Alarmin HMGB1 is released in the small intestine of gnotobiotic piglets infected with enteric pathogens and its level in plasma reflects severity of sepsis.

OBJECTIVES: Alarmin high mobility group box 1 (HMGB1) is essential for correct DNA folding and transcription. It can be released from damaged cells or secreted by stimulated cells. HMGB1 has been detected in serum or plasma as a late marker of sepsis, but its suitability as a marker of sepsis has been disputed. METHODS: One-week-old germ-free piglets were orally infected/colonized with enteric bacterial pathogens (Salmonella Typhimurium or Escherichia coli O55) or with probiotic bacteria (E. coli Nissle 1917) for 24 h. The transcriptions of HMGB1, interleukin (IL)-8, tumor necrosis factor (TNF)-α, and IL-10 (quantitative reverse transcription and polymerase chain reaction), their protein levels (ELISA), and clinical state of the piglets (somnolence, anorexia, diarrhea, tachycardia, tachypnea, and tremor) were estimated. RESULTS: The piglets infected with enteric pathogens suffered from infections. HMGB1 was transcribed in the terminal ileum constitutively, regardless of any bacterial presence. In contrast, the transcription of cytokines was upregulated by virulent bacteria. HMGB1, IL-8, and TNF-α levels in the ileum were increased by both enteric pathogens, while IL-10 levels increased in E. coli O55-infected piglets only. HMGB1 significantly increased in the plasma of piglets infected with virulent E. coli only, but cytokine levels were in most cases increased by both virulent bacteria. HMGB1 and cytokine levels in ileum lavages and plasma of piglets colonized with probiotic E. coli remained comparable to those of the non-stimulated germ-free piglets. CONCLUSION: The local and systemic expression of HMGB1, its relationship to the inflammatory cytokines, and clinical findings showed HMGB1 as a suitable marker of severity of sepsis in the gnotobiotic piglet infection model.

Modulation of natural immunity in the gut by Escherichia coli strain Nissle 1917.

The beneficial effect of probiotic Escherichia coli strain Nissle 1917 (EcN) suggests the gut epithelium plays a basic role in immune interactions with bacteria. Contrary to other commensal strains of Escherichia coli, EcN profoundly modulates the gut barrier to elevate its resistance to microbial pathogens. The present review documents the properties of EcN that have led to the protection of gnotobiotic pigs against lethal enteric infections. This effect could be important in light of the growing number of acquired deficiencies that paralyze gut immunity in humans.

Cross-talk of human gut with bifidobacteria.

The gut constitutes a prominent part of the immune system. Its commensal microflora plays an important role in defense and in tolerance to diet allergens. Disturbances in immune regulations may lead to food allergy. Among commensal bacteria, bifidobacteria are able to induce mechanisms of immune tolerance. Comprehension of their mutual cross-talk with the host is necessary for understanding their role in the diet and in food supplements.

Susceptibility of germ-free pigs to challenge with protease mutants of Salmonella enterica serovar Typhimurium.

Salmonella protease mutants, clpP and especially htrA, are candidate live oral vaccines in humans. A functional and mature immune system is, however, required to cope with them in mice. Here, we test the cytokine response of highly susceptible germ-free pigs to infection with Salmonella Typhimurium clpP and htrA mutants. Cytokine levels (IL-4, IL-10, IL-18 and IFN-gamma) were measured by ELISA in plasma and washes from the terminal small bowel 24h after oral challenge. Unlike the infection with the wild type strain, no IFN-gamma response and low IL-18 intestinal levels were found in pigs infected with the protease mutants. Despite this and regardless of partially reduced ability of htrA and clpP mutants to invade and multiply in a 3D4 porcine macrophage-like cell line, both the mutants were as virulent as was the wild type LT2 strain and caused fatal septicaemia in germ-free pigs. IFN-gamma and IL-18 response therefore did not correlate with the virulence of Salmonella Typhimurium. Our results indicate that htrA and clpP attenuations should be used with caution in populations in which an increased number of immunocompromised individuals can be expected.

Effect of bacterial virulence on IL-18 expression in the amnion infected with Escherichia coli.

PROBLEM: The upregulation of inflammatory substances threatens pregnancy. Interleukin-18 (IL-18) is elevated in women who miscarried. The purpose of this study was to develop a pig model of chorioamnionitis to study the effect of bacterial virulence on IL-18 response in experimentally infected amnion. METHOD OF STUDY: A total of 20,000 colony-forming units of Escherichia coli (an enteropathogenic O55 strain, EPEC or O86 non-pathogenic strain) were administered into the amniotic cavity of pig fetuses at 70% of gestation for 10 hr. Fetal amniotic fluid samples were analyzed for IL-18 levels by enzyme-linked immunosorbent assay. The expression of IL-18 was studied also by immunohistochemistry on cryostat sections through amniotic membranes and pathological changes were observed by electron microscopy. RESULTS: Both E. coli strains propagated in amniotic fluids and reached similar counts. Only EPEC, however, caused a significant increase of IL-18 amniotic fluid levels (P < 0.001) and cytokine expression in the amniotic epithelium. CONCLUSIONS: The levels of IL-18 in infected amniotic fluids correlated with bacterial virulence and pathological changes in the amnion.

