The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tien, M.-T.
Right arrow Articles by Pédron, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tien, M.-T.
Right arrow Articles by Pédron, T.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
The Journal of Immunology, 2006, 176: 1228-1237.
Copyright © 2006 by The American Association of Immunologists

Anti-Inflammatory Effect of Lactobacillus casei on Shigella-Infected Human Intestinal Epithelial Cells1

Meng-Tsung Tien2,*,{dagger}, Stephen E. Girardin2,*, Béatrice Regnault{dagger}, Lionel Le Bourhis{ddagger}, Marie-Agnès Dillies{dagger}, Jean-Yves Coppée{dagger}, Raphaëlle Bourdet-Sicard§, Philippe J. Sansonetti3,* and Thierry Pédron*

* Pathogénie Microbienne Moléculaire Unit, Institut National de la Santé et de la Recherche Médicale U389, {dagger} DNA Chip Platform, Genopole, and {ddagger} Imunité Innée et Signalisation, Pasteur Institute, Paris, France; and § Danone Vitapole, Nutrivaleur, Palaiseau, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-{kappa}B pathway, particularly through stabilization of I-{kappa}B{alpha}. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-{kappa}B{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
More than 400 species of bacteria are present in the human gut, ranging from 105 to 107 CFU/g of intestinal contents in the jejunum to 1011 to 1012 CFU/g in the colon (1). To maintain the homeostatic mechanisms responsible for tolerance of the intestinal mucosa vis-a-vis its commensal flora, and at the same time to permit the development of an innate response to pathogenic bacteria, intestinal epithelial cells (IECs),4 which are in close contact with luminal bacteria, are expected to play a key regulatory role (2, 3). Intestinal homeostasis has been shown to be under the control of TLRs. Indeed, mice deficient in TLR2, TLR4, or the adaptor molecule MyD88 are more prone to develop intestinal inflammation after dextran sodium sulfate-induced colitis (4). Tolerance to the commensal flora is similarly dysregulated in the case of inflammatory bowel disease (IBD) (5).

Lactobacilli and Bifidobacteria are natural components of the colonic microbiota, and as probiotic agents, they have been tested in the prevention and treatment of IBD (6, 7). A strain of Lactobacillus casei, DN-114 001, has been shown to decrease the secretion of TNF-{alpha} from the inflammed ileum of Crohn’s disease patients (8, 9). The beneficial effect of probiotics is proposed to be due, at least in part, to interference with the innate immune system and possibly the orientation of adaptive immunity. Indeed, the probiotic preparation VSL#3 (a mixture of eight different Gram-positive bacteria) as well as different lactobacilli strains were shown to increase production of the anti-inflammatory cytokine IL-10 by dendritic cells (10, 11). This anti-inflammatory effect was also observed using DNA extracted from these probiotics; VSL#3 DNA was able to reduce IL-8 production by epithelial cells exposed to pro-inflammatory stimuli via a mechanism involving I-{kappa}B stabilization (12). Using a nonvirulent Salmonella strain, Neish and colleagues showed that regulation of epithelial responses occurs by inhibition of I-{kappa}B{alpha} ubiquitination (13), which is likely mediated by the Salmonella-secreted protein AvrA (14). Finally, regulation of multiple intestinal functions, such as nutrient absorption, mucosal barrier enhancement, and angiogenesis, is also modulated by the commensal Bacteroides thetaiotaomicron (15, 16).

Shigella flexneri, an entero-invasive, Gram-negative bacterium, causes the inflammatory destruction of the intestinal epithelium (17), leading to bacillary dysentery, an acute recto-colitis causing lethal complications, mostly in infants and toddlers in the most impoverished areas of the planet. The annual mortality attributed to bacillary dysentery is about one million. The mechanisms leading to acute mucosal inflammation need to be understood to develop efficient methods of prevention and cure. A number of studies have demonstrated that S. flexneri infection leads to the induction of several markers of acute inflammation, such as the chemokines IL-8 and CCL20. These pro-inflammatory genes are under the control of the NF-{kappa}B pathway (18), and recently, the mechanism of NF-{kappa}B activation in Shigella-infected cells has been uncovered. Indeed, our group has demonstrated that after infection, epithelial cells sense the presence of invading S. flexneri through Nod1, an intracellular pattern recognition molecule that specifically detect peptidoglycan from Gram-negative bacteria (19, 20).

Because probiotics such as L. casei represent a subclass of commensals that modulate mucosal innate responses and possibly exhibit an anti-inflammatory function (8), we aimed to analyze the effect of L. casei on epithelial inflammatory signaling induced by S. flexneri. Our results demonstrate that L. casei potently modulates pro-inflammatory pathways induced by S. flexneri, and by using global microarray analyses, we identify the ubiquitin/proteasome complex as the main target of L. casei action. Together, this study allows for a better understanding of how the commensal microflora contributes to the homeostasis of the host intestinal tract.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells lines, bacterial strains, and infections

Caco-2 and HEK293T epithelial cells lines were cultured with DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% nonessential amino acids (Invitrogen Life Technologies).

Invasive colonies of the S. flexneri serotype 5a strain M90T were isolated on Congo red agar plates (Congo red-positive colonies). One colony was then grown overnight in 7 ml of Trypticase soy broth (Difco) medium at 37°C under rotation. After dilution in fresh Trypticase soy broth, a subculture was then performed for 2 h to bring the culture to the exponential phase (OD, 600 nm; O.4). The strain SC301, a derivative of M90T after its transformation with plasmid pIL22 (encoding the afimbrial adhesin AfaE) from uropathogenic Escherichia coli, was used in this study (21).

The probiotic strain L. casei DN-114 001 was provided by Danone Vitapole. A colony of L. casei isolated on a Mann-Rogosa-Sharpe (MRS) agar plate was picked and cultured overnight at 37°C in 10 ml of MRS broth without agitation in absence of oxygen.

To obtain a polarized monolayer of IECs, Caco-2 cells were grown to confluency and then for 2 additional weeks to reach full differentiation and polarization. The culture medium was changed every 2 d. For the infection step, Caco-2 cells were incubated overnight without serum, with L. casei at a multiplicity of infection (MOI) of 100, before the addition of strain M90T-AfaE at the same MOI for 4 h. For the time-course experiment, nonpolarized Caco-2 cells were incubated at a MOI of 100 with L. casei for 2, 6, or 24 h. Biological duplicates were done for this time-course experiment. After washing Caco-2 cells with cold PBS, RNAs were extracted with the RNEasy mini kit (Qiagen). The quantity and quality of RNA preparations were determined, respectively, by absorbance lecture and electrophoresis using the Agilent nanochip technology.

