Intravenous injection of endogenous microbial components abrogates DSS-induced colitis.

BACKGROUND: The etiology of inflammatory bowel diseases (IBD) is largely unknown, but appears to be perpetuated by uncontrolled responses to antigenic components of the endogenous flora. Tolerance to antigenic stimulation can be achieved by exposure to a given antigen in high amounts (high dose tolerance). Colitis induced by feeding of Dextran Sodium Sulfate (DSS) is an often-used animal model mimicking clinical and histological features of human IBD. AIMS: We investigated whether treatment with high doses of endogenous bacterial components can affect the response to these antigenic components and thus impact the course of the inflammatory response induced by DSS. METHODS: 129/SvEv mice were injected intravenously in the tail vein with lysates prepared from fecal material of conventionally-raised mice. Control mice received a solution of bacterial antigen-free lysates prepared from fecal material of germ-free mice. Seven days later, colitis was induced in these mice by introducing DSS (3.5%) in the drinking water for 5 days. Onset and course of the inflammatory response was monitored by assessment of weight loss. Mice were sacrificed at day 7 post colitis induction and tested for histopathologic injury, intestinal cytokine release, and systemic response to bacterial antigens. RESULTS: Intravenous injection with fecal lysates reduced intestinal and antigen-stimulated systemic pro-inflammatory cytokine release and prevented DSS-induced weight loss and intestinal injury. CONCLUSION: Pretreatment with high amount of endogenous bacterial components has a profound tolerogenic effect on the systemic and mucosal immune responses resulting in reduced intestinal inflammation and abrogates colitis-induced weight loss.

Neonatal exposure to fecal antigens reduces intestinal inflammation.

BACKGROUND: A role for bacterial antigens in the pathogenesis of inflammatory bowel disease (IBD) has been established in enhanced humoral and cellular immune response to ubiquitous antigens of the enteric flora. However, we have recently shown that bacterial antigens in the absence of live bacteria cannot initiate an intestinal inflammation in IBD-prone interleukin (IL)-10 gene-deficient mice. The objective was to investigate whether neonatal exposure to antigens of their own endogenous flora can tolerize mice to bacterial antigens. METHODS: IL-10 gene-deficient neonates were injected intraperitoneally within 72 hours of birth with a sterile solution of bacterial lysates prepared from fecal material of either conventionally raised mice (contains bacterial antigens) or axenic mice (lacks bacterial antigens). The onset of intestinal inflammation was monitored as the appearance of occult blood in the stool in weekly hemoccult analysis. Mice were sacrificed between age 15 and 19 weeks and tested for histopathologic injury, intestinal inflammation, and systemic response to bacterial antigens. RESULTS: In mice neonatally exposed to bacterial antigens the onset of intestinal inflammation was delayed and the incidence of histopathologic injury at age 18 weeks was reduced. In addition, mice injected with lysates from conventionally raised mice exhibited decreased release of proinflammatory cytokines (interferon gamma [IFN-γ] and IL-17) in intestinal tissue and demonstrated reduced bacteria-stimulated systemic responses when compared to mice injected with lysates derived from bacteria-free, axenic mice. CONCLUSIONS: Neonatal intraperitoneal injection of antigens from the commensal flora causes long-lasting changes in systemic and mucosal immune responses resulting in delayed onset of intestinal inflammation and injury in IBD-prone IL-10 gene-deficient mice.

Oral administration of curcumin emulsified in carboxymethyl cellulose has a potent anti-inflammatory effect in the IL-10 gene-deficient mouse model of IBD.

Curcumin is a tumeric-derived, water-insoluble polyphenol with potential beneficial health effects for humans. It has been shown to have preventive as well as therapeutic effects in chemically induced murine models of colitis. To investigate whether curcumin exerts a similar effect on the spontaneous colitis in interleukin (IL)-10 gene-deficient mice, we gavaged these mice daily for 2 weeks with 200 mg/kg per day curcumin emulsified in carboxymethyl cellulose, a food additive generally used as a viscosity modifier. Mice fed the curcumin/carboxymethyl cellulose mixture and those receiving carboxymethyl cellulose alone demonstrated similar reductions in histological injury score and colon weight/length ratio compared to water-fed controls. However, significant reductions in pro-inflammatory cytokine release in intestinal explant cultures were only seen in mice treated with the curcumin mixture. Our data demonstrate that in IL-10 gene-deficient mice, both oral curcumin and carboxymethyl cellulose, appear to have modifying effects on colitis. However, curcumin has additional anti-inflammatory effects mediated through a reduced production of potent pro-inflammatory mucosal cytokines.

HCV induces oxidative and ER stress, and sensitizes infected cells to apoptosis in SCID/Alb-uPA mice.

