Human tetraspanin CD81 facilitates invasion of Salmonella enterica into human epithelial cells.

Human CD81 and CD9 are members of the tetraspanin family of proteins characterized by a canonical structure of four transmembrane domains and two extracellular loop domains. Tetraspanins are known as molecular facilitators, which assemble and organize cell surface receptors and partner molecules forming clusters known as tetraspanin-enriched microdomains. They have been implicated to play various biological roles including an involvement in infections with microbial pathogens. Here, we demonstrate an important role of CD81 for the invasion of epithelial cells by Salmonella enterica. We show that the overexpression of CD81 in HepG2 cells enhances invasion of various typhoidal and non-typhoidal Salmonella serovars. Deletion of CD81 by CRISPR/Cas9 in intestinal epithelial cells (C2BBe1 and HT29-MTX-E12) reduces S. Typhimurium invasion. In addition, the effect of human CD81 is species-specific as only human but not rat CD81 facilitates Salmonella invasion. Finally, immunofluorescence microscopy and proximity ligation assay revealed that both human tetraspanins CD81 and CD9 are recruited to the entry site of S. Typhimurium during invasion but not during adhesion to the host cell surface. Overall, we demonstrate that the human tetraspanin CD81 facilitates Salmonella invasion into epithelial host cells.

The Salmonella virulence protein PagN contributes to the advent of a hyper-replicating cytosolic bacterial population.

Salmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella-containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by Salmonella in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of pagN affects Salmonella replication by affecting the cytosolic bacterial population. This study presents the first example of a Salmonella virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of Salmonella cytosolic hyper-replication.

Alterations in anthropometric, inflammatory and mental health parameters during Ramadan intermittent fasting in a group of healthy people: a prospective cohort study.

Fasting has been practiced with different time span in different areas of the world and for various reasons. One of the types of fasting regimens is Ramadan intermittent fasting (RIF), which is described as intermittent dry fasting and known as the most commonly practiced form of religious fasting. Different studies have shown its effects on body composition parameters and mental health, fatigue and quality of life (QoL). Elucidating the relationship of RIF on biological parameters would also be of importance to show its mechanism. Therefore, we evaluated several biological mediators related to mental health, such as ß-nerve growth factor (ß-NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and insulin-like growth factor-1 (IGF-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and matrix-metalloproteinase-9 (MMP-9). This study consisted of fasting (FG; n = 25) and non-fasting group (NFG; n = 25). Four different time points were assessed for FG: one week before (T1), mid (T2), last days (T3), and one week after (T4) RIF. T1 and T3 were the assessment time points for NFG. Biological mediators were determined from serum samples by using Human Magnetic Luminex and enzyme-linked immunosorbent assay. Furthermore, we then performed correlation analyses between biological mediators and our previously published clinical parameters including body composition and mental health parameters at all time points. Significant alterations were shown in FG for ß-NGF (T2vsT3, p < 0.05; T2vsT4, p < 0.05), GDNF (T1vsT4, p < 0.05; T2vsT4, p < 0.05), IL-8 (T2vsT3, p < 0.05; T3vsT4, p < 0.05), TNF-α (T1vsT3, p < 0.05; T1vsT4, p < 0.001; T2vsT4, p < 0.001), and MMP-9 (T1vsT4, p < 0.01). There were no statistically significant differences between FG and NFG in all biological mediators at T1 and T3. Correlation analysis showed that MMP-9 levels had negative correlation with body mass index (BMI) at T3. At T3 BDNF levels had negative correlation with Epworth Sleepiness Scale (ESS) as one of measured QoL parameters. ß-NGF, GDNF, TNF-α, and MMP-9 had positive correlation with some of body composition and mental health parameters. Findings demonstrate that RIF altered different biological mediators could give benefit to health. Its benefit is mediated by the alteration of biological mediators.

Infection of porcine enteroids and 2D differentiated intestinal epithelial cells with rotavirus A to study cell tropism and polarized immune response.