To detect and quantify cell invasion, overnight culture of HEK cells seeded on coverslips, in the presence or absence of L. casei, were infected with M90T-AfaE as described above. After 1 h of infection, cells were washed in PBS and fixed with 4% paraformaldehyde. To identify intracellular vs extracellular Shigella, after saturation with BSA (1% in PBS), a first staining was made in the absence of permeabilization with a rabbit polyclonal Ab against S. flexneri LPS, followed by the addition, after washing, of a rhodamine-labeled goat Ab against rabbit Ig. The cells were then permeabilized with 0.1% Triton X-100. The same incubation with the Ab was performed, with a FITC-labeled goat Ab. Once labeled intracellular bacteria appeared green-labeled, whereas twice-labeled extracellular bacteria appeared yellow-labeled. Fluorescence detection was done using an Olympus BX50 microscope (at a x400 magnification) and a Leica DC350F camera. Pictures were analyzed using the Leica IM50 software (version 1.2; (Leica Microsystems). Determination of the green and red pixels were performed using Photoshop 7.0 software.

Macroarrays hybridization and analysis

The PCR products of 1050 human genes were spotted in duplicate on positively charged nylon membranes as described previously (22). The macroarray design can be found on the ArrayExpress web site (www.ebi.ac.uk/arrayexpress) with the accession number A-MEXP-141. cDNA labeling and hybridization scanning were also as described previously (22).

After recording of the signals for each gene with ArrayVision and quality control of hybridizations using the luciferase signal intensity, data corresponding to all of the membranes were transformed in a log2 scale and normalized by a method derived from the variance analysis (ANOVA) to give to all membranes an equal mean signal. This statistical method estimates the weight and significance of variability sources on experimental data. For each condition, eight biological replicates were performed and hybridized onto macroarrays in which PCR products were spotted in duplicate; 16 signals for one gene were used for one experimental condition. Comparative analyses between baseline (noninfected cells) and experiment (infected cells) were done with the dChip software (23), using an unpaired Welch t test with a p-value threshold of 0.05. This software was also used for hierarchical clustering using Euclidian distance and average as a linkage method. Before clustering, the expression values for one gene across all samples are standardized to have a mean of zero. Increased or decreased values are then ranged compared with this mean. In the clustering picture, each row represents a gene and each column represents a sample. For the color scale, blue and red represent, respectively, low and high signal expression values.

GeneChip hybridization and analysis

Starting from 5 µg of total RNA, the first strand of cDNA was synthetized using T7-(dT24) oligonucleotides and Superscript II reverse transcriptase (Invitrogen Life Technologies) for 1 h at 37°C. The double-strand cDNA was then synthetized at 16°C for 2 h by the addition of DNA polymerase, DNA ligase, and RNaseH. A terminal step by T7 DNA polymerase was performed for 5 min. The resulting DNA was used to synthetize biotin-labeled cRNA via an in vitro transcription labeling kit (Enzo Diagnostics). Ten micrograms of fragmented cRNA were hybridized with the GeneChip U133A for 16 h at 60 rpm in a 45°C hybridization oven. The chips were washed and stained with streptavidin PE (Molecular Probes) in the Affymetrix fluidics station. To amplify staining, an anti-streptavidin biotinylated Ab was introduced between two streptavidin PE staining steps. After hybridization, staining, and scanning (Agilent), the chip images were analyzed for quality control before signal analysis. The chips were then normalized using the scaling factor method (with a target intensity of 100). The analysis was performed using the statistical algorithms from the MAS-5 software (Affymetrix) to obtain absolute signals, detection calls, and the associated p values. Transcripts that were absent both in control and experimental conditions were removed from further analysis. Comparative analysis, between control and experimental conditions, which give a signal log ratio, a change call, and an associate p value, was performed with MAS-5 software using the t test and Mann-Whitney U test. Because the experiments were done in duplicate, to choose the modulated genes, we selected the probe set, the change call (increase or decrease) of which appeared to be four times (each experimental point vs each control point). Hierarchical clustering was performed using dChip software with the same parameters than those described above.

PCR analysis

cDNAs were synthetized from a template of 5 µg of total RNA using oligo(dT) primers (Promega) and Superscript II (Invitrogen Life Technologies) for 1 h at 42°C. PCR was performed using 5 µl of a 1/20 dilution of the cDNA as a template in a final volume of 50 µl. The standard program used was as follows: denaturation for 5 min at 94°C, 35 cycles of 45 s treatment at 94°C, 45 s at 55°C, and 90 s at 72°C, followed by a final elongation for 7 min at 72°C. One unit of Eurobiotaq (Eurobio) was used for one PCR. After electrophoresis in a 2% agarose gel containing ethidium bromide, PCR products were quantified using the ImageQuant software (Amersham Biosciences). The primers used are described in Table I.


View this table:
[in this window]
[in a new window]
  		Table I. PCR primers used for PCRs

 
NF-{kappa}B reporter gene assays and I-{kappa}B{alpha} evaluation

Experiments were conducted as reported previously (19). Briefly, 5 x 105 HEK293 cells were transiently transfected using 75 ng of the NF-{kappa}B luciferase reporter system, and NF-{kappa}B-dependent luciferase activity was measured 20 h after transfection. NF-{kappa}B-dependent luciferase assays were performed in duplicate, and data represent at least three independent experiments. Data show mean ± SE.

For experiments in which L. casei was added before stimulation with either S. flexneri or TNF-{alpha}, the following procedure was set up: HEK293 cells were seeded at 5 x 105 cells/ml and either left unstimulated (for Western blotting experiments) or transfected with 75 ng of the NF-{kappa}B luciferase reporter gene (for NF-{kappa}B-dependent luciferase assays) as described above. Twenty hours later, the incubation medium was changed and replaced by fresh medium containing either MRS medium alone or L. casei grown in MRS buffer (5 x 107 bacteria/ml). After an overnight incubation, cells were rinsed three times with PBS, and fresh medium was added. HEK293 cells were then left unstimulated, stimulated with TNF-{alpha} (100 ng/ml), or infected with S. flexneri M90T-AfaE (5 x 107 bacteria/ml) for variable periods (4 h in the case of NF-{kappa}B luciferase reporter assays), before lysis of the cells.

