Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice.

One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms - bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization.

BACKGROUND: The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. METHODS: We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. RESULTS: Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. CONCLUSION: Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

Pivotal Advance: Bifidobacteria and Gram-negative bacteria differentially influence immune responses in the proinflammatory milieu of celiac disease.

CD is a chronic inflammatory disorder of the small intestine that presents in genetically predisposed individuals following gluten consumption. In this study, the effects of Bifidobacterium (Bifidobacterium bifidum IATA-ES2 and Bifidobacterium longum ATCC15707) and Gram-negative bacteria (Bacteroides fragilis DSM2451, Escherichia coli CBL2, and Shigella CBD8 isolated from CD patients), alone and in the presence of CD triggers (gliadins and/or IFN-gamma) on surface marker expression and cytokine production by PBMCs, were determined. These effects were also evaluated in cocultures of PBMCs and Caco-2 cells. The Gram-negative bacteria induced higher secretion of Th1-type proinflammatory cytokines (IL-12 and/or IFN-gamma) than the Bifidobacterium strains. Shigella CBD8 and E. coli CBL2 up-regulated mainly HLA-DR and CD40 expression involved in Th1 activation, and Bifidobacterium strains up-regulated CD83 expression. Specific interactions among the studied bacteria, gliadins, and IFN-gamma, which favored the CD immune features, were also detected. Therefore, intestinal bacteria could be additional factors that regulate the ability of monocytes recruited to the mucosa to respond to gliadins and IFN-gamma in CD patients, influencing the course of the disease.

Monocolonization with Bacteroides ovatus protects immunodeficient SCID mice from mortality in chronic intestinal inflammation caused by long-lasting dextran sodium sulfate treatment.

This study was aimed to evaluate the role of commensal Gram-negative bacterium Bacteroides ovatus in murine model of chronic intestinal inflammation. The attempt to induce chronic colitis was done in Bacteroides ovatus-monoassociated, germ-free and conventional mice either in immunocompetent (BALB/c) mice or in mice with severe combined immunodeficiency (SCID), using 2.5 % dextran-sodium sulfate (DSS) in drinking water (7 days DSS, 7 days water, 7 days DSS). Conventional mice developed chronic colitis. Some of germ-free BALB/c and the majority of germ-free SCID mice did not survive the long-term treatment with DSS due to massive bleeding into the intestinal lumen. However, monocolonization of germ-free mice of both strains with Bacteroides ovatus prior to long-term treatment with DSS protected mice from bleeding, development of intestinal inflammation and precocious death. We observed that though DSS-treated Bacteroides ovatus-colonized SCID mice showed minor morphological changes in colon tissue, jejunal brush-border enzyme activities such as gamma-glutamyltranspeptidase, lactase and alkaline phosphatase were significantly reduced in comparison with DSS-untreated Bacteroides ovatus-colonized mice. This modulation of the enterocyte gamma-glutamyltranspeptidase localized to the brush border membrane has been described for the first time. This enzyme is known to reflect an imbalance between pro-oxidant and anti-oxidant mechanisms, which could be involved in protective effects of colonization of germ-free mice with Bacteroides ovatus against DSS injury.

Commensal intestinal bacterial strains trigger ankylosing enthesopathy of the ankle in inbred B10.BR (H-2(k)) male mice.

Joint disease ankylosing enthesopathy (ANKENT) naturally occurs in inbred mice with C57Bl/10 genetic background. ANKENT has many parallels to human ankylosing spondylitis (AS) and represents an animal model for AS. Environmental conditions (i.e., microbial load of the organism) are among the risk factors for ANKENT, similar to AS. The role of microflora in the development of ANKENT was investigated. ANKENT was tested in four experimental groups of germ-free mice associated with different numbers of various intestinal microbes and three control groups: germ-free, specific pathogen-free, and conventional (CV) mice. Mice were colonized either with anaerobic bacteria isolated from the intestine of a CV mouse or with bacterial strains obtained from the collection of microorganisms. Microbes were characterized and checked by microbiological cultivation methods and with the use of polymerase chain reaction amplification and rDNA sequence analysis. Joint disease developed in GF mice colonized with a mixture containing Bacteroides spp. and Enterococcus sp., and/or Veillonella sp. and Staphylococcus sp. No ANKENT appeared in males colonized with probiotic bacterium Lactobacillus sp. In control groups ANKENT occurred in SPF and CV animals; the GF animals remained healthy. The results confirmed that the germ-free conditions protect from joint inflammation, and thus microbes are necessary for ANKENT development. In colonized mice the ANKENT was triggered by luminal anaerobic bacteria, which are common components of intestinal microflora.