Intestinal epithelial cell interactions with enteric pathogens have been incompletely elucidated owing to the lack of model systems that recapitulate the cellular diversity, architecture and functionality of the intestine. To analyze rotavirus (RV) infection and the subsequent innate immune response, we established cultures of differentiated porcine intestinal epithelial cells in three different variations: basolateral-out enteroids, apical-out enteroids and two-dimensional (2D) filter-grown intestinal epithelial cells. Application of specific antibodies for fluorescent staining indicated that enteroids and enteroid-derived cell cultures contain multiple intestinal epithelial cell types. Infection studies indicated that both apical-out enteroids and 2D intestinal epithelial cells are susceptible to porcine RV infection. However, 2D intestinal epithelial cells are more useful for a detailed characterization and comparison of apical and basolateral infection than apical-out enteroids. Virus-induced apoptosis was observed in apical-out enteroids at 24 h post infection but not at earlier time points after infection. RV infected not only enterocytes but also goblet cells and Paneth cells in apical-out enteroids and 2D intestinal epithelial cells. Interestingly, despite the lack of significant differences in the efficiency of infection after apical and basolateral infection of 2D intestinal epithelial cells, stronger innate immune and inflammatory responses were observed after basolateral infection as compared to infection via the apical route. Therefore, apical-out enteroids and 2D intestinal epithelial cells provide useful primary cell culture models that can be extended to analyze invasion and replication strategies of agents implicated in enteric diseases or to study immune and inflammatory responses of the host induced by enteric pathogens.

Acetyl-CoA-Carboxylase 1-mediated de novo fatty acid synthesis sustains Lgr5+ intestinal stem cell function.

Basic processes of the fatty acid metabolism have an important impact on the function of intestinal epithelial cells (IEC). However, while the role of cellular fatty acid oxidation is well appreciated, it is not clear how de novo fatty acid synthesis (FAS) influences the biology of IECs. We report here that interfering with de novo FAS by deletion of the enzyme Acetyl-CoA-Carboxylase (ACC)1 in IECs results in the loss of epithelial crypt structures and a specific decline in Lgr5+ intestinal epithelial stem cells (ISC). Mechanistically, ACC1-mediated de novo FAS supports the formation of intestinal organoids and the differentiation of complex crypt structures by sustaining the nuclear accumulation of PPARδ/ß-catenin in ISCs. The dependency of ISCs on cellular de novo FAS is tuned by the availability of environmental lipids, as an excess delivery of external fatty acids is sufficient to rescue the defect in crypt formation. Finally, inhibition of ACC1 reduces the formation of tumors in colitis-associated colon cancer, together highlighting the importance of cellular lipogenesis for sustaining ISC function and providing a potential perspective to colon cancer therapy.

STAT1 coordinates intestinal epithelial cell death during gastrointestinal infection upstream of Caspase-8.

Intestinal homeostasis and the maintenance of the intestinal epithelial barrier are essential components of host defense during gastrointestinal Salmonella Typhimurium infection. Both require a strict regulation of cell death. However, the molecular pathways regulating epithelial cell death have not been completely understood. Here, we elucidated the contribution of central mechanisms of regulated cell death and upstream regulatory components during gastrointestinal infection. Mice lacking Caspase-8 in the intestinal epithelium are highly sensitive towards bacterial induced enteritis and intestinal inflammation, resulting in an enhanced lethality of these mice. This phenotype was associated with an increased STAT1 activation during Salmonella infection. Cell death, barrier breakdown and systemic infection were abrogated by an additional deletion of STAT1 in Casp8ΔIEC mice. In the absence of epithelial STAT1, loss of epithelial cells was abolished which was accompanied by a reduced Caspase-8 activation. Mechanistically, we demonstrate that epithelial STAT1 acts upstream of Caspase-8-dependent as well as -independent cell death and thus might play a major role at the crossroad of several central cell death pathways in the intestinal epithelium. In summary, we uncovered that transcriptional control of STAT1 is an essential host response mechanism that is required for the maintenance of intestinal barrier function and host survival.

Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine.

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

Proteoglycan-Dependent Endo-Lysosomal Fusion Affects Intracellular Survival of Salmonella Typhimurium in Epithelial Cells.

