METHODS FOR DIAGNOSIS AND TREATMENT OF SOLAR LENTIGO

Abstract

The present invention relates to the diagnosis and treatment of solar lentigo (SL). The inventors established an in vitro model of primary fibroblasts isolated from SL (FL) and perilesional (FS) biopsies, which were collected from a cohort of 10 volunteers. Then, the inventors defined morphological and functional characteristics of both dermal cells. The inventors demonstrated by immunofluorescence studies differential morphological features with FL displaying elongated shape, a thin epidermis, disorganized basement membrane, intense melanin deposition and elongated rete ridges collapsing into the dermis and FS presented flattened morphology. Moreover, both fibroblasts demonstrated distinct functional characteristics with FL exhibiting a lower proliferation rate and migration capacity, senescent-like phenotype as well as a higher ability to secrete KGF, HGF, SCF, IL-13 and TGF1. Thus, the present invention relates to a method of identifying a subject having or at risk of having or developing solar lentigo, comprising measuring the expression level of IL-13, TGF1, HGF, KGF and SCF. The present invention also relates to an IL-13 inhibitor compound for use in the treatment of solar lentigo.

Claims

1. A method of treating solar lentigo in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an IL-13 inhibitor.

2. The method according to claim 1 wherein said IL-13 inhibitor is an IL-13 signaling pathway inhibitor.

3. The method according to claim 2 wherein said IL-13 signaling pathway inhibitor is selected from the group consisting of IL-13R1 antagonist, IL-13R1 expression inhibitor, IL-4R antagonist, IL-4R expression inhibitor, IL-13R2 antagonist, IL-13R2 expression inhibitor, JAK1 inhibitor, JAK1 expression inhibitor, JAK2 inhibitor, JAK2 expression inhibitor, STAT6 inhibitor, STAT6 expression inhibitor, TYK2 inhibitor, TYK2 expression inhibitor, TGF inhibitor and TGF expression inhibitor.

4. The method according to claim 1, wherein the IL-13 inhibitor is administered in combination with one or more of a Hepatocyte Growth Factor (HGF) inhibitor, a Keratinocyte Growth Factor (KGF) inhibitor and a Stem Cell Factor (SCF) inhibitor.

5-8. (canceled)

9. A method of screening a candidate compound for use as a drug for the treatment of solar lentigo, wherein the method comprises the steps of: (i) providing an IL-13, one or more IL-13 receptors, and one or more IL-13 downstream effectors, or a cell, tissue sample or organism expressing the IL-13, the one or more IL-13 receptors and the one or more 1L-13 downstream effectors, (ii) contacting the IL-13. the one or more IL-13 receptors, and the one or more IL-13 downstream effectors, or the cell, tissue sample or organism expressing the IL-13, the one or more IL-13 receptors and the one or more IL-13 downstream effectors with a candidate compound, (iii) measuring the IL-13 activity, and (iv) selecting positively candidate compounds that inhibit IL-13 activity.

10. A method of identifying and treating a subject having or at risk of having or developing solar lentigo, comprising the steps of measuring in a sample obtained from said subject the expression level of IL-13; comparing the expression level with a reference value, wherein a differential in the expression level of the IL-13 in the sample and the reference value indicates that the subject has or is at risk of having or developing solar lentigo; and administering a therapeutically effective amount of an IL-13 inhibitor to a subject whose measurement is indicative of having or having a risk of having or developing solar lentigo.

11. The method of claim 10 further comprising a step of measuring in the sample obtained from said subject the expression level of at least one biomarker selected from the group consisting of TGF1, HGF, KGF and SCF.

12. (canceled)

13. The method of claim 10, further comprising monitoring the progression of solar lentigo in the subject before, during or after treatment.

14. (canceled)

15. The method of claim 9, wherein the candidate compound is a small organic molecule, a peptide, a polypeptide, an aptamer, an oligonucleotide, an antibody or an intra-antibody.

16. The method of claim 1, wherein the treatment is a cosmetic treatment of the subject.

Description

FIGURES

[0164] FIG. 1: Differential cellular features between adjacent- and solar lentigo-isolated fibroblasts. Heterogeneous morphologies of fibroblasts isolated from peri-lesional (FS) and solar lentigo (FL) skin biopsies after examination by phase contrast microscopy (n=10 FS/FL couples) and representative immuno fluorescence images of FS and FL fibroblasts (n=10) showing F-actin labelling and Hoechst (nuclei) was performed. (a) F-actin staining was quantified in both FS (n=119) and FL (n=102) and graphed. Based on elliptical form parameter, quantitative analyses of cellular body (b) and nucleus (c) shapes of both FS and FL fibroblasts are shown; ***p<0.0001.