Western blot analysis

After overnight incubation of Caco-2 cells in the presence or absence of L. casei, the cells were stimulated either with M90T-AfaE or with TNF before lysis by the addition of 200 µl of Laemmli solution. After heating 5 min at 90°C, 10 µl of lysate were loaded in a 10% acrylamide SDS-PAGE. After migration, proteins were transferred onto nitrocellulose by semidry transfer. After blocking by PBS/5% milk, the membrane was incubated overnight with anti-I-{kappa}B{alpha}, antiphosphorylated I-{kappa}B{alpha}, anti-Rbx1, or anti-poly-ubiquitin Abs (all purchased from Santa Cruz Biotechnology) or with anti-{beta}-tubulin (Sigma-Aldrich) at 1/500 in PBS/milk. After three washes in PBS/Tween, the membrane was incubated with a peroxydase-labeled secondary Ab (1/1000) for 1 h. After washing, the membrane was incubated for 5 min with ECL+ chemiluminescence reagent (Amersham Biosciences). Kodak film was then exposed for different time periods to the membrane.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Gene expression patterns in Caco-2 cells during infection with Shigella

Until recently, GeneChip studies have been hampered by some technical concerns, including the absence of key genes and redundancy. In an effort to characterize with precision the global response of cells infected by a bacterial pathogen, we have developed a macroarray-based procedure in which PCR products of 1050 genes of interest, including cytokines, chemokines, transcription factors, etc. (22), have been spotted. Using this tool, we aimed to identify the repertoire of genes modulated after infection of polarized Caco-2 cells with the invasive S. flexneri strain M90T-AfaE. Radioactive cDNA was hybridized onto macroarray nylon membranes, and after normalization plus comparative analysis using dChip, 36 genes modulated during infection with M90T with statistical significance (p < 0.05) were identified (Table II). Interestingly, genes encoding chemoattractant chemokines with pro-inflammatory functions such as CXCL1–3 and CCL20 were found up-regulated after Shigella infection. Importantly, results obtained by macroarray analyses confirm and further expand results obtained during Shigella infection of nonconfluent Caco-2 cells using Affimetrix technology (24).


View this table:
[in this window]
[in a new window]
  		Table II. Macroarray analysis of polarized Caco-2 cell gene expression during Shigella infectiona

 
Modulation of the response of Caco-2 cells infected with Shigella by L. casei

To observe the effect of L. casei DN-114 001 on the inflammatory process occurring during Shigella infection of IECs, polarized Caco-2 cells were preincubated overnight with L. casei before infection with M90T-AfaE. After RNA extraction, hybridizations to the macroarray membranes were performed. Interestingly, hierarchical clustering identified L. casei-mediated modulation of Shigella-induced responses of epithelial cells (Fig. 1). Among these, some key pro-inflammatory genes were found to be down-regulated when Caco-2 cells were preincubated with the probiotic bacteria before infection with Shigella. This was the case for the expression of CXCL1 and CXCL2, two chemokines involved in chemoattraction of polymorphonuclear cells, and for the expression of the dendritic cell chemoattractant CCL20.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 1. Hierarchical clustering modulated genes during infection of Caco-2 cells with Shigella, in the presence or absence of L. casei. Caco-2 cells were incubated overnight in the presence or absence of L. casei and challenged for 3 h with M90T-AfaE, the virulent strain of S. flexneri. Radioactive nucleotides were incorporated during cDNA synthesis. The labeled probes were hybridized onto macroarrays consisting of 1050 human genes represented by PCR products spotted onto nylon membranes. After washings and scanning, signal detection and normalization were performed allowing group comparisons. Modulated genes were then clustered using dChip software. Each row represents a gene, and each column represents the mean expression for each replicate. The red color represents an expression level above the mean expression of a gene across all samples, and the blue color represents an expression level lower than the mean. For each condition, eight biological replicates were made.

 
When macroarray-based technologies are used, it is crucial to further confirm the results obtained by individual RT-PCR. Using this approach, we could validate that expression of CXCL1 and CXCL2 was down-regulated (both by ~50%) in conditions of coinfection with L. casei and M90T-AfaE compared with M90T-AfaE alone (Fig. 2). Moreover, in a previous study (24), we demonstrated that the expressions of ICAM-1 and amphiregulin were up-regulated during Shigella infection. In this study, we report that preincubation of Caco-2 cells with L. casei prevented increased transcription of these genes after infection with Shigella (Fig. 2).



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 2. Anti-inflammatory effect of L. casei. The cells were treated as described in Material and Methods and in the legend to Fig. 1. After RNA extraction, cDNA was synthetized using Oligo-dT and reverse transcriptase enzyme. Five microliters of a 1/20 dilution were used as a template for the PCR. A, Confluent, polarized Caco-2 cells were used. B, Densitometric quantification of PCR products ((gene/GAPDH) x 100).

 
The above results pointed to a role of L. casei in the specific modulation of a selected repertoire of Shigella-induced genes. However, to ascertain that these observations were not an indirect consequence of the experimental set-up used, we aimed to investigate whether coculture of Caco-2 cells with L. casei interferes with Shigella invasion. Using double-fluorescence labeling, followed by counting of intracellular vs extracellular Shigella, we observed that regardless of the absence or presence of L. casei in the culture medium, the ratio of intracellular vs extracellular Shigella remained unchanged (1.91 ± 0.74 or 1.70 ± 0.66, respectively).

Together, these results demonstrate that L. casei selectively down-regulates the expression of some key factors induced after infection of IECs with Shigella.

L. casei blocks NF-{kappa}B activation induced by S. flexneri

Because a number of the Shigella-induced genes identified above are under the control of the NF-{kappa}B transcription complex, we aimed to investigate whether L. casei acted directly on this signaling pathway. Strikingly, using a NF-{kappa}B-dependent luciferase reporter gene assay, we observed that overnight preincubation of HEK293 cells with L. casei repressed NF-{kappa}B activation induced by Shigella (Fig. 3). We then investigated the nature of this blockage at the molecular level. In epithelial cells, the signaling pathway leading to the activation of NF-{kappa}B after S. flexneri infection was recently identified. It involves the intracellular peptidoglycan-recognition molecule Nod1 and a signaling cascade involving Rip2/RICK/Cardiak, the IKK complex, and the inhibitory protein I-{kappa}B{alpha} (25). We analyzed whether preincubation with L. casei could modulate the expression of selected members of this signaling cascade. Although the expressions of Rip2 and IKK{alpha} were not modified by pretreatment with L. casei (Fig. 4A), we noticed a substantial increase of I-{kappa}B{alpha} expression after overnight coculture of HEK293 cells with L. casei (Fig. 4A).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3. Inhibition of Shigella-induced NF-{kappa}B activation by L. casei. Overnight preincubation of HEK293 cells with L. casei blocks NF-{kappa}B activation induced by the invasive bacteria S. flexneri. CTR, Control.

