A newborn mouse model for the study of intestinal pathogenesis of shigellosis

Shigella infection is characterized by the induction of acute inflammation, which is responsible for the massive tissue destruction of the intestinal mucosa. A murine model would be a valuable tool for gaining a better understanding of the physiopathology of shigellosis and the host immune response to Shigella infection, but adult mice do not develop disease upon oral inoculation. We therefore attempted to develop a model of infection in newborn mice. Four-day-old mice inoculated with 50 microl of 5 x 10(9) invasive wild-type Shigella flexneri 5a were susceptible to bacterial infection, but mice inoculated with the non-invasive strain BS176 were not. Histologically, 4-day-old mice infected with the invasive strain presented intestinal lesions and inflammation similar to those described in patients with shigellosis. Moreover, cytokine and chemokine responses consistent with inflammation were observed. Lower bacterial inocula induced less severe intestinal damage. In contrast, 5-day-old mice inoculated with either the invasive or the non-invasive strain were not infected. We have thus established a mouse model that is suitable for the study of the pathogenesis of intestinal Shigella infection.     

Effect of CD14 blockade in rabbits with Escherichia coli pneumonia and sepsis

CD14, a pattern recognition receptor found on myeloid cells, is a critical component of the innate immune system that mediates local and systemic host responses to Gram-negative and Gram-positive bacterial products. Previous studies in normal animals have tested the effect of CD14 blockade on the systemic response to i.v. LPS. The goals of the study were to determine whether CD14 blockade protected against the deleterious systemic response associated with Escherichia coli pneumonia and to determine whether this strategy affected the pulmonary response to tissue infection. Rabbits were pretreated with either anti-CD14 mAb or isotype control mAb at 2.5 mg/kg. E. coli (1 x 109 CFU) was inoculated into the lungs, and the animals were observed for either 4 or 24 h. The blockade of CD14 improved the mean arterial blood pressure (p = 0.001) and decreased the i.v. fluid requirements (p = 0.01). Although this therapy protected the vascular compartment, rabbits treated with anti-CD14 mAb had increased bacterial burdens in the bronchoalveolar lavage fluid recovered from the instilled lung (p = 0.005) and widened alveolar-arterial oxygen difference. Blockade of CD14 prevents the deleterious systemic responses that occur in sepsis; however, other measures are necessary to control bacterial proliferation at the primary site of infection.     

Neuromodulation of experimental Shigella infection reduces damage to the gut mucosa

Bacillary dysentery arises when Shigella invades the colonic and rectal mucosae of the human gut and elicits a strong inflammatory response, which may lead to life-threatening complications. Hence, downregulation of the host inflammatory response is an appealing therapeutical alternative. The gastrointestinal tract is densely innervated, and nerve endings are often found in the vicinity of leukocytes. We have assessed the impact of experimental Shigella infection on levels of neuropeptides in the intestinal mucosa of rabbits. Ligated small intestinal loops were created in rabbits, and either live, pathogenic Shigella flexneri, a nonpathogenic mutant of Shigella, or NaCl was injected into the loops. Infection was allowed to proceed for 8 or 16 h, after which the rabbits were sacrificed and intestinal biopsies collected. Tissue destruction, fluid secretion and degree of bacterial invasion were monitored. Intestinal biopsies were homogenized, and levels of the neuropeptides calcitonin gene-related peptide, substance P, peptide YY (PYY), vasoactive intestinal peptide, somatostatin, galanin, motilin and neurotensin were measured by radioimmunoassay. Loops exposed to invasive Shigella had 5.7 times lower levels of PYY (P = 0.0095) than loops exposed to NaCl, after 16 h of infection. The levels of the other neuropeptides tested were unchanged. Inhibition of nicotinic cholinergic neurotransmission partly protected the intestinal mucosa from destruction elicited by invasive Shigella. These findings indicate that a tissue-invasive bacterium such as Shigella, which is strictly localized to the intestinal mucosa, activates intramural nerve reflexes that presumably involve a nicotinic synapse as well as the neuropeptide PYY.     

CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells

Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid-phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half- maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella . Pretreatment of cells with an anti-CD44 monoclonal antibody resulted in inhibition of Shigella -induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB–CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.     

Expression of Toll-like receptor 2 (TLR2), TLR4, and CD14 in biopsy samples of patients with inflammatory bowel diseases: upregulated expression of TLR2 in terminal ileum of patients with ulcerative colitis.

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 in biopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis.     

IL-10 regulates movement of intestinally derived CD4+ T cells to the liver

Diseases that affect the intestine may have hepatic manifestations, but the mechanisms involved in establishing hepatic disease secondarily remain poorly understood. We previously reported that IL-10 knockout (KO) mice developed severe necrotizing hepatitis following oral infection with Trichinella spiralis. In this study, we used this model of intestinal inflammation to further examine the role of IL-10 in regulating hepatic injury. Hepatic damage was induced by migrating newborn larvae. By delivering the parasite directly into the portal vein, we demonstrated that an ongoing intestinal immune response was necessary for the development of hepatitis. Intestinally derived CD4+ cells increased in the livers of IL-10 KO mice, and Ab-mediated blockade of MAdCAM-1 inhibited the accumulation of CD4+alpha(4)beta(7)+ cells in the liver. Moreover, adoptive transfer of intestinally primed CD4+ T cells from IL-10 KO mice caused hepatitis in infected immunodeficient animals. Conversely, transfer of wild-type donor cells reduced the severity of hepatic inflammation in IL-10 KO recipients, demonstrating regulatory activity. Our results revealed that IL-10 prevented migration of intestinal T cells to the liver and inhibited the development of hepatitis.     

Shigella's ways of manipulating the host intestinal innate and adaptive immune system: a tool box for survival?

Shigella, a Gram-negative invasive enteropathogenic bacterium, causes the rupture, invasion and inflammatory destruction of the human colonic epithelium. This complex and aggressive process accounts for the symptoms of bacillary dysentery. The so-called invasive phenotype of Shigella is linked to expression of a type III secretory system (TTSS) injecting effector proteins into the epithelial cell membrane and cytoplasm, thereby inducing local but massive changes in the cell cytoskeleton that lead to bacterial internalization into non-phagocytic intestinal epithelial cells. The invasive phenotype also accounts for the potent pro-inflammatory capacity of the microorganism. Recent evidence indicates that a large part of the mucosal inflammation is initiated by intracellular sensing of bacterial peptidoglycan by cytosolic leucine-rich receptors of the NOD family, particularly NOD1, in epithelial cells. This causes activation of the nuclear factor kappa B and c-JunNH(2)-terminal-kinase pathways, with interleukin-8 appearing as a major chemokine mediating the inflammatory burst that is dominated by massive infiltration of the mucosa by polymorphonuclear leukocytes. Not unexpectedly, this inflammatory response, which is likely to be very harmful for the invading microbe, is regulated by the bacterium itself. A group of proteins encoded by Shigella, which are injected into target cells by the TTSS, has been recently recognized as a family of potent regulators of the innate immune response. These enzymes target key cellular functions that are essential in triggering the inflammatory response, and more generally defense responses of the intestinal mucosa. This review focuses on the mechanisms employed by Shigella to manipulate the host innate response in order to escape early bacterial killing, thus ensuring establishment of its infectious process. The escape strategies, the possible direct effect of Shigella on B and T lymphocytes, their impact on the development of adaptive immunity, and how they may help explain the limited protection induced by natural infection are discussed.     

