COMPOSITIONS FOR TREATING EPITHELIAL BARRIER FUNCTION DISORDERS

Abstract

The present disclosure relates to novel pharmaceutical compositions comprising recombinantly engineered probiotic bacteria that can be used, inter alia, in the treatment of gastrointestinal inflammatory diseases and epithelial barrier function disorders. The probiotic bacteria preferably contain a nucleic acid encoding the heterodimeric protein of SEQ ID NO:1 and 2, or homologous sequences thereof sharing at least 80% identity with said sequences. In some embodiments, the pharmaceutical compositions described herein have particular application in the treatment or prevention of disease states associated with abnormally permeable epithelial barriers as well as inflammatory bowel diseases or disorders.

Claims

1. A recombinant nucleic acid encoding a heterodimeric protein comprising a first polypeptide whose amino acid sequence is SEQ ID NO: 1 or a homologous sequence thereof having the same biological function, and a second polypeptide whose amino acid sequence is SEQ ID NO: 2 or a homologous sequence thereof having the same biological function.

2. The recombinant nucleic acid of claim 1, encoding a heterodimeric protein whose first and second polypeptides have a sequence having at least 80% sequence identity with SEQ ID NO:1 and SEQ ID NO:2 respectively.

3. The recombinant nucleic acid of claim 1, comprising a first polynucleotide having the sequence SEQ ID NO:3 and a second polynucleotide having the sequence SEQ ID NO:4, or homologous sequences thereof, in the same Open Reading Frame.

4. The recombinant nucleic acid of claim 1, comprising the polynucleotide having the sequence SEQ ID NO:8, or an homologous thereof sharing at least 80% identity with said sequence and having the same biological function.

5. The recombinant nucleic acid of claim 1, comprising the polynucleotide having the sequence SEQ ID NO:5, or an homologous thereof sharing at least 80% identity with said sequence and having the same biological function.

6. The recombinant nucleic acid of claim 3, wherein said first and second polynucleotides are operably linked to regulatory sequences allowing their expression in a host cell.

7. An expression vector or cassette comprising the recombinant nucleic acid as defined in claim 1.

8. (canceled)

9. A recombinant host cell, comprising the expression vector or cassette of claim 7.

10. The recombinant host cell of claim 9, comprising a polynucleotide having the sequence SEQ ID NO:5 a polynucleotide having the SEQ ID NO:8.

11. The recombinant host cell of claim 9, wherein it is a genetically engineered prokaryotic bacterium.

12. The recombinant host cell of claim 11, wherein it is a non-pathogenic bacteria chosen from the group consisting of: Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia coli, Lactobacillus, Lactococcus and Saccharomyces, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactococcus lactis, and Saccharomyces boulardii.

13. The recombinant host cell of claim 11, wherein it is a Gram-negative non-pathogenic bacterium.

14. (canceled)

15. A pharmaceutical composition comprising the recombinant host cell of claim 11 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising at least one muropeptide chosen from the group consisting of: ##STR00007## ##STR00008## and a pharmaceutically acceptable carrier.

17. A method for: reducing gastrointestinal inflammation, reducing intestinal mucosal inflammation, increasing wound healing, increasing intestinal epithelial cell proliferation, or for treating or preventing epithelial barrier function disorders, said method comprising the step of administering the recombinant host cell of any of claim 8 to a patient in need thereof.

18. The method of claim 17, wherein said epithelial barrier function disorder is chosen in the group consisting of: inflammatory bowel disease, ulcerative colitis, pediatric UC, Crohn's disease, pediatric Crohn's disease, short bowel syndrome, mucositis GI mucositis, oral mucositis, mucositis of the esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon), and/or rectum, chemotherapy-induced mucositis, radiation-induced mucositis, necrotizing enterocolitis, pouchitis, a metabolic disease, celiac disease, irritable bowel syndrome, or chemotherapy associated steatohepatitis (CASH).

