TREATMENT OF RESPIRATORY DISEASES WITH A BACTERIUM OF THE GENUS LACTOBACILLUS

Abstract

The present invention relates to the use of a bacterium of the genus Lactobacillus in the treatment of respiratory diseases in humans as well as in animals. in particular, a particular strain of this bacterium and pharmaceutical compositions comprising it are described.

Claims

1. A bacterium of the genus Lactobacillus for use in the treatment and/or prevention of inflammation associated with a respiratory disease, the bacterium comprising a polynucleotide having a sequence which has at least 98% identity with the sequence of SEQ ID NO: 1.

2. The bacterium for use as claimed in claim 1, characterized in that the bacterium is a Lactobacillus animalis or a Lactobacillus murinus.

3. The bacterium for use as claimed in any one of claim 1 or 2, characterized in that the bacterium is inactivated.

4. The bacterium for use as claimed in any one of claims 1 to 3, characterized in that the treatment and/or prevention comprises a decrease in leukocyte infiltration and an increase in pulmonary populations of Th17 cells as well as Tregs cells.

5. The bacterium for use as claimed in claim 4, characterized in that the Tregs are iTregs.

6. The bacterium for use as claimed in claim 5, characterized in that the iTregs are biTregs.

7. The bacterium for use as claimed in any one of claims 1 to 6, characterized in that the respiratory disease is tuberculosis.

8. The bacterium for use as claimed in any one of claims 1 to 6, characterized in that the bacterium is the strain Lactobacillus sp. deposited at the Collection nationale des cultures de microorganisms (CNCM, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) under the number I-5314.

9. A strain of Lactobacillus sp. deposited at the Collection nationale des cultures de microorganisms (CNCM, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) under number I-5314.

10. A pharmaceutical composition comprising the strain of claim 9 and at least one pharmaceutically acceptable excipient.

11. The composition of claim 10 characterized in that the strain is inactivated.

12. The composition of claim 11 characterized in that the strain is heat inactivated.

13. The composition of claim 11 characterized in that the strain is present in the form of extracts.

Description

LEGENDS OF THE FIGURES

[0064] FIG. 1: Growth kinetics of strain CNCM I-5314. Growth characteristics of lactobacilli. The growth analysis of strains CNCM I 4968, CNCM I-5314 and CNCM I 4967 was carried out by measurements on fresh cultures made from 1/100 dilutions of precultures by measurements of optical density (OD) at 600 nm and of colony forming units (CFU). These measurements allow the preparation of inocula at the desired concentration from an OD measurement using the concordance factor between OD and CFU. The representation of the LN(OD) as a function of time also makes it possible to determine the generation time of the bacteria, calculated from the slope of the curve in exponential phase. Each point represents the average of two OD measurements. A representative experiment of 2-3 independent experiments is shown.

[0065] FIG. 2: Experimental protocol used to study the effect of administration of bacteria isolated from the lung microbiota on Mycobacterium tuberculosis (Mtb) infection. Six-week-old female C57BL/6 mice were intranasally (i.n.) administered 10.sup.7 bacteria (CNCM I 4968, or CNCM I-5314 or CNCM I 4967 or PBS) in 25 μL of PBS 3 times a week for 2 weeks. Depending on the experiment, they are then sacrificed (FIG. 2) or infected with 10.sup.3 CFU of Mtb (other figures). For infected mice, lactobacilli administration is continued until sacrifice at a rate of twice a week (groups treated before and after infection, denoted “bef/aft”). Alternatively, lactobacilli may be administered only after infection (CNCM group I-5314 ap) and not before. After sacrifice, the lungs are used either whole for histology analysis of immunopathology or homogenized to determine bacterial load and characterize the local immune response by flow cytometry.

