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1.  New insights into probiotic mechanisms 
Gut Microbes  2013;4(2):94-100.
There has been continued and expanding recognition of probiotic approaches for treating gastrointestinal and systemic disease, as well as increased acceptance of probiotic therapies by both the public and the medical community. A parallel development has been the increasing recognition of the diverse roles that the normal gut microbiota plays in the normal biology of the host. This advance has in turn has been fed by implementation of novel investigative technologies and conceptual paradigms focused on understanding the fundamental role of the microbiota and indeed all commensal bacteria, on known and previously unsuspected aspects of host physiology in health and disease. This review discusses current advances in the study of the host-microbiota interaction, especially as it relates to potential mechanisms of probiotics. It is hoped these new approaches will allow more rational selection and validation of probiotic usage in a variety of clinical conditions.
doi:10.4161/gmic.23283
PMCID: PMC3595083  PMID: 23249742
probiotics; microbiota; Lactobacilli; intestinal disorders; nutrition; prebiotics; metagenomics; metabolism; immunity
2.  The Salmonella effector AvrA mediates bacterial intracellular survival during infection in vivo 
Cellular microbiology  2011;14(1):28-39.
SUMMARY
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 Salmonella 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 signaling. 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.
doi:10.1111/j.1462-5822.2011.01694.x
PMCID: PMC3240734  PMID: 21899703
Salmonella; AvrA; JNK; Apoptosis; Innate immunity
3.  Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair 
N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1–/–IEC and AnxA1–/– mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.
doi:10.1172/JCI65831
PMCID: PMC3533303  PMID: 23241962
4.  Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-κB activation 
Molecular Microbiology  2010;78(5):1232-1245.
Summary
Enteric bacterial pathogens have evolved sophisticated strategies to evade host immune defences. Some pathogens deliver anti-inflammatory effector molecules into the host cell cytoplasm via a type III secretion system (T3SS). Enteropathogenic Escherichia coli (EPEC) inhibits inflammation by an undefined, T3SS-dependent mechanism. Two proteins encoded outside of the EPEC locus of enterocyte effacement (LEE) pathogenicity island, non-LEE-encoded effector H1 (NleH1) and H2 (NleH2), display sequence similarity to Shigella flexneri OspG, which inhibits activation of the pro-inflammatory transcription factor NF-κB. We hypothesized that the anti-inflammatory effects of EPEC were mediated by NleH1 and NleH2. In this study, we examined the effect of NleH1/H2 on the NF-κB pathway. We show that NleH1/H2 are secreted via the T3SS and that transfection of cells with plasmids harbouring nleH1 or nleH2 decreased IKK-β-induced NF-κB activity and attenuated TNF-α-induced degradation of phospho-IκBα by preventing ubiquitination. Serum KC levels were higher in mice infected with ΔnleH1H2 than those infected with WT EPEC, indicating that NleH1/H2 dampen pro-inflammatory cytokine expression. ΔnleH1H2 was cleared more rapidly than WT EPEC while complementation of ΔnleH1H2 with either NleH1 or NleH2 prolonged colonization. Together, these data show that NleH1 and NleH2 function to dampen host inflammation and facilitate EPEC colonization during pathogenesis.
doi:10.1111/j.1365-2958.2010.07400.x
PMCID: PMC3325542  PMID: 21091507
5.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background
Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4.
Methods
Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels.
Results
One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data.
Conclusions
These data are not only consistent with previous functional studies of KLF4’s role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4’s functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
6.  Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts 
Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions.
PMCID: PMC3165197  PMID: 21892412
KLF4; microarray; MEF; DAVID; GSEA; IPA; SAM; FDR
7.  Lactobacillus rhamnosus blocks inflammatory signaling in vivo via reactive oxygen species generation 
Free radical biology & medicine  2009;47(8):1205-1211.