 


View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 4. L. casei infection results in up-regulation of I-{kappa}B{alpha} protein and inhibits Shigella-induced I-{kappa}B{alpha} degradation. A, Overnight preincubation of HEK293 cells with L. casei induces the up-regulation of I-{kappa}B{alpha} protein, while leaving other proteins of the Nod1-dependent signaling pathway unchanged. B, Overnight (O/N) preincubation of HEK293 cells with L. casei blocks I-{kappa}B{alpha} degradation induced by the invasive bacteria S. flexneri. CTR, Control.

 
Infection of epithelial cells with S. flexneri induces rapid and dramatic degradation of I-{kappa}B{alpha}, resulting in the release of a free NF-{kappa}B complex that translocates to the nucleus to induce transcription of NF-{kappa}B-dependent pro-inflammatory genes. Indeed, we could confirm these observations in a kinetic study of I-{kappa}B{alpha} degradation ranging from 10 to 40 min after infection (Fig. 4B, top panel). In similar conditions, overnight preincubation of HEK293 cells with L. casei resulted in a strong inhibition of S. flexneri-induced I-{kappa}B{alpha} degradation. Instead, degradation of I-{kappa}B{alpha} was observed only at later time points and remained partial (Fig. 4B, bottom panel). Interestingly, a slower migrating band likely corresponding to a phosphorylated form of I-{kappa}B{alpha} was transiently detected, but only in cells preincubated with L. casei. I-{kappa}B{alpha} degradation is mediated by the ubiquitin-proteasome system and the phosphorylation of I-{kappa}B{alpha} is a signal that targets the protein to degradation. In unstimulated cells, phospho-I-{kappa} B{alpha} is hardly detectable because the protein is immediately targeted for degradation. Because in epithelial cells incubated with L casei we could detect phospho-I-{kappa}B{alpha} (Fig. 5), we anticipated that the effect of L. casei on I-{kappa}B{alpha} may be related to altered degradation of the protein by the ubiquitin-proteasome system.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 5. L. casei infection results in up-regulation of the phosphorylated form of the I-{kappa}B{alpha} protein. Overnight (O/N) preincubation of HEK293 cells with L. casei (ranging from 2.5 106 to 5.107 bacteria/ml) induced a dose-dependent increase of I-{kappa}B{alpha} protein phosphorylation as determined by Western blotting using a specific polyclonal anti-human phospho-I-{kappa}B{alpha} Ab. CTR, Control.

 
These results suggested that the anti-inflammatory effect of L. casei is largely mediated through specific modulation of the I-{kappa}B{alpha} protein level. However, I-{kappa}B{alpha} lays relatively downstream in the NF-{kappa}B signaling cascade, and several pro-inflammatory stimuli lead to its degradation. We therefore investigated whether L. casei could down-regulate NF-{kappa}B activation induced by TNF-{alpha}, another well characterized pro-inflammatory stimulus. Indeed, we could observe that overnight preincubation of HEK293 cells with L. casei potently repressed NF-{kappa}B activation induced by TNF-{alpha} (Fig. 6A). Moreover, a kinetic study of I-{kappa}B{alpha} degradation ranging from 15 to 45 min after stimulation with TNF-{alpha} showed that I-{kappa}B{alpha} degradation was strongly decreased if HEK293 cells had been preincubated overnight with L. casei (Fig. 6B). Together, these results strongly suggest that the anti-inflammatory properties of L. casei largely depend on the modulation of I-{kappa}B{alpha} expression and/or stability.



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 6. Inhibition of TNF-{alpha}-induced NF-{kappa}B activation by L. casei. A, HEK293 cells were preincubated overnight with L. casei or MRS buffer (control medium) before stimulation for 4 h with TNF-{alpha}. Three bacterial concentrations were used: 5.106, 2.107, or 5.107 bacteria/ml. B, HEK293 cells were preincubated overnight with L. casei or MRS buffer (control medium) before stimulation for various times (15, 30, or 45 min) with TNF-{alpha}, followed by lysis of the cells. The I-{kappa}B{alpha} protein content was determined by Western blotting using a polyclonal anti-human I-{kappa}B{alpha} Ab. CTR, Control.

 
Global transcriptomic analysis of epithelial cells incubated with L. casei

The above results demonstrate a role for L. casei in the specific modulation of NF-{kappa}B-driven genes induced after Shigella infection. However, it remains possible that other key signaling pathways are targets of L. casei-induced modulatory properties. To gain more insight into the global effects mediated by L. casei on host cell signaling, we performed a large-scale gene expression study (14,500 well characterized human genes; Affymetrix Genechips U133A) on Caco-2 cells cocultured with the probiotic strain for 2, 6, or 24 h. The entire set of results is available online (supplemental Table S1) 5, and Table III represents the number of probe sets modulated during Caco-2 cell culture in the presence of L. casei. Interestingly, most of the modulation was observed after 24 h of incubation. Because all of the results are available online, and to avoid to present an extensive list of modulated genes, we have focused in this study on four functional clusters (defined using NettAffyx, the database from Affymetrix) that appeared to be modulated most extensively by L. casei (Table IV and see below).


View this table:
[in this window]
[in a new window]
  		Table III. Determination of modulated probe sets during time course culture of Caco-2 cells with L. caseia

 

View this table:
[in this window]
[in a new window]
  		Table IV. Gene expression modulation during L. casei time coursea

 

View this table:
[in this window]
[in a new window]
  		Table IVA. Continued

 
Group 1: genes involved in control of the cell cycle. L. casei induced both positive and negative modulation of expression of genes involved in the regulation of the cell cycle. Indeed, tumor suppressor genes, such as tumor rejection Ag (gp96) and sel-1 suppressor of lin-12, were up-regulated during incubation with L. casei, whereas the expression of the gene encoding prohibitin, a negative regulator of cell proliferation, was down-regulated.

