Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Regulated MIP-3alpha/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity

Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3alpha/CCL20 is chemotactic for immature dendritic cells and CD45RO(+) T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappaB target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3alpha. The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.     

Genes in the Salmonella pathogenicity island 2 and the Salmonella virulence plasmid are essential for Salmonella-induced apoptosis in intestinal epithelial cells

Intestinal epithelial cells are an important site of the host's interaction with enteroinvasive bacteria. Genes in the chromosomally encoded Salmonella pathogenicity island 2 (SPI 2) that encodes a type III secretion system and genes on the virulence plasmid pSDL2 of Salmonella enteritica serovar Dublin (spv genes) are thought to be important for Salmonella dublin survival in host cells. We hypothesized that genes in those loci may be important also for prolonged Salmonella growth and the induction of apoptosis induced by Salmonella in human intestinal epithelial cells. HT-29 human intestinal epithelial cells were infected with wild-type S. dublin or isogenic mutants deficient in the expression of spv genes or with SPI 2 locus mutations. Neither the spv nor the SPI 2 mutations affected bacterial entry into epithelial cells or intracellular proliferation of Salmonella during the initial 8 h after infection. However, at later periods, bacteria with mutations in the SPI 2 locus or in the spv locus compared to wild-type bacteria, manifested a marked decrease in intracellular proliferation and a different distribution pattern of bacteria within infected cells. Epithelial cell apoptosis was markedly increased in response to infection with wild-type, but not the mutant Salmonella. However, apoptosis of epithelial cells infected with wild-type S. dublin was delayed for approximately 28 h after bacterial entry. Apoptosis was preceded by caspase 3 activation, which was also delayed for approximately 24 h after infection. Despite its late onset, the cellular commitment to apoptosis was determined in the early period after infection as inhibition of bacterial protein synthesis during the first 6 h after epithelial cell infection with wild-type S. dublin, but not at later times, inhibited the induction of apoptosis. These studies indicate that genes in the SPI 2 and the spv loci are crucial for prolonged bacterial growth in intestinal epithelial cells. In addition to their influence on intracellular proliferation of Salmonella, genes in those loci determine the ultimate fate of infected epithelial cells with respect to caspase 3 activation and undergoing death by apoptosis.     

Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta

Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ) genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.     

Cross-talk between enteric pathogens and the intestine

Enteric pathogens finely regulate the expression of virulence genes in reply to stimuli generated by the intestinal environment. This minireview focuses on recently discovered strategies developed by enteric bacteria to cause intestinal secretion through the elaboration of factors that share structure and function with specific host counterparts. Such bacterial antigens appear to interfere largely with the epithelial cell signalling that physiologically regulates the numerous and, as yet not fully elucidated, mechanisms controlling both the transcellular and the paracellular secretion pathways. Heat-stable enterotoxins (STs) elaborated by enterotoxigenic Escherichia coli and the enteroaggregative E. coli enterotoxin (EAST1) are both typical examples of enteric toxins that activate the transcellular secretion pathway by mimicking guanylin, the endogenous modulator of cGMP signalling. Alternative strategies have been developed by Salmonella to induce intestinal secretion through the elaboration of a factor (SopB) that resembles at least two of the host cell 4-phosphatases, enzymes that activate the Ca-dependent transcellular secretion pathway. Finally, Vibrio cholerae has developed innovative tactics to activate the paracellular secretion pathway through the elaboration of Zonula occludens toxin (Zot), a factor that mimics a recently described physiological modulator of intercellular tight junctions.     

Lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive Escherichia coli strains.

Intestinal epithelial cells are able to differentially interact with commensal or pathogenic microorganisms, triggering a physiological or destructive inflammation, respectively. To mimic commensal-enteroinvasive bacteria-host cell interaction, we infected Caco-2 cells with noninvasive Escherichia coli HB101 and with recombinant invasive E. coli HB101(pRI203). Using DNA microarray mRNA profiling and ELISA assays, we studied the expression of several cytokine and cytokine-related genes in infected Caco-2 cells in the absence or presence of bovine lactoferrin (bLf). Infection of Caco-2 cells with the noninvasive strain induced a slight increase in the expression of interleukin 8 (IL-8), whereas infection with invasive E. coli HB101(pRI203) induced a significant increase in the expression of IL-8 as well as other pro-inflammatory cytokines. The addition of bLf, in native- or holo-form, did not influence expression of cytokine genes by uninfected Caco-2 cells, but it decreased expression of IL-8 by cells infected with E.coli HB101. Moreover, except for IL-8, bLfs dramatically downregulated pro-inflammatory cytokines upexpressed by Caco-2 cells infected with the invasive strain. Although IL-8 was decreased by bLfs, it remained upregulated, suggesting that it could be a signal of persistence of intracellular bacteria. The bLf ability to reduce expression of some pro-inflammatory cytokines, which appears independent of its iron saturation, might represent an important natural mechanism in regulating epithelial cell responses to pathogenic bacteria and in limiting cell damage and the spread of infections.     