[0066] FIG. 3: Modification of the pulmonary immune system of uninfected mice by bacteria isolated from the lung microbiota. C57BL/6 mice are intranasally administered 10.sup.7 bacteria (CNCM I4968 or CNCM I-5314 or CNCM I4967 or PBS) in 25 μL of PBS 3 times a week for 2 weeks. After sacrifice, a cell suspension is obtained by enzymatic and mechanical dissociation of the lungs. The proportions of CD4.sup.+ T cell subpopulations are determined by flow cytometry. A. Analysis strategy. After exclusion of doublets and dead cells, CD4.sup.+ T cells are selected. The proportion of different subpopulations is determined by selecting cells expressing a specific intracellular factor of interest (bottom panel) and not its control isotype (top panel). B. Proportion of CD4.sup.+ T cells expressing Foxp3 factor (called Treg), or producing TNF-α (Th1-like cells) or IL-17 (Th17). The graphs represent the median obtained by grouping 3 experiments with 4-7 mice each. Statistic: Kruskal-Wallis test: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

[0067] FIG. 4: Impact of administration of bacteria isolated from the lung microbiota on Mtb infection. C57BL/6 mice are intranasally administered 10.sup.7 bacteria (CNCM I-4968 or CNCM I-5314 or CNCM I-4967 or PBS) in 25 μL of PBS 3 times a week for 2 weeks. They are then infected with 1000 bacteria of Mtb strain H37Rv by intranasal route. After infection, the mice are given the lung bacteria as before twice a week for 30 days. After sacrifice the lungs are dissociated to estimate the bacterial load or fixed to assess tissue damage. A. Estimation of bacterial load measured by agar medium plating of ground lung tissue. Each graph represents an independent experiment comparing 2 groups of treated mice with a control group, n=3-7 mice per group. B. Tissue damage estimated by observation of hematoxylin-eosin (HE) staining performed on histological sections of lungs fixed in 10% formalin and embedded in paraffin. Representative images from 2 independent experiments n=3 mice per group.

[0068] FIG. 5: Experimental scheme for studying cytokine production by mouse lung explants in the presence of strain CNCM I-5314.

[0069] FIG. 6: Lactate dehydrogenase (LDH) assay. Bacterium 20: CNCM 5314; bacterium 11: known cytotoxic bacterium (positive control).

[0070] FIG. 7: Characterization of the impact of lactobacillus CNCM I-5314 on cytokine secretion in mouse lung explants. Mouse lung explants are placed in the presence or absence of strain CNCM I-5314 at 37° C. After 16 hours of incubation, cytokines are assayed by the Luminex technique. A. Pro-Th1/Th1 cytokines. B. Pro-Th2/Th2 cytokines. C. Pro-Th17/Th17/Th22 cytokines. D. Th9/Treg/Prolif cytokines. E. Pro-inflammatory cytokines. Strain CNCM I-5314 is listed as bacterium 20.

[0071] FIG. 8: Characterization of the impact of Lactobacillus I-5314 administration on Mtb infection. C57BL/6 mice receive PBS (white bars) or 10.sup.7 of lactobacillus intranasally before and after infection (CNCM I5314 bef/aft group, gray bars) per 1000 CFU of Mtb strain H37Rv intranasally, or only after infection (CNCM I-5314 aft group, hatched bars). A. Estimated bacterial load 42 days post-infection, measured by agar media plating of ground lung tissue. The graph shows a representative experiment from 2 independent experiments, n=6 mice per group. B. Tissue damage estimated by observation (left panel) and quantification of leukocyte infiltrates (right panel) on hematoxylin-eosin stains performed on histological sections of lungs fixed in 10% formalin and embedded in paraffin 42 days after infection. Percentage infiltration is the ratio of the area occupied by leukocyte infiltrates to the total area of the lungs. 2-5 experiments with 4-5 mice per group are shown. C.D. Characterization of lung CD4.sup.+ T cells present 42 days after infection by flow cytometry. The proportions of CD4.sup.+ TC expressing different transcription factors (C.) (T-bet, characteristic of Th1, RORγt for Th17 and Foxp3 for Treg) or producing cytokines (D.) after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin and brefeldin A (IFN-γ and TNF-α for Th1, IL-17 for Th17, IL-10 and TGF-β for Treg) are shown. E.F.G. Characterization of Tregs expressing Foxp3. The origin of Tregs, natural (nTreg, expressing Helios factor, top) or induced (iTreg, not expressing it, bottom) (E.), their proliferation (characterized by Ki67 antigen expression) (F.) and cytokine production (G.) is detected by flow cytometry as in C and D. Statistics: graphs corresponding to I-4 independent pooled experiments with 4-7 mice per group for panels C and D. The median of each group is shown and a Kruskal-Wallis test compares treated mice with control mice: * p<0.05; ** p<0.01; **** p<0.0001.