[0165] FIG. 2: Differential proliferation rates and migration capacities between FL and FS. Equal number of FS and FL were seeded at time 0. Proliferation rate was determined after counting cells 96 hours later (a; p<0.002; n=10) and metabolic activity was assessed using MTT assay after 24 hours (b; **p<0.003; n=10). FS and FL migration capacity was determined after a wound healing assay (c). Percentage of wound closure was calculated for each time point and average of percentages in 3 FS/FL samples were plotted; ****p<0.0001.

[0166] FIG. 3: Differential secretion profiles for HGF, KGF, SCF, IL-13 and TGF1 between FL and FS. FL- and FS-conditioned media (n=10 couples) were subjected to quantification for 3 growth factors using ELISA (a) and 31 other soluble factors using V-Plex (b). After normalization to FS or FL number, concentration of these factors was determined and graphed (HGF ***p<0.0005, KGF *p<0.05, SCF *p<0.05, IL-13 *p<0.0294 and TGF1 **p<0.0076).

[0167] FIG. 4: -SMA staining intensity is similar between FS and FL. Immunofluorescence images of FS and FL fibroblasts (n=10) showing -SMA labelling and Hoechst (nuclei) was performed. -SMA staining was quantified in both FS (n=119) and FL (n=102) and graphed. ns: non significant.

[0168] FIG. 5: Differential morphological and functional features between non lesional- and solar lentigo-explanted fibroblasts. Images of representative senescence-associated -galactosidase (SA--Gal) staining of 2 FNL/FL couples and normal fibroblasts subjected (FUV) or not (FNUV) to repeated-UVB exposures. Quantification of -Gal positive fibroblasts showed that FL (n=87/388) were more senescent than FNL cells (n=27/514) (**** p<0.0001); FNUV (n=5/458) and FUV (n=269/353) were used as controls.

EXAMPLES

Example 1

[0169] Morphological and Functional Characterizations of Fibroblasts Extracted from Solar Lentigo

[0170] Material & Methods

[0171] Phosphate buffer solution (PBS), Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), gentamycin, trypsin and all cell culture plastics were purchased from Dutscher (Brumath, France); paraformaldehyde (PFA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), dimethylformamide (DMF), glycine, acetone, triton X-100, bovine serum albumin (BSA), mouse -SM actin and goat anti-mouse IgG-FITC antibodies, rhodamine-conjugated phalloidin, Hoechst were purchased from Sigma-Aldrich (St Quentin Fallavier, France).

[0172] Human Skin Biopsies

[0173] Skin specimens were obtained from 13 volunteers on the back of the hands (Caucasian females, 67-89 years of age) after written informed consent (Pr. P. Humbert, University hospital of Besancon, France). Two biopsies of 3-mm punches were collected from the same patient with one solar lentigo lesional skin and one non-pigmented peri-lesional skin.

[0174] Histological Detection of Melanin

[0175] Briefly, Solar Lentigo (SL) and Peri-lesional (PL) biopsies were fixed in 3% PFA solution, dehydrated and embedded in paraffin. Seven-m thick sections were cut using a rotary microtome (Leitz 1512; Leica) and stained with Fontana-Masson following the manufacturer's instructions (Diapath). After mounting, histological features of the skin sections were examined by optical microscopy (Axioskop 40, Zeiss).

[0176] Cell Cultures

[0177] Primary culture of human dermal fibroblasts obtained from SL (FL) and PL (FS) of 10 patients were maintained in DMEM supplemented with 10% FCS and 1% Gentamycin in a humidified 5% CO2 atmosphere at 37 C. FL and FS cells were maintained until the monolayer cultures reached 80% confluency and were passaged twice using a trypsin (0.05%)/EDTA (0.02%) solution.

[0178] Cell Proliferation

[0179] Proliferation of FL and FS was determined by measuring metabolic activity (colorimetric MTT assay) 30 and by counting viable cells (Trypan blue exclusion assay).