CD4+ T cells mediate murine syngeneic graft-versus-host disease-associated colitis

Syngeneic graft-vs-host disease (SGVHD) develops following lethal irradiation, reconstitution with syngeneic bone marrow, and treatment with a 21-day course of the immunosuppressive agent cyclosporin A (CsA). Following cessation of CsA, this inducible disease is characterized by weight loss, diarrhea, and development of inflammation in the colon and liver. Although nonspecific effector cells and Th1 cytokines have been shown to participate in disease induction, the role of T cells has not been fully elucidated. Initial studies demonstrated significant increases in CD4+ T cells, but not other T cell populations in the colons of diseased animals relative to transplant control animals. To demonstrate a functional linkage between increases in colonic CD4+ T cells and disease induction, in vivo T cell depletion studies were performed. Beginning on the day of bone marrow transplantation, groups of control and CsA-treated animals were treated with mAb against either CD4 or CD8 for 21 days. Treatment with anti-CD4, but not anti-CD8, eliminated clinical symptoms and colon pathology. Interestingly, neither anti-CD4 nor anti-CD8 therapy affected the development of liver pathology associated with SGVHD. These findings demonstrated that CD4+ T cells initiate development of the intestinal inflammation associated with murine SGVHD.     

Expression of clusterin in Crohn's disease of the terminal ileum

Crohn's disease (CD) is a chronic inflammatory intestinal disorder with disturbance and injury of the intestinal mucosal barrier, in which various proinflammatory molecules as well as molecules with antiinflammatory activity and cytoprotective function are found to be expressed. We investigated whether clusterin, a multifunctional cytoprotective protein, is upregulated in Crohn's disease, because augmented expression of clusterin is seen in many organs following various forms of tissue injury. Human actively and inactively inflamed ileal tissues from CD patients as well as normal intestinal specimens from control patients (normal ileum) were investigated by Western blot analysis, immunohistochemisty and in situ hybridization. As compared with controls, a strongly enhanced expression of clusterin was found in CD tissues, correlating with disease activity. Immunohistochemistry and in situ hybridization analysis revealed foci of crypts almost completely lined by clusterin expressing enterocytes in CD, a feature that was never seen in controls. Such crypts appeared especially within the morphologically intact mucosa apart from erosive or ulcerative lesions. Besides epithelia, clusterin was also expressed by inflammatory mononuclear cells. Enhanced expression of clusterin by crypt epithelia might reflect a cytoprotective function of the protein in order to prevent further injury of the intestinal mucosal barrier in CD.     

Role of MutS homolog 2 (MSH2) in intestinal myofibroblast proliferation during Crohn's disease stricture formation

Tissue remodeling and mesenchymal cell accumulation accompanies chronic inflammatory disorders involving joints, lung, vasculature, and bowel. Chronic inflammation may alter DNA-mismatch repair (MMR) systems in mesenchymal cells, but is not defined in Crohn's disease (CD) and its associated intestinal remodeling and stricture formation. We determined whether DNA-MMR alteration plays a role in the pathogenesis of CD tissue remodeling. Control and CD bowel tissues were used to generate primary cultures of muscularis mucosa myofibroblasts, which were assessed directly or following stimulation with TNF-alpha/LPS or H2O2. MutS homolog (MSH)2, MSH3, and MSH6 expression in tissues and myofibroblasts was determined. Immunohistochemical staining revealed an increased expression of MSH2 in CD muscularis mucosa and submucosal tissues compared with controls or uninvolved CD tissue, and MSH2 expression was increased in CD myofibroblasts compared with control cells. TNF-alpha/LPS and H2O2 further enhanced MSH2 expression in both control and CD cells, which were decreased by simvastatin. There were no significant changes in MSH3 and MSH6 expression. Proliferating cell nuclear antigen and Ki67 staining of CD tissue revealed increased proliferation in the muscularis mucosa and submucosa of chronically inflamed tissues, and enhanced proliferation was seen in CD myofibroblasts compared with controls. Simvastatin reversed the effects of inflammatory stress on the DNA-MMR and inhibited proliferation of control and CD myofibroblasts. Gene silencing with MSH2 siRNA selectively decreased CD myofibroblast proliferation. These data demonstrate a potential role for MSH2 in the pathogenesis of nonneoplastic mesenchymal cell accumulation and intestinal remodeling in CD chronic inflammation.     

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.     

