NEW STRATEGY TO TREAT AND PREVENT DISEASES CAUSED BY ENTEROBACTERIAE

Abstract

The present invention relates to the treatment of diseases induced by Enterobacteriae. The inventors evaluated, in a multicellular in vitro model associating cells representing human enterocytes (Caco-2 cells), goblet mucus secreting cells (HT29-MTX) and M cells, whether Bacteroides fragilis, a non-enterotoxigenic strain, could be useful to limit the severity of the Salmonella Heidelberg infection, with an hypermutator phenotype, by analyzing their impact on growth and mucosal translocation. Thus, the present invention relates to a Bacteroides fragilis strain for use in the treatment of diseases induced by Enterobacteriae in a subject in need thereof.

Claims

1. A method of treating a diseases caused by Enterobacteriae in a subject in need thereof comprising, administering to the subject a therapeutically effective amount of a Bacteroides fragilis strain.

2. The method according to claim 1 wherein the Enterobacteriae is a Salmonella selected from the group consisting of Salmonella Heidelberg, Escherichia coli, Yersinia pestis, Klebsiella and Shigella.

3. The method according to claim 2 wherein the Enterobacteriae is a Salmonella or Escherichia coli.

4. The method according to claim 1, wherein the Bacteroides fragilis strain is administered as a probiotic.

5. The method according to claim 1, wherein the disease caused by Enterobacteriae is Salmonellosis, typhoid fever, diarrhea Crohn's disease, travelers' diarrhea or ulcerative colitis.

6. The method according claim 1, wherein the subject is an animal.

7. The method according to claim 6 wherein the animal is a fish.

8. A method of treating an Inflammatory Bowel Disease and/or an Irritable Bowel Syndrome in a subject in need thereof comprising, administering to the subject a therapeutically effective amount of a Bacteroides fragilis strain.

9. The method according to claim 8, wherein the Inflammatory Bowel Disease is Crohn's disease or ulcerative colitis.

10. The method according to claim 1, wherein the Bacteroides fragilis strain is a non-toxigenic strain.

11. A therapeutic composition comprising a Bacteroides fragilis strain.

12-13. (canceled)

Description

FIGURES

[0052] FIG. 1: Epithelial integrity during cells differenciation of Caco-2, Caco-2/HT29 MTX (double co-culture), Caco-2/HT29 MTX/M (triple co-culture): (A) Transepithelial electrical resistance (TEER) analysis; (B) Lucifer Yellow (LY) permeability crossing different monolayers, expressed in pmol/cm2/s in basal compartment (*p<0.05).

[0053] FIG. 2: Interaction between the in vitro co-culture models (Caco-2, double and triple) with bacteria (Salmonella Heidelberg (Salmonella) and Bacteroides fragilis (B.fragilis)): translocation of bacteria in basal compartment : (A) Salmonella; (B) B. fragilis; (C) impact of Salmonella and B. fragilis on TEER and (D) LY permeability after 21 days of culture. *p<0.05.

[0054] FIG. 3: Impact of B. fragilis or its cell free supernatant on Salmonella host cells interaction: (A) translocation of Salmonella; (B) evaluation of Salmonella growth (cfu/ml) during 3 h and 24 h of co-culture; (C) TEER evaluations on triple co-culture model; (D) mRNA expression changes in tight junction protein genes (occludin and ZO-1) in the triple coculture model infected by Salmonella Heidelberg, Bacteroides fragilis or both. *p<0.05.

[0055] FIG. 4: Impact of B. fragilis or its cell free supernatant on E. coli translocation after 3 h of incubation.

EXAMPLE

Material & Methods

Cell Lines and Growth Culture

[0056] Caco-2 cells, obtained from American Type Culture Collection (ATCC), were cultivated in complete medium consisting of Dulbecco's modified Eagle medium (DMEM) supplemented with 20% fetal bovine serum, 1% L-glutamine and 1% penicillin and streptomycin. HT29-MTX cells were kindly provided by CRB CelluloNet (SFR Biosciences, CNRS UMS 3444, Inserm US 8, Université Claude-Bernard, Lyon, France) and were grown in the same medium as Caco-2 with only 10% of fetal bovine serum under a 5% CO2 water saturated atmosphere [17]. The Raji B (ECACC 85011429), issued from human Burkitt's lymphoma cell-line, were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% L-glutamin and 1% penicillin and streptomycin, at 37° C. in a 5% CO2 water saturated atmosphere.