Cytokines in host defense against Salmonella.

Cytokines are key communication molecules between host cells in the defense against the enteric pathogen, Salmonella. Infection with Salmonella induces expression of multiple chemokines and proinflammatory cytokines in cultured intestinal epithelial cells and macrophages. In animal models, protective roles have been shown for IL-1alpha, TNFalpha, IFN-gamma, IL-12, IL-18 and IL-15, whereas IL-4 and IL-10 inhibit host defenses against Salmonella.     

Ameliorating effect of hepatocyte growth factor on inflammatory bowel disease in a murine model

Hepatocyte growth factor (HGF), a multifunctional cytokine, accelerates intestinal epithelial proliferation. We studied the effects of HGF in mice with trinitrobenzene sulfonic acid-induced colitis, which shows clinical and molecular resemblance to Crohn's disease. Mice with colitis repeatedly were transfected intramuscularly with human HGF cDNA. Weight, survival, histopathology, proinflammatory cytokine mRNAs, and leukocyte infiltration were assessed. Treatment with HGF cDNA induced tyrosine phosphorylation of intestinal c-Met/HGF receptors, inhibited apoptosis, and promoted mitosis in intestinal epithelial cells, accelerating intestinal epithelial restoration and suppressing inflammation. Transfection with HGF cDNA markedly suppressed intestinal mRNA expression of T-helper 1 cytokines such as interleukin-12 and -1beta, interferon-gamma, and tumor necrosis factor-alpha. Numbers of total and CD4-positive T cells, neutrophils, and myloperoxidase activity in intestinal epithelium were diminished by HGF gene transfer, which also prevented weight loss, and improved survival. HGF might prove useful for controlling inflammatory bowel disease.     

Gene expression patterns of epithelial cells modulated by pathogenicity factors of Yersinia enterocolitica

Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray technology we have profiled epithelial cell gene expression upon interaction with Yersinia enterocolitica. Yersinia enterocolitica wild-type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv), which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22 283 probesets (14,239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF-kappa B-regulated factors such as cytokines and adhesion molecules. Yersinia virulence plasmid (pYV)-encoded factors counter regulated Inv-induced gene expression. Thus, YopP repressed Inv-induced NF-kappa B regulated genes at 2 h post infection whereas other pYV-encoded factors repressed host cell genes at 4 and 8 h post infection. Chromosomally encoded factors of Yersinia, other than Inv, induced expression of genes known to be induced by TGF-beta receptor signalling. These genes were also repressed by pYV-encoded factors. Only a few host genes were exclusively induced by pYV-encoded factors. We hypothesize that some of these genes may contribute to pYV-mediated silencing of host cells. In conclusion, the data demonstrates that epithelial cells express a limited number of genes upon interaction with enteric Yersinia. Both Inv and YopP appear to modulate gene expression in order to subvert epithelial cell functions involved in innate immunity.     

The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo

The enteric pathogen Salmonella typhimurium secretes the preformed AvrA effector protein into host cells. This acetyltransferase has been shown to modulate mammalian intestinal immune and survival responses by inhibition of JNK MAPK. To study the role of this effector in natural enteric infection, we used a mouse model to compare wild-type S. typhimurium to an isogenic AvrA null Salmonella mutant. Salmonella lacking AvrA induced increased intestinal inflammation, more intense systemic cytokine responses, and increased apoptosis in epithelial cells. Increased apoptosis was also observed in extra epithelial macrophages. AvrA null-infected mice consistently showed higher bacterial burden within mucosal lymphoid tissues, spleen and liver by 5 days post infection, which indicated a more severe clinical course. To study the molecular mechanisms involved, recombinant adenoviruses expressing AvrA or mutant AvrA proteins were constructed, which showed appropriate expression and mediated the expected inhibition of JNK signalling. Cultured epithelial cells and macrophages transduced with AvrA expressing adenovirus were protected from apoptosis induced by exogenous stimuli. In conclusion, the results demonstrated that Salmonella AvrA modulates survival of infected macrophages likely via JNK suppression, and prevents macrophage death and rapid bacterial dissemination. AvrA suppression of apoptosis in infected macrophages may allow for establishment of a stable intracellular niche typical of intracellular pathogens.