Use of a polypeptide for effecting immune signalling and/or affecting intestinal barrier function and/or modulating metabolic status

Abstract

It has been found that an extracellular polypeptide derived from Akkermansia municiphila is capable of modulating and/or promoting gut mucosal immune system function and/or maintaining and/or restoring metabolic status and/or increasing the physical integrity of the gut mucosal barrier in a mammal. The polypeptide or host cells comprising such polypeptide may be employed to prevent and/or treat a variety of conditions that benefit from an increased physical integrity of the gut mucosal barrier and/or an improved gut mucosal immune system function and metabolic status.

Claims

1. A composition comprising an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1 over the entire length of SEQ ID NO: 1, said polypeptide being capable of effecting immune signaling and/or affecting intestinal barrier function and/or affecting glucose and/or cholesterol and/or triglyceride homeostasis, and a pharmaceutically or alimentary acceptable carrier, wherein the pharmaceutically or alimentary acceptable carrier is formulated for oral delivery and is encapsulated with an enteric coating.

2. The composition according to claim 1, which is a nutritional composition or a pharmaceutical composition.

3. The composition of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.

4. The composition according to claim 1, wherein the polypeptide retains activity while passing through the stomach.

5. The composition according to claim 4, wherein the pharmaceutically or alimentary acceptable carrier further comprises one or more controlled release agents.

6. The composition according to claim 4, wherein the pharmaceutically or alimentary acceptable carrier further comprises a mucosal binding agent.

7. The composition according to claim 1, wherein the polypeptide comprises between one and ten amino acid substitutions relative to SEQ ID NO:1.

Description

DESCRIPTION OF THE FIGURE

(1) FIG. 1 shows: A) Total body weight gain (g) (n=8-10). B) Total fat mass gain (g) measured by Time domain-Nuclear magnetic resonance (n=8-10). C) daily food intake. D) Plasma VLDL, LDL and HDL cholesterol levels (n=8-10). E) Plasma glucose (mg dl.sup.−1) profile and F) mean area under the curve (AUC) measured between −30 and 120 min after glucose loading (mg.dr.sup.1.min.sup.−1; n=8-10). G) Ratio of the control and insulin-stimulated p-IRβ on the loading control as measured by densitometry (n=3-5). H and I) Ratio of the control and insulin-stimulated p-Akt.sup.thr308 and p-Akt.sup.ser473 on the loading control as measured by densitometry (n=3-5).

SEQUENCE LISTING

(2) SEQ ID NO:1: Amino acid sequence of the Amuc-1100 polypeptide

(3) SEQ ID NO: 2: Nucleotide sequence encoding the Amuc-1100 polypeptide

(4) SEQ ID NO:3: Amino acid sequence of the predicted N-terminal signal sequence of Amuc-1100 polypeptide

(5) SEQ ID NO:4: Nucleotide sequence of the predicted N-terminal signal sequence of Amuc-1100 polypeptide

EXAMPLES

Example 1: Generation of Bacteria Genetically Modified to Produce Amuc-1100 Proteins

(6) Method:

(7) The polynucleotide encoding the mature Amuc-1100 (nucleotide sequence of SEQ ID NO:2) was cloned into E. coli TOP10 with a C-terminal His-Tag under control of the inducible T7 promoter of pET28-derivatives and introduced into E. coli BL23(DE3) for overproduction. For this purpose an ATG start codon was added to the nucleotide sequence of SEQ ID NO; 2, so that the resulting polypeptide started with the amino acid sequence MIVNS. All constructs were confirmed by Sanger sequence analysis. The constructs carrying the overexpressed Amuc-1100 resulted in overproduction of soluble Amuc-1100 proteins that were purified to apparent homogeneity by Ni-column affinity chromatography and used in a concentration of 100-300 ug/ml. The purified Amuc-1100 was used to generate antibodies in rabbits essentially as described previously (Reunanen J et al. 2012, Appl Environ Microbiol 78:2337-44).

(8) Results:

(9) The results show that E. coli transformed with the polynucleotide of the invention (SEQ ID NO:2) was able to produce the Amuc-1100 protein in a soluble form that could be isolated easily using Ni-column chromatography as described (Tailford L E et al. 2015, Nat Commun. 6:7624).

Example 2: Interaction and Stimulation of the TLR2 Signalling Pathway

(10) Method:

(11) In order to test the ability of Amuc-1100 to bind the TLR2 and other TLR receptors and subsequently stimulate the TLR2 and other TLR signalling pathways, reporter cell lines expressing TLR2 and TLR4 receptors were prepared. The ability of Amuc-1100 to bind cell lines expressing TLR2 or TLR4 and thereafter stimulate the TLR2 and/or TLR4 signaling pathway in said cells was tested in vitro by measuring the production of NK-kB from the reporter cells.