[0057] Upon confluence, cells were harvested with trypsin-EDTA and a predetermined amount of cells of each type were mixed prior to seeding to yield cell ratio of 9:1 (Caco-2:HT29-MTX) on the apical chamber of polycarbonate Transwell® inserts and maintained as described by Schimpel et al., 2014 [16]. After 14 days of culture, Raji B cells are added to the basolateral chamber to induce the differentiation of Caco-2 cells into M cells [16]. Caco-2/HT29-MTX and RajiB co-culture were maintained for 7 days in DMEM.

TEER Measurements and Paracellular Permeability Study

[0058] The integrity of the polarized epithelial co-culture (Caco-2:HT29-MTX: M cells) was evaluated by measuring the transepithelial electrical resistance (TEER) using an Ohm/voltmeter (EVOM2; World Precision Instruments). The resistance obtained from a cell free culture insert was subtracted from resistance measured across each well and resistance values were calculated in Ohms (Ω).Math.cm2 by multiplying the resistance values by the filter surface area.

[0059] The integrity of polarized cells was checked also by measuring the Lucifer yellow (LY) transport rate. Regarding the paracellular permeability study, LY solution at 10 μM was prepared in DMEM, then added to the apical side of the insert while only DMEM was added in the basolateral side. After incubation for the periods indicated for TEER study, the solution in the basal compartment was collected and the fluorescence intensity of LY was measured using POLARstar Omega Microplate Reader. Results were expressed in pmol/cm2/s in kinetic differentiation or as percentage of LY permeability inhibition compared to insert without cells.

Transmission Electron Microscopy

[0060] Transmission electron microscopy (TEM) was performed on polarized cells after 21 days of growth on polycarbonate Transwell® cell culture inserts. After several washing with PBS, samples were fixed for 2 hours in room temperature in 2.5% glutaraldehyde dissolved in 0.1 M cacodylate, postfixed in 1% osmium tetroxide for 1 hour at room temperature, rinsed in cacodylate buffer, and dehydrated in an ascending series of ethanol. The polycarbonate membrane contained in the inserts and on which the cells grown was recovered, then cut into thin strips. Samples were then infiltrated with an ascending concentration of Epon resin in ethanol mixtures. Finally, they were placed in fresh Epon for several hours and then embedded in Epon for 48 hours at 60° C. Resins blocks were sectioned into 80 nm ultrathin sections using LEICA UC7 ultramicrotome (LEICA Systems, Vienna, Austria): cut sections were performed so that it allowed to visualize transversally Transwell® membrane with cells layer. These sections were mounted on copper grids and stained. Grids were observed using a TEM JEOL-JEM 1400 (JEOL Ltd, Tokyo, Japan) at an accelerating voltage of 120 kV and equipped with a Gatan Inc. Orius 1000 camera.

Quantification of Selected Genes Expression Level by Quantitative Reverse Transcription PCR (RT-qPCR)

[0061] After 21 days of co-culture, RNA were extracted from the upper chamber using Total RNA and Protein isolation kit (Macherey-Nagel) according to the manufacturer's instructions. Afterwards, High-Capacity cDNA Reverse Transcription Kit (Applied biosystems) was used to reverse-transcribe the RNA into cDNA. Then, the selected genes specific for each cells were relatively quantified using StepOnePlus (Applied Biosystems) with the SYBR Green PCR Master Mix (Applied Biosystems) [18]. Genes playing essential roles for each cells were selected: sucrase isomaltase (SI) which is specific of Caco-2, mucin-2 (muc2) secreted by HT29-MTX, and glycoprotein 2 (GP2) associated to M cells. Primers used for these selected genes were described in table 1. Each gene was normalized to the TBP (TATA box binding protein) mRNA expression level before calculation of the fold-change values. Relative gene expression was calculated by the 2-ΔΔCT method [19].

Bacteria and Growth Conditions

[0062] Strain of Salmonella Heidelberg B182 (S. Heidelberg), with a hypermutator phenotype (deletion of 12 bp in mutS) was grown overnight at 37° C. as we previously described [18]. The non-toxigenic Bacteroides fragilis strain (B. fragilis), ATCC 25285 [20], [21], was purchased from the American Type Culture Collection. To mimic the in vivo scenario of gut lumen, S. Heidelberg and B. fragilis were applied simultaneously to the apical side of the Transwell® system at a ratio of 1:99 respectively. They were cultured in DMEM medium containing 20% SVF, 1% L-glutamin and 1% L-cystein. To separate supernatant and bacterial pellets, the media were centrifuged at 3000×g for 5 min. Supernatant samples were sterilized in 0, 22 μm filter. B. fragilis supernatant was used to evaluate its impact on S. Heidelberg growth and translocation.