Effects of monocolonization with Escherichia coli strains O6K13 and Nissle 1917 on the development of experimentally induced acute and chronic intestinal inflammation in germ-free immunocompetent and immunodeficient mice.

Germ-free immunocompetent (BALB/c) and immunodeficient (SCID) mice were colonized either by E. coli O6K13 or by E. coli strain Nissle 1917 and intestinal inflammation was induced by administering 2.5% dextran sulfate sodium (DSS) in drinking water. Controls were germ-free mice which demonstrated only mild inflammatory changes after induction of an acute intestinal inflammation with DSS as compared with conventional mice in which acute colitis of the colon mucosa similar to human ulcerative colitis is elicited. In mice monocolonized with the nonpathogenic E. coli Nissle 1917 the inflammatory disease did not develop (damage grade 0) while animals monocolonized with uropathogenic E. coli O6K13 exhibited inflammatory changes similar to those elicited in conventionally reared mice (damage grade 3). In the chronic inflammation model, immunocompetent BALB/c mice monocolonized with E. coli Nissle 1917 showed no conspicuous inflammatory changes of the colon mucosa whereas those monocolonized with E. coli O6K13 developed colon inflammation associated with marked infiltration of inflammatory cells. In contrast to germ-free immunodeficient SCID mice that died after application of DSS, the colon mucosa of SCID mice monoassociated with E. coli Nissle 1917 exhibited only moderate inflammatory changes which were less pronounced than changes of colon mucosa of SCID mice monoassociated with E. coli O6K13.

Interaction of mucosal microbiota with the innate immune system.

Organisms live in continuos interaction with their environment; this interaction is of vital importance but at the same time can be life threatening. The largest and most important interface between the organism and its environment is represented by surfaces covered with epithelial cells. Of these surfaces, mucosae comprise in humans approximately 300 m2, and the skin covers approximately 1.8 m2 surface of the human body. Mucosal tissues contain two effector arms of the immune system, innate and adaptive, which operate in synergy. Interaction with commensal bacteria, which outnumber the nucleated cells of our body, occurs physiologically on epithelial surfaces; this interaction could pose the risk of inflammation. The mucosal immune system has developed a complex network of regulatory signalling cascades that is a prerequisite for proper activation but also for a timely inactivation of the pathway. As demonstrated in gnotobiotic animal models of human diseases, impaired regulation of mucosal responses to commensal bacteria plays an important role in the development of several inflammatory and autoimmune diseases.

Experimental colitis does not increase the prevalence of ANKENT, a spontaneous joint disease in mice.

A possible relationship between intestinal inflammation and joint disease development was investigated. Clinical symptoms of colitis--diarrhea and rectal bleeding--were confirmed by findings of inflammatory processes in the colon in dextran sodium sulfate-treated mice and joint ankylosing enthesopathy (ANKENT) developed in 12.8 % mice with chronic colitis and 13.6 % mice in the control group. Consequently no significant difference in ANKENT frequency was found between mice with and without chronic colitis and the occurrence of ANKENT in both groups was typical for conventional conditions. ANKENT cannot be triggered solely a generalized inflammatory process in the gut.

Differential effect of Bacillus firmus on immune response and enterocyte brush-border enzyme levels in BALB/c and B10.BR mice.

A nonpathogenic bacterium of external environment possessing remarkable immunomodulatory activity, Bacillus firmus (BF) inactivated with formaldehyde, was given intragastrically to two genetically different mouse strains BALB/c (H-2d) and B10.BR/SnPh (B10.BR, H-2k) reared in conventional (CV) and B10.BR strain also in germ-free (GF) conditions. Repeated intragastric administration of BF (500 micrograms every other day over two weeks, starting at the age of 3 months) significantly enhanced intestinal IgA levels in CV BALB/c mice but did not affect intestinal IgA in CV B10.BR mice. In GF B10.BR mice, IgG levels in sera and intestinal washings increased after BF administration compared to CV B10.BR mice. In CV BALB/c mice, specific activity of enterocyte brush-border enzymes (lactase, gamma-glutamyltransferase, alkaline phosphatase) decreased after BF treatment; sucrase (sucrose alpha-glucosidase) activity was not affected. On the other hand, in B10.BR mice, specific activity of gamma-glutamyltransferase and dipeptidyl peptidase IV were higher after administration of BF in both CV and GF groups relative to untreated controls. The activities of lactase and glucoamylase (glucan 1,4-alpha-glucosidase) were significantly stimulated only in the group of GF B10.BR mice treated with formolized BF. The stimulation of immunoglobulin production after BF treatment was accompanied by changes in the levels of enterocyte brush-border enzymes; this responsiveness to BF treatment was genetically regulated.

