Compositions useful for the treatment of immune-related diseases

Abstract

The present invention relates to a gingival fibroblast-derived product for use in the treatment or prevention of an immune-related disease in an individual.

Claims

1. A method for treating an immune-related disease in an individual in need thereof, wherein the disease is selected from the group consisting of an allergy, an inflammatory skin disease and asthma, comprising administering to the individual a therapeutically effective amount of a gingival fibroblast-derived product.

2. The method of claim 1, wherein the inflammatory skin disease is atopic dermatitis.

3. The method of claim 1, wherein the gingival fibroblast-derived product is selected from the group consisting of gingival fibroblast whole cells, a gingival fibroblast culture, a gingival fibroblast extract and a gingival fibroblast conditioned medium.

4. The method of claim 1, wherein the gingival fibroblast-derived product is obtained from gingival fibroblasts taken from the individual.

5. The method of claim 1, comprising: taking gingival fibroblasts from the individual; culturing the gingival fibroblasts; obtaining a gingival fibroblast-derived product from the cultured gingival fibroblasts; administering the gingival fibroblast-derived product to the individual.

6. The method of claim 1, wherein the gingival fibroblast-derived product is associated with at least one agent intended for the treatment of inflammatory skin disease.

7. The method of claim 1, wherein the gingival fibroblast-derived product is associated with at least one agent intended for the treatment of inflammatory skin disease selected from the group consisting of a corticosteroid, a calcineurin inhibitor, an emollient, a moisturizer.

8. The method of claim 1, wherein the gingival fibroblast-derived product is administered topically.

9. A method for treating an immune-related disease in an individual in need thereof, wherein the disease is selected from the group consisting of an allergy, an inflammatory skin disease and asthma, comprising administering to the individual a therapeutically effective amount of a composition comprising a gingival fibroblast-derived product, and comprising at least one agent intended for the treatment of an inflammatory skin disease, and comprising at least one pharmaceutically acceptable carrier or excipient.

Description

DESCRIPTION OF THE FIGURES

(1) FIGS. 1A and 1B represent the level of secretion (vertical axis pg/ml) of CCL26 (FIG. 1A) and TSLP (FIG. 1B) by keratinocytes in a simultaneous treatment. The experiment was conducted in low inflammatory condition. Dexamethasone (black bars) has been tested at 10 μM, 100 μM and 500 μM compared to the control (hatched bar).

(2) FIG. 2 represents the level of secretion (vertical axis pg/ml) of CCL26 by keratinocytes in a simultaneous treatment. The experiment was conducted in low inflammatory condition. Two batches of conditioned medium from various gingival fibroblasts donors have been used GF009 (dotted bar) and GF010 (hatched bar) compared to the control (grey bar). The two batches of conditioned medium have been tested at the concentration 5×.

(3) FIGS. 3A, 3B and 3C represent the level of secretion (vertical axis pg/ml) of CCL26 (FIG. 3A), TSLP (FIG. 3B) and IL-1β (FIG. 3C) by keratinocytes in a simultaneous treatment. The experiment was conducted in high inflammatory condition. Two batches of conditioned media from various gingival fibroblasts donors have been used GF009 (dotted bar) and GF010 (hatched bar) compared to the control (grey bar). The two batches of conditioned media have been tested at the concentration 5×.

(4) FIGS. 4A and 4B represent the level of secretion (vertical axis pg/ml) of CCL26 by keratinocytes in a deferred treatment. The experiment was conducted in low inflammatory condition. Two batches of conditioned media from various gingival fibroblasts donors have been used GF009 (dotted bar) and GF015 (hatched bar) compared to the control (grey bar). The two batches of conditioned media have been tested in two different concentrations: 5× and 50× and collected on day 2 (FIG. 4A) and day 5 (FIG. 4B).

EXAMPLE

(5) A. Material and Methods

(6) 1. Human Gingival Fibroblasts

(7) 1.1. Culture Cell

(8) Human gingival fibroblasts (GF) were obtained from healthy donors. After enzymatic dissociation of gingival biopsies (collagenase, dispase), gingival fibroblasts were cultured in a serum-free medium in presence of platelet lysate (Doucet et al. (2005) J Cell Physiol. 205:228-36).

(9) 1.2. Preparation of Gingival Fibroblast Conditioned Medium

(10) The culture medium of confluent GF was discarded. After rinsing (PBS), fresh medium was added (without platelet lysate and without antibiotics). Cells were incubated at 37° C. for 24 hours. The conditioned medium (CM) was collected, aliquoted and stored at −80° C. These steps can be performed with different passage cells.

(11) 1.3 Concentration of CM

(12) The media were centrifuged on a concentration membrane (Millipore, Amicon 3K) and then stored at −80° C.

(13) 2. Human Keratinocytes

(14) Keratinocytes were obtained from breast reconstruction of healthy donors. For expansion, cells were seeded into irradiated feeders in a suitable medium.

(15) 3. Protocol

(16) 3.1. Induction of Inflammation

(17) A same combination of pro-inflammatory agents (IL-4, IL-13, TNFα, polyinosinic:polycytidylic acid (polyI:C) was used at 2 different concentrations: Low concentration: IL-4 10 ng/ml, IL-13 10 ng/ml, TNFα 5 ng/ml, polyI:C 5 μg/ml; High concentration: IL-4 100 ng/ml, IL-13 100 ng/ml, TNFα 20 ng/ml, polyI:C 10 μg/ml.

