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- MATERIALS AND METHODS
Intestinal flora plays a critical role in the initiation and perpetuation of inflammatory bowel disease. This study examined whether live fecal bacteria were necessary for the initiation of this inflammatory response or whether sterile fecal material would provoke a similar response. Three preparations of fecal material were prepared: (1) a slurry of live fecal bacteria, (2) a sterile lysate of bacterial antigens, and (3) a sterile filtrate of fecal water. Each preparation was introduced via gastric gavage into the intestines of axenic interleukin-10 gene-deficient mice genetically predisposed to develop inflammatory bowel disease. Intestinal barrier integrity and degrees of mucosal and systemic inflammations were determined for each preparation group. Intestinal barrier integrity, as determined by mannitol transmural flux, was altered by both live fecal bacterial and sterile lysates of bacterial antigens, although it was not altered by sterile filtrates of fecal water. However, only live fecal bacteria initiated mucosal inflammation and injury and a systemic immune response. Fecal bacterial antigens in the presence of live bacteria and sterile fecal bacterial antigens have different effects on the initiation and perpetuation of intestinal inflammation.
Evidence from the last several decades has revealed that the body's indigenous flora plays an important role in the pathogenesis of inflammatory bowel diseases (IBDs).1–3 Although there is no specific disease-causing bacterium, monoassociation studies have demonstrated that certain individual bacteria can by themselves cause an inflammation in susceptible rodents, whereas other bacteria cannot.4–8 The disease pathology depends on the animal model used and the specific inherent or induced defect of the model. Furthermore, it has been shown that humoral and cellular responses to bacterial antigens drive the perpetuation of the inflammatory state.9 Feeding of fecal material, which includes the whole array of endogenous bacteria, induces a systemic response to various bacterial antigens.10 In the case of monoassociation, the response is directed against antigens from the inoculating bacteria.8
Recently, individual bacterial components have been identified as immunogens involved in intestinal inflammation. Among the bacterial antigens identified so far are bacterial flagellins, histones, superantigens, and peptidoglycans of bacterial cell-wall material.11–14 Colonic intramural or subserosal injection of bacterial cell-wall peptidoglycans can, for instance, cause chronic, spontaneously relapsing enterocolitis in rats.15,16 In addition, bacterial oligonucleotides have been reported to have modulating effects on intestinal inflammation.17
However, it is not clear whether these bacterial antigens can, in the absence of live bacteria, initiate a sustained inflammatory response. To investigate whether oral exposure of bacterial antigens alone may lead to a mucosal and/or systemic immune response resulting in a sustained inflammation, we gavaged axenic interleukin-10 (IL-10) gene-deficient mice with filter-sterilized fecal slurry containing all of the bacterial antigens and compared the results of this exposure to those of an exposure to the same live fecal bacteria.
- Top of page
- MATERIALS AND METHODS
In this study, we have shown for the first time that inoculation of axenic IL-10 gene-deficient mice with either sterile fecal water or sterile fecal bacterial antigens does not initiate an intestinal injury or inflammation. This is the case despite the fact that a sterile lysate of fecal bacterial antigens, but not the sterile filtrate of fecal water, was able to cause a defect in intestinal epithelial barrier integrity. In contrast, inoculation of axenic IL-10 gene-deficient mice with a slurry of live fecal bacteria will rapidly induce both a defect in barrier integrity and subsequent intestinal inflammation and injury.
IL-10 gene-deficient mice are genetically susceptible to develop enterocolitis; however, the initiation of the disease appears to depend on the presence of the “right” luminal bacteria. Enterocolitis can be initiated rapidly within 3 days, reaching a maximum intensity in 7 days, in axenic mice gavaged with a slurry of live fecal bacteria that contains the array of endogenous bacteria normally seen in fecal samples.10 Nevertheless, trying to identify which single or group of bacteria from this fecal sample initiates the inflammation has been difficult. Monoassociation studies on axenic IL-10 gene-deficient mice have demonstrated that a limited number of bacterial strains are able to initiate any type of intestinal inflammation when present as the single bacterial species in the gut. Currently, only 3 bacteria have been shown to cause enterocolitis in this genetically modified strain of mice: Enterococcus faecalis, Escherichia coli, and Enterobacter cloacae.4,5 Other tested strains that do not cause disease in a time frame of up to 30 weeks include Viridans group streptococcus, C sordellii, B vulgatus, Helicobacter hepaticus, Pseudomonas fluorescens, Candida albicans, and various Lactobacillus species.4,5,7,8
The reason why some bacteria cause intestinal inflammation, whereas others do not, is largely unknown. The difference may be rooted in the varying effects that individual bacterial species have on the immune system. For example, Gram-positive strains belonging to the genera Lactobacillus, Streptococcus, and Eubacterium have little immunogenic effect in stimulating an immunoglobulin A response or release of IFN-γ in monoassociated mice, whereas Gram-negative strains such as Bacteroides and Escherichia are highly stimulatory.8,23 In addition, Umesaki et al24 have shown that bacteria-induced changes differ between the small and large intestines of mice monoassociated with segmented, filamentes bacteria and/or Clostridium bacteria.