Hepatitis C virus (HCV) is a blood-borne pathogen and a major cause of liver disease worldwide. Gene expression profiling was used to characterize the transcriptional response to HCV H77c infection. Evidence is presented for activation of innate antiviral signaling pathways as well as induction of lipid metabolism genes, which may contribute to oxidative stress. We also found that infection of chimeric SCID/Alb-uPA mice by HCV led to signs of hepatocyte damage and apoptosis, which in patients plays a role in activation of stellate cells, recruitment of macrophages, and the subsequent development of fibrosis. Infection of chimeric mice with HCV H77c also led an inflammatory response characterized by infiltration of monocytes and macrophages. There was increased apoptosis in HCV-infected human hepatocytes in H77c-infected mice but not in mice inoculated with a replication incompetent H77c mutant. Moreover, TUNEL reactivity was restricted to HCV-infected hepatocytes, but an increase in FAS expression was not. To gain insight into the factors contributing specific apoptosis of HCV infected cells, immunohistological and confocal microscopy using antibodies for key apoptotic mediators was done. We found that the ER chaperone BiP/GRP78 was increased in HCV-infected cells as was activated BAX, but the activator of ER stress-mediated apoptosis CHOP was not. We found that overall levels of NF-kappaB and BCL-xL were increased by infection; however, within an infected liver, comparison of infected cells to uninfected cells indicated both NF-kappaB and BCL-xL were decreased in HCV-infected cells. We conclude that HCV contributes to hepatocyte damage and apoptosis by inducing stress and pro-apoptotic BAX while preventing the induction of anti-apoptotic NF-kappaB and BCL-xL, thus sensitizing hepatocytes to apoptosis.

CD4+CD25+ regulatory T cells have divergent effects on intestinal inflammation in IL-10 gene-deficient mice.

The regulatory effect of murine CD4+CD25+ T-cells in vivo appears to be dependent on the secretion of IL-10. The lack of IL-10 in the IL-10 gene-deficient mouse has a profoundly negative effect on the mouse's regulation of the response to intestinal bacteria, resulting in severe enterocolitis. We investigated the effect of neonatal injection with wild-type CD4+CD25+ T-cells on the intestinal immune response in IL-10 gene-deficient mice. At the time of analysis, 8-15 weeks later, all mice demonstrated an increased, antigen-stimulated systemic response. However, the intestinal response was divergent with about half of the mice developing an intestinal inflammation with a high injury score, the other half demonstrating a remarkable reduction in injury score with a marked decrease in intestinal IFNgamma release. Our data demonstrate that CD4+CD25+ T-cells can be activated in IL-10 gene-deficient mice and that this stimulation under stringent conditions has the potential to reduce intestinal inflammation.

Association with selected bacteria does not cause enterocolitis in IL-10 gene-deficient mice despite a systemic immune response.

Resident bacteria have been implicated to play a major role in the development of inflammatory bowel disease. While luminally sterile IL-10 gene-deficient mice remain disease-free, their conventionally raised littermates develop enterocolitis associated with increased numbers of luminal and mucosal adherent bacteria. To investigate the role of defined bacteria on the initiation and development of this enterocolitis, we associated luminally sterile IL-10 gene-deficient mice with pure strains of resident bacteria. Axenic, luminally sterile mice were either monoassociated with viridans group Streptococcus or Clostridium sordellii or co-associated with Bacteroides vulgatus and Clostridium sordellii. Seven to 22 weeks later the mice were analyzed for intestinal histologic injury, epithelial permeability, and an inflammatory immune response to bacterial antigens. Despite optimal colonization none of the tested bacteria caused intestinal inflammation, release of inflammatory cytokines from the epithelia, or disruption of the epithelial barrier integrity. However, in the case of association with Bacteroides vulgatus and Clostridium sordellii, a systemic immune response to bacterial-derived antigens was measured, with a magnitude similar to that seen in conventional sick Il-10 gene-deficient mice. This response was not detected in mice associated with viridans group Streptococcus. We conclude that colonization of the intestinal lumen with individual bacterial species may not be sufficient to alter epithelial barrier integrity, increase intestinal cytokine release, or cause intestinal inflammation in susceptible IL-10 gene-deficient mice, despite the ability of these same bacteria to stimulate a systemic response.

Normal Breast Milk Limits the Development of Colitis in IL-10-Deficient Mice.

BACKGROUND AND AIMS: This study examined the role of breast milk in neonatal bacterial colonization of the colon and disease progression in IL-10-deficient mice. METHODS: IL-10-deficient mice were cross-fostered at birth and raised until weaning with a normal mother. Results were compared with normal pups cross-fostered to an IL-10-deficient mother. Mice were examined at various ages for histologic disease, levels of colonic bacteria, and proinflammatory cytokine secretion. RESULTS: IL-10-deficient mice that had been cross-fostered to a normal mother demonstrated normal levels of colonic adherent bacteria and reduced TNFalpha and IFN gamma secretion at 2 to 12 weeks of age. Histologic disease was significantly reduced up to 12 weeks of age. Normal mice cross-fostered to an IL-10-deficient mother had increased levels of adherent bacteria at 2 and 4 weeks and increased IFN gamma secretion. This group also demonstrated slight inflammation up until 12 weeks of age. CONCLUSION: Breast milk has a role in neonatal bacterial colonization. Changing the luminal environment of IL-10-deficient mice during the neonatal period alters the natural disease course.