Differences between the fecal microbiota of coeliac infants and healthy controls

Coeliac disease (CD) is an immune-mediated enteropathy with a multifactorial aetiology, characterized by chronic inflammation of the small intestinal mucosa. Although evidence suggests that the gut microbiota contributes to other chronic inflammatory disorders, its possible role in CD has not been determined. In this study, the composition of the fecal microbiota of coeliac children and age-matched controls was investigated by culture-dependent and -independent methodologies, using fluorescent in situ hybridization (FISH). The levels of Bacteroides, Clostridium and Staphylococcus were significantly higher (p < 0.05) in fecal samples from coeliac patients than in healthy subjects when analysed by culture methods. The numbers of Bacteroides-Prevotella, Clostridium histolyticum, Eubacterium rectale-C. coccoides, Atopobium, and sulfate reducing bacterial groups were also significantly higher (p < 0.05) in fecal samples from coeliac infants when analysed by FISH. The counts of Bifidobacterium tended to be higher in healthy controls by the two type of analysis but the differences were not significant. This is the first report on the identification of the specific bacterial groups responsible for alterations in the intestinal microecology of children with active CD. The bacterial pattern detected in coeliac patients, correlates with the epidemiological data and metabolic deviations associated with CD, and involve bacterial groups link to other chronic inflammatory disorders.     

Antigen-specific CD8+ T cell responses in intestinal tissues during murine listeriosis

Infection of mice with Listeria monocytogenes induces a strong CD8+ T cell response, which is critical for the control of bacteria and for protection against re-infection. We analyzed the CD8+ T cell response in different intestinal tissues following oral and intravenous (i.v.) L. monocytogenes infection. After oral infection, bacterial titers in small intestine and large intestine, and the listeria-specific CD8+ T cell response in the mucosa of both parts of the intestine, were highly correlated. Oral infection of CD28-deficient mice revealed that this response was strictly dependent on CD28 costimulation. Significant listeria-specific CD8+ T cell responses also occurred in all intestinal tissues analyzed after i.v. infection or after DNA vaccination, indicating that the accumulation of listeria-specific CD8+ T cells in these tissues only partially depends on local antigen presentation and inflammation.     

Immunohistological characterization of leukocytes in the lungs of healthy mice and after bacterial intratracheal infection

Leukocytes in the peripheral lung parenchyma of mice have not been characterized histologically during bacterial infection. The aim of this study was to investigate (a) the immunohistological characteristics of healthy murine lungs and (b) the cell kinetics during acute inflammation. BALB/c and MF1 mice were examined; as well as transgenic mice with the gene defect of cystic fibrosis (CF) in the airways as an animal model for this disease. MF1 mice served as controls for the transgenic animals. Lavaged and perfused lungs were snap frozen. B and T lymphocytes, CD4+ and CD8+ cells, dendritic cells, neutrophils and a subset of macrophages were enumerated on cryostat lung sections. The lung tissue and bronchoalveolar lavage (BAL) of BALB/c mice, infected intratracheally with Haemophilus influenzae type b (Hib), were studied at different time points after infection. In the lungs of healthy mice, including CF mice, the largest population was that of T cells, CD4+ cells being always more frequent than CD8+ cells. During acute inflammation the number of neutrophils in the lung parenchyma and BAL increased strongly within the first hours after bacterial instillation and reached baseline levels within one week. This study provides a semi-quantitative analysis of immunocompetent cells in normal and infected murine lung tissue. Differences in cell numbers are found between different strains. Moreover, the cellular reaction during Hib infection in mouse lungs is dominated by neutrophils, as expected in a primary immune response. In uninfected CF mice the numbers and distribution of immune cells in the lung tissue are normal, indicating that the cellular defense is adequate.     

Critical role of lipopolysaccharide-binding protein and CD14 in immune responses against gram-negative bacteria

LPS-binding protein (LBP) and CD14 potentiate cell activation by LPS, contributing to lethal endotoxemia. We analyzed the contribution of LBP/CD14 in models of bacterial infection. Mice pretreated with mAbs neutralizing CD14 or LBP showed a delay in TNF-alpha production and died of overwhelming infection within 24 h, after a challenge with 250 CFU of virulent Klebsiella pneumoniae. Blockade of TNF-alpha also increased lethality, whereas pretreatment with TNF-alpha protected mice, even in the presence of LBP and CD14 blockade. Anti-LBP or anti-CD14 mAbs did not improve or decrease lethality with a higher inoculum (10(5) K. pneumoniae) and did not affect outcome following injections of low or high inocula of Escherichia coli O111. These results point to the essential role of LBP/CD14 in innate immunity against virulent bacteria.     