It is well established that loss of tolerance to antigens of the endogenous flora plays a major role in the development of intestinal inflammation. Loss of tolerance occurs as a result of an imbalance in the homeostatic environment and leads to overstimulation of inflammatory cytokines.25 Chandran et al26 hypothesize that quorum-sensing molecules, when secreted in high concentration by bacteria, may alter the state of mucosal tolerance through modulation of the immune system. Moreover, studies on the sera of Crohn's disease patients have revealed that loss of tolerance to the various microbial antigens appears to be patient specific.27 Thus, responsiveness to individual bacterial antigens may in part be dependent on immunological genetic makeup.
Recently, much effort has been concentrated on identifying bacterial components that are recognized by pattern recognition molecules. Bacterial compounds such as bacterial cell-wall material, nucleic acid, flagellin, and superantigens such as lipopolysaccharide have been studied in detail. Their effects on the immune system through interaction with toll-like receptors (TLRs) found on intestinal epithelial cells can greatly influence the immunological homeostasis of the intestinal environment, making them prime candidates for involvement in IBD pathogenesis. Bacterial flagellins have been demonstrated to induce epithelial proinflammatory gene expression after binding to TLR5,28 whereas bacterial DNA has been shown to exert anti-inflammatory effects through interaction with TLR9.29 The gene products of the NOD2/CARD15 gene also are involved in bacterial antigen recognition. The protein product of NOD2 is thought to be a cytoplasmic receptor for muramyl dipeptide, which is produced after the lysozyme-mediated breakdown of the polysaccharide component of bacterial cell-wall peptidoglycans.30,31NOD2/CARD15 is believed to act through the NF-κB pathway to regulate production of tumor necrosis factor and other proinflammatory cytokines.32
In IBD pathogenesis, the intestinal inflammatory response is frequently accompanied by a loss of epithelial integrity. Although it is uncertain whether this defect results from or is the cause of the inflammatory response, it is believed that this defect allows easier passage of bacteria and their antigens; thus, it perpetuates a bacteria-stimulated response in the underlying tissue.
Metabolic stress of the intestinal epithelia in the form of chemicals such as trinitrobenzene sulfonic acid and dextran sodium sulfate can cause immunological imbalances that lead to intestinal inflammatory response.33–35 This response also is characterized by a loss of tolerance for the endogenous flora.36 In the IL-10 gene-deficient mouse, an epithelial defect preexists the onset of the disease.22 This defect appears to be initiated shortly after weaning, when the mice acquire their intestinal flora. In the SCID transfer model, the epithelial defect is detected after the transfer of colitogenic T cells.37 Thus, the defect may require the stimulation of lymphocytes by bacterial antigens.
Our results demonstrate that loss of epithelial integrity can occur in the absence of live bacteria. Stimulation with bacterial components alone, in the absence of any added chemicals, was sufficient to affect the epithelial integrity. However, only the sterile lysate of fecal bacterial antigens that contained sufficient cell-wall material had an effect on epithelial permeability. Although we cannot exclude the possibility that there also were higher concentrations of other bacterial products such as proteins and/or nucleic material in the sterile lysate of fecal bacterial antigens compared with the sterile filtrate of fecal water preparation, these results suggest that loss of epithelial integrity may be dependent on the inclusion of cell-wall material.
Despite the influence that bacterial antigens can exert on the mucosal immune response, it appears that bacterial antigens alone are not sufficient to initiate intestinal inflammation. There are several possible reasons why oral bacterial antigens do not induce inflammation. These reasons include the inaccessibility of the gut-associated intestinal tissue to bacterial antigens, the lack of stimulation of anti-inflammatory responses, and generation of oral tolerance. It is conceivable that in healthy individuals, bacterial antigens may be denied access to the underlying tissue as a result of the glycocalyx coating of the cells. In the absence of live bacteria, bacterial antigens are not newly generated and therefore may have a limited lifetime in the intestinal environment and may not be circulated in the bloodstream in a sufficient amount, consistent with our finding that no systemic response was generated. Alternatively, bacterial antigens may stimulate anti-inflammatory responses that suppress any proinflammatory responses. Haller et al38 have demonstrated that nonpathogenic bacteria can induce anti-inflammatory effects in epithelial cells through the inhibition of NF-κB activation and the induction of transforming growth factor-β production. In this context, it is interesting to note that IL-4 is produced in consistently higher amounts in spleen cells from IL-10 gene-deficient mice inoculated with a sterile lysate of fecal bacterial antigens than in spleen cells from axenic mice (B.C.S., unpublished observation). Finally, feeding of antigens in high concentrations has been shown to induce peripheral deletion of antigen-specific T cells after an initial increase in reactive T cells.39 A large amount of bacterial antigens given orally in the absence of live bacteria may thus stimulate a form of oral tolerance rather than stimulate an inflammatory response.
In summary, we have shown that sterile lysates of fecal bacterial antigens can cause a defect in intestinal barrier integrity but that they cannot initiate intestinal inflammation or injury in mice genetically predisposed to developing IBD. This is in contrast to live fecal bacteria, which both cause a defect in intestinal barrier integrity and lead to the development of intestinal inflammation and injury.