EXAMPLES

Materials and Methods

Bacterial Strains, Media, Growth Conditions

[0072] The pulmonary bacterial strains were isolated from mouse lung homogenates with a homogenizer (ULTRA-TURRAX (IKA) or TissueLyser (Qiagen)). They were then cultured on yhBHI, M17, MRS, or Mannitol Salt Agar (BD biosciences) for 24 to 48 h at 37° C. under aerobic conditions or 5 days at 37° C. in a Freter chamber under anaerobic conditions. Isolated strains were frozen at −80° C. in 16% glycerol. The identity of each strain was confirmed by mass spectrophotometry and PCR sequencing of 16S RNA. The selected strains were deposited at the Collection Nationale des Cultures de Microorganisms (CNCM). The three strains used here are lactobacilli strains deposited under the references CNCM I-5314, (CNCM I-4967) and (CNCM I-4968). These bacteria are cultured in MRS liquid medium for 24 h at 30° C. (pre-culture) or 3-4 h at 37° C. (for instillations) without agitation.

Material and Method for Sequencing

[0073] The bacterial pellet from a 15 mL culture of strain I-5314 (exponential phase) was centrifuged (1550 g, 5 min) and frozen at −20° C. This bacterial pellet was taken up in saline solution (30 mM NaCl, 2 mM EDTA (pH=8.0) then centrifuged and resuspended in lysis buffer (20 mM Tris HCl pH 8.0, 2 mM EDTA, 1.2% Triton® X-100) enriched in lysozyme (20 mg/mL, Sigma-Aldrich #L6876) for 2 h at 37° C. Proteinase K (DNeasy® Blood Et Tissue kit) and RNase A were added for an additional 1 h at 55° C. The same volume of buffer “AL” (DNeasy® Blood Et Tissue kit) was added to the lysate, vortexed and incubated 30 min at 56° C. After incubation, the same volume of 100% ethanol was added. DNA was extracted using the DNeasy Mini spin columns kit (Qiagen) and following the vendor's instructions.

DNA Sequencing.

[0074] Sequencing was produced by the “GeT-PlaGe” platform (INRA, Castanet-Tolosan, France). The DNA was fragmented by sonication to obtain fragments of 200 to 1000 base pairs (bp). These fragments were added to Illumina adapters and sequenced using the “Illumina HiSeq 3000” method.

Genome Assembly and Annotation.

[0075] The raw sequences were put in fastp, version 0.19.4 format to remove Illumina-like adaptor sequences and low-quality sequences. The sequences were assembled by “Unicycler version, v0.4.7” and the quality of the assembly was verified by QUAST v5.0.2, b7350347c. The assembled genome is visualized by “Bandage v0.8.1” and annotated by Prokka v1.13.

Bacterial Growth Kinetics

[0076] The growth profile was determined by spectrophotometry (Spectronic instruments 20; Genesys) which allows the measurement of the optical density (OD) from the culture medium containing the bacteria. To this end, the bacteria were pre-cultured on agar plates and then subcultured in 10 mL of Hiveg BHI medium (bacterium 20=CNCM I-5314). Growth was monitored by measuring the OD at 600 nm of the bacterial culture every hour from 0 h to 9 h+a reading at 24 h.

Murine Model of Tuberculosis Infection and Probiotic Treatment

[0077] All experiments performed on animals were approved by the French Ministry of Higher Education and Research after review by the Regional Ethics Committee (APAFIS Approval 5704). The mice used were C57BL/6 6-to-8-week-old females from Charles River Laboratories.

[0078] For each in vivo administration of one of the lactobacilli, a bacterial suspension containing 4.0×10.sup.8 CFU/mL was prepared in phosphate-buffered saline (PBS) from fresh cultures in exponential phase. Mice received 25 μL of PBS containing 1.0×10.sup.7 CFU or 25 μL of PBS (control group) intranasally (i.n.) under gas anesthesia (4% isoflurane, Virbac Denmark). This is repeated 3 times per week for 2 weeks and then the mice are either sacrificed (experiments on uninfected mice) or infected (procedures described below), and receive commensal bacteria administration again twice per week until sacrifice. In certain experiments, lactobacilli are administered only after infection and not before and after infection (see FIG. 1).