Proteoglycans (PGs) are glycoconjugates which are predominately expressed on cell surfaces and consist of glycosaminoglycans (GAGs) linked to a core protein. An initial step of GAGs assembly is governed by the ß-D-xylosyltransferase enzymes encoded in mammals by the XylT1/XylT2 genes. PGs are essential for the interaction of a cell with other cells as well as with the extracellular matrix. A number of studies highlighted a role of PGs in bacterial adhesion, invasion, and immune response. In this work, we investigated a role of PGs in Salmonella enterica serovar Typhimurium (S. Typhimurium) infection of epithelial cells. Gentamicin protection and chloroquine resistance assays were applied to assess invasion and replication of S. Typhimurium in wild-type and xylosyltransferase-deficient (ΔXylT2) Chinese hamster ovary (CHO) cells lacking PGs. We found that S. Typhimurium adheres to and invades CHO WT and CHO ΔXylT2 cells at comparable levels. However, 24 h after infection, proteoglycan-deficient CHO ΔXylT2 cells are significantly less colonized by S. Typhimurium compared to CHO WT cells. This proteoglycan-dependent phenotype could be rescued by addition of PGs to the cell culture medium, as well as by complementation of the XylT2 gene. Chloroquine resistance assay and immunostaining revealed that in the absence of PGs, significantly less bacteria are associated with Salmonella-containing vacuoles (SCVs) due to a re-distribution of endocytosed gentamicin. Inhibition of endo-lysosomal fusion by a specific inhibitor of phosphatidylinositol phosphate kinase PIKfyve significantly increased S. Typhimurium burden in CHO ΔXylT2 cells demonstrating an important role of PGs for PIKfyve dependent vesicle fusion which is modulated by Salmonella to establish infection. Overall, our results demonstrate that PGs influence survival of intracellular Salmonella in epithelial cells via modulation of PIKfyve-dependent endo-lysosomal fusion.

Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Inflammatory Bowel Disease-associated GP2 Autoantibodies Inhibit Mucosal Immune Response to Adherent-invasive Bacteria.

Adherent-invasive Escherichia coli have been suggested to play a pivotal role within the pathophysiology of inflammatory bowel disease (IBD). Autoantibodies against distinct splicing variants of glycoprotein 2 (GP2), an intestinal receptor of the bacterial adhesin FimH, frequently occur in IBD patients. Hence, we aimed to functionally characterize GP2-directed autoantibodies as a putative part of IBD's pathophysiology. Ex vivo, GP2-splicing variant 4 (GP2#4) but not variant 2 was expressed on intestinal M or L cells with elevated expression patterns in IBD patients. The GP2#4 expression was induced in vitro by tumor necrosis factor (TNF)-α. The IBD-associated GP2 autoantibodies inhibited FimH binding to GP2#4 and were decreased in anti-TNFα-treated Crohn's disease patients with ileocolonic disease manifestation. In vivo, mice immunized against GP2 before infection with adherent-invasive bacteria displayed exacerbated intestinal inflammation. In summary, autoimmunity against intestinal expressed GP2#4 results in enhanced attachment of flagellated bacteria to the intestinal epithelium and thereby may drive IBD's pathophysiology.

Persistent Salmonella enterica Serovar Typhimurium Infection Induces Protease Expression During Intestinal Fibrosis.