19. (canceled)

20. A recombinant heterodimeric protein comprising a first polypeptide whose amino acid sequence is SEQ ID NO: 1, and a second polypeptide whose amino acid sequence is SEQ ID NO: 2, or homologous sequences thereof sharing at least 80% identity with said sequences and having the same biological function.

21. The recombinant heterodimeric protein of claim 20, comprising the amino acid sequence of SEQ ID NO:9 or a homologous sequence thereof sharing at least 80% identity with SEQ ID NO:9 and having the same biological function.

22. The recombinant heterodimeric protein of claim 20, wherein said biological function is measured by analysing the presence of the muropeptides as defined in claim 16 in the supernatant of cells overexpressing said recombinant heterodimeric protein.

23. The recombinant heterodimeric protein of claim 20, wherein said biological function is measured by contacting the supernatant of cells overexpressing said heterodimeric protein with dendritic cells, and observing the upregulation of IL-genes in said cells, or the secretion of IL-10 by said cells.

Description

FIGURE LEGENDS

[0174] FIG. 1: AlphaLISA. The supernatant from the complemented F4D5:MsbA1-A2 clone (A1A2) is able to stimulate IL10 secretion from DCs (results obtained using whole supernatants).

[0175] FIG. 2: Fractionation of the supernatant of the clone F4 on HPLC (HyperCarb column). Fractions were recovered, dried to eliminate acetonitrile, resuspended in water and tested on HEK Null NF-kB reporter cells.

[0176] FIG. 3: AlphaLISA. F4 fractions positive on HEK Null NF-kB reporter cells (cf. FIG. 2) were tested by Alpha LISA on IL10 secretion by DCs.

[0177] FIG. 4: Gene expression analysis on HT-29 cells treated with the HPLC-HILIC fraction of F4D5:A1A2 (positive clone) and F4D5-pBAD (negative control). TNFα (10 ng/ml), LPS (10 ng/ml), Na-Butyrate (2 mM).

[0178] FIG. 5: TEER measurement 4H post-infection. F−=Fraction from negative control (FADS-pBAD); F+=Fraction from positive F4D5:MsbA1-A2 (A1A2).

[0179] FIG. 6: Hematoxylin and eosin (H&E) staining at Objective ×40, on tissues infected with LF82-GFP bacteria, treated or not with F− or F+. F−=Fraction from negative control (pBAD); F+=Fraction from positive F4D5:MsbA1-A2 (A1A2).

[0180] FIG. 7: A. Transformed Epi300 bacteria with A1A2 transporter secrete EB7020/EB7021/EB7022 muropeptide precursors very efficiently (10 μM range). B. LC-MS analysis of the supernatant of the clone F4.

[0181] FIG. 8: Chemically synthesized EB7020 (F13) generates IL-10 secretion from human DCs.

[0182] FIG. 9: Synergy between TLR activators and EB7020/EB7021/EB7022 in generating IL10. LPS 100 ng/ml, FIMH 10 μg/ml, Mean of 3 MoDC donors

EXAMPLES

[0183] 1. Material and Methods

[0184] 1.1 Cell Culture

[0185] HEK Null NF-κB/SEAP reporter cells (Invivogen) were used to follow-up the purification of target compounds. The cells were maintained in RPMI 1640 medium (Sigma) with 2 mM L-glutamine, 50 IU/mL penicillin, 50 mg/mL streptomycin, 10 mM Hepes and 10% heat-inactivated foetal calf serum (FCS-Lonza) in a humidified 5% CO2 atmosphere at 37° C.

[0186] In activation tests, 30,000 reporter cells/well were seeded in RPMI in 96 wells plates 24h before activation (and kept in a humidified 5% CO2 atmosphere at 37° C.). Pre-purified, or HPLC-eluted, fractions were added to reporter cells at 10% vol/vol to a final volume of 100 μl. SEAP in the supernatant was revealed 24 h after cell stimulation using Quanti-Blue™ reagent (Invivogen) according to the manufacturer's protocol and quantified as OD at 655 nm. Measurements were performed using a Epoch microplate reader (Biotek). Cytotoxicity of target compounds during the purification process was measured (when appropriate) using the The CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega).