Spontaneous in vitro IL-6 production in various intestinal segments in patients with inflammatory bowel disease

Interleukin-6 (IL-6) plays an important role in regulation of intestinal inflammatory processes in inflammatory bowel disease (IBD). The levels of IL-6 in media from cultured biopsy samples were determined by ELISA in 14 Crohn’s disease (CD) patients, 17 patients with ulcerative colitis (UC), and 24 healthy controls in terminal ileum, cecum, and rectum. Results were confirmed by measuring mRNA expression in selected patients. In CD patients, there were increased levels of IL-6 (expressed in picograms per milligram of biopsy tissue mass) in terminal ileum compared with controls (median, 617 vs. 90.4; p < 0.001). High IL-6 levels were found in the rectum of CD patients with active disease but normal endoscopic findings (791 vs. 131; p < 0.05). This result was confirmed by mRNA expression. There was a substantial increase of IL-6 levels in cultured cecal (median, 327 vs. 94.0; p < 0.001) and rectal mucosa (median, 282 vs.131; p < 0.05) but not in ileal mucosa of UC patients. In conclusion, IL-6 production was higher in IBD patients than in controls; it correlated with disease activity and varied among different intestinal segments. In clinically active CD patients without rectal involvement, high IL-6 levels in cultured rectal mucosa suggest immune stimulation even in the absence of macroscopic inflammation.     

Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia

CD4+CD25+FoxP3+ regulatory T cells are decreased in patients infected with HIV and have been shown to be critical in mediating Ag tolerance in the lung. Because a subset of Pneumocystis-infected individuals develop substantial lung injury, which can be modeled in immune reconstituted scid mice, we used mouse models of Pneumocystis carinii to investigate the role of regulatory T cells in opportunistic infection and immune reconstitution. In this study, we show that CD4+CD25+FoxP3+ cells are part of the host response to Pneumocystis in CD4+ T cell-intact mice. Moreover, lung injury and proinflammatory Th1 and Th2 cytokine levels in the bronchoalveolar lavage fluid and lung homogenate were increased following CD4+CD25- immune reconstitution in Pneumocystis-infected SCID mice but not in CD4+CD25+ T cell-reconstituted animals. The ability of CD4+CD25+ T cells to control inflammation and injury during the course of Pneumocystis was confirmed by treatment of wild-type C57BL/6 mice with anti-CD25 mAb. These data show that CD4+CD25+ T cells control pulmonary inflammation and lung injury associated with Pneumocystis infection both in the setting of immune reconstitution as well as new acquisition of infection.     

Enteric infection with Citrobacter rodentium induces coagulative liver necrosis and hepatic inflammation prior to peak infection and colonic disease

Acute and chronic forms of inflammation are known to affect liver responses and susceptibility to disease and injury. Furthermore, intestinal microbiota has been shown critical in mediating inflammatory host responses in various animal models. Using C. rodentium, a known enteric bacterial pathogen, we examined liver responses to gastrointestinal infection at various stages of disease pathogenesis. For the first time, to our knowledge, we show distinct liver pathology associated with enteric infection with C. rodentium in C57BL/6 mice, characterized by increased inflammation and hepatitis index scores as well as prominent periportal hepatocellular coagulative necrosis indicative of thrombotic ischemic injury in a subset of animals during the early course of C. rodentium pathogenesis. Histologic changes in the liver correlated with serum elevation of liver transaminases, systemic and liver resident cytokines, as well as signal transduction changes prior to peak bacterial colonization and colonic disease. C. rodentium infection in C57BL/6 mice provides a potentially useful model to study acute liver injury and inflammatory stress under conditions of gastrointestinal infection analogous to enteropathogenic E. coli infection in humans.