BACKGROUND: Intestinal fibrosis is a common and serious complication of Crohn's disease characterized by the accumulation of fibroblasts, deposition of extracellular matrix, and formation of scar tissue. Although many factors including cytokines and proteases contribute to the development of intestinal fibrosis, the initiating mechanisms and the complex interplay between these factors remain unclear. METHODS: Chronic infection of mice with Salmonella enterica serovar Typhimurium was used to induce intestinal fibrosis. A murine protease-specific CLIP-CHIP microarray analysis was employed to assess regulation of proteases and protease inhibitors. To confirm up- or downregulation during fibrosis, we performed quantitative real-time polymerase chain reaction (PCR) and immunohistochemical stainings in mouse tissue and tissue from patients with inflammatory bowel disease. In vitro infections were used to demonstrate a direct effect of bacterial infection in the regulation of proteases. RESULTS: Mice develop severe and persistent intestinal fibrosis upon chronic infection with Salmonella enterica serovar Typhimurium, mimicking the pathology of human disease. Microarray analyses revealed 56 up- and 40 downregulated proteases and protease inhibitors in fibrotic cecal tissue. Various matrix metalloproteases, serine proteases, cysteine proteases, and protease inhibitors were regulated in the fibrotic tissue, 22 of which were confirmed by quantitative real-time PCR. Proteases demonstrated site-specific staining patterns in intestinal fibrotic tissue from mice and in tissue from human inflammatory bowel disease patients. Finally, we show in vitro that Salmonella infection directly induces protease expression in macrophages and epithelial cells but not in fibroblasts. CONCLUSIONS: In summary, we show that chronic Salmonella infection regulates proteases and protease inhibitors during tissue fibrosis in vivo and in vitro, and therefore this model is well suited to investigating the role of proteases in intestinal fibrosis.

Schistosome Eggs Impair Protective Th1/Th17 Immune Responses Against Salmonella Infection.

Countries with a high incidence of helminth infections are characterized by high morbidity and mortality to infections with intracellular pathogens such as Salmonella. Some patients with Salmonella-Schistosoma co-infections develop a so-called "chronic septicemic salmonellosis," with prolonged fever and enlargement of the liver and spleen. These effects are most likely due to the overall immunoregulatory activities of schistosomes such as induction of Tregs, Bregs, alternatively activated macrophages, and degradation of antibodies. However, detailed underlying mechanisms are not very well investigated. Here, we show that intraperitoneal application of live Schistosoma mansoni eggs prior to infection with Salmonella Typhimurium in mice leads to an impairment of IFN-γ and IL-17 responses together with a higher bacterial load compared to Salmonella infection alone. S. mansoni eggs were found in granulomas in the visceral peritoneum attached to the colon. Immunohistological staining revealed IPSE/alpha-1, a glycoprotein secreted from live schistosome eggs, and recruited basophils around the eggs. Noteworthy, IPSE/alpha-1 is known to trigger IL-4 and IL-13 release from basophils which in turn is known to suppress Th1/Th17 responses. Therefore, our data support a mechanism of how schistosomes impair a protective immune response against Salmonella infection and increase our understanding of helminth-bacterial co-infections.

Lactate oxidation facilitates growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis (Mtb) uses alveolar macrophages as primary host cells during infection. In response to an infection, macrophages switch from pyruvate oxidation to reduction of pyruvate into lactate. Lactate might present an additional carbon substrate for Mtb. Here, we demonstrate that Mtb can utilize L-lactate as sole carbon source for in vitro growth. Lactate conversion is strictly dependent on one of two potential L-lactate dehydrogenases. A knock-out mutant lacking lldD2 (Rv1872c) was unable to utilize L-lactate. In contrast, the lldD1 (Rv0694) knock-out strain was not affected in growth on lactate and retained full enzymatic activity. On the basis of labelling experiments using [U-13C3]-L-lactate as a tracer the efficient uptake of lactate by Mtb and its conversion into pyruvate could be demonstrated. Moreover, carbon flux from lactate into the TCA cycle, and through gluconeogenesis was observed. Gluconeogenesis during lactate consumption depended on the phosphoenolpyruvate carboxykinase, a key enzyme for intracellular survival, showing that lactate utilization requires essential metabolic pathways. We observed that the ΔlldD2 mutant was impaired in replication in human macrophages, indicating a critical role for lactate oxidation during intracellular growth.

Salmonella enterica serovar Typhimurium ΔmsbB triggers exacerbated inflammation in Nod2 deficient mice.