[0187] 1.2 AlphaLISA

[0188] Dendritic Cells (DCs) were prepared from PBMC (Buffy Coat) and differentiated with GM-CSF (D 1:10000; =5 μl/50 ml) and IL-4 (D 1:5000; =10 μl/50 ml).

[0189] In this test, 30,000 cells/well were seeded in complete IMDM media (2 mM L-glutamine, 50 IU/mL penicillin, 50 mg/mL streptomycin, 10 mM Hepes and 10% heat-inactivated foetal calf serum) in 96 wells plates 24h before activation (and kept in a humidified 5% CO2 atmosphere at 37° C.). Pre-purified, or HPLC-eluted, fractions as well as EB7020 chemically synthetized and various controls were added to reporter cells at 10% vol/vol to a final volume of 100 μl.

[0190] AlphaLisa (Perkin Elmer) was realized on supernatant according the supplier protocol (for complete references: AlphaLISA® Research Reagents Human Interleukin 10 (IL10) Kit Product No.: AL218 C/F sold by PerkinElmer).

[0191] 1.3. Cloning of MsbA1-A2: Primer List

[0192] Target genes were amplified using Phusion High Fidelity DNA Polymerase (New England BioLabs) and primers A1EcoRfw and A2Xbarv (MsbA1-A2 gene).

TABLE-US-00002 A1EcoRfw (SEQ ID NO: 6) (TTTTGAATTCTTTAGGAGGttttttatgtttcagttaaagtgggtgtgg aagcag) A2XbArv (SEQ ID NO: 7) (TTTTTCTAGAtcagctctccagctgggccgtgtacagatggtaataata tc)

[0193] Said forward primer contains the AGGAGG RBS consensus sequence.

[0194] 1.4. Complementation of F4D5 Mutation (Epi300 Transformation)

[0195] E. coli Epi300 electrocompetent cells were co-transformed with F4D5 fosmid together with pBAD-MsbA1-A2 plasmid by electroporation. Transformants were selected onto LB agar plates containing 12.5 ug/ml Chloramphenicol, 50 ug/ml Kanamycin and 100 ug/ml Ampicillin.

[0196] 1.5. Array (DCs)

[0197] DCs from three different donors were seeded at 1.0×10.sup.5 DCs/well in a 48 well plate in 0.5 ml of complete IMDM. The effect of the active fractions of the F4 clone was compared to the same fractions purified from the negative control (pBAD-fractions). TNF-α (10 ng/ml), PHA (3 μg/ml), LPS (3 ng/ml), PAM3CSK4 (10 ng/ml), and MDP 5 μg/ml were used as control.

[0198] Data were generated and analyzed using Spotfire software.

[0199] 1.6. RT-Q-PCR

[0200] HT-29 were seeded on 48 wells plate at 150,000 cells/well in 0.5 ml of complete RPMI. Cells were stimulated in a final volume of 0.3 ml with 30 ul of 10× agonists (or pre-purified fractions) for 24 h with: TNF-α (10 ng/ml), LPS (10 ng/ml), Na-butyrate (2 mM), F4 and pBAD-F4 HPLC-Hilic fraction. Before lysis (0.35 ml/well RTL buffer, Qiagen) cells were washed with PBS.

[0201] RNA was extracted using the Rneasy minikit (Qiagen). RNA was eluted in 20 μl of MQ water and quantified by Qubit. Reverse Transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem) using 1 μg RNA/reaction.

[0202] Data were analyzed through the Bio-Rad CFX Manager 3.1 software.