[0079] A fresh culture of Mtb strain H37Rv (grown in 7H9 liquid medium (Difco) supplemented with 0.5% glycerol, 10% ADC (Middlebrook) and 0.05% tyloxapol) is used to infect the mice. Each mouse receives i.n. 1.0×10.sup.3 CFU of Mtb in 25 μL of PBS under isoflurane anesthesia. Mice are sacrificed by cervical dislocation (under isoflurane anesthesia) after 42 days of infection.

Histological Analysis

[0080] Whole lungs from mice dedicated to histological analysis are used. They are inflated and fixed for 5 days at 4° C. with a solution containing 10% formalin (Formalin solution, Sigma-Aldrich) and embedded in paraffin. Hematoxylin-eosin (HE) staining was performed to visualize leukocyte infiltrates, which were quantified, after scanning, with the CaseViewer software (3DHISTECH).

Preparation of Lung Homogenates and Determination of Bacterial Load

[0081] Whole lungs from mice were collected in a sterile manner, homogenized with a gentleMACS dissociator before (C-tubes, cycle m_lung_01, Miltenyi) and after (cycle m_lung_02) 30 min incubation at 37° C. with collagenase D (2 mg/mL, Roche) and DNase I (0.1 mg/mL, Roche). A portion of this homogenate is serially diluted in PBS and then spread on 7H10 agar medium (Difco) supplemented with peptone and OADC (Middlebrook). After 2-3 weeks, the count of Mtb colonies obtained allows the estimation of the lung bacterial load. The remaining homogenates were passed through a 70 μm filter to destroy aggregates, and centrifuged at 329×g for 5 min. The supernatants were passed twice through 0.2 μm filters and stored at −80° C. for analysis of cytokines present in the lungs. Red blood cells in the pellet are lysed for 5 min with a solution containing 150 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA (pH 7.2), neutralized by addition of RPMI medium containing 10% fetal calf serum (FCS). The cell suspension thus obtained is filtered through a 40 μm filter (removal of lysed red cell aggregates) for flow cytometry analysis.

Flow Cytometry

[0082] Analysis of the different CD4.sup.+ T helper populations is performed by flow cytometric detection of transcription factors and cytokines characteristic of these subpopulations by labelling the cells present in the cell suspension obtained as described in the previous section. A portion of the cell suspension is incubated in RPMI containing 50 ng/mL phorbol myristate acetate (PMA, Sigma Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich) to induce cytokine production by lymphocytes as well as brefeldin A (Golgi plug 1/1000, BD Biosciences) and monensin (Golgi Stop 1/2000, BD Biosciences) to segregate these cytokines in the Golgi apparatus for 4 h at 37° C. and 5% CO.sub.2. The rest of the cell suspension, used for transcription factor labelling, is kept in cell staining buffer (CSB, Biolegend) for 4 h at 4° C. Extracellular labeling of these two fractions is performed in CSB for 30 min at 4° C. in the dark using an anti-cluster differentiation 16/32 antibody (CD16/CD32, Biolegend) to limit aspecific labeling, a viability marker (live/dead fixable blue dead cell stain kit, Invitrogen), an anti-CD452 BV711 (clone 104, BD Biosciences), an anti-CD3 FITC (17A2, Biolegend) or anti-T cell receptor beta (TCRb) Alexa 700 (H57-597, Biolegend) and an anti-CD4 BV786 (Sk3, BD Biosciences). For intracellular labeling, cells are then fixed for 30 min at room temperature (RT), permeabilized for 15 min at RT (Foxp3/transcription factor staining buffer set, eBioscience) and incubated for 45 min at RT with a panel of antibodies including an anti-RORgt PE-CF594 (Q31-378, BD Biosciences) anti-T-bet PE-Cy7 (eBio4BIO, eBiosciences), anti-Foxp3 APC (FJK-16s, eBioscience), anti-Helios APC-eFluor 780 (22F6, eBiosciences), anti-Ki67 Alexa 700 (SolA15, eBioscience) or an anti-interleukin 10 (IL-10) FITC (JES5-16E3, BD Biosciences), an anti-IL-17 PE (TC11-18H10, BD Biosciences), an anti-IFNγ PE-Dazzle (XMG1.2, Biolegend), an anti-TNFα PE Cy7 (MP6-XT22, BD Biosciences), the anti-Foxp3 APC and an anti-TGF-β BV421 (TW7-16B4, BD Biosciences). For experiments performed on Mtb-infected mice, cells are fixed for 2 h in 4% paraformaldehyde (PFA) at RT. Data are acquired with a FACS LSRII or Fortessa (BD Biosciences) and analyzed on FlowJo V10 software. Doublets (FSH-H vs. FSC-W and SSC-H vs. SSC-W) and dead cells (live/dead positive) are excluded at the beginning of each analysis.

