Probiotics, secretory IgA and inflammation

Abstract

The present invention relates generally to the field of nutrition, health and wellness. In particular the present invention relates to probiotics and ways to increase their effectiveness. One embodiment of the present invention relates to a combination of probiotics with SIgA and possible uses of this combination. For example a use of a composition comprising SIgA and at least one probiotic for the preparation of a product to treat or prevent inflammation is disclosed.

Claims

1. A method for treating inflammation, the method comprising: administering a therapeutically-effective amount of a composition comprising an isolated secretory IgA (SIgA) of 0.0001 mg to 10 mg per daily dose and at least one probiotic of 10.sup.2 to 10.sup.10 cells per daily dose, wherein the SIgA and the at least one probiotic are associated by a chemical bond as complexes in the composition, to a subject having the inflammation.

2. The method of claim 1 wherein the composition generates, improves or reinforces homeostasis and oral tolerance.

3. The method of claim 1 wherein the at least one probiotic is selected from the group consisting of Bifidobacterium, Lactobacillus, Streptococcus, Saccharomyces and mixtures thereof.

4. The method of claim 1, wherein the composition is selected from the group consisting of a food product, an animal food product and a pharmaceutical composition.

5. The method of claim 1, wherein the subject is a human.

6. The method of claim 1, wherein the composition controls and/or alleviates inflammatory reaction.

7. The method of claim 1, wherein the composition comprises at least one other kind of other food grade bacteria.

8. The method of claim 1, wherein the composition further contains at least one prebiotic.

9. The method of claim 1 wherein the at least one probiotic is selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Enterococcus faecium, Saccharomyces boulardii, Lactobacillus reuteri and mixtures thereof.

10. The method of claim 1, wherein the composition comprises at least one other kind of other food grade bacteria selected from the group consisting of lactic acid bacteria, bifidobacteria, enterococci and mixtures thereof.

11. The method of claim 1, wherein the at least one probiotic is selected from the group consisting of Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818).

12. The method of claim 1, wherein the SIgA and the at least one probiotic micro-organism are present in a stoichiometric ratio of at least 10:1.

Description

(1) Further advantages and features of the present invention are apparent from the following Examples and Figures.

(2) FIG. 1 shows schematically how SIgA is believed to improve the effects of commensal bacteria, when associated with them by increasing the interaction with the intestinal mucosa of the host. Depicted are possible and documented routes of interaction of SIgA associated with commensal bacteria with the host intestinal mucosa.

(3) FIG. 2 shows the result of experiments testing the binding properties of two probiotic strains, Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818), representative for the two main genders Lactobacilli and Bifidobacteria to epithelial cells. Data are expressed as means CFU per 100 Caco-2 cells±SEM.

(4) FIG. 3 shows the result of experiments testing the binding properties of two probiotic strains, Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818), representative for the two main genders Lactobacilli and Bifidobacteria to epithelial cells and the influence of secretory IgA (SIgA) or secretory component (SC). Data are expressed as means CFU per 100 Caco-2 cells±SEM.

(5) FIG. 4 shows the result of an experiment testing the effect of two probiotic strains, Lactobacillus rhamnosus NCC4007 (LPR) and Bifidobacterium lactis NCC2818 (BL818), representative for the two main genders Lactobacilli and Bifidobacteria, alone or in combination with SIgA or SC, on transepithelial electrical resistance (TER) measuring epithelial permeability. Data are expressed as means ohms per cm.sup.2±SEM.

(6) FIG. 5 shows the result of an experiment testing the effect of LPR, combined or not with SIgA or SC, on NF-κB activation in Caco-2 cell monolayer. Decrease in NF-kB binding activity is indicative of attenuated inflammatory pathway(s) within the Caco-2 cell.