[0203] 1.7. Ex Vivo Experiments

[0204] Human ileal explants were mounted on Ussing chambers and left untreated or pre-treated for 1 h at 37° C. with: [0205] pre-purified fractions of F4D5:MsbA1-A2 clone (designed as F+ in the “Result” section) [0206] pre-purified fractions of its negative control F4D5-pBAD (designed as F− in the “Result” section)

[0207] After the 1 h pre-incubation period at 37° C. in a CO2 incubator bacteria were added into the apical compartment of the Ussing chambers at 4×10.sup.7 and 1×10.sup.9 bacteria/mL for Salmonella typhimurium and E. coli LF82-gfp, respectively.

[0208] The integrity/tightness of the human explants during the incubation was evaluated through Transepithelial Electrical Resistance (TEER) measurement using Millicell-ERS (Electrical Resistance System) Voltohmmeter (Millipore).

[0209] At the end of the incubation (6 h for Patient 1 and 4 h for Patient 2), human explants were washed 6-times with 2 mL of PBS, were removed from Ussing chambers and were fixed using PBS PFA 4% solution at 4° C. during 24 h. Fixed human explants were then processed for microscopy and histological analysis (H&E staining).

[0210] 1.8. LC-MS Analysis

[0211] The analysis was carried out using a UPLC system (Vanquish, Thermo Fisher Scientific) coupled with a high-resolution quadrupole-orbitrap mass spectrometer (Q Exactive™ HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific). An electrospray ionization interface was used as ionization source. Analysis was performed in negative ionization mode. A QC sample was analysed in MS/MS mode for identification of compounds. The UPLC was performed using a slightly modified version of the protocol described by Catalin et al. (UPLC/MS Monitoring of Water-Soluble Vitamin Bs in Cell Culture Media in Minutes, Water Application note 2011, 720004042en). Peak areas were extracted using TraceFinder 4.1 (Thermo Scientific). Identification of MtriDAP-MP were performed using retention time (compared against an authentic standard) and accurate mass (with an accepted deviation of 3 ppm), and for UDP-MtriDAP and UDP-MtetraDAP using accurate mass alone (with an accepted deviation of 3 ppm).

[0212] 2. Results

[0213] 2.1. Identification of the F4 Clone

[0214] The F4 clone has been identified by screening 5000 metagenomic Escherichia coli clones from a healthy donor library, because this clone demonstrated activity on AP-1 and NE-KB reporter system in HT-29 cells.

[0215] Sequencing analysis demonstrated that this clone F4 encodes a contig of about 41 kb coming from a Firmicutes Gram-positive bacterium. By systematically mutating the F4 clone it was shown that the component responsible of said biological activity is a putative MsbA1-MsbA2-like heterodimeric transporter.

[0216] The sequences encoding said transporter were identified (SEQ ID NO:3 and SEQ ID NO:4). In the contig, the two genes were found to be consecutive, in the same Open Reading Frame (ORF) (SEQ ID NO:8).

[0217] 2.2. Subcloning of the Target Genes of Interest

[0218] The F4 clone was shown to be very unstable and to easily loose its biologic activity. The sequence of the genes of interest, encoding the MsbA1-MsbA2-like transporter, were therefore subcloned so as to complement the loss of activity of the parental clone. To do that, the sequence encoding the MsbA1-MsbA2-like transporter (SEQ ID NO:8) was amplified from the original F4 fosmid and cloned into the arabinose induced pBAD30 vector (containing the arabinose PBAD promoter of the araBAD “arabinose” operon). The construction was verified by sequencing (see methods for details).

[0219] This new construction was called pBAD-MsbA1-A2. It was used to complement the F4D5 transposed clone (containing the inactive msbA1-msbA2-like genes). The complemented and functional clone is called F4D5:MsbA1-A2 clone (also referred as “A1A2”).

[0220] The appropriate transformants were selected on LB-agar plates containing Ampicillin (100 μg/ml), Kanamycin (50 μg/ml) and Chloroamphenicol (12.5 μg/ml). Various L-Arabinose percentages were tested, to induce the Ara promoter and to effectively produce active compounds into the liquid bacterial culture. Importantly, complementation of the transposed clone (F4D5) with the construction encoding the MsbA1-MsbA2-like transporter allowed to restore the original activity of the F4 clone, confirming the key role of said genes for the biological activity of these bacteria. For this reason, the complemented F4D5:MsbA1-A2 clone (A1A2) was used for all the experiments described hereafter. The F4D5-pBAD clone (F4D5 clone transformed with an empty vector) was also used as negative control.