Statistical Analysis

[0083] The statistical analysis of the results was performed with GraphPad Prism 7 software. On the graphs, each point represents a different mouse. The median of each group is represented by the bars. A Mann-Whitney test (comparison of 2 groups) or a Kruskal-Wallis test (comparison of 3 groups) was used to compare the values. Significance is represented by: * p<0.05; ** p<0.01; *** p<0.001; and **** p<0.0001.

Precision-Cut Lung Slices

[0084] Precision-cut lung slices (PCLS) were obtained from fresh lungs using a Krumdieck Md. 6000 microtome (Alabama Research and Development, Munford, Ala., USA). The lungs were filled with 1.5% low-melting agarose in RPMI (Invitrogen, Villebon sur Yvette, France) heated to 37° C. via the trachea. After 1 min for solidification, the lungs were placed in the Krumdieck microtome chamber filled with cold PBS and cut to a thickness of 200 μm. Two of the PCLS per well were then placed at 37° C., 5% CO.sub.2, in P24 well plates (Nunc, Sigma-Aldrich, Lyon, France) with 1 mL of RPMI 1640 (Gibco, Sigma-Aldrich, Lyon, France) supplemented with 10% heat-inactivated fetal calf serum (Gibco) and 2 mM L-glutamine (Gibco). The medium was changed every 30 min for 2 h to remove agarose, as well as one last time after overnight incubation. PCLS were then co-incubated for 24 h with lung bacteria.

Assay of Lactate Dehydrogenase

[0085] The lactate dehydrogenase (LDH) assay is used to determine the potential cytotoxicity of bacteria on lung explants. Indeed, the release of LDH is associated with cell death. The LDH assay was performed on explant lysates and on supernatants recovered 16 h post-culture with bacteria. For this assay, LDH substrate (Promega) is added to each well containing either explant lysates or post-culture supernatants. Lysis buffer is used as a blank for the explant lysates. Incubation is done at room temperature in the dark for 30 to 40 minutes. The reaction is then stopped with 100 μL of stop solution, the plate is then read at 490 nm with a plate reader (TECAN Infinite M200 pro) using the “I-control” software.

[0086] The assay of LDH on explant lysates and supernatants is used to determine the cytotoxicity of bacteria on lung explants. For LDH assayed on explant lysates, the blank must be subtracted from the values obtained.

[0087] Cytotoxicity represents the percentage ratio between the LDH released, i.e., present in the lung explant supernatant, and the total LDH present (in the supernatants+in the lung explant lysates), represented by the following calculation

[00001]$\mathrm{Cytoxicity}\left(\%\right)=\frac{\mathrm{ODLDH}\mathrm{released}\left(\mathrm{supernatants}\right)}{\begin{array}{c}\mathrm{ODLDH}\mathrm{released}\left(\mathrm{supernatants}\right)+\\ \left(\mathrm{ODLDH}\left(\mathrm{explant}\mathrm{lysates}\right)-\mathrm{blank}\right)\end{array}}\times 100$

Cytokine Assay

[0088] Cytokines were assayed using the Luminex technique. The Luminex technique uses magnetic beads with their own fluorescence, which allows a large number of cytokines to be assayed at the same time. Here, the magnetic bead has anti-IL-2 capture antibodies. Thus, when the supernatants are brought into contact with the beads in the wells, this bead will bind specifically to IL-2 via its multiple capture antibodies present on its surface. This identifies the cytokine bound to the bead. The detection antibody also recognizes the cytokine but is bound to the streptavidin-PE. The concentration of the assayed sample is directly proportional to the fluorescence intensity of the PE.