Control of intestinal inflammation by regulatory T cells.

Transfer of CD4+ T cells to immune-deficient mice in the absence of the CD25+ subset leads to the development of colitis, indicating that regulatory cells capable of controlling a bacteria-driven inflammatory response are present in normal mice. Cells with this function are present in the thymus as well as in the periphery of germ-free mice, suggesting they may be reactive with self-antigen. These cells resemble CD4+CD25+ cells that inhibit organ-specific autoimmunity, suggesting that a similar subset of regulatory T cells may control responses to self and foreign antigens. Development of colitis is dependent on accumulation of activated CD134L+ dendritic cells (DC) in the mesenteric lymph nodes, which is inhibited by CD4+CD25+ cells, indicating that regulatory T cells may control DC activation in vivo. Whilst inhibition of T-cell activation in vitro by CD4+CD25+ cells does not involve interleukin-10 and transforming growth factor-beta, these cytokines are required for the suppression of colitis. It may be that control of responses that activate the innate immune system requires multiple mechanisms of immune suppression. Recently, we identified CD4+CD25+ cells with immune suppressive activity in the thymus and peripheral blood of humans, raising the possibility that dysfunction in this mechanism of immune regulation may be involved in the development of autoimmune and inflammatory diseases.

The role of microflora in the development of intestinal inflammation: acute and chronic colitis induced by dextran sulfate in germ-free and conventionally reared immunocompetent and immunodeficient mice.

One-week dextran sulfate treatment of conventional (CV) immunodeficient (SCID) mice gave rise to acute colitis in the colon mucosa; germ-free (GF) SCID mice did not exhibit any changes in colon morphology. Dextran sulfate application to CV immunocompetent (BALB/c) mice did induce substantial changes in the colon mucosa (grade 4); GF BALB/c mice showed mild changes in the colon morphology (grade 1) only. GF SCID mice and CV SCID mice died during the second round of dextran sulfate treatment suffering from chronic colitis; GF BALB/c mice exhibited mild crypt distortion while CV BALB/c mice showed a complete loss of the surface epithelium (grade 4), accompanied by T and B lymphocyte infiltration.

Germ-free mice do not develop ankylosing enthesopathy, a spontaneous joint disease.

Ankylosing enthesopathy (ANKENT) is a naturally occurring joint disease in mice with numerous parallels to human ankylosing spondylitis (AS). Similarities between AS and ANKENT include not only affected tissue (joint entheses) but also association of the disease with genetic background, including MHC genes, gender, and age. Young males with the C57Bl/10 background have been described to suffer from ANKENT and, among H-2 congenic strains, high frequency of afflicted joints has been recorded in B10.BR (H-2(k)) males. Interestingly, the incidence of ANKENT is higher in conventional (CV) males that in their specific-pathogen-free (SPF) counterparts. The latter finding suggests that microbes could play a role as an ANKENT-triggering agent. To further examine this hypothesis we have established a germ-free (GF) colony of B10.BR mice and observed ANKENT incidence in both GF males and their conventionalized (ex-GF) male littermates; 20% of ex-GF males developed ANKENT before 1 year of age. In contrast, no joint disease was observed under GF conditions (p < 0.0001). Our results show that live microflora is required in ANKENT pathogenesis.

Humoral and cellular immune responses in gluten-treated suckling or hand-fed rats.

We analyzed the immune response to gliadin in suckling rats and rats hand-fed with an artificial milk formula, an animal model of gluten enteropathy. Animals of both groups were intragastrically given either gliadin or albumin (control animals) or gliadin from birth till day 55. When compared to the controls, spleen lymphocytes from both groups of gliadin-treated rats cultivated in vitro exhibited a significant increase of spontaneous 3H-thymidine incorporation. Moreover, the proliferation of spleen and mesenteric lymph node (MLN) lymhocytes from both groups of gliadin-treated suckling and hand-fed rats was specifically increased by the in vitro gliadin challenge. Spleen B cells from gliadin-treated rats spontaneously produced higher amounts of gliadin-specific antibodies than those from the controls, however, in vitro stimulation by gliadin caused no further increase in antibody production. Apoptotic DNA fragmentation in MLN cells was higher in gliadin-treated rats than in albumin-treated ones, independently of the milk diet during the suckling period.