The intracellular pathogen Salmonella enterica serovar Typhimurium causes intestinal inflammation characterized by edema, neutrophil influx and increased pro-inflammatory cytokine expression. A major bacterial factor inducing pro-inflammatory host responses is lipopolysaccharide (LPS). S. Typhimurium ΔmsbB possesses a modified lipid A, has reduced virulence in mice, and is being considered as a potential anti-cancer vaccine strain. The lack of a late myristoyl transferase, encoded by MsbB leads to attenuated TLR4 stimulation. However, whether other host receptor pathways are also altered remains unclear. Nod1 and Nod2 are cytosolic pattern recognition receptors recognizing bacterial peptidoglycan. They play important roles in the host's immune response to enteric pathogens and in immune homeostasis. Here, we investigated how deletion of msbB affects Salmonella's interaction with Nod1 and Nod2. S. Typhimurium Δ msbB-induced inflammation was significantly exacerbated in Nod2-/- mice compared to C57Bl/6 mice. In addition, S. Typhimurium ΔmsbB maintained robust intestinal colonization in Nod2-/- mice from day 2 to day 7 p.i., whereas colonization levels significantly decreased in C57Bl/6 mice during this time. Similarly, infection of Nod1-/- and Nod1/Nod2 double-knockout mice revealed that both Nod1 and Nod2 play a protective role in S. Typhimurium ΔmsbB-induced colitis. To elucidate why S. Typhimurium ΔmsbB, but not wild-type S. Typhimurium, induced an exacerbated inflammatory response in Nod2-/- mice, we used HEK293 cells which were transiently transfected with pathogen recognition receptors. Stimulation of TLR2-transfected cells with S. Typhimurium ΔmsbB resulted in increased IL-8 production compared to wild-type S. Typhimurium. Our results indicate that S. Typhimurium ΔmsbB triggers exacerbated colitis in the absence of Nod1 and/or Nod2, which is likely due to increased TLR2 stimulation. How bacteria with "genetically detoxified" LPS stimulate various innate responses has important implications for the development of safe and effective bacterial vaccines and adjuvants.

Insulin-producing intestinal K cells protect nonobese diabetic mice from autoimmune diabetes.

BACKGROUND & AIMS: Type 1 diabetes is caused by an aberrant response against pancreatic ß cells. Intestinal K cells are glucose-responsive endocrine cells that might be engineered to secrete insulin. We generated diabetes-prone non-obese diabetic (NOD) mice that express insulin, via a transgene, in K cells. We assessed the effects on immunogenicity and diabetes development. METHODS: Diabetes incidence and glucose homeostasis were assessed in NOD mice that expressed mouse preproinsulin II from a transgene in K cells and nontransgenic NOD mice (controls); pancreas and duodenum tissues were collected and analyzed by histology. We evaluated T cell responses to insulin, levels of circulating autoantibodies against insulin, and the percentage of circulating antigen-specific T cells. Inflammation of mesenteric and pancreatic lymph node cells was also evaluated. RESULTS: The transgenic mice tended to have lower blood levels of glucose than control mice, associated with increased plasma levels of immunoreactive insulin and proinsulin. Fewer transgenic mice developed diabetes than controls. In analyses of pancreas and intestine tissues from the transgenic mice, insulin-producing K cells were not affected by the immune response and the mice had reduced destruction of endogenous ß cells. Fewer transgenic mice were positive for insulin autoantibodies compared with controls. Cells isolated from mesenteric lymph nodes of the transgenic mice had significantly lower rates of proliferation and T cells from transgenic mice tended to secrete lower levels of inflammatory cytokines than from controls. At 15 weeks, transgenic mice had fewer peripheral CD8(+) T cells specific for NRP-V7 than control mice. CONCLUSIONS: NOD mice with intestinal K cells engineered to express insulin have reduced blood levels of glucose, are less likely to develop diabetes, and have reduced immunity against pancreatic ß cells compared with control NOD mice. This approach might be developed to treat patients with type 1 diabetes.

Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice.

A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium (S. Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S. Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1(+/+) and Nramp1(-/-) mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S. Typhimurium to systemic sites. We observed that Nramp1(+/+) mice showed lower numbers of S. Typhimurium in the caecum compared with Nramp1(-/-) mice at all times analysed. Acute inflammation was much more pronounced in Nramp1(+/+) mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-gamma, TNF-alpha and MIP-1alpha and a massive infiltration of neutrophils and macrophages, compared with Nramp1(-/-) animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S. Typhimurium infection in Nramp1(+/+) mice.