[0089] Cytokines were determined from the supernatant recovered 16 h post-culture with the bacteria. We used a Luminex kit to determine the concentration of fifteen cytokines in a single assay (Thermofisher).

TABLE-US-00001 TABLE 1 Cytokines measured. The cytokines assayed correspond to the cytokines released during different immune responses such as the type 1, 2, 9 or 17 and 22 response. Pro- Th1/Pro Th17/ Cell Immunity inflammatory Th1 Th2 Th22 Th9 proliferation Cytokines GM-CSF IFN-γ IL-4 IL-17A IL-9 IL-2 IL-1β IL-12p70 IL-5 IL-22 TNF-α IL-18 IL-10 IL-6 IL-13

[0090] The plate is read on the Luminex Magpix using the “Xponent” software and then analyzed using the “Bioplex Manager” software (Biorad version 6).

Results

[0091] The identification of bacteria from the lung microbiota capable of modifying the immune response during Mtb infection is based on a bank of primary colonizing bacteria from the lung of mice isolated and identified by Dr Langella's team (Probihôte—MICALIS—INRA), and in particular by Aude Remot and Muriel Thomas. One of these bacteria, a strain of Enterococcus sp. deposited at the CNCM under the number I-4969 is able to modulate the susceptibility of mice to allergic asthma (see WO 2017/12987 and [8]). Among these bacteria, 3 are lactobacilli that are Generally Recognized As Safe (GRAS) and have been deposited at the CNCM under the numbers: CNCM I-4968, CNCM I-5314 and CNCM I-4967, respectively.

[0092] The genome of the strain deposited under number I-5314 has been sequenced. Using sequence homology analysis software (Blast), it was found that the DNA sequence encoding the 16S rRNA (SEQ ID NO: 1) has more than 98% homology with reference strains of L. animalis and strains of L. murinus.

[0093] The growth profile of each of the three strains, CNCM I-4968, CNCM I-5314 and CNCM I-4967, was determined by measuring the OD at 600 nm. It is presented in FIG. 1.

[0094] To determine the probiotic potential of these 3 lactobacilli for the prevention and treatment of tuberculosis, different protocols summarized in FIG. 2 were used in a mouse model. The administration of 10.sup.7 bacteria is performed intranasally for two weeks before the mice are sacrificed (FIG. 3) or infected with Mycobacterium tuberculosis (Mtb) (other figures). In these experiments the bacteria are administered before and after infection (bef/aft groups) or only after infection (aft group, FIG. 5).

[0095] As a first step, the ability of bacteria to modify the local immune system of uninfected mice was assessed by flow cytometric analysis of the expression of intracellular markers characteristic of different subpopulations of CD4.sup.+ helper T cells (analysis strategy in FIG. 3A), key orchestrators of the anti-Mtb immune response: Th1 (TNF-α producing), Th17 (IL-17A producing) and regulatory T cells (Treg expressing Foxp3) [16, 17, 18]. Our results show that the three lactobacilli isolated from the lung microbiota have strong immunomodulatory capabilities, with an anti-inflammatory profile (even outside an infectious context). Strain CNCM I-5314 is that which induces the strongest decrease in Th1, increase in Th17 and pulmonary Treg (FIG. 3B), while these three species belong to the same bacterial genus.

[0096] To determine whether these bacteria (and in particular strain CNCM I-5314) induce a sufficient anti-inflammatory response to decrease the immunopathology associated with tuberculosis, the bacteria were administered as before intranasally, 15 days prior to infection with Mtb and then throughout the infection (FIG. 2). In this model, the three bacteria did not alter the bacterial load of Mtb (FIG. 4A). In contrast, CNCM I-5314 appears to confer significant protection against leukocyte infiltration of the lungs (less lung surface area occupied by immune cells) leading to immunopathology in the late stages of infection (and the other two bacteria do not) (FIG. 4B).