[0180] MTT Assay

[0181] FL and FS were seeded into 96-well plates (5103 cells/well) and incubated with DMEM supplemented for 24 h. After removing supernatant and washing by PBS 1, 10 l of MTT solution (0.5 mg/mL in DMEM) were added for 4 h at 37 C. The formazan-blue crystals were dissolved overnight at 37 C. in 100 L of extraction medium (10% SDS/0.04% DMF in H2O). The optical density was read at 570 nm with a Multiskan FCTM spectrophotometer

[0182] (Fisher Scientific).

[0183] Trypan Blue Exclusion Assay

[0184] FL and FS were seeded at a density of 7105 cells/T75 flask and were incubated with DMEM supplemented for 4 days. Cells were trypsinized and count by Trypan blue stain.

[0185] Fluorescence Staining

[0186] Primary FL and FS were seeded in Lab-Tek chamber slides at 1104 cells/chamber and maintained in culture at 37 C. for 24 h. They were then fixed 10 min in 3% PFA solution and permeabilized for 10 min with cold acetone at 20 C. Nonspecific adhesion sites were blocked by a 10 min incubation in 1% glycine solution followed by a 1 h incubation in BSA solution (3% BSA/10% serum/0.1% triton X-100 in PBS). Primary antibody directed against human -SMA was then added (1: 100 in PBS/1%BSA/0.1% triton X-100) and incubated overnight at 4 C. in a moist chamber. Cells were then incubated with the FITC conjugated secondary antibody (1: 40 in PBS) for 1 h and further 30 min with rhodamine-conjugated phalloidin for filamentous actin (F-actin) staining (2.5 g/ml). Cellular nuclei were countermarked with Hoechst solution. Cells were then mounted in Dako fluorescent medium, observed under fluorescence microscopy (Axioskop 40, Zeiss) and further analyzed using Metamorph software (Molecular Devices).

[0187] Wound Healing Assay

[0188] Primary FL and FS were grown on 12-well Falcon plates for 24 h to 48 h to reach 100% cell confluent monolayers. Wounds were done using a sterile 10 l-pipette tip across each well. After gently washes, cells were incubated with supplemented DMEM w/o Red phenol, covered with mineral oil and placed in the incubator at 37 C. with 5% CO2 of the BD Pathway 855 Bioimaging (Becton Dickinson, USA). Image captures at 1 h-regular intervals using the AttoVision Software (Becton Dickinson) allowed calculating the percentage of wound closure for each time point (ImageJ Software).

[0189] ELISA and V-Plex Assays

[0190] Secreted factors in paired FL/FS-conditioned media were quantified by ELISA kits (Quantikine for KGF, HGF, SCF and TGF-1; R&D Systems) or by MSD 30-PLEX Proinflammatory Panell, Cytokine Panell, Chemokine Panell kits and TGF1 kit, according to the manufacture's protocols.

[0191] Statistical Analysis

[0192] Statistical significance was assessed using paired Student's t test (Prism5.0, Graph Pad Software). P values were considered significant with the following degrees: *p<0.05; **p<0.01; ***p<0.001.

[0193] Results

[0194] Melanin Staining of Human SL and Peri-Lesional Skin Biopsies

[0195] To check that human skin fibroblasts used in this study were isolated from peri-lesional and lesional skins, biopsies from 3 volunteers who presented solar lentigo were subjected to Fontana-Masson staining. Peri-lesional (PL) and lesional (SL) tissues displayed different histological features. SL showed a thin epidermis, disorganized basement membrane, a high melanin staining of the basal layer, intense melanin deposition and elongated rete ridges collapsing into the dermis. Altogether, microscopic analysis validated the SL origin of our in vitro fibroblastic model.

[0196] Differential Morphological Features Between Isolated Fibroblasts from Peri-Lesional and SL Skin Biopsies

[0197] Heterogeneous morphologies of fibroblasts isolated from peri-lesional (FS) and solar lentigo (FL) skin biopsies after examination by phase contrast microscopy (n=10 FS/FL couples) and immunofluorescence images of FS and FL fibroblasts (n=10) showing F-actin labelling and Hoechst (nuclei) was performed (data not shown).