Group 2: genes involved in the control of apoptosis. Expression of two genes encoding proteins involved in the inhibition of apoptosis (Fas apoptotic inhibitory molecule and cytokine-induced apoptosis inhibitor) were down-regulated, and expression of BCL2/adenovirus E1B was increased. It should be noted that the expression of Nalp2, encoding a part of the inflammasome (26), decreased during the incubation of Caco-2 cells with L. casei, suggesting its potential involvement in the anti-inflammatory properties of the probiotic bacteria. In addition, the expression of programmed cell death 4, a tumor suppressor (27), was positively modulated.

Group 3: genes involved in hypoxia. Expression of two genes under the control of the hypoxia-inducible factor-1 (HIF-1) transcription factor (hypoxia up-regulated 1 and HIF-1 response RTP801) was induced during the incubation of Caco-2 cells with L. casei.

Group 4: genes belonging to the ubiquitination/degradation pathway. Twenty-five genes involved in the protein ubiquitination and/or degradation process were found to be positively or negatively modulated during incubation of Caco-2 cells with L. casei. The transcription of genes encoding three ubiquitin-conjugating enzymes, E2D, E2L, and E2N, and four subunits of the 26S proteasome were down-regulated. Another gene, the expression of which decreased during this incubation, was rbx-1/roc-1/hrt-1, which encodes a protein belonging to the E3 ligase complex. We confirmed by individual PCR on a set of these selected genes their down-regulated expression after treatment with L. casei (Fig. 7A). It is striking to note that, among all of the genes modulated by L. casei, the group of genes implicated in ubiquitination/degradation (group 4) is, by far, the most represented. This observation suggests that L. casei is capable of establishing a program leading to the down-regulation of the ubiquitin-mediated degradation of proteins. Moreover, because the ubiquitin system is responsible for the degradation of I-{kappa}B{alpha}, this observation strongly supports our conclusions that modulating the stability of I-{kappa}B{alpha} represents a major target of L. casei.



View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 7. L. casei induced a down-regulation of genes involved in ubiquitination/degradation processes. Caco-2 cells were cultured overnight with L. casei with a MOI of 100. A, After washes with PBS and RNA extraction with the Rneasy mini kit, cDNA was synthetized using oligo-d(T) and reverse transcriptase. PCRs were performed as described in the legend to Fig. 2. B, After washes with PBS and lysis with Laemmli buffer, aliquots of the lysates were loaded on 15 or 10% SDS-PAGE for the subsequent detection of Rbx-1 or tubulin, respectively. The Rbx-1 or tubulin protein contents were determined by Western blotting using a polyclonal anti-human Rbx-1 or a monoclonal anti-human tubulin Ab, respectively. C, HEK cells were treated overnight with L. casei or for 6 h with 50 µM of the proteasome inhibitor MG-132 before incubation with or without TNF. The I-{kappa}B{alpha} protein content and global ubiquitinated proteins were determined by Western blotting (WB) using a polyclonal anti-human I-{kappa}B{alpha} Ab or monoclonal anti-ubiquitin Ab, respectively.

 
Among the 25 genes of the ubiquitination/degradation pathway that were identified, we noticed that Rbx-1 expression was down-regulated to a greater extent (~4-fold decrease) by L. casei than the others (Table IV). This prompted us to investigate whether the effects of L. casei on the NF-{kappa}B pathway could be accounted for by the specific down-regulation of Rbx-1. First, the expression of Rbx-1 at the protein level was monitored in L. casei-treated cells and was found to be reduced by ~50% (Fig. 7B). Second, overexpression of exogenous epitope-tagged Rbx-1 in L. casei-treated cells was performed, and TNF-mediated (as well as Shigella-mediated) degradation of I-{kappa}B{alpha} was monitored (data not shown). However, restoring Rbx-1 expression was found to be insufficient to recapitulate the effects of L. casei on the ubiquitin/degradation pathway. As a consequence, it is likely that L. casei down-regulates this cellular process via the targeting of multiple effectors. Accordingly, we observed that the global pattern of ubiquitinated proteins after L. casei treatment closely resembles the one of cells treated with MG132, a broad inhibitor of multiple ubiquitin/proteasome-dependent pathways (Fig. 7C). Together, this large-scale analysis of L. casei-modulated genes in Caco-2 cells strongly supports the conclusion that this probiotic bacteria displays anti-inflammatory properties through the targeting of the ubiquitin/proteasome system.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have investigated the effect of coculture of IECs with the probiotic bacteria L. casei. We were interested in two questions of fundamental importance: 1) Does this probiotic bacteria display protective properties on IECs infected with a pathogenic entero-invasive bacteria? and 2) What is the pattern of transcriptional responses of the epithelial cells cocultured for various periods with L. casei? To gain insight into these questions, we embarked on a project to identify the global repertoire of host responses to L. casei using, for the first time on this topic, the technologies of macroarray analysis (1,050 genes selected) associated with DNA chip hybridization (Affymetrix; 14,500 genes) and biochemical analysis. Our results clearly identify a role for L. casei in the specific modulation of a repertoire of genes associated with the ubiquitin/proteasome pathway. Importantly, the down-regulation of this set of ubiquitin system-associated genes appears to play a crucial role in the modulation of pro-inflammatory pathways in IECs. Indeed, when pro-inflammatory stimuli, such as Shigella infection or TNF treatment, were used to stimulate these cells, we observed a key role of L. casei preculture in dampening these responses. Moreover, we could pinpoint the molecular basis of this modulation by the observation that L. casei specifically targets the stability of I-{kappa}B{alpha}, the specific NF-{kappa}B inhibitor, thereby shutting down this major pro-inflammatory pathway.

Our study confirms and also extends the hypothesis that certain commensal microorganisms have the potential to actively influence the homeostatic control of intestinal inflammation (2). Indeed, coculture of human tissues with L. casei significantly reduced TNF-{alpha} release from Crohn’s disease patients’ inflamed mucosa (8) even in presence of a nonpathogenic E. coli that alone enhanced TNF-{alpha} release from inflamed tissues (9). In addition, in a mouse model of skin hypersensitivity, ingestion of L. casei was demonstrated to reduce skin inflammation induced by dinitrofluorobenzene (28). Exposure of IECs to nonpathogenic Salmonella spp. could mediate anti-inflammatory signals by preventing ubiquitination of I-{kappa}B{alpha} (13). Recently, another anti-inflammatory mechanism induced by a commensal bacteria strain has been described. B. thetaiotaomicron attenuates pro-inflammatory cytokine expression by inducing the nuclear export of complexes formed by NF-{kappa}B and peroxisome proliferator-activated receptor-{gamma} (29). This set of results indicates that preincubation with certain probiotic strains such as L. casei decreases NF-{kappa}B-driven activation of pro-inflammatory genes. In contrast, it has been shown that other commensals, such as E. coli MG 1655, induce NF-{kappa}B activation in IECs through the TLR5 signaling pathway (30). It must be noted that, in the present study, the two bacterial strains used, L. casei and S. flexneri, are not flagellated; thus, signal transduction via flagellin and TLR5, a major pro-inflammatory pathway described at the intestinal epithelial level (30), does not occur.