[0097] Cytokine production induced by strain CNCM I-5314 was determined according to the experimental scheme in FIG. 5 (see also Remot et al., 2017). Precision-cut slices of 6-day-old mouse lung were cultured in the presence or absence of CNCM I-5314. After 16 hours of culture, secreted cytokines were assayed in the medium, while cell viability was assessed by LDH assay (FIG. 6). The LDH assay showed no decrease in lung explant viability, indicating that strain CNCM I-5314 is not toxic (unlike a control strain). In particular, the cytokine assay showed induction of GM-CSF, IL-17a and TNFα(FIG. 7). These data made it possible to establish the immunomodulatory profile of strain CNCM I-5314 with respect to mouse lung explants.

[0098] To better characterize the protective effect of strain CNCM I-5314, the same experiment was repeated (CNCM I-5314 bef/aft group) including analyses to determine the composition of the lung immune infiltrate. In addition, the capacity of this bacterium to exert its protective effect in a treatment strategy (as opposed to the prophylactic approach described above) was evaluated by adding a group for which the administration of the bacterium began only after infection (CNCMI 5314 aft group) (details of the different groups in FIG. 2). These experiments confirm that the anti-inflammatory effect induced by this bacterium does not allow Mtb to multiply uncontrollably (FIG. 8A) while it grants a significant protection (for the group having received the bacterium in pre-treatment, CNCM I-5314 bef/aft) against the immunopathology induced by the infection with a reduction of the leukocytic infiltrates (FIG. 8B). Although lung infiltration of pro-inflammatory Th1 lymphocytes (expressing T-bet and producing TNF-α or IFN-γ) varies little between groups, strain CNCM I-5314 induces an increase in both Th17 (expressing RORγt and producing IL-17A) and Treg (expressing Foxp3, producing IL-10 and TGF-β) lymphocytes at 42 days post-infection (FIG. 8C). However, further analysis reveals that the increase in Tregs observed with administration of the bacterial strain (whether with a prophylactic or treatment approach) is not related to an increase in conventional Tregs (Foxp3.sup.+RORγt.sup.−), but to that of a recently described, double-positive Foxp3.sup.+RORγt.sup.+ population called bi-Treg. These cells possess both pro- and anti-inflammatory functions and produce IL-17 but also IL-10, TGF-β and IL-35 (not analyzed here) which make them key regulators of inflammation [17, 18]. They could therefore be responsible for the decrease in leukocyte infiltrates (observed in FIG. 8B) and thus the promising effect of this bacterium for the prevention and treatment of tuberculosis. The proportion of Th17 (Foxp3.sup.−RORγt.sup.+) also appears to be increased. In the case of an environment rich in IL-10 and TGF-β (as is the case here), Th17/IL-17 activity is biased and these cells exert anti-inflammatory and protective functions with respect to tissue damage, mainly via IL-22 production (production not measured in our experiments), suggesting a beneficial role of these cells in our context [16, 17]. Tregs enhanced by CNCM I-5314, and in particular biTregs, do not express the transcription factor Helios, indicating that they are induced in the mucous membranes (iTreg) as opposed to naturally occurring Tregs (nTreg), i.e., generated in the thymus, suggesting that this effect is specific and related to peripheral tolerance (FIG. 8E). The increase in these cells does not appear to be related to increased proliferation since Ki67 antigen expression is decreased (FIG. 8F). They seem to be able to have the pro- and anti-inflammatory effects described in the literature since the Tregs observed produce IL-17, IL-10 but above all TGF-β (the production of which is also higher in the two groups of mice having received the administration of this bacterial strain) (FIG. 8G).

[0099] We show here for the first time that a bacterial strain from the lung microbiota (CNCM I-5314) is capable of modifying the immune response to Mtb, in particular through the induction of biTregs that could better control the immune balance (pro-/anti-inflammatory) and thus reduce the immunopathology induced by the infection. These results thus present the present strain of Lactobacillus (CNCM I-5314) isolated from the pulmonary microbiota as a promising probiotic for tuberculosis. Preliminary results obtained with the “CNCM I-5314 aft” group also suggest applications for the treatment of this disease. As other respiratory diseases are related to inflammation, we assume that its beneficial role would not be limited to tuberculosis.

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