Evaluation of Bacterial Translocation

[0063] After 21 days of cellular culture, in order to study the cells/bacteria interaction, S. Heidelberg and B. fragilis after overnight culture were recovered and added on washed intestinal epithelial cell layer at an MOI of 10 and incubated at 37° C. for 3 h. Following incubation, basal and apical medium were separately collected and CFU enumeration was performed. The number of translocated bacteria recovered in the lower chambers was expressed as a ratio between this number and number of bacteria counted in the upper chamber.

Statistical Analysis

[0064] Experiments were performed at least in triplicates and data were analyzed using Student's t-test. Data presented as mean±SD and p-value less than 0.05 was considered as significant.

Results

Characterization of Caco-2/HT29-MTX/M Cells Co-Culture

[0065] In this study, we developed an original in vitro triple co-culture model composed of enterocytes (Caco-2), goblets cells (HT29-MTX) secreting mucus and M cells. M cells were obtained by inducing the differentiation of enterocytes of the Caco-2 cell line cultured in close contact with B lymphocytes of the Raji B cell line already reported by Schimpel et al., 2014 [16].

[0066] We investigated the cell morphologies by transmission electron microscopy (TEM) in a triple co-culture of 21 days in a Transwell® membrane. TEM allowed to recognize M cells through the particular shape of their apical membrane exhibiting short and irregular microvilli, whereas Caco-2 cells showed a typical brush border (data not shown). Goblet cells, randomly distributed, were visualized by their typical mucus containing vesicles (data not shown). To further characterize the triple co-culture model, we checked the level of expression of genes that are molecular markers for each cell type. At the differentiation state (21 days), in the double (Caco-2/HT29-MTX) and triple (Caco-2/HT29-MTX/M) co-culture models, muc2 expression was significantly upregulated: 4, 36±1, 01 and 3, 9±0, 37 fold higher than Caco-2 alone respectively (data not shown). The gene expression of the M-like cells markers GP2 increased significantly when the co-cultures were grown with Raji-B cells in the basolateral chamber compared to Caco-2 alone (9.6±085 fold higher than Caco-2). Sucrase isomaltase (SI) mRNA relative expression was not significantly different between all conditions.

[0067] To investigate the permeability of this in vitro triple co-culture model, we evaluated the epithelial barrier integrity by measuring TEER. The TEER values increased with time for all cells (FIG. 1A). At the end of the 21 days of differentiation process, Caco-2 monoculture presented the highest value (390±37 Ω.Math.cm2) followed by double co-culture (Caco-2/HT29-MTX) (364±20 Ω.Math.cm2) while triple co-culture (Caco-2/HT29-MTX/RajiB) showed a value of 264±99 Ω.Math.cm2. Those values were not significantly different from Caco-2 alone (p>0.05).

[0068] The cell permeability was investigated by measuring the paracellular efflux of a fluorescent tracer, Lucifer yellow (LY), across our models. After 7 days of culture, LY permeability decreased for all conditions. These results matched with TEER values as at 7 days.

[0069] After 21 days of differentiation, there was no detectable amount of LY in the basal chamber whereas in insert without cells, LY could be found (FIG. 1B).

B. fragilis and S. Heidelberg Translocation Across Triple Co-Culture Model

[0070] To evaluate the impact of M cells on bacteria translocation in the triple co-culture model, we evaluated the translocation rate of two different bacteria strains across this model. Those were a commensal, B. fragilis a non-toxigenic strain, and a pathogen, S. Heildeberg [9]. We compared data with measurement within the Caco-2 model and the double cell type model (Caco-2 and HT29-MTX without M Cells). For this purpose, we enumerated each strain of bacteria in basal compartment. S. Heidelberg translocated with the highest efficiency across triple co-culture model (5.9%±1.9) after 3 h of incubation whereas the translocation rate was 0.0003% ±0.00006 in Caco-2 alone and 0.002%±0.001 in double co-culture model (FIG. 2A). Concerning B. fragilis, the translocation was very weak through all cell models even in triple co-culture model (0.005%±0.006) (FIG. 2B).

[0071] The impact of bacteria exposure on the integrity of the different models was evaluated by measuring TEER (FIG. 2C). When double and triple co-culture models were exposed to B. fragilis, no significant modification of the TEER was shown (double and triple co-culture with a TEER of 303±42 Ω.Math.cm2 and 327±9 Ω.Math.cm2 respectively). In presence of S. Heidelberg, TEER decreased significantly (p<0.05) in double (160±36 Ω.Math.cm2) and triple co-culture (121±30 Ω.Math.cm2) compared to model without bacteria (302±36 Ω.Math.cm2 and 296±9 Ω.Math.cm2 respectively). In double co-culture model, TEER is of 160±36 Ω.Math.cm2 but only 0.002%±0.001 of S. Heidelberg translocated in basal compartment whereas in triple co-culture model where TEER is 121±30 Ω.Math.cm2, the translocation was of 5.9%±1.9.