Among the genes that have been found to be modulated by L. casei through the Affymetrix study, we have identified a set of genes encoding proteins that control the cell cycle. Hence, the expressions of sel-1 and gp96, two tumor suppressor genes, were found to be up-regulated during incubation with L. casei. A high level of Sel-1 has been suggested to correlate with the decrease of breast tumor growth (31), and gp96 not only elicits immune responses to tumors by inducing a CD8+ T cell response (32) but also plays an adjuvant role in antitumor immune responses (33).

Another important pathway modulated by L. casei was shown to involve hypoxia-related genes. The level of transcription of vegf was found increased during incubation of Caco-2 cells with L. casei. This observation could prove of importance because VEGF is a growth factor that plays a key role in angiogenesis (34). Transcription of vegf is under the control of the transcription factor HIF-1 (35). Interestingly, two other hypoxia-responsive genes, both involved in apoptosis, were found up-regulated when Caco-2 cells were incubated with L. casei: the hypoxia up-regulated 1 gene (ORP150), which displays a protective function in hypoxia-conditioned cells (36), and RTP801 gene, which inhibits hypoxia-mediated apoptosis (37). The transcription of other genes known to be under the control of HIF-1, such as erythropoietin, was not modulated during such incubation. This is likely due to the restricted transcriptional program of Caco-2 cells. Significance as well as the mechanisms involved in the establishment of hypoxia sensing by Caco-2 cells during incubation with L. casei remain to be further analyzed. In particular, it will be of interest to determine whether these microorganisms actively induce this process or whether cellular hypoxia is an indirect consequence of extracellular consumption of oxygen or Fe3+ by the bacteria.

Because the expression of genes involved in different pathways (i.e., cell cycle, response to hypoxia, NF-{kappa}B-dependent transcription) was found to be modified during incubation of Caco-2 cells with L. casei, we investigated whether a common signaling pathway could account for these diverse effects. Of interest, these three pathways are regulated by a similar ubiquitination/proteasome system (38, 39, 40, 41). In our time-course study using Affymetrix technology, we observed that a large set of genes involved in the ubiquitination/degradation pathways was modulated by L. casei. In light of our results, it appears very likely that the specific modulation of the ubiquitin/proteasome system represents a main target of action of L. casei on host cells and that this effect would in turn be responsible for the alteration of several signaling pathways, such as those involving the cell cycle and hypoxia.

In agreement with our findings that L. casei affects the ubiquitin/proteasome pathway, Caco-2 cells coincubated with L. casei showed stabilization of I-{kappa}B{alpha} even after subsequent stimulation by Shigella or TNF-{alpha}. Thus, the anti-inflammatory effects of L. casei are likely mediated by its effects on the ubiquitin/proteasome system and the consequent dampening of NF-{kappa}B-driven pro-inflammatory signals.

Convergent pieces of evidence suggest that, through their ability to modulate inflammatory pathways, some commensal bacteria contribute to the homeostasis of the intestinal epithelium. Our observations, and also the results from other groups, will impact on the understanding of the mechanisms responsible for some of the beneficial effects of probiotics on IBDs. This knowledge will contribute to offer, in the near future, new therapeutic means to counteract the inflammatory disorders observed in human pathologies, such as ulcerative colitis and Crohn’s disease.


   Acknowledgments
 
The Ab for ubiquitin detection was a gift from Dr. Claude Parsot (Pasteur Institute). We thank Drs. Armelle Phalipon, Dana Philpott, and Régis Tournebize for critical reading of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Danone-Vitapole (Palaiseau, France), the APEX program of assistance to exceptional proposals from Institut National de la Santé et de la Recherche Médicale, and the Genopole of Pasteur Institute. The Affymetrix station of the Pasteur Institute was purchased with a donation from Dr. R. Nunnikhoven. M.-T.T. was supported by fellowships from the French Research Ministry and Taïwan Chao-Tung University. P.J.S. is a Howard Hughes Medical Institute scholar. Back

2 M.-T.T. and S.E.G. contributed equally to this work. Back

3 Address correspondence and reprint requests to Prof. Philippe J. Sansonetti, Pathogénie Microbienne Moléculaire Unit, Institut National de la Santé et de la Recherche Médicale U389, Pasteur Institute, 28 rue du Docteur Roux, 75015 Paris, France. E-mail address: psanson{at}pasteur.fr Back

4 Abbreviations used in this paper: IEC, intestinal epithelial cell; IBD, inflammatory bowel disease; MRS, Mann-Rogosa-Sharpe; MOI, multiplicity of infection. Back