Uncontrolled inflammatory responses in the immature gut may play a role in the pathogenesis of many intestinal inflammatory syndromes that present in newborns or children such as necrotizing enterocolitis (NEC), idiopathic inflammatory bowel diseases (IBD), or infectious enteritis. Consistent with previous reports that murine intestinal function matures over the first 3 weeks of life, we show that inflammatory signaling in neonatal mouse gut increases during postnatal maturation with peak responses occurring at 2-3 weeks. Probiotic bacteria can block inflammatory responses in cultured epithelia by inducing the generation of reactive oxygen species (ROS) which inhibit NF-κB activation through oxidative inactivation of the key regulatory enzyme Ubc12. We now report for the first time that the probiotic Lactobacillus rhamnosus GG (LGG) can induce ROS generation in intestinal epithelia in vitro and in vivo. Intestines from immature mice gavage fed LGG exhibited increased GSH oxidation and cullin-1 deneddylation reflecting local ROS generation and its resultant Ubc12 inactivation, respectively. Furthermore, prefeeding LGG prevented TNF-α induced intestinal NF-κB activation. These studies indicate that LGG can reduce inflammatory signaling in immature intestines by inducing local ROS generation and may be a mechanism by which probiotic bacteria can prevent NEC in premature infants or reduce severity of IBD in children.
doi:10.1016/j.freeradbiomed.2009.07.033
PMCID: PMC2760264  PMID: 19660542
Necrotizing enterocolitis; inflammatory bowel disease; inflammation; reactive oxygen species; probiotics; lactobacillus
8.  Microbes in Gastrointestinal Health and Disease 
Gastroenterology  2008;136(1):65-80.
Most, if not all, animals coexist with a complement of prokaryotic symbionts that confer a variety of physiologic benefits. In humans, the interaction between animal and bacterial cells is especially important in the gastrointestinal tract. Technical and conceptual advances have enabled rapid progress in characterizing the taxonomic composition, metabolic capacity, and immunomodulatory activity of the human gut microbiota, allowing us to establish its role in human health and disease. The human host coevolved with a normal microbiota over millennia and developed, deployed, and optimized complex immune mechanisms that monitor and control this microbial ecosystem. These cellular mechanisms have homeostatic roles beyond the traditional concept of defense against potential pathogens, suggesting these pathways contribute directly to the well-being of the gut. During their coevolution, the bacterial microbiota has established multiple mechanisms to influence the eukaryotic host, generally in a beneficial fashion, and maintain their stable niche. The prokaryotic genomes of the human microbiota encode a spectrum of metabolic capabilities beyond that of the host genome, making the microbiota an integral component of human physiology. Gaining a fuller understanding of both partners in the normal gut-microbiota interaction may shed light on how the relationship can go awry and contribute to a spectrum of immune, inflammatory, and metabolic disorders and may reveal mechanisms by which this relationship could be manipulated toward therapeutic ends.
doi:10.1053/j.gastro.2008.10.080
PMCID: PMC2892787  PMID: 19026645
9.  The Bacterial Fermentation Product Butyrate Influences Epithelial Signaling via Reactive Oxygen Species-Mediated Changes in Cullin-1 Neddylation1 
The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-κB pathway by blockade of IκB-α ubiquitination, a process catalyzed by the E3-SCFβ-TrCP ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-κB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3-SCFβ-TrCP ubiquitin ligase, the NF-κB inhibitor IκB-α. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.
PMCID: PMC2872164  PMID: 19109186
10.  Advances in understanding the interaction between the gut microbiota and adaptive mucosal immune responses 
Commensal gut bacteria are necessary for the complete development of mucosal innate and adaptive immunity and thus may influence intestinal and systemic immune disorders. Recent work has advanced our understanding of this association by identification of a single taxon of the murine microbiota which can stimulate T-cell development and differentiation. It is hoped that further characterization of the mechanisms involved will enable targeted manipulation of the microbiota-immune system relationship.
doi:10.3410/B2-27
PMCID: PMC2948354  PMID: 20948806
11.  Toll-Like Receptor 5-Dependent Regulation of Inflammation in Systemic Salmonella enterica Serovar Typhimurium Infection▿  
Infection and Immunity  2009;77(9):4121-4129.