(12) Briefly, hTLR2 and hTLR4 cell lines (Invivogen, Calif., USA) were used. Stimulation of the receptors with the corresponding ligands activates NF-κB and AP-1, which induces the production of Secreted embryonic alkaline phosphatase (SEAP), the levels of which can be measured by spectrophotometer (Spectramax). All cell lines were grown and subcultured up to 70-80% of confluency using as a maintenance medium Dulbecco's Modified Eagle Medium (DMEM) supplemented with 4.5 g/I D-glucose, 50 U/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml Normocin, 2 mM L-glutamine, and 10% (v/v) of heat-inactivated Fetal Bovine Serum (FBS). For each cell line, an immune response experiment was carried out by adding 20 μl of Amuc-1100 suspensions. The reporter cells were incubated with Amuc-1100 for 20-24 h at 37° C. in a 5% CO2 incubator. Receptor ligands Pam3CSK4 (10 ng/ml for hTLR2) and LPS-EB (50 ng/ml for hTLR4) were used as positive control whereas maintenance medium without any selective antibiotics was used as negative control. SEAP secretion was detected by measuring the OD600 at 15 min, 1 h, 2 h, and 3 h after addition of 180 μL of QUANTI-Blue (Invivogen, Calif., USA) to 20 μL of induced hTLR2 and hTLR4 supernatant. Experiments were performed in triplicate.

(13) Results:

(14) The results show that Amuc-1100 was able to interact with TLR2. Further, the results show that Amuc-1100 exerted immune-stimulatory effects on reporter cells expressing TLR2, i.e. Amuc-1100 was capable of stimulating the release of NF-κB from reporter cells.

Example 3. Stimulation of Cytokine Release from Peripheral Blood Mononuclear Cells

(15) Method:

(16) The ability of Amuc-1100 to stimulate cytokine production or release from peripheral blood mononuclear cells (PBMCs) was tested in vitro. Briefly, peripheral blood of three healthy donors was received from the Sanquin Blood Bank, Nijmegen, The Netherlands. Peripheral blood mononuclear cells (PBMCs) were separated from the blood of healthy donors using Ficoll-Paque Plus gradient centrifugation according to the manufacturer's protocol (Amersham biosciences, Uppsala, Sweden). After centrifugation the mononuclear cells were collected, washed in Iscove's Modified Dulbecco's Medium (IMDM)+Glutamax (Invitrogen, Breda, The Netherlands) and adjusted to 0.5×10.sup.6 cells/ml in IMDM+Glutamax supplemented with penicillin (100 U/ml) (Invitrogen), streptomycin (100 μg/ml) (Invitrogen), and 10% heat inactivated FBS (Lonza, Basel, Switzerland). PBMCs (0.5×10.sup.6 cells/well) were seeded in 48-well tissue culture plates. For each donor, a negative control (medium only) was used.

(17) The PBMCs were stimulated with A. muciniphila cells (1:10 ratio to PBMCs) either alive or heated for 10 min at 99° C.) or Amuc-1100 for 1 day and subsequently the production of cytokine IL-6, IL-8, IL-10, TNF-α, IL-113 and IL-12p70 was measured in culture supernatants using multiple analysis (Human inflammation CBA kit, Becton and Dickinson) according to the manufacturer's protocol on a FACS CantoII (Becton Dickinson) and analysed using BD FCAP software (Becton Dickinson). The detection limits according to the manufacturer were as follows: 3.6 pg/ml IL-8, 7.2 pg/ml IL-1β, 2.5 pg/ml IL-6, 3.3 pg/ml IL-10, 3.7 pg/ml TNF-α, 1.9 pg/ml IL-12p70.

(18) Results

(19) The results show that, compared to the control situation (medium only), Amuc-1100 was able to stimulate the production of cytokines, i.e. increased levels of IL-113, IL-6, IL-8, IL-10 and TNF-α were observed. The level of cytokine induced by 4.5 μg/ml Amuc-1100 was at a similar level as that of 5×10.sup.6 cells of A. muciniphila either alive or in a heat-killed form (see Table 1 below).