5 The online version of this article contains supplemental material. Back

Received for publication March 25, 2005. Accepted for publication October 25, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Isolauri, E., S. Salminen, A. C. Ouwehand. 2004. Microbial-gut interactions in health and disease: probiotics. Best Pract. Res. Clin. Gastroenterol. 18: 299-313. [Medline]
  2. Sansonetti, P. J.. 2004. War and peace at mucosal surfaces. Nat. Rev. Immunol. 4: 953-964. [Medline]
  3. Mahida, Y. R.. 2004. Microbial-gut interactions in health and disease: epithelial cell responses. Best Pract. Res. Clin. Gastroenterol. 18: 241-253. [Medline]
  4. Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, R. Medzhitov. 2004. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118: 229-241. [Medline]
  5. McCracken, V. J., R. G. Lorenz. 2001. The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota. Cell Microbiol. 3: 1-11. [Medline]
  6. Gionchetti, P., F. Rizzello, U. Helwig, A. Venturi, K. M. Lammers, P. Brigidi, B. Vitali, G. Poggioli, M. Miglioli, M. Campieri. 2003. Prophylaxis of pouchitis onset with probiotic therapy: a double-blind, placebo-controlled trial. Gastroenterology 124: 1202-1209. [Medline]
  7. Cui, H. H., C. L. Chen, J. D. Wang, Y. J. Yang, Y. Cun, J. B. Wu, Y. H. Liu, H. L. Dan, Y. T. Jian, X. Q. Chen. 2004. Effect of probiotic on intestinal mucosa of patients with ulcerative colitis. World J. Gastroenterol. 10: 1521-1525. [Medline]
  8. Borruel, N., M. Carol, F. Casellas, M. Antolin, F. de Lara, E. Espin, J. Naval, F. Guarner, J.R. Malagelada. 2002. Increased mucosal tumour necrosis factor {alpha} production in Crohn’s disease can be downregulated ex vivo by probiotic bacteria. Gut 51: 659-664. [Abstract/Free Full Text]
  9. Borruel, N., F. Casellas, M. Antolin, M. Llopis, M. Carol, E. Espiin, J. Naval, F. Guarner, J. R. Malagelada. 2003. Effects of nonpathogenic bacteria on cytokine secretion by human intestinal mucosa. Am. J. Gastroenterol. 98: 865-870. [Medline]
  10. Drakes, M., T. Blanchard, S. Czinn. 2004. Bacterial probiotic modulation of dendritic cells. Infect. Immun. 72: 3299-3309. [Abstract/Free Full Text]
  11. Christensen, H. R., H. Frokiaer, J. J. Pestka. 2002. Lactobacilli differentially modulate expression of cytokines and maturation surface markers in murine dendritic cells. J. Immunol. 168: 171-178. [Abstract/Free Full Text]
  12. Jijon, H., J. Backer, H. Diaz, H. Yeung, D. Thiel, C. McKaigney, C. De Simone, K. Madsen. 2004. DNA from probiotic bacteria modulates murine and human epithelial and immune function. Gastroenterology 126: 1358-1373. [Medline]
  13. Neish, A. S., A. T. Gewirtz, H. Zeng, A. N. Young, M. E. Hobert, V. Karmali, A. S. Rao, J. L. Madara. 2000. Prokaryotic regulation of epithelial responses by inhibition of I{kappa}B-{alpha} ubiquitination. Science 289: 1560-1563. [Abstract/Free Full Text]
  14. Collier-Hyams, L. S., H. Zeng, J. Sun, A. D. Tomlinson, Z. Q. Bao, H. Chen, J. L. Madara, K. Orth, A. S. Neish. 2002. Salmonella AvrA effector inhibits the key proinflammatory, anti-apoptotic NF-{kappa}B pathway. J. Immunol. 169: 2 846-2850. [Abstract/Free Full Text]
  15. Hooper, L. V., M. H. Wong, A. Thelin, L. Hansson, P. G. Falk, J. I. Gordon. 2001. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884. [Abstract/Free Full Text]
  16. Stappenbeck, T. S., L. V. Hooper, J. I. Gordon. 2002. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc. Natl. Acad. Sci. USA 99: 15451-15455. [Abstract/Free Full Text]
  17. Sansonetti, P. J.. 2001. Rupture, invasion and inflammatory destruction of the intestinal barrier by Shigella, making sense of prokaryote-eukaryote cross-talks. FEMS Microbiol. Rev. 25: 3-14. [Medline]
  18. Philpott, D. J., S. Yamaoka, A. Israel, P. J. Sansonetti. 2000. Invasive Shigella flexneri activates NF-{kappa}B through a lipopolysaccharide-dependent innate intracellular response and leads to IL-8 expression in epithelial cells. J. Immunol. 165: 903-914. [Abstract/Free Full Text]
  19. Girardin, S. E., R. Tournebize, M. Mavris, A. L. Page, X. Li, G. R. Stark, J. Bertin, P. S. DiStefano, M. Yaniv, P. J. Sansonetti, D. J. Philpott. 2001. CARD4/Nod1 mediates NF-kappaB and JNK activation by invasive Shigella flexneri. EMBO Rep. 2: 736-742. [Medline]
  20. Girardin, S. E., I. G. Boneca, L. A. Carneiro, A. Antignac, M. Jehanno, J. Viala, K. Tedin, M. K. Taha, A. Labigne, U. Zahringer, et al 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300: 1584-1587. [Abstract/Free Full Text]
  21. Clerc, P., P. J. Sansonetti. 1987. Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect. Immun. 55: 2681-2688. [Abstract/Free Full Text]
  22. Petit-Bertron, A.-F., T. Pedron, U. Gross, J.-Y. Coppee, P. J. Sansonetti, J.-M. Cavaillon, M. Adib-Conquy. 2005. Adherence modifies the regulation of gene expression induced by interleukin-10. Cytokine 29: 1-12. [Medline]
  23. Li, C., W. H. Wong. 2001. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc. Natl. Acad. Sci. USA 98: 31-36. [Abstract/Free Full Text]
  24. Pedron, T., C. Thibault, P. J. Sansonetti. 2003. The invasive phenotype of Shigella flexneri directs a distinct gene expression pattern in the human intestinal epithelial cell line Caco-2. J. Biol. Chem. 278: 33878-33886. [Abstract/Free Full Text]
  25. Kufer, T. A., J. H. Fritz, D. J. Philpott. 2005. NACHT-LRR proteins (NLRs) in bacterial Infect. Immun. Trends Microbiol. 13: 381-388.
  26. Tschopp, J., F. Martinon, K. Burns. 2003. NALPS: a novel protein family involved in inflammation. Nat. Rev. Mol. Cell Biol. 4: 95-104. [Medline]
  27. Goke, R., C. Gregel, A. Goke, R. Arnold, H. Schmidt, B. Lankat-Buttgereit. 2004. Programmed cell death protein 4 (PDCD4) acts as a tumor suppressor in neuroendocrine tumor cells. Ann. NY Acad. Sci. 1014: 220-221. [Medline]
  28. Chapat, L., K. Chemin, B. Dubois, R. Bourdet-Sicard, D. Kaiserlian. 2004. Lactobacillus casei reduces CD8+ T cell-mediated skin inflammation. Eur. J. Immunol. 34: 2520-2528. [Medline]
  29. Kelly, D., J. I. Campbell, T. P. King, G. Grant, E. A. Jansson, A. G. Coutts, S. Pettersson, S. Conway. 2004. Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-{gamma} and RelA. Nat. Immunol. 5: 104-112. [Medline]
  30. Bambou, J. C., A. Giraud, S. Menard, B. Begue, S. Rakotobe, M. Heyman, F. Taddei, N. Cerf-Bensussan, V. Gaboriau-Routhiau. 2004. In vitro and ex vivo activation of the TLR5 signaling pathway in intestinal epithelial cells by a commensal Escherichia coli strain. J. Biol. Chem. 279: 42984-42992. [Abstract/Free Full Text]
  31. Orlandi, R., M. Cattaneo, F. Troglio, P. Casalini, C. Ronchini, S. Menard, I. Biunno. 2002. SEL1L expression decreases breast tumor cell aggressiveness in vivo and in vitro. Cancer Res. 62: 567-574. [Abstract/Free Full Text]
  32. Udono, H., D. L. Levey, P. K. Srivastava. 1994. Cellular requirements for tumor-specific immunity elicited by heat shock proteins: tumor rejection antigen gp96 primes CD8+ T cells in vivo. Proc. Natl. Acad. Sci. USA 91: 3077-3081. [Abstract/Free Full Text]
  33. Baker-LePain, J. C., M. Sarzotti, T. A. Fields, C. Y. Li, C. V. Nicchitta. 2002. GRP94 (gp96) and GRP94 N-terminal geldanamycin binding domain elicit tissue nonrestricted tumor suppression. J. Exp. Med. 196: 1447-1459. [Abstract/Free Full Text]
  34. Bussolati, B., A. Ahmed, H. Pemberton, R. C. Landis, F. Di Carlo, D. O. Haskard, J. C. Mason. 2004. Bifunctional role for VEGF-induced heme oxygenase-1 in vivo: induction of angiogenesis and inhibition of leukocytic infiltration. Blood 103: 761-766. [Abstract/Free Full Text]
  35. Skinner, H. D., J. Z. Zheng, J. Fang, F. Agani, B. H. Jiang. 2004. Vascular endothelial growth factor transcriptional activation is mediated by hypoxia-inducible factor 1{alpha}, HDM2, and p70S6K1 in response to phosphatidylinositol 3-kinase/AKT signaling. J. Biol. Chem. 279: 45643-45651. [Abstract/Free Full Text]
  36. Ozawa, K., K. Kuwabara, M. Tamatani, K. Takatsuji, Y. Tsukamoto, S. Kaneda, H. Yanagi, D. M. Stern, Y. Eguchi, Y. Tsujimoto, et al 1999. 150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death. J. Biol. Chem. 274: 6397-6404. [Abstract/Free Full Text]
  37. Shoshani, T., A. Faerman, I. Mett, E. Zelin, T. Tenne, S. Gorodin, Y. Moshel, S. Elbaz, A. Budanov, A. Chajut, et al 2002. Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol. Cell. Biol. 22: 2283-2293. [Abstract/Free Full Text]
  38. Ben-Neriah, Y.. 2002. Regulatory functions of ubiquitination in the immune system. Nat. Immunol. 3: 20-26. [Medline]
  39. Tanaka, K., T. Kawakami, K. Tateishi, H. Yashiroda, T. Chiba. 2001. Control of IkappaBalpha proteolysis by the ubiquitin-proteasome pathway. Biochimie 83: 351-356. [Medline]
  40. Krek, W.. 2000. VHL takes HIF’s breath away. Nat. Cell. Biol. 2: E121-E123. [Medline]
  41. Kamura, T., D. M. Koepp, M. N. Conrad, D. Skowyra, R. J. Moreland, O. Iliopoulos, W. S. Lane, W. G. Kaelin, Jr, S. J. Elledge, R. C. Conaway, et al 1999. Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science 284: 657-661. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 MicrobiologyHome page
N. Jandu, Z. J. Zeng, K. C. Johnson-Henry, and P. M. Sherman
Probiotics prevent enterohaemorrhagic Escherichia coli O157 : H7-mediated inhibition of interferon-{gamma}-induced tyrosine phosphorylation of STAT-1
Microbiology, February 1, 2009; 155(2): 531 - 540.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
C. Cosseau, D. A. Devine, E. Dullaghan, J. L. Gardy, A. Chikatamarla, S. Gellatly, L. L. Yu, J. Pistolic, R. Falsafi, J. Tagg, et al.
The Commensal Streptococcus salivarius K12 Downregulates the Innate Immune Responses of Human Epithelial Cells and Promotes Host-Microbe Homeostasis
Infect. Immun., September 1, 2008; 76(9): 4163 - 4175.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. A. Hamon, E. Batsche, B. Regnault, T. N. Tham, S. Seveau, C. Muchardt, and P. Cossart
Histone modifications induced by a family of bacterial toxins
PNAS, August 14, 2007; 104(33): 13467 - 13472.
[Abstract] [Full Text] [PDF]