The effect of intestinal colonization of germ-free pigs with Escherichia coli on calprotectin levels in plasma, intestinal and bronchoalveolar lavages.

Calprotectin levels were measured by ELISA in plasma, terminal small bowel lavage and bronchoalveolar lavage from 8-day-old germ-free piglets or gnotobiotic piglets 24 h after colonization with one of the following Escherichia coli strains: non-pathogenic O86, probiotic Nissle 1917 or enteropathogenic O55. The concentration of calprotectin in plasma was about 30 ng/ml only in germ-free piglets and piglets associated with non-pathogenic E. coli. Piglets infected with O55 showed a significant increase of plasma calprotectin and the highest mean level of calprotectin in the bronchoalveolar lavage, which was coincident with septicaemia. However, in the lumen of the small intestine, E. coli Nissle 1917 alone elicited a significant increase of the calprotectin level which was confirmed by immunofluorescence and APAAP immunohistochemistry on cryostat sections through the small bowel. The relevance of this finding to the therapeutic effect of E. coli Nissle 1917 in inflammatory bowel disease is discussed.

Protection of gnotobiotic pigs against Salmonella enterica serotype Typhimurium by rough mutant of the same serotype is accompanied by the change of local and systemic cytokine response.

We have demonstrated that severe systemic disease caused by virulent LT2 strain Salmonella enterica serotype Typhimurium in gnotobiotic piglets can be alleviated by oral inoculation with an avirulent rough (R) mutant of the same serotype 24 h before challenge with the virulent strain. Protected piglets had no signs of enteritis. The concentrations of TNF-alpha, IL-1beta, IL-8 and IL-10 were measured by ELISA in ileal washings and plasma of uninfected and infected pigs. The cytokines were not detected in plasma of germ-free piglets, and low concentrations of IL-1beta and IL-8 were found in their ileal washings. The pre-inoculation of the rough mutant induced an increase in IL-8 and decrease in IL-1beta and IL-10 in plasma. The virulent LT2 strain induced very high TNF-alpha concentrations in the ileum which were reduced in the pigs pre-inoculated with the R mutant.

Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium.

Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals. Germ-free animals, however, enable to study response against infection without background effect of other microorganisms. Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA. In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups. TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection. In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella. These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis.

Mucosal immunity: its role in defense and allergy.

The interface between the organism and the outside world, which is the site of exchange of nutrients, export of products and waste components, must be selectively permeable and at the same time, it must constitute a barrier equipped with local defense mechanisms against environmental threats (e.g. invading pathogens). The boundaries with the environment (mucosal and skin surfaces) are therefore covered with special epithelial layers which support this barrier function. The immune system, associated with mucosal surfaces covering the largest area of the body (200-300 m(2)), evolved mechanisms discriminating between harmless antigens and commensal microorganisms and dangerous pathogens. The innate mucosal immune system, represented by epithelial and other mucosal cells and their products, is able to recognize the conserved pathogenic patterns on microbes by pattern recognition receptors such as Toll-like receptors, CD14 and others. As documented in experimental gnotobiotic models, highly protective colonization of mucosal surfaces by commensals has an important stimulatory effect on postnatal development of immune responses, metabolic processes (e.g. nutrition) and other host activities; these local and systemic immune responses are later replaced by inhibition, i.e. by induction of mucosal (oral) tolerance. Characteristic features of mucosal immunity distinguishing it from systemic immunity are: strongly developed mechanisms of innate defense, the existence of characteristic populations of unique types of lymphocytes, colonization of the mucosal and exocrine glands by cells originating from the mucosal organized tissues ('common mucosal system') and preferential induction of inhibition of the responses to nondangerous antigens (mucosal tolerance). Many chronic diseases, including allergy, may occur as a result of genetically based or environmentally induced changes in mechanisms regulating mucosal immunity and tolerance; this leads to impaired mucosal barrier function, disturbed exclusion and increased penetration of microbial, food or airborne antigens into the circulation and consequently to exaggerated and generalized immune responses to mucosally occurring antigens, allergens, superantigens and mitogens.

Lipopolysaccharide induces inflammatory cytokines in the pig amnion.

Inflammatory mediators that are induced by gram-negative bacteria in the course of intrauterine infections threaten successful pregnancy. To compare the effect of two different routes of cytokine induction, bacterial lipopolysaccharide (LPS) was administered in vivo either into the cord vein or into the amniotic cavity of pig fetuses in the second half of gestation for 20 h and cytokines were detected in the amnion.Tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were induced in the amniotic epithelium after intra-amniotic but not after intra-venous administration of LPS. The presence of IL-8 was confirmed by RT-PCR. In contrast, transforming growth factor-beta1 (TGF-beta1) was expressed constitutively and was found in all samples of the amniotic epithelium. Amniotic fluid contained only minute levels of TNF-alpha. IL-8 levels in amniotic fluid increased after the treatment with LPS and the highest IL-8 levels were found in dead LPS-treated fetuses.