[0221] 2.3. Biological Activity of the Polypeptides Encoded by the Target Genes of Interest

[0222] Surprisingly, the clone F4D5:MsbA1-A2 was shown to be able to produce a supernatant displaying anti-inflammatory properties.

[0223] 2.3.1. This Supernatant Contains Compounds that Enhance the Secretion of IL10 by Dendritic Cells (DCs)

[0224] As shown on FIG. 1, the clone F4D5:MsbA1-A2 was shown to increase the secretion of IL10 by DCs.

[0225] HPLC fractionation of pre-purified supernatant through a Hypercarb column and an Acetonitril-TFA gradient allowed to identify three biologically active fractions (F13, F22 and F25) when tested on HEK Null reporter cells (FIG. 2). These three fractions were then tested on DCs and two of these (F13 and F25) were able to stimulate IL-10 secretion and to induce an upregulation of CD80, CD83, and CD86 differentiation clusters. (FIG. 3).

[0226] The bioactive compounds present in these fractions were then characterized.

[0227] 2.3.2. This Supernatant Contains Bioactive Anti-Inflammatory Muropeptide Precursors.

[0228] The purification of the two active fractions (F13 & F25) resulted in the identification of three compounds corresponding to the following muropeptide precursors:

##STR00005## ##STR00006##

[0229] EB7020 is a M-triDAP monophosphate having the formula C.sub.26H.sub.44N.sub.5O.sub.18P. Its exact mass is 745,2419; its Molecular weight is of 745,6240.

[0230] EB7021 is a UDP-M-Tri-DAP monophosphate having the formula C.sub.35H.sub.55N.sub.7O.sub.26P.sub.2. Its exact mass is 1051,267; its Molecular weight is of 1051,790.

[0231] EB7022 is a UDP-M-tetrapeptide monophosphate having the formula C.sub.38H.sub.60N.sub.8O.sub.27P.sub.2. Its exact mass is 1122,304; its Molecular weight is of 1122,868.

[0232] The quantity of the three muropeptides that are secreted has been evaluated by LC-MS (FIG. 7). EPI300 bacteria that are transformed with the vector of the invention encoding the heterodimeric transporter are able to secrete between 5 and 20 μM of each muropeptide precursor (FIG. 7A).

[0233] These muropeptide precursors participate in the protective effect of the epithelium which is specifically observed in inflammatory circumstances.

[0234] 2.3.3. F4D5-MsbA1-A2 Transformed Epi300 Bacteria Secrete these Bioactive Anti-Inflammatory Muropeptide Precursors.

[0235] M-TriDAP-MP EB7020 was quantified from F4D5-MsbA1-A2 and F4D5-pBAD supernatants by LC-MS using a calibration curve generated with chemically synthesized M-TriDAP-MP (FIG. 7).

[0236] UDP-M-TriDAP EB7021 and UDP-M-TetraDAP EB7022 amounts were extrapolated from their known mass using the same calibration curve.

[0237] Very high yield, about 10 μM range for M-Tri-DAP-MP and UDP-M-TetraDAP have also been obtained.

[0238] Pre-purified fractions from Epi300 bacteria transformed with F4D5:A1A2, but not those transformed with negative control F4D5-pBAD, are able to enhance the production of IL10, when combined with TLR activators (FIG. 9) and to protect human ileal resections against loss of TEER (FIG. 5).

[0239] Many studies were then performed to investigate the anti-inflammatory activity of these muropeptide precursors: [0240] Array on Dendritic Cells stimulated by the IL10 positive fractions of the F4D5-MsbA1-A2 clone [0241] Evaluation of IL-10 secretion induced by chemically synthesized EB7020 in presence of bacteria supernatant or LPS [0242] RT-Q-PCR on HT-29 epithelial cells [0243] Ex-vivo studies.