Brush border enzyme activities in the small intestine after long-term gliadin feeding in animal models of human coeliac disease.

Coeliac disease is a human, genetically linked, disorder which develops in gluten-sensitive persons. The aim of this study was to investigate the effect of prolonged feeding of gliadin, a major fraction of gluten, on enzyme activities of enterocyte brush border membrane enzymes in rats, mice and pigs. Brush-border membranes were isolated from mucosal scrapings of the small intestine of 21-d-old rat pups hand-fed with formula milk diet, two-month-old nu/nu and +/+ BALB/c mice and two-month-old piglets fed three times a week starting at birth with high doses of gliadin. Activities of lactase, sucrase and dipeptidyl peptidase IV (DPP IV) were determined. Individual animal models differed in their response to gliadin feeding. In comparison with albumin fed controls the activities of DPP IV and lactase were decreased in rat pups, nu/nu BALB/c mice and piglets. DPP IV activity was mostly affected in the ileum of rats and piglets fed with gliadin starting at birth. On the other hand, lactase and sucrase activities of nu/nu BALB/c mice and piglets decreased to the largest extent in jejunum.

Differences in development of lymphocyte subpopulations from gut-associated lymphatic tissue (GALT) of germfree and conventional rats: effect of aging.

The aim of the study was to compare the phenotype of lymphocyte subpopulations of the GALT (gut-associated lymphatic tissue) in germfree (GF) and conventionally (CV) reared rats, i.e. to analyze the effect of microbial colonization on the development of intestinal lymphocyte subsets. Surface marker characteristics were studied in cell suspensions isolated from Peyer's patches, mesenteric lymph nodes, spleen and the intraepithelial lymphocyte compartment of 2- and 12-month old inbred AVN rats. The pattern of T lymphocyte phenotypes in Peyer's patches, mesenteric lymph nodes and spleen determined by FACS analysis did not reveal differences between GF and CV rats. In contrast, a 2-month conventionalization of GF rats led to substantial changes in the composition of intestinal intraepithelial lymphocyte subsets (IELs): increase of CD4+, CD8 alpha+, CD8 beta+, TcR alpha/beta+ bearing lymphocytes was observed after colonization of rats with normal microflora. Surprisingly, the relative numbers of lymphocytes bearing TcR gamma/delta+ did not change during conventionalization. The effect of aging was also studied and differences in IELs composition of aged (GF) and (CV) rats were found to be more pronounced: 6.6% and 30% of lymphocytes bearing TcR alpha/beta were present among IELs in two-month old GF and CV rats, respectively. 30% of IELs in 2-month old GF rats, 80% of IEL from 12-month old CV rats were found to bear TcR alpha/beta. This finding demonstrates that during conventionalization and aging the TcR alpha/beta bearing population of IELs substantially expands. It suggests that mainly this lymphocyte subset responds to microflora stimuli and is probably involved in the protection of the epithelial cell layer of intestinal mucosa.

Autoimmunity, immunodeficiency and mucosal infections: chronic intestinal inflammation as a sensitive indicator of immunoregulatory defects in response to normal luminal microflora.