Home page
 J Med MicrobiolHome page
L. Yin and B. A. Dale
Activation of protective responses in oral epithelial cells by Fusobacterium nucleatum and human {beta}-defensin-2
J. Med. Microbiol., July 1, 2007; 56(7): 976 - 987.
[Abstract] [Full Text] [PDF]

Home page
 Poult. Sci.Home page
M. Chichlowski, J. Croom, B. W. McBride, L. Daniel, G. Davis, and M. D. Koci
Direct-Fed Microbial PrimaLac and Salinomycin Modulate Whole-Body and Intestinal Oxygen Consumption and Intestinal Mucosal Cytokine Production in the Broiler Chick
Poult. Sci., June 1, 2007; 86(6): 1100 - 1106.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
B. Corthesy, H. R. Gaskins, and A. Mercenier
Cross-Talk between Probiotic Bacteria and the Host Immune System
J. Nutr., March 1, 2007; 137(3): 781S - 790S.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Fillon, K. Soulis, S. Rajasekaran, H. Benedict-Hamilton, J. N. Radin, C. J. Orihuela, K. C. El Kasmi, G. Murti, D. Kaushal, M. W. Gaber, et al.
Platelet-Activating Factor Receptor and Innate Immunity: Uptake of Gram-Positive Bacterial Cell Wall into Host Cells and Cell-Specific Pathophysiology
J. Immunol., November 1, 2006; 177(9): 6182 - 6191.
[Abstract] [Full Text] [PDF]

Home page
 Evid Based Complement Alternat MedHome page
A. Vojdani and J. Erde
Regulatory T Cells, a Potent Immunoregulatory Target for CAM Researchers: Modulating Allergic and Infectious Disease Pathology (II)
Evid. Based Complement. Altern. Med., June 1, 2006; 3(2): 209 - 215.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tien, M.-T.
Right arrow Articles by Pédron, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tien, M.-T.
Right arrow Articles by Pédron, T.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS