Martin F. Kagnoff

IKKβ Links Inflammation and Tumorigenesis in a Mouse Model of Colitis-Associated Cancer

A link between inflammation and cancer has long been suspected, but its molecular nature remained ill defined. A key player in inflammation is transcription factor NF-kappaB whose activity is triggered in response to infectious agents and proinflammatory cytokines via the IkappaB kinase (IKK) complex. Using a colitis-associated cancer model, we show that although deletion of IKKbeta in intestinal epithelial cells does not decrease inflammation, it leads to a dramatic decrease in tumor incidence without affecting tumor size. This is linked to increased epithelial apoptosis during tumor promotion. Deleting IKKbeta in myeloid cells, however, results in a significant decrease in tumor size. This deletion diminishes expression of proinflammatory cytokines that may serve as tumor growth factors, without affecting apoptosis. Thus, specific inactivation of the IKK/NF-kappaB pathway in two different cell types can attenuate formation of inflammation-associated tumors. In addition to suppressing apoptosis in advanced tumors, IKKbeta may link inflammation to cancer.

A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion.

Pathogenic bacteria that penetrate the intestinal epithelial barrier stimulate an inflammatory response in the adjacent intestinal mucosa. The present studies asked whether colon epithelial cells can provide signals that are important for the initiation and amplification of an acute mucosal inflammatory response. Infection of monolayers of human colon epithelial cell lines (T84, HT29, Caco-2) with invasive strains of bacteria (Salmonella dublin, Shigella dysenteriae, Yersinia enterocolitica, Listeria monocytogenes, enteroinvasive Escherichia coli) resulted in the coordinate expression and upregulation of a specific array of four proinflammatory cytokines, IL-8, monocyte chemotactic protein-1, GM-CSF, and TNF alpha, as assessed by mRNA levels and cytokine secretion. Expression of the same cytokines was upregulated after TNF alpha or IL-1 stimulation of these cells. In contrast, cytokine gene expression was not altered after infection of colon epithelial cells with noninvasive bacteria or the noninvasive protozoan parasite, G. lamblia. Notably, none of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, IL-12p40, IFN-gamma, or significant levels of IL-1 or IL-10 in response to the identical stimuli. The coordinate expression of IL-8, MCP-1, GM-CSF and TNF alpha appears to be a general property of human colon epithelial cells since an identical array of cytokines, as well as IL-6, also was expressed by freshly isolated human colon epithelial cells. Since the cytokines expressed in response to bacterial invasion or other proinflammatory agonists have a well documented role in chemotaxis and activation of inflammatory cells, colon epithelial cells appear to be programmed to provide a set of signals for the activation of the mucosal inflammatory response in the earliest phases after microbial invasion.

Differential cytokine expression by human intestinal epithelial cell lines: Regulated expression of interleukin 8

BACKGROUND To characterize the role of intestinal epithelial cells in mucosal host defense, we have examined constitutive cytokine expression and regulated expression of interleukin (IL)-8 by human colonic epithelial cells. METHODS Cytokine expression by the human colonic epithelial cell lines, T84, Caco-2, SW620, and HT29 was assessed by using polymerase chain reaction amplification of reverse-transcribed RNA. Regulated IL-8 expression was analyzed by nuclear run-off assays, Northern blot analysis, and enzyme-linked immunosorbent assay. RESULTS The cell lines constitutively expressed messenger RNA (mRNA) for IL-8 and transforming growth factor beta 1. In addition, some cell lines expressed mRNA for IL-1 alpha, IL-1 beta, IL-10 and tumor necrosis factor alpha (TNF alpha). None of the cell lines expressed mRNA for IL-2, IL-4, IL-5, IL-6, or interferon gamma. Cell lines secreted IL-8 either constitutively or after stimulation with the physiological agonists TNF alpha, IL-1 beta, or lipopolysaccharide. Increased IL-8 secretion after TNF alpha stimulation of T84 cells was accompanied by increased IL-8 mRNA levels and an increased transcription rate of the IL-8 gene. IL-8 was preferentially secreted at the basolateral surface of polarized T84 cells. In further studies, freshly isolated human colon epithelial cells also secreted IL-8. CONCLUSIONS These results support the notion of bidirectional communication between intestinal epithelial cells and mucosal immune and inflammatory cells.

Maintenance of colonic homeostasis by distinctive apical TLR9 signalling in intestinal epithelial cells

The mechanisms by which commensal bacteria suppress inflammatory signalling in the gut are still unclear. Here, we present a cellular mechanism whereby the polarity of intestinal epithelial cells (IECs) has a major role in colonic homeostasis. TLR9 activation through apical and basolateral surface domains have distinct transcriptional responses, evident by NF-κB activation and cDNA microarray analysis. Whereas basolateral TLR9 signals IκBα degradation and activation of the NF-κB pathway, apical TLR9 stimulation invokes a unique response in which ubiquitinated IκB accumulates in the cytoplasm preventing NF-κB activation. Furthermore, apical TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges. IECs in TLR9-deficient mice, when compared with wild-type and TLR2-deficient mice, display a lower NF-κB activation threshold and these mice are highly susceptible to experimental colitis. Our data provide a case for organ-specific innate immunity in which TLR expression in polarized IECs has uniquely evolved to maintain colonic homeostasis and regulate tolerance and inflammation.

Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis.

Chlamydia species infect epithelial cells at mucosal surfaces, and are major causes of sexually transmitted diseases. Infection is characterized by inflammation which is exacerbated upon reinfection, ultimately leading to tissue damage and scarring. Although central for the development of disease manifestations, little is known about the mechanisms that initiate and sustain the inflammatory response to Chlamydia. Infection of cervical and colonic epithelial cells with Chlamydia trachomatis and Chlamydia psittaci is shown in the present studies to upregulate mRNA expression and secretion of the proinflammatory cytokines IL-8, GRO alpha, GM-CSF, and IL-6. In contrast to the rapid, but transient, cytokine induction following infection with other invasive bacteria, the epithelial cytokine response to Chlamydia was delayed until 20-24 h after infection, persisted throughout the chlamydial growth cycle (2-4 d), and required bacterial protein synthesis. Moreover, epithelial cell lines and primary endocervical epithelial cells released IL-1alpha after Chlamydia infection, and increased secretion of the proinflammatory cytokines could be inhibited by anti-IL-1alpha. This suggests that IL-1alpha, released following lysis of infected epithelial cells, may amplify the inflammatory response by stimulating additional cytokine production by noninfected neighboring cells. These findings suggest a novel pathophysiologic concept wherein the acute host response to Chlamydia at mucosal surfaces is primarily initiated and sustained by epithelial cells, the first and major targets of chlamydial infection.

The two faces of IKK and NF-kappaB inhibition: prevention of systemic inflammation but increased local injury following intestinal ischemia-reperfusion.

We studied the role of NF-κB in acute inflammation caused by gut ischemia-reperfusion through selective ablation of IκB kinase (IKK)-β, the catalytic subunit of IKK that is essential for NF-κB activation. Ablation of IKK-β in enterocytes prevented the systemic inflammatory response, which culminates in multiple organ dysfunction syndrome (MODS) that is normally triggered by gut ischemia-reperfusion. IKK-β removal from enterocytes, however, also resulted in severe apoptotic damage to the reperfused intestinal mucosa. These results show the dual function of the NF-κB system, which is responsible for both tissue protection and systemic inflammation, and underscore the caution that should be exerted in using NF-κB and IKK inhibitors.

Celiac disease: pathogenesis of a model immunogenetic disease.

Celiac disease is characterized by small-intestinal mucosal injury and nutrient malabsorption in genetically susceptible individuals in response to the dietary ingestion of wheat gluten and similar proteins in barley and rye. Disease pathogenesis involves interactions among environmental, genetic, and immunological factors. Although celiac disease is predicted by screening studies to affect approximately 1% of the population of the United States and is seen both in children and in adults, 10%-15% or fewer of these individuals have been diagnosed and treated. This article focuses on the role of adaptive and innate immune mechanisms in the pathogenesis of celiac disease and how current concepts of immunopathogenesis might provide alternative approaches for treating celiac disease.

Apoptosis of human intestinal epithelial cells after bacterial invasion.

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.

Cell Differentiation Is a Key Determinant of Cathelicidin LL-37/Human Cationic Antimicrobial Protein 18 Expression by Human Colon Epithelium

ABSTRACT Antimicrobial peptides are highly conserved evolutionarily and are thought to play an important role in innate immunity at intestinal mucosal surfaces. To better understand the role of the antimicrobial peptide human cathelicidin LL-37/human cationic antimicrobial protein 18 (hCAP18) in intestinal mucosal defense, we characterized the regulated expression and production of this peptide by human intestinal epithelium. LL-37/hCAP18 is shown to be expressed within epithelial cells located at the surface and upper crypts of normal human colon. Little or no expression was seen within the deeper colon crypts or within epithelial cells of the small intestine. Paralleling its expression in more differentiated epithelial cells in vivo, LL-37/hCAP18 mRNA and protein expression was upregulated in spontaneously differentiating Caco-2 human colon epithelial cells and in HCA-7 human colon epithelial cells treated with the cell differentiation-inducing agent sodium butyrate. LL-37/hCAP18 expression by colon epithelium does not require commensal bacteria, since LL-37/hCAP18 is produced with a similar expression pattern by epithelial cells in human colon xenografts that lack a luminal microflora. LL-37/hCAP18 mRNA was not upregulated in response to tumor necrosis factor alpha, interleukin 1α (IL-1α), gamma interferon, lipopolysaccharide, or IL-6, nor did the expression patterns and levels of LL-37/hCAP18 in the epithelium of the normal and inflamed colon differ. On the other hand, infection of HCA-7 cells with Salmonella enterica serovar Dublin or enteroinvasive Escherichia coli modestly upregulated LL-37/hCAP18 mRNA expression. We conclude that differentiated human colon epithelium expresses LL-37/hCAP18 as part of its repertoire of innate defense molecules and that the distribution and regulated expression of LL-37/hCAP18 in the colon differs markedly from that of other enteric antimicrobial peptides, such as defensins.

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.