[0198] Then, we undertook the morphological characterization of the fibroblasts isolated from non-lesional (FS) and solar lentigo (FL) skin biopsies. Global microscopic examination revealed that both types of primary fibroblasts exhibited variable shapes and sizes (data not shown). Fluorescent phalloidin staining showed that FL exhibited a more heterogeneous network of F-actin with a much denser labelling of stress fibers (data not shown). Quantification of the F-actin intensity staining in FS (n=119) and FL (n=102) revealed that FL staining level was significantly higher relative to FS (FIG. 1a). Moreover, analysis of the cellular body and nuclear appearances showed that FL displayed more elongated shapes (FIG. 1b) with an elliptical shaped nucleus (FIG. 1c) as compared with FS. In contrast, immunofluorescence analysis for the -SMA, a hallmark of the myofibroblastic phenotype, did not show difference in its intensity between FS and FL (FIG. 4).

[0199] Altogether, these data demonstrate distinct morphological characteristics between both fibroblastic cell types without any difference in their myofibroblastic feature.

[0200] Differential Functional Characteristics Between Isolated Fibroblasts from Peri-Lesional and SL Skin Biopsies

[0201] To functionally explore both types of fibroblasts, we first analysed their proliferation rate by seeding equal number of FS and FL and counting them at 96 h of culture. As shown in the FIG. 2a, cell number was significantly lower in FL relative to FS. Accordingly, measurement of metabolically intact cells demonstrated that FL proliferation was significantly reduced as compared to FS (FIG. 2b). Next, we examined the migration capacity of both fibroblastic types by wound healing assay and calculated the percentage of wound closure for each time point. Results presented in the FIG. 2c demonstrated that migration kinetics of FL were significantly lower relative to FS. Finally, secretion capacity of both types of fibroblasts was determined by quantifying cytokines and growth factors present in their respective conditioned media by ELISA and V-plex assays. As shown in FIG. 3a, HGF, KGF and SCF were detected in both FS and FL but significantly at higher rates in FL. We further investigated the secretion pattern of 31 additional soluble factors that were chosen based on their role in inflammation. Interestingly, the cytokine IL-13 and the growth factor TGF1 were significantly increased in the FL media as compared to FS (FIG. 3b); the concentration of the 29 other soluble factors was neither differentially secreted (Eotaxin, Eotaxin-3, GM-CSF, IFN-, IL-10, IL-12p70, IL-16, IL-17A, IL-4, IL-6, IL-7, IL-8/IL-8 (HA), IP-10, MCP-4, MDC, MIP-1, MIP-1, TARC, TNF-, VEGF-A) nor detectable (IL-12/IL-23p40, IL-15, IL-1, IL-1, IL-2, IL-5, IL8 cck, MCP-1, TNF-).

[0202] Collectively, functional study of the fibroblasts isolated from SL demonstrated that their proliferation rate, metabolic activity and migration capacity were lower as compared to their intra-individual counterparts whereas their secretion profiles were higher for some soluble factors that are involved in tissue fibrosis.

[0203] Discussion

[0204] To our knowledge, this study constitutes the first description of the isolation of primary fibroblasts from peri-lesional and SL biopsies that were issued and compared from the same volunteer. By staining human peri-lesional and SL skin samples for melanin, we validated the origin of the FS and FL and identified some of their morphological and functional features.

[0205] It is now accepted that fibroblasts represent a heterogeneous population of cells found in most of the tissues (6, 7). Accordingly, our microscopic observations revealed that FS and FL exhibit heterogeneous shapes and sizes with FL characterized by more elongated shapes and elliptical shaped nuclei. Given that actin isoforms are markers of the fibroblast heterogeneity with their differential expression and organization of cytoskeletal proteins (31, 32), we looked at the F-actin iso form. Spread FS and FL showed bundles that were quite similar but not identical in terms of cables length, number and distribution. Indeed, FL showed noticeable actin stress fibers in their cytoplasm that were linked to higher quantity of F-actin, suggesting that both types of fibroblasts have different actin-based cytoskeletal frameworks. Looking at the -SM actin isoform, similar quantities of this myofibroblast marker were found in FS and FL. This observation was in agreement with the absence of difference between peri-lesional and SL skin in terms of -SMA-positive cells in the dermis (33). Despite evidence describing that fibroblasts isolated from tissues continually differentiate into myofibroblast-like cells with different degrees of -SMA expression once they are grown in culture (34, 35), its is tempting to speculate that our primary SL-fibroblast model behaves, in terms of myofibroblast differentiation, like the fibroblasts described in SL biopsies.