Despite the fact that target antigens and the genetic basis of several autoimmune diseases are now better understood, the initial events leading to a loss of tolerance towards self-components remain unknown. One of the most attractive explanations for autoimmune phenomena involves various infections as possible natural events capable of initiating the process in genetically predisposed individuals. The most accepted explanation of how infection causes autoimmunity is based on the concept of "molecular mimicry" (similarity between the epitopes of an autoantigen and the epitopes in the environmental antigen). Infectious stimuli may also participate in the development of autoimmunity by inducing an increased expression of stress proteins (hsp), chaperones and transplantation antigens, which leads to abnormal processing and presentation of self antigens. Superantigens are considered to be one of the most effective bacterial components to induce inflammatory reactions and to take part in the development and course of autoimmune mechanisms. It has long been known that defects in the host defense mechanism render the individual susceptible to infections caused by certain microorganisms. Impaired exclusion of microbial antigens can lead to chronic immunological activation which can affect the tolerance to self components. Defects in certain components of the immune system are associated with a higher risk of a development of autoimmune disease. The use of animal models for the studies of human diseases with immunological pathogenesis has provided new insights into the influence of immunoregulatory factors and the lymphocyte subsets involved in the development of disease. One of the most striking conclusion arising from work with genetically engineered immunodeficient mouse models is the existence of a high level of redundancy of the components of the immune system. However, when genes encoding molecules involved in T cell immunoregulatory functions are deleted, spontaneous chronic inflammation of the gut mucosa (similar to human inflammatory bowel disease) develops. Surprisingly, when such immunocompromised animals were placed into germfree environment, intestinal inflammation did not develop. Impairment of the mucosal immune response to the normal bacterial flora has been proposed to play a crucial role in the pathogenesis of chronic intestinal inflammation. The use of immunodeficient models colonized with defined microflora for the analysis of immune reactivity will shed light on the mode of action of different immunologically important molecules responsible for the delicate balance between luminal commensals, nonspecific and specific components of the mucosal immune system.

Differences in enterocyte brush border enzyme activities in ageing rats reared in germ-free and conventional conditions.

The aim of this study was to evaluate the levels of disaccharidase and dipeptidyl peptidase i.v. activities in rat jejunal enterocytes under the influence of long-term germ-free conditions. We found that the brush-border lactase and dipeptidyl peptidase i.v. activities were two to three times higher in 2-month-old germ-free rats in comparison with their conventional counterparts. The highest effect of germ-free condition was observed on lactase activity in 6-month-old and dipeptidyl peptidase i.v. in 2-month-old rats. No difference between germ-free and conventional rats in sucrase and glucoamylase activities was found in 2-month-old rats. The difference develops with increasing age, sucrase activity becoming significantly higher in 6- and 12-month-old rats and glucoamylase in 12-month-old germ-free rats.

Immunomodulatory effects of Bacillus firmus.

3-day-old miniature piglets were stimulated in vivo with Bacillus firmus by the intraperitoneal or intragastric route for 1 d. Cells containing IgA and IgG2 were detected in the ileum in all stimulated but not in control animals. The frequency of blood CD3+ cells increased after intraperitoneal administration of B. firmus, the ratio of polymorphonuclears to lymphocytes increased in all stimulated piglets. B. firmus induced antitumor immunity in rats with transplanted Yoshida sarcoma cells. Granular lymphocytes and dead tumor cells were found in peritoneal exudate of stimulated animals. B. firmus induced IFN-gamma synthesis in human blood lymphocytes stimulated in vitro for 1 d. The amount of TNF-alpha produced by these stimulated human peripheral blood mononuclears (PBMC) was lower than that of PBMC stimulated with some other bacterial immunomodulators. Cells containing TGF-beta or IL-8 were not found in human PBMC stimulated with B. firmus.

Changes in jejunal mucosa after long-term feeding of germfree rats with gluten.

BACKGROUND: Coeliac disease (CD) or gluten-sensitive enteropathy is a chronic gastrointestinal disease of children and adults. An experimental model using inbred germfree rats has been developed to study the effects of intragastric gliadin application on intestinal mucosa. METHODS: AVN strain Wistar rats (inbred F 87)-germfree were used. Gliadin was applied by intragastric probe from birth until day 63 (0.5-5 mg of gliadin per immunization). Intraepithelial lymphocytes (IEL) were separated from the jejunum, and surface marker characterization was performed using flow cytometry. Isolated IEL were labelled with fluorescein isothiocyanate and injected into control jejunal loops. After 1 h and 6 h the abdominal cavity was reopened. The samples of jejunum were fixed. RESULTS: Prolonged application of gliadin led to the shortening of jejunal villi, crypt hyperplasia, increased number of mitoses in the crypt epithelium, and increased number of IEL-characteristic CD8+, RGL-1+, and TcR alpha/beta +. Transfer of IEL separated from rats fed with gliadin into the intestinal loops of untreated rats led to tight junctions in the enterocytes of the intestinal loops. The IEL isolated from controls (albumin-treated) induced no mucosal changes in intestinal loops. CONCLUSION: These data suggest that IEL isolated from gliadin-treated rats transfer mucosal damage and that gluten-induced enteropathy has an autoimmune component.