(7) FIG. 6 shows the result of an experiment testing the effect of LPR, combined or not with SIgA or SC, on S. flexneri invasion of Caco-2 cell. Two monoclonal SIgA molecules were used: a non-specific SIgA (SIgAnon-specific) recognizing a Salmonella epitope, and a specific anti-S. flexneri SIgA (SIgAC5). Data are expressed as means CFU per transwell filter±SEM.

(8) FIG. 7 shows the result of an experiment testing the effect of probiotics on polymeric Ig receptor (pIgR) expression in a Caco-2 monolayer. (A) Different treatments were tested at 16 h, including combination of probiotics with non-specific SIgA and combination of S. flexeneri with specific anti-S. flexneri SIgA (western blot). (B) Semi-quantitative analysis of pIgR expression levels normalized to β-actin by densitometric analysis of the bands identified in the gels in A. (C) Kinetic of pIgR expression over 24 h incubation of Caco-2 cells with various preparations (ELISA).

EXAMPLE 1

(9) Binding to Epithelial Cells

(10) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 16 h at 37° C. with different doses of bacteria, indicated in the figure legend in absence of antibiotic or FCS. Fresh overnight cultures of LPR, BL818 and E. coli TG-1 bacteria were used. Cells were then washed prior to enumeration. Bound bacteria were counted by plating on MRS or LB plates. For each experiment, triplicate tests were performed. Data were expressed as means of bound bacteria per 100 Caco-2 cells±SEM. Triplicates were performed for each experiment. In a subsequent experiment, cells were incubated with 2×10.sup.7 bacteria for 16 hours at 37° C., in the presence of increasing doses of either SIgA or SC as indicated in the legend to FIG. 3. Cells were then washed prior to enumeration. Bound bacteria were counted by plating on MRS or LB plates. For each experiment, triplicate tests were performed. Data were expressed as means of bound bacteria per 100 Caco-2 cells±SEM. Triplicates were performed for each experiment.

(11) A preferential binding to polarized Caco-2 cells of LPR and BL818 is observed in comparison to E. coli TG-1 (FIG. 2). There is a dose-dependent binding capacity of probiotics to intestinal epithelial cells. It can be observed that binding properties could be differentiated between the two strains.

(12) For subsequent experiments 2×10.sup.7 CFU of probiotics were used, as this amount did not lead to any pH change in the medium on one hand, and showed an efficient binding ratio on the other hand.

(13) Increasing the dose of monoclonal SIgA potentiated the capacity of both LPR and BL818 to bind to polarized Caco-2 cell monolayers. Secretory component by itself did not exhibit such properties (FIG. 3). The 1 μg dose of SIgA that confers a significant improvement in probiotic binding capacity was selected for subsequent experiments. This dose enables a final complex constituted of 50,000 to 100,000 units of SIgA for 1 bacterium.

(14) Results are shown in FIGS. 2 and 3.

EXAMPLE 2

(15) Barrier Function in Polarized Caco-2 Cell Monolayer

(16) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 24 h at 37° C. with 2×10.sup.7 CFU of bacteria in absence of antibiotic or FCS. Bacteria were tested either alone or in combination with SIgA or SC at concentrations indicated in the legend to FIG. 4. Transepithelial electrical resistance (TER) was measured at 3, 6, 9, 15 and 24 h. Controls include incubation with SIgA and SC alone. Triplicates were performed for each experiment.

(17) A 20-25% increase in transepithelial electrical resistance (TER) resulted from the incubation of polarized Caco-2 cell monolayer with LPR or BL818 alone, suggesting that probiotics potentiated epithelial barrier function. This remained true when the bacteria were combined with SIgA or SC (FIG. 4). SIgA or SC by themselves did not lead to any TER change.

(18) Results are shown in FIG. 4.

EXAMPLE 3

(19) NF-κB Activation in Polarized Caco-2 Cell Monolayer

(20) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm2 Transwell filter. Cells were incubated for 16 h at 37° C. with 2×10.sup.7 CFU of LPR in absence of antibiotic or FCS. Bacteria were tested either alone or in combination with SIgA or SC at concentrations indicated in the legend to FIG. 5. S. flexneri, S. typhi and H. pylori (2×10.sup.7 CFU) were used as pathogenic controls. Nuclear and cytoplasmic extracts were prepared and analysed by electrophoretic mobility shift assay (EMSA) and Western blot using anti-IκBα-specific monoclonal antibody. Triplicates were performed for each experiment.