[0206] Functional studies demonstrated that both fibroblasts displayed differential proliferation rate with a lower level for FL. Apparent conflicting immunohistochemistry data, generated with the proliferation marker Ki67, were brought back together by demonstrating that expression of this marker is modulated during SL development. Indeed, Ki67-positive cells are strongly reduced at the later stages relative to early stages (23). Despite the association between Ki67-positive cells and supra-basal keratinocytes in tissue sections (23) and, given that our SL samples were probably of the later SL stages, our results strongly suggested that fibroblasts contribute to the cellular quiescent status observed in advanced SL.

[0207] Another biological function of the fibroblast consists on its migration capacity that is related to actin cytoskeleton, integrin adhesion molecules and extracellular matrix proteins (36, 37). According to the different cell morphology and cytoplasmic structural organization between FL and FS, cell mobility was also different between FS and FL. The decrease of FL motility relative to FS could be explained by impaired integrin or extracellular matrix protein expression.

[0208] The last function studied was the secretion profile of FS and FL, which contributes to cellular functional crosstalks. Indeed, melanocytes and their neighbouring keratinocytes regulate mutually their functions through networks of factors (10, 27, 38, 39). Moreover, recent evidence points out functional implication of dermal fibroblast secretion in modulating not only constitutive pigmentation (10, 11, 40-42) but also the development of various hyper-pigmented disorders, via the secretion of growth factors such as SCF (43-45), HGF (43, 45) and KGF (46). In this context, we first quantified these melanogenic factors in our primary fibroblastic models that were issued from peri-lesional and SL biopsies. Higher secretion levels of SCF, HGF and KGF detected in FL media supported published data showing higher immuno-detection levels of these 3 growth factors in the upper dermis of SL skin (28). This strongly suggests that the secretion capacity of our in vitro primary fibroblast model was close to fibroblasts located in the upper dermis of SL.

[0209] In line with the chronic inflammatory context of the SL (8, 25, 26), our findings also demonstrated that TGF1 and IL-13 (47, 48) were differentially secreted between FS and FL with a higher level for FL. While the ubiquitous TGF1 has been reported to inhibit in vivo keratinocyte proliferation (47, 49) and in vitro melanocyte differentiation 2, the endogenous secretion of the cytokine IL-13 by dermal fibroblasts, melanocytes and keratinocytes has never been described. Nevertheless, further mechanism studies using neutralizing antibodies should clarify the role of these soluble factors in SL progression.

[0210] In conclusion, isolation of fibroblasts from adjacent- and SL-skin biopsies allowed establishing primary dermal cell models with some specific morphological and functional features that are similar to those described in SL biopsies. By identifying FS and FL secretion signatures, new investigations are required to clarify the autocrine/paracrine roles of the differential concentrations of soluble factors on melanocyte and keratinocyte functions. It is tempting to speculate that our primary fibroblast model might constitute a good cellular tool to test compounds for the development of whitening and anti-aging agents in topical treatments.

REFERENCES

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Example 2

[0261] Differential Morphological and Functional Features Of Fibroblasts Explanted from Solar Lentigo.

[0262] Materials and methods

[0263] Materials

[0264] Phosphate buffer solution (PBS), Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS), gentamycin, trypsin and all cell culture plastics were purchased from Dutscher (Brumath, France); paraformaldehyde (PFA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), dimethylformamide (DMF), glycine, acetone, triton X-100, bovine serum albumin (BSA), mouse anti--SM actin (clone 1A4, Sigma-Aldrich) and goat anti-mouse IgG-FITC antibodies, rhodamine-conjugated phalloidin, Hoechst were purchased from Sigma-Aldrich (St Quentin Fallavier, France).

[0265] Preparation and Cell Culture of Primary Fibroblasts from Skin Biopsies

[0266] Skin specimens were obtained from 10 volunteers (Caucasian females, 67-89 years of age) after written informed consent (Pr. P. Humbert, University hospital of Besanon, France). Two biopsies of 3-mm punches (20 mm apart) were collected on the back of the hand from the same patient; one containing a solar lentigo lesional macula and one excluding any visible pigmented spot (A FL and a FNL explant derived from one patient are defined in the text as couple). After surgery, both skin pieces were kept at 4 C. in a sterilized compress previously soaked in 0.9% sterile solution of sodium chloride in water. Within one hour from the sampling, the skin pieces were washed for 10 minutes with 1% penicillin/streptomycin antibiotics cocktails. Thereafter, explants were maintained in cell culture dishes containing DMEM supplemented with 10% FCS and 1% Gentamycin for fibroblasts extraction. Installed vertically to dry for 1 hour in a humidified 5% CO2 atmosphere at 37 C., cell culture dishes were then slowly horizontally settled to enable the explants to be in contact with the medium. Fibroblasts obtained from SL spot (FL) and non-pigmented skin (FNL) of 10 patients started to migrate out from the biopsies after 1 or 2 weeks. FL and FNL were maintained until the monolayer cultures reached 80% confluency and were passaged twice using a trypsin (0.05%)/EDTA (0.02%) solution. Cells were used for experiments at passages 2-7.