[0244] Due to the complexity of the purification process and the difficulties to produce sufficient amounts of purified fractions, most of the experiments were realized using samples submitted to several pre-purification steps (ultra-filtration+acetone precipitation+HPLC-HILIC fractionation). Moreover, the EB7020 compound has been successfully synthetically produced.

[0245] 2.3.4. This supernatant contains compounds that are involved in the maintaining of epithelial integrity.

[0246] Transcriptomics was carried out on PBMC derived DCs from 3 donors.

[0247] DCs were stimulated (6h) with: [0248] TNFα, PHA, LPS, PAM3CSK4, MDP=Positive control [0249] F13, F25=HPLC fractions obtained from the complemented and functional F4D5-MsbA1-A2 clone [0250] F4D5-pBAD-F13, F4D5-pBAD-F25=HPLC fractions obtained from the F4D5-pBADclone (negative control)

[0251] Data analysis was realised using TIBCO Spotfire software. The F4 fractions (from both positive and negative clones) were analysed individually and compared to known agonists (TNFα, PHA, LPS, PAM3CSK4, MDP).

[0252] As shown in Table 2, most of the upregulated genes when DCs are contacted with these fractions are involved in the maintaining of epithelial integrity (PERP, PMP22, SLC39A14,) suggesting a protective role of the compounds present in these fractions. This hypothesis is reinforced by the fact that most pro-inflammatory genes (like those encoding IL6, CCL1, CCL8, TNF-α) are downregulated, as shown in Table 3.

TABLE-US-00003 TABLE 2 List of Up-regulated genes by F25 fraction. GeneSymbol TNF-α PHA LPS PAM MDP F25/pBAD PERP 1.0 1.7 1.8 2.6 1.6 2.0 PMP22 0.8 0.7 0.8 1.0 1.0 2.4 SLC39A14 1.2 1.0 1.0 1.6 0.9 2.3

[0253] Up-regulated genes (all of them are involved in epithelial integrity). Data of F25 from positive clone are normalized versus the corresponding fraction from the negative clone.

TABLE-US-00004 TABLE 3 List of Down-regulated genes by F25 fraction. GeneSymbol TNF-α PHA LPS PAM MDP F25/pBAD IL6 32.9 525.8 756.8 718.1 9.4 0.3 CCL1 4.2 25.4 38.6 30.6 3.1 0.6 CCL8 11.2 193.6 271.6 64.8 4.1 0.5 TNF-α 5.6 17.5 22.6 28.4 3.5 0.7

[0254] Down-regulated genes (pro-inflammatory cytokine and chemokines). Data of F25 are normalized versus F25 pBAD30 values.

[0255] Because modulation of genes linked to cellular adhesion (PERP) was observed in DCs, the effect of the bioactive supernatant was investigated on the modulation of several genes regulating tight junctions in the intestinal epithelial cell line HT-29 (see methods for details).

[0256] For this experiment pre-purified fractions from positive F4D5-MsbA1-A2 and from F4D5-pBAD negative clones) were used.

[0257] As shown in FIG. 4, the tested fraction of the F4D5-MsbA1-A2 clone is able to upregulate various genes linked to tight junctions in epithelial cells. This means that the bioactive compounds present in the supernatant of this clone have a protective role on the epithelial barrier.

[0258] 2.3.5. Chemically Synthesized EB7020 Generates IL-10 Secretion from Human DCs.

[0259] As shown in FIG. 8, IL-10 secretion is induced by chemically synthesized EB7020(F13) in presence of LPS.

[0260] 2.3.6. Synergy Between the Pre-Purified Supernatants from Transformed Bacteria and TLR Activators.

[0261] According to the literature NOD1 and NOD2 agonists synergise with LPS which is a TLR4 ligand.