(18) 3.2. Anti-Inflammatory Treatment

(19) 3.2.1. Reference Products

(20) Dexamethasone (from 100 μM to 500 μM) (Sigma Aldrich)

(21) 3.2.2. Gingival Fibroblast Conditioned Medium

(22) The gingival fibroblast conditioned medium was concentrated for use at a final concentration from 5× to 50×. Three batches of conditioned media from various gingival fibroblasts donors have been used (GF009, GF010 and GF015)

(23) 3.3. Optimising the Time of Treatment

(24) For the efficacy tests of CM, keratinocytes were seeded without feeder in 6-well plates with a medium suitable for cell culture without feeder (KSFM medium, Gibco).

(25) 4. Test Procedure

(26) 4.1. Simultaneous Treatment for the Induction of Inflammation

(27) When the keratinocyte culture reached confluence, the culture medium of keratinocytes was replaced by a medium containing the pro-inflammatory agents (day 0) and the anti-inflammatory treatment (reference product or gingival fibroblast conditioned medium). The medium was collected on day 1 and stored at −80° C. until analysis.

(28) 4.2. Deferred Treatment for the Induction of Inflammation

(29) When the keratinocyte culture reached confluence, the culture medium of keratinocytes was replaced by a medium containing pro-inflammatory agents (day 0). On day 1, the gingival fibroblast conditioned medium was directly added in the medium containing pro-inflammatory cytokines. The medium was collected on day 2 or day 5 and stored at −80° C. until analysis.

(30) 4.3. Analysis

(31) The inflammatory condition of keratinocytes was determined by measuring inflammatory cytokine levels in their culture media. The studied cytokines were CCL26, TSLP and IL-1β. These cytokines were quantified with an ELISA assay (Duoset kit, R&D system).

(32) B. Results

(33) The present Example is based on an in vitro model of atopic dermatitis comprising: (i) growing keratinocytes until they reach confluence, (ii) stimulating them using a cocktail of pro-inflammatory agents, including PolyI:C, tumour necrosis factor (TNF-α), IL-4 and IL-13, known to be involved in atopic dermatitis inducement (Castex-Rizzi et al. (2014) British Journal of Dermatology 170 (suppl. S1):12-18; Le et al. (2009) Allergy 64:1226-1235) and (iii) determining the level of inflammatory cytokines/chemokines (TSLP, CCL26 and IL-1β) known to be involved in the pathogenesis of atopic dermatitis (Castex-Rizzi et al. op. cit., Le et al. op. cit., Cianferoni et al. op. cit. Kagami et al. op. cit. Sera et al. op. cit.) in the presence or absence of gingival fibroblast (GF) conditioned medium.

(34) 1. Anti-Inflammatory Effect of Reference Products (Simultaneous Treatment)

(35) Results presented in FIGS. 1A-1B show that dexamethasone inhibits the secretion of CCL26 (FIG. 1A) and TSLP (FIG. 1B).

(36) This model is thus relevant and dexamethasone can serve as reference product to evaluate the efficacy of gingival fibroblast conditioned medium to inhibit secretion of pro-inflammatory cytokines.

(37) 2. Anti-Inflammatory Effect of the Gingival Fibroblast Conditioned Medium (Simultaneous Treatment)

(38) 2.1. Induction of Inflammation with a Low Concentration of Inflammatory Agents

(39) Results presented in FIG. 2 show that the two tested batches of gingival fibroblast conditioned medium (GF009 and GF010) inhibit the secretion of CCL26 by inflammatory keratinocytes at the concentration 5×. Similar results were obtained for the secretion of TSLP and IL-1β.

(40) 2.2. Induction of Inflammation with a High Concentration of Inflammatory Agents

(41) FIGS. 3A-3C show that gingival fibroblast conditioned medium inhibits the secretion of CCL26 (FIG. 3A), TSLP (FIG. 3B) and IL-1β (FIG. 3C)

(42) 2.3. Conclusion

(43) Gingival fibroblast conditioned medium has an inhibitory effect on the secretion of inflammatory cytokines CCL26, TSLP and IL-1β by keratinocytes exposed to inflammatory conditions, even under highly inflammatory conditions.

(44) 3. Anti-Inflammatory Effect of the Gingival Fibroblast Conditioned Medium (Deferred Treatment)

(45) 3.1. Induction of Inflammation

(46) The two tested batches of gingival fibroblast conditioned medium (GF009 and GF010) inhibit the secretion of inflammatory cytokine CCL26 by inflammatory keratinocytes either at the concentrations 5× or 50× when collected on day 2 (FIG. 4A) and on day 5 (FIG. 4B).

(47) 3.2. Conclusion

(48) Gingival fibroblast conditioned medium has a long term inhibitory effect on the secretion of inflammatory cytokines by keratinocytes exposed to inflammatory conditions, even when the inflammatory conditions are established for 24 hours before the keratinocytes are treated by the conditioned medium.