[0267] Fluorescence Staining

[0268] Primary FNL and FL fibroblasts were seeded in Lab-Tek chamber slides at 110.sup.4 cells/chamber and maintained in culture at 37 C. for 24 h. They were then fixed 10 min in 3% PFA solution and permeabilized for 10 min with cold acetone at 20 C. Nonspecific adhesion sites were blocked by 10 min incubation in 1% glycine solution followed by 1 h incubation in BSA solution (3% BSA/10% serum/0.1% triton X-100 in PBS). Primary antibody directed against human -SMA was then added (1: 100 in PBS/1%BSA/0.1% triton X-100) and incubated overnight at 4 C. in a moist chamber. Cells were then incubated with the FITC conjugated secondary antibody (1: 40 in PBS) for 1 h and further 30 min with rhodamine-conjugated phalloidin for filamentous actin (F-actin) staining (2.5 g/ml). Cellular nuclei were countermarked with Hoechst solution. Cells were then mounted in Dako fluorescent medium, observed under fluorescence microscopy (Axioskop 40, Zeiss) and further analyzed using MetaMorph software (Molecular Devices). Elliptical Fournier analysis (EFA Harmonic 2, available within the MetaMorph package) was used to describe and quantify the cellular body shape of FNL and FL. Elliptical Form Factor was calculated by the ratio of the object breadth (the caliper width of the object, perpendicular to the longest chord) and its length (the span of the longest chord through the object) for 109 FNL and 102 FL cells explanted from 5 skin couples.

[0269] Cell Proliferation

[0270] Proliferation of FNL and FL was determined by measuring metabolic activity (colorimetric MTT assay) (1) and by counting viable cells (Trypan blue exclusion assay).

[0271] MTT Assay

[0272] FNL and FL were seeded into 96-well plates (510.sup.3 cells/well) and incubated with DMEM supplemented for 24 h. After removing supernatant and washing by PBS 1, 10 l of MTT solution (0.5 mg/mL in DMEM) were added for 4 h at 37 C. The formazan-blue crystals were dissolved overnight at 37 C. in 100 L of extraction medium (10% SDS/0.04% DMF in H.sub.2O). The optical density was read at 570 nm with a Multiskan FC spectrophotometer (Fisher Scientific).

[0273] Trypan Blue Exclusion Assay

[0274] FNL and FL were seeded at a density of 7105 cells/T75 flask and were incubated with DMEM supplemented for 4 days. Cells were trypsinized and counted by Trypan blue stain.

[0275] Senescence-Associated -Galactosidase Activity

[0276] FNL and FL from 2 SL patients, as well as normal fibroblasts explanted from one biopsy collected after abdominal plastic surgery, were seeded at a density of 7000 cells/cm.sup.2 in fibroblasts medium for 72 hours. Normal fibroblasts were subjected (FUV) or not (FNUV) to repeated-UVB exposures (200 mJ/cm.sup.2) with a VL-6.M tube (6W, 312 nm, Fischer Scientific, Massachusetts, USA). Senenescence-associated -Galactosidase activity was detected using the SA--Gal staining kit (Sigma Aldrich, France) according to the manufacturer's guidelines. Fibroblasts were then analysed by phase contrast on an Olympus microscope. Fibroblasts with blue cytoplasmic staining were scored as positive. The ratio of SA--Gal positive cells over total cell numbers was determined by blind counting 3 fields/well in the triplicated FNL (27/514 cells), FL (87/388 cells), FNUV (5/458 cells), FUV (269/353 cells).

[0277] ELISA and V-Plex Assays

[0278] Secreted factors in paired FNL/FL-conditioned media were quantified by ELISA kits (Quantikine for KGF, HGF, SCF and TGF-1; R&D Systems) or by MSD 30-PLEX Proinflammatory Panell, Cytokine Panell, Chemokine Panell kits and TGF1 kit, according to the manufacturer's protocols.

[0279] Scratch Assay

[0280] Primary FNL and FL were grown on 12