(21) Exposure to pathogenic bacteria led to much more pronounced activation of nuclear NF-κB compared to non-pathogenic bacteria (FIG. 5).

(22) Disappearance of IκBα (lower panel) reflects activation of the pathway leading to nuclear translocation of NF-κB. In that respect, while LPR alone has a mild effect on NF-κB activation, combination of LPR with SIgA or SC reduced NF-κB activation in Caco-2 cells (BL818 not tested). Incubation of epithelial cells with pathogenic S. flexneri led to total disappearance of IκBα expression.

(23) Results are shown in FIG. 5.

EXAMPLE 4

(24) Anti-Pathogenic Activity

(25) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 16 h at 37° C. with 2×10.sup.7 CFU of LPR in absence of antibiotic or FCS. LPR was tested alone or in combination with either 0.2 μg of SC, 1 μg of polyclonal SIgA or 1 μg of specific anti-S. flexneri LPS SIgAC5. After incubation with LPR cells were washed and then incubated with 10.sup.7 S. flexneri for 6 hours, washed again and incubated with 50 mg/ml gentamycin for 45 min. Finally, cells were lysed and intracellular S. flexneri were enumerated on LB agar plates. Triplicates were performed for each experiment.

(26) Addition of LPR reduced infection of polarized Caco-2 cell monolayer by S. flexneri in a dose dependent manner. The effect was highly enhanced upon combination with SIgA. Full prevention of infection was achieved when S. flexneri LPS-specific SIgAC5 antibody was used (FIG. 6).

(27) Results are shown in FIG. 6.

EXAMPLE 5

(28) Expression of Polymeric IG Receptor in Polarized Caco-2 Cell Monolayer

(29) Approximately 10.sup.6 Caco-2 cells were seeded per 1 cm.sup.2 Transwell filter. Cells were incubated for 16 h at 37° C. with 2×10.sup.7 CFU of LPR or BL818 in absence of antibiotic or FCS. Probiotics were tested alone or in combination with either 0.2 μg of SC, 1 μg of polyclonal SIgA. Control S. flexneri was tested alone or in combination with 1 μg of specific anti-S. flexneri LPS SIgAC5. After washing, Caco-2 cells were directly recovered from the Transwell filters and lysed. Nuclei were removed and cell debris as well as cytoplasms were analysed by Western blot using anti-pIgR antibody and antisera to human SC and β-actin as controls. Triplicates were performed for each experiment.

(30) In a subsequent experiment cells were incubated following the same procedure and then recovered from the transwell filter at 8, 16 and 24 h of incubation. Quantitative analysis of pIgR was performed by ELISA on cell debris/cytoplasm fractions. Total proteins were determined by BCA protein assay. Values were normalized to protein content and data expressed as means of ng pIgR/mg of total protein±SEM.

(31) pIgR expression in epithelial cells was normalized to β-actin expression. As revealed by Western blot (upper panel) and densitometric analysis of the respective bands (lower panel) there was an increase of pIgR level following overnight exposure of polarized Caco-2 monolayers to combinations of LPR or BL818 with either SIgA or SC compared to probiotics alone (FIG. 7a). Specific anti-S. flexneri LPS SIgAC5 prevented interaction of the pathogen with the Caco-2 cell polarized monolayer, thus explaining the decrease in pIgR expression when compared to S. flexneri treatment alone.

(32) The results further showed a time-dependent increase of polymeric Ig receptor (pIgR) level following exposure of polarized Caco-2 cell monolayers to probiotic combinations with SIgA or SC (FIG. 7b).

(33) Results are shown in FIG. 7.