Sesquiterpene lactones inhibit Yersinia invasin protein-induced IL-8 and MCP-1 production in epithelial cells.

Enteric Yersinia bacteria trigger transcription and secretion of the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in epithelial cells. Both chemokines are controlled by NF-kappaB. The NF-kappaB-binding site in the IL-8 promoter preferentially binds Rel p65/p65 homodimers and p50/p65 heterodimers while the NF-kappaB-binding motifs of the MCP-1 promoter preferably bind p50/p65 heterodimers and p50/p50 homodimers. Sesquiterpene lactones inhibit the transcription factor NF-kappaB by alkylating the p65 subunit. In this study we investigated the inhibitory effects of sesquiterpene lactones and the NF-kappaB inhibitor SN50 on NF-kappaB p50 and p65 subunits in Yersinia-triggered IL-8 and MCP-1 production. The sesquiterpene lactones blocked Yersinia-triggered IL-8 and MCP-1 production in a dose-dependent manner. In contrast, SN50 inhibited IL-8 production at high concentrations whereas it diminished the amount of secreted MCP-1 significantly already at low concentrations. By means of electrophoretic mobility shift assays we demonstrate that sesquiterpene lactones inhibit Yersinia-triggered activation of NF-kappaB by inhibiting Rel p65, but not Rel p50. Our results also demonstrate that SN50 is useful for inhibition of nuclear translocation of the NF-kappaB p50 subunit but cannot be considered a general NF-kappaB inhibitor.

Bartonella henselae inhibits apoptosis in Mono Mac 6 cells.

Bartonella henselae causes the vasculoproliferative disorders bacillary angiomatosis and peliosis probably resulting from the release of vasculoendothelial growth factor (VEGF) from infected epithelial or monocytic host cells. Here we demonstrate that B. henselae in addition to VEGF induction was also capable of inhibiting the endogenous sucide programme of monocytic host cells. Our results show that B. henselae inhibits pyrrolidine dithiocarbamate (PDTC)-induced apoptosis in Mono Mac 6 cells. B. henselae was observed to be present in a vacuolic compartment of Mono Mac 6 cells. Direct contact of B. henselae with Mono Mac 6 cells was crucial for inhibition of apoptosis as shown by the use of a two-chamber model. Inhibition of apoptosis was paralleled by diminished caspase-3 activity which was significantly reduced in PDTC-stimulated and B. henselae-infected cells. The anti-apoptotic effect of B. henselae was accompanied by (i) the activation of the transcription factor NF-kappaB and (ii) the induction of cellular inhibitor of apoptosis proteins-1 and -2 (cIAP-1, -2). Our results suggest a new synergistic mechanism in B. henselae pathogenicity by (i) inhibition of host cell apoptosis via activation of NF-kappaB and (ii) induction of host cell VEGF secretion.

Interaction of Yersinia enterocolitica with epithelial cells: invasin beyond invasion.

The chromosomally encoded inv gene product is an outer membrane protein that is functionally expressed in the enteropathogenic Yersinia species Yersinia enterocolitica and Yersinia pseudotuberculosis. Invasin protein is a high-affinity ligand for beta1 integrins and especially important in the early phase of intestinal infection for efficient translocation through the M cells located in the follicle-associated epithelium overlying the Peyer's patches. In addition to bacterial internalization, Yersinia invasin mediates proinflammatory epithelial cell reactions. Epithelial cells exhibit immunological functions including production of cytokines thereby signaling to the immune system the presence of invasive or pathogenic bacteria. Several other enteropathogenic bacteria also induce cytokine production in epithelial cells. However, the signaling pathways by which this reaction is accomplished differ for various pathogens. Binding of invasin-expressing Yersinia to beta1 integrin receptors of epithelial cells induces activation of a signal cascade involving Rac1, MAP kinases, activation of the transcription factor NF-kappaB, and the subsequent production of chemotactic cytokines. The Yersinia invasin-triggered inflammatory epithelial cell reaction may lead to the recruitment of phagocytes followed by tissue disruption which may be part of the strategy of the pathogen to promote its dissemination in the host tissue.