[0262] Pre-purified fractions from both positive (F4D5:MsbA1-A2) and negative (F4D5-pBAD) clones were tested in co-stimulation with either LPS or FimH (a bacterial adhesin) for their capacity to induce IL-10 secretion from DCs. The results are shown in FIG. 9.

[0263] It is observed a strong synergy of the fractions of the invention with TLR4 agonists such as LPS or FIMH on IL-10 secretion. This is consistent with literature pertaining to the MoA of muropeptide precursors.

[0264] 2.3.7. Ex Vivo Experiments.

[0265] Ex vivo experiments were also performed to study the properties of the F4 supernatant on a different and more physiological system. The effect of the bioactive compounds was assessed on ileal explants treated with live bacteria (E. coli LF82 and Salmonella thyphimurium) in order to mimic an intestinal inflammation.

[0266] Human ileal explants were obtained from two patients and treated within 3 h from the surgery (only the non-inflamed part of the tissue is considered for the experiment). Explants were pre-treated for 1h with the control or active fractions before being loaded with live bacteria. For this experiment, pre-purified samples were used (10 kDa filtered+acetone precipitation+C18 SPE). Infection was done during 4h. For each patient, measurement of transepithelial resistance (TEER), and histology were realized.

[0267] In the following and in FIGS. 5 and 6, the fraction obtained from the active F4D5:MsbA1-A2 clone is indicated as “F+” while the fraction obtained from the negative control (F4D5-pBAD) is indicated as “F−”.

[0268] Measurement of TEER was found to be comparable between the two patients. In both cases, the positive sample exerted a protective function on the tissue loaded with the pathogenic bacteria (FIG. 5).

[0269] In parallel with the measurement of TEER on ex vivo human explants, a comparative histology study was performed on patient 2, for which the tissue at TO was found non-inflammatory, with a mucosa of normal thickness. The mucosa and the submucosa stand perfectly.

[0270] Infection with LF82-GFP Bacteria (FIG. 6).

[0271] After 4 hours of incubation with culture media, the thickness remained normal and the mucosa was normal at the level of crypts and on the first third of the villosities. The normal thickness of the mucosa at the level of the crypt showed that the tissue was not inflammatory.

[0272] The explants incubated with F+ gave similar structure compared to control at T=4h.

[0273] However, after 4 hours of incubation with the LF82-GFP bacteria, a strong release of the submucosa and a strong secretion of mucus were observed, which resulted in a strong desquamation of the mucosa at the level of the villosities. At the level of the crypts, the thickness of the mucosa greatly diminished, probably due to an important inflammatory state.

[0274] After 4 hours of incubation with the LF82-GFP bacteria in the presence of the fraction F−, no differences were observed with respect to the bacteria alone. Significant and greater desquamation at the level of the villosities could be due to an irritating effect of the fraction F-which was added to an installed inflammatory state caused by the LF82-GFP bacteria.

[0275] On the other hand, the fraction F+ clearly improved the general state of the tissue. It was found that the treated tissue corresponded to the T 4h control without infection. Moreover, the bioactive compounds present in the fraction F+ blocked the inflammatory effect caused by LF82-GFP infection.

[0276] Infection with Salmonella typhimurium

[0277] As with infection with E. coli LF82-GFP, the infection by Salmonella typhimurium bacteria caused a strong destruction of the mucosa at the level of villosities. The addition of the compound F+ reduced the desquamation to the level of control state, with reappearance of a continuous mucosa around the submucosa even when the submucosa was lost.

[0278] In conclusion, a clear protective effect was observed for the bioactive compounds present in F+ on the infection caused by LF82-GFP or Salmonella typhimurium bacteria. Indeed, the bioactive compounds present in F+ strongly reduced the desquamation of the mucosa and therefore the inflammatory state of the tissue caused by the bacterial infection. On the other hand, the compounds present in F− had no effect on bacterial infections, either with LF82-GFP or with Salmonella typhimurium.