Salmonella enterica, a gram-negative pathogen, causes a spectrum of human infections including enterocolitis and typhoid fever. We previously showed that Salmonella flagellin played a role in suppressing intestinal mucosal inflammation in a murine model of acute enterocolitis. In this study, we examined the role of flagellin in the typhoid-like systemic murine Salmonella infection by measuring bacterial proliferation, inflammation, leukocyte recruitment, and cellular apoptosis in Peyer's patches (PPs), mesenteric lymph node (MLN), and spleen. We found that relative to an isogenic wild-type (WT) strain, aflagellate Salmonella exhibited increased proliferation at 4 days postinfection in PPs and MLN but not spleen. The aflagellate mutant also elicited increased local and systemic secretion of inflammatory cytokines such as interleukin-1β, gamma interferon, and tumor necrosis factor alpha and enhanced surface expression of ICAM-1 on macrophages and dendritic cells (DCs). Furthermore, the recruitment of macrophages and DCs in PPs and MLN, but not spleen, was enhanced upon infection with aflagellate Salmonella. The relative differences between WT and aflagellate Salmonella were highly attenuated in Toll-like receptor 5-deficient (TLR5−/−) mice, indicating involvement of TLR5-dependent signaling. Interestingly, infection with the aflagellate mutant also resulted in decreased levels of T-cell apoptosis in PPs relative to infection with WT Salmonella. We postulate that the initial lack of detection of the aflagellate mutant in the mucosa permits increased proliferation within the host and enhances inflammatory signaling in nonepithelial cell types, which subsequently promotes leukocyte recruitment. In contrast, lack of difference in any disease parameter measured in the spleen likely reflects that Salmonella expression of flagellin is downregulated in this organ. Thus, the characteristic inflammatory pathology of Salmonella infection occurs only in PPs and to a lesser extent in MLN during the initial phases of infection and these early responses are dependent on TLR5.
doi:10.1128/IAI.00656-09
PMCID: PMC2738035  PMID: 19596770
12.  The Probiotic Lactobacillus GG May Augment Intestinal Host Defense by Regulating Apoptosis and Promoting Cytoprotective Responses in the Developing Murine Gut 
Pediatric research  2008;64(5):511-516.
Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in preterm infants. Although its pathogenesis is poorly understood, inappropriate apoptosis of the mucosal epithelia has been implicated. Recent clinical trials have shown probiotics may reduce the incidence of NEC, and probiotics have been shown to suppress intestinal epithelial apoptosis in cultured cells. However, little is known about their mechanism of action in the developing intestine in vivo. Here, we confirm that the probiotic Lactobacillus rhamnosus GG (LGG) reduces chemically induced intestinal epithelial apoptosis in vitro. Furthermore, we report for the first time that LGG administered orally to live animals can reduce chemically induced epithelial apoptosis ex vivo, as measured by staining for active caspase 3 and TUNEL. Utilizing cDNA microarray analysis from the intestine of live, orally inoculated mice, we show that LGG upregulates of a battery of genes with known and likely cytoprotective effects. These studies indicate that probiotics such as LGG may augment intestinal host defenses in the developing intestine by stimulating anti-apoptotic and cytoprotective responses. Since apoptosis may be a precursor to NEC, understanding the mechanism behind probiotic modulation of apoptotic pathways may allow for development of more specifically targeted therapies or preventive strategies in the future.
doi:10.1203/PDR.0b013e3181827c0f
PMCID: PMC2694849  PMID: 18552706
Necrotizing enterocolitis; probiotics; Lactobacillus; apoptosis; intestinal epithelia
13.  Toll-Like Receptor 5-Deficient Mice Have Dysregulated Intestinal Gene Expression and Nonspecific Resistance to Salmonella-Induced Typhoid-Like Disease▿ †  
Infection and Immunity  2008;76(3):1276-1281.
The recognition of flagellin by Toll-like receptor 5 (TLR5) is the dominant means by which model intestinal epithelia activate proinflammatory gene expression in response to Salmonella enterica. The role of the flagellin-TLR5 interaction in vivo has been addressed primarily via studies that use flagellar mutants. Such studies suggest that host recognition of flagellin promotes rapid neutrophil recruitment that protects the host from this pathogen. However, these works do not directly address the role of TLR5 and are subject to the caveat that flagellar mutations may broadly affect Salmonella gene expression. Thus, we examined the role of the flagellin-TLR5 interaction via the use of TLR5-deficient (TLR5KO) mice. We utilized both the traditional model of murine Salmonella infection, wherein low-dose oral infection of mice with Salmonella enterica subsp. enterica serovar Typhimurium results in systemic typhoid-like disease, and a more recently characterized model in which mice are pretreated with streptomycin to result in gut-restricted acute enteritis. In the enteritis model, TLR5KO mice had more severe gut pathology, thus “phenocopying” previous results obtained with Salmonella mutants. In contrast, TLR5KO mice were resistant to Salmonella-induced typhoid-like disease. However, such resistance was not specific for flagellated serovar Typhimurium, but rather, TLR5KO mice were also resistant to challenges by flagellin-deficient serovar Typhimurium. Such resistance associated with elevations in the microbiota was ablated by antibiotic pretreatment and correlated with basal elevations in intestinal host defense gene expression. All together, these results indicate that the resistance of TLR5KO mice to Salmonella-induced typhoid-like illness resulted from alterations in their basal phenotype rather than from the lack of TLR5 ligation during the infection per se.
doi:10.1128/IAI.01491-07
PMCID: PMC2258833  PMID: 18195036
14.  Deletion of TLR5 results in spontaneous colitis in mice 
The Journal of Clinical Investigation  2007;117(12):3909-3921.
Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.
doi:10.1172/JCI33084
PMCID: PMC2075480  PMID: 18008007
15.  Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation 
Journal of Clinical Investigation  2007;117(3):703-711.
A major adaptive pathway for hypoxia is hypoxic preconditioning (HPC), a form of endogenous protection that renders cells tolerant to severe challenges of hypoxia. We sought to define the antiinflammatory properties of HPC. cDNA microarray analysis of lung tissue from mice subjected to hypoxia or HPC identified a cluster of NF-κB–regulated genes whose expression is attenuated by HPC. Studies using an NF-κB luciferase reporter assay confirmed a significant suppression of NF-κB activation during HPC. HPC-elicited activity was conferrable, as a soluble supernatant from HPC-treated cells, and the active fraction was purified and identified as adenosine (Ado). Guided by recent studies demonstrating bacterial inhibition of NF-κB through cullin-1 (Cul-1) deneddylation, we found a dose-dependent deneddylation of Cul-1 by Ado receptor stimulation predominantly mediated by the Ado A2B receptor subtype. Further, siRNA-mediated repression of CSN5, a subunit of the COP9 signalosome responsible for deneddylation of Cul-1, partially reversed HPC-mediated inhibition of NF-κB. Cul-1 deneddylation was evident in a murine model of HPC and lost in animals lacking extracellular Ado (Cd73–/– mice). Taken together, these results demonstrate that HPC induces extracellular accumulation of Ado and suppresses NF-κB activity through deneddylation of Cul-1. These results define a molecular regulatory pathway by which Ado provides potent antiinflammatory properties.
doi:10.1172/JCI30049
PMCID: PMC1797604  PMID: 17318263
16.  Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response 
Journal of Clinical Investigation  2001;107(1):99-109.
This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model epithelia stimulated with other inducers of IL-8 secretion (TNF-α or carbachol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using microsequencing to be the protein flagellin. In support of this finding, flagellin-deficient S. typhimurium mutants did not secrete detectable levels of PIF (i.e., a bioactivity that induced IL-8 secretion when placed basolaterally on model epithelia). Furthermore, viable flagellin-deficient mutant organisms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apically to model intestinal epithelia. These findings indicate that translocation of flagellin across epithelia, subsequent to apical epithelial–S. typhimurium interaction, is likely a major means of activating a mucosal inflammatory response.
PMCID: PMC198545  PMID: 11134185
17.  Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-κB pathway 
Interactions between the enteric pathogen Salmonella typhimurium and the luminal surface of the intestine provoke an acute inflammatory response, mediated in part by epithelial cell secretion of the chemokine IL-8 and other proinflammatory molecules. This study investigated the mechanism by which this pathogen induces IL-8 secretion in physiologically polarized model intestinal epithelia. IL-8 secretion induced by both the prototypical proinflammatory cytokine TNF-α and S. typhimurium was NF-κB dependent. However, NF-κB activation and IL-8 secretion induced by S. typhimurium, but not by TNF-α, was preceded by and required an increase in intracellular [Ca2+]. Additionally, agonists that increased intracellular [Ca2+] by receptor-dependent (carbachol) or independent (thapsigargin, ionomycin) means also induced IL-8 secretion. Furthermore, the ability of S. typhimurium mutants to induce IκB-α degradation, NF-κB translocation, and IL-8 transcription and secretion correlated precisely with their ability to induce an intracellular [Ca2+] increase in model intestinal epithelia, but not with their ability to invade these cells. Finally, S. typhimurium, but not TNF-α, induced a Ca2+-dependent phosphorylation of IκB-α. These results indicate that S. typhimurium–induced activation of NF-κB–dependent epithelial inflammatory responses proceeds by a Ca2+-mediated activation of an IκB-α kinase. These observations raise the possibility that pharmacologic intervention of the acute inflammatory response can be selectively matched to the specific class of initiating event.
PMCID: PMC382586  PMID: 10619864

Results 1-17 (17)