory cytokines, which were dependent on EspA (except for TNF-alpha) and intimin, while IL-10 was induced by EspA and intimin mutants. In contrast, spleen lymphocytes (systemic compartment) were unable to produce IL-1beta and TNF-alpha. These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium. Furthermore, this cytokine profile, including IL-6 and IL-1beta, which may be involved in the diarrhea produced by EPEC, depends on intimin.

SUBMITTER: Ramirez K

PROVIDER: S-EPMC538993 | biostudies-literature | 2005 Jan

REPOSITORIES: biostudies-literature

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Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits.

Ramirez Karina K Huerta Rocio R Oswald Eric E Garcia-Tovar Carlos C Hernandez Jose Manuel JM Navarro-Garcia Fernando F

Infection and immunity 20050101 1

Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent. In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC). Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7). At day 6 postinoculation, animals were analyzed for the induction of proinflammatory ...[more]

PMID: 15618145

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Project description:Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Project description:Strains of Shiga toxin (Stx)-producing Escherichia coli, also called enterohemorrhagic E. coli (EHEC), are important food-borne pathogens for humans. Most EHEC strains intimately adhere to the intestinal mucosa in a characteristic attaching and effacing (A/E) pattern, which is mediated by the bacterial adhesin intimin. Subsequent release of Stx1 and/or Stx2 leads to the frequent development of hemorrhagic colitis and, less commonly, to hemolytic-uremic syndrome. The aim of the present study was to develop an attenuated A/E E. coli strain for use as a vaccine against EHEC infection encoding a truncated intimin lacking adhesive capacity, but which would still express somatic antigens, other products of the locus of enterocyte effacement pathogenicity island, and an immunogenic remnant of the intimin molecule. A single-nucleotide deletion was generated in the eae gene in the prototype rabbit A/E E. coli strain RDEC-1 (O15:H-), which resulted in truncation of intimin by 81 C-terminal residues (860 to 939 amino acids) containing a disulfide loop. Inoculation of rabbits with large doses of the truncated intimin mutant (RDEC-1Deltaeae(860-939)) was well tolerated, as observed by the absence of clinical signs of disease or evidence of intestinal A/E lesions. The efficacy of RDEC-1Deltaeae(860-939) as a vaccine was evaluated by orogastric inoculation of rabbits with RDEC-1Deltaeae(860-939) followed by challenge with the virulent strain RDEC-H19A, an Stx1-producing derivative of wild-type RDEC-1 capable of inducing hemorrhagic colitis in rabbits. Following RDEC-H19A challenge, nonimmunized control rabbits exhibited characteristic weight loss with watery to bloody diarrhea and demonstrated intimate bacterial attachment, effacement of microvilli, submucosal edema, mucosal heterophile infiltrates, and Shiga toxin-induced vascular lesions. In contrast, the RDEC-1Deltaeae(860-939)-immunized rabbits showed no clinical signs of disease, maintained normal weight gain, had reduced fecal shedding of challenge organisms, and showed an absence of gross or microscopic lesions in the intestinal mucosa. Serum antibodies specific to intimin were detected among rabbits immunized with RDEC-1Deltaeae(860-939), indicating that truncation of the intimin functional domain not only attenuated bacterial virulence, but also retained at least some of the immunogenicity of native intimin. Although it is not possible to gauge the exact contribution of residual intimin immunity to protection, this attenuation strategy for A/E E. coli strains shows promise for the development of effective vaccines to prevent EHEC infection in humans and animals.

Project description:BackgroundAnisakiasis is an emerging public health problem, caused by Anisakis spp. nematode larvae. Anisakiasis presents as variable and unspecific gastrointestinal and/or allergic clinical symptoms, which accounts for the high rate of misdiagnosed cases.Methodology/principal findingsThe aim of this study was to characterize the early cellular (6-72 h p.i.) and molecular (6 h p.i.) immune response and general underlying regulatory mechanism in Anisakis infected rats. Each Sprague-Dawley rat was infected with 10 Anisakis spp. larvae by gastric intubation. Tissues with visible lesions were processed for: i) classic histopathology (HE), immunofluorescence (CD3, iNOS, S100A8/A9), and transmission electron microscopy (TEM); ii) target genes (Il1b, Il6, Il18, Ccl3, Icam1, Mmp9) and microRNA (Rat Immunopathology MIRN-104ZF plate, Quiagen) expression analysis; and iii) global DNA methylation. Histopathology revealed that Anisakis larval migration caused moderate to extensive hemorrhages in submucosal and epimysial/perimysial connective tissue. In stomach and muscle, moderate to abundant mixed inflammatory infiltrate was present, dominated by neutrophils and macrophages, while only mild infiltration was seen in intestine. Lesions were characterized by the presence of CD3+, iNOS+, and S100A8/A9+ cells. The greatest number of iNOS+ and S100A8/A9+ cells was seen in muscle. Il6, Il1b, and Ccl3 showed particularly strong expression in stomach and visceral adipose tissues, but the order of expression differed between tissues. In total, three miRNAs were differentially expressed, two in stomach (miRNA-451 and miRNA-223) and two in intestine (miRNA-451 and miRNA-672). No changes in global DNA methylation were observed in infected tissues relative to controls.Conclusions/significanceAnisakis infection induces strong immune responses in infected rats with marked induction of specific proinflammatory cytokines and miRNA expression. Deciphering the functional role of these cytokines and miRNAs will help in understanding the anisakiasis pathology and controversies surrounding Anisakis infection in humans.

Dataset Information

Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits.

Publications

Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits.

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