(20) TABLE-US-00001 TABLE 1 Levels of cytokine induced by Amuc-1100 and Akkermansia muciniphila either alive or in a heat-killed form Live Heat-killed Amuc-1100 Cytokine (pg/ml) A. muciniphila A. muciniphila (4.5 μg/ml) IL-1β 894 ± 298 392 ± 71 504 ± 227 IL-6 18029 ± 309  13477 ± 2014 12508 ± 2362  IL-8 60018 ± 18229 54230 ± 9030 45432 ± 12507 IL-10 823 ± 310  638 ± 118 526 ± 180 TNF-α 1920 ± 349   957 ± 568 1317 ± 885  IL-12p70 <2 <2 <2

Example 4: Modulation of the Transepithelial Resistance (TER)

(21) Method:

(22) The ability of Amuc-1100 to promote the integrity of gut epithelial cell layer was assessed by measuring the ability of Amuc-1100 to stimulate or increase TER of Caco-2 cells in vitro. Briefly, Caco-2 cells (5×10.sup.4 cells/insert) were seeded in Millicell cell culture inserts (3 μm pore size; Millipore) and grown for 8 days. Bacterial cells were washed once with RPMI 1640, and applied onto the inserts at OD600 nm of 0.25 (approximately 10.sup.8 cells) in RPMI 1640. Purified Amuc-1100 was applied onto the inserts at concentrations of 0.05, 0.5 and 5 μg/ml. The transepithelial resistance was determined with a Millicell ERS-2 TER meter (Millipore) from cell cultures at time points 0 h, and 24 h after addition of Amuc-1100.

(23) Results:

(24) The results showed that already 0.05 μg/ml of Amuc-1100 was able to significantly increase TER after 24 h of co-cultivation with the Caco-2 cells at a similar level of approximately 10.sup.8 A. muciniphila cells.

Example 5: Modulation of Diet-Induced Metabolic Dysfunction

(25) A cohort of 10-11 week-old C57BL/6J mice (n=10 per subset) was fed a control diet (ND) or an HF diet (HFD; 60% fat and 20% carbohydrates (kcal/100 g) D12492i, Research Diet, New Brunswick, N.J., USA) as previously described by Everard et al. (2013. PNAS. Vol. 110(22):9066-9071). A. muciniphila Muc.sup.T was grown on a synthetic medium (containing per liter deionized water: 0.4 g KH.sub.2PO.sub.4, 0.669 g Na.sub.2HPO.sub.4.2H.sub.2O, 0.3 g NH.sub.4Cl, 0.3 g NaCl, 0.1 g MgCl.sub.2.6H.sup.2O, 10 g Casitone, 1 mM L-threonine, 1 ml trace mineral solution, 5 mM L-fucose and 5 mM D-glucose) as described by Lucovac et al. (2014, mBio 01438-14) and concentrated, formulated in PBS containing 25% glycerol, and stored at −80° C. as described by Everard et al. supra. A subset of mice receiving HFD additionally received, daily and by oral gavage, 2×10.sup.8 cfu/0.15 ml A. muciniphila suspended in sterile anaerobic PBS (HFD Akk)—since this included a 10-fold dilution of the A. muciniphila, a final concentration of 2.5% glycerol was obtained. The ND and HFD groups were treated daily with an oral gavage of an equivalent volume of sterile anaerobic PBS containing 2.5% glycerol, as previously described by Everard et al., supra. A further subset of mice receiving HFD additionally received Amuc-1100 peptide delivered by daily oral gavage of 3.1 μg of the protein Amuc_1100 in an equivalent volume of sterile PBS containing 2.5% glycerol. Treatment of HFD-fed mice with Amuc-1100 caused a similar or even more prominent decrease in body weight and fat mass gain when compared to the live A. muciniphila bacterium (FIGS. 1 A and B), without affecting food intake (FIG. 1 C). Treatment with A. muciniphila or Amuc-1100 also corrected the HFD-induced hypercholesterolemia, with a significant decrease in serum HDL-cholesterol and a similar trend for LDL-cholesterol (FIG. 1 D).

(26) Remarkably, treatment with Amuc-1100 led to a significant decrease of serum triglycerides when compared to untreated HFD-fed mice. Moreover, Amuc-1100 treatment also reduced the adipocyte mean diameter from 38 micrometer in HFD-fed mice to 29 micrometer, a similar diameter as found in untreated mice (27 micrometer).

(27) Interestingly, administration of Amuc-1100 reduced glucose intolerance with the same potency as the live bacterium (FIG. 1 E-F).

(28) To further investigate glucose metabolism we investigated insulin sensitivity by injecting insulin in the portal vein. We analyzed insulin-induced phosphorylation of the insulin receptor (IR) and its downstream mediator Akt in the liver at the threonine (Akt.sup.thr) and serine (Akt.sup.ser) sites (FIG. 1 G). Administration of the HFD led to a decreased phosphorylation of all proteins when compared to mice fed a control chow, reaching significance in the case of Akt.sup.thr (FIG. 1 H). Treatment with live A. muciniphila or Amuc-1100 counteracted these effects, with significantly higher levels of p-IR and p-Akt.sup.thr in mice treated with Amuc-1100 (FIG. 1 G-H) and significantly higher levels of p-Akt.sup.ser in mice treated with the live bacterium (FIG. 1 I) when compared to the untreated HFD-fed mice.