[0072] Measuring the paracellular transport of LY across the different models infected by S. Heidelberg or B. fragilis, no significant increase of LY permeability was observed, compared to untreated cells (FIG. 2D). Thus, it appears that infection with S. Heidelberg significantly decreased the TEER of the triple model, but not enough to compromise the barrier integrity.

[0073] Enteric pathogens are known to perturb the intestinal epithelial barrier by modifying tight junctional proteins: zonula occludens (ZO) and occludin. Occludin and ZO-1 mRNA analysis showed that only occludin gene was significantly increased compared to uninfected cells in presence of S. Heidelberg. However, in presence of B. fragilis, the genes expression was the same as in the case of uninfected cells.

[0074] S. Heidelberg Translocation is Inhibited in Triple Co-Culture Model in Presence of B. fragilis

[0075] To evaluate whether B. fragilis can modify S. Heidelberg translocation rate, we mixed the two bacteria (S. Heidelberg/B. fragilis) in the upper chamber of the triple co-culture model. After a 3 h incubation, we quantified the presence of S. Heidelberg and B. fragilis in the basal compartment. We found that 89% of S. Heidelberg translocation was significantly (p<0.05) inhibited in presence of B. fragilis (FIG. 3A). Enumeration of each bacteria in the upper compartment at 3 h and 24 h (FIG. 3B) showed that there was no significant impact of B. fragilis on S. Heidelberg growth in the upper chamber. B. fragilis growth was also not impacted by the presence of S. Heidelberg (FIG. 3B).

[0076] Then, we analyzed the impact of a bacterial S. Heidelberg and B. fragilis co-culture on TEER and compared it to the model infected with only one of the bacteria (FIG. 3C). TEER in triple co-culture model infected by both bacteria was the same as when the co-cultures were exposed to S. Heidelberg alone and was lower than in cells infected by B. fragilis alone. The LY transfer rate was not modified in this condition (data not shown). Occludin and ZO-1 expression were the same as when this model was infected with S. Heidelberg alone (FIG. 3D).

B. fragilis Supernatant Inhibited S. Heidelberg Translocation

[0077] In order to study the role of bacterial secreted substances on the inhibition of S. Heidelberg translocation, we examined whether B. fragilis supernatant could abrogate the effects of S. Heidelberg on intestinal cells. Firstly, we explored whether treatment with B. fragilis supernatant could reduce S. Heidelberg growth. The results presented in FIG. 3B showed that B. fragilis supernatant did not affect the growth of S. Heidelberg. When triple co-culture model was exposed to S. Heidelberg mixed to B. fragilis supernatant, a significant inhibition of Salmonella translocation was shown (FIG. 3A) without significantly affecting TEER or occludin gene expression (FIG. 3C). The level of S. Heidelberg translocation inhibition by B. fragilis supernatant was in the same range that the inhibition observed when S. Heidelberg was mixed with B. fragilis (86% and 89% respectively).

B. fragilis and Its Cell Free Supernatant Inhibited Other Enterobacteriae Translocation Such as E. coli

[0078] In order to investigate if B. fragilis or its cell free supernatant impact translocation of other enterobacteria, we have used Escherichia coli (E. coli ATCC11775). When triple co-culture model was exposed to E. coli mixed to B. fragilis or its free supernatant, an inhibition of E. coli translocation was shown (FIG. 4).

Conclusion

[0079] By using an original triple co-culture model including Caco-2 cells (representing human enterocytes), HT29-MTX (representing mucus-secreting goblet cells), and M cells differentiated from Caco-2 by addition of Raji B lymphocytes, bacterial translocation was evaluated. The data showed that S. Heidelberg could translocate in the triple co-culture model with high efficiency, whereas for B. fragilis a weak translocation was obtained. When cells were exposed to both bacteria, S. Heidelberg translocation was inhibited. The cell-free supernatant of B. fragilis also inhibited S. Heidelberg translocation without impacting epithelial barrier integrity. This supernatant did not affect the growth of S. Heidelberg, demonstrating that the effects of growth (i.e. increasing number of bacteria over the time) and the effects of translocation (i.e. passage of bacteria across the intestinal epithelium) have to be differentiated in this study. The non-toxigenic B. fragilis confers health benefits to the host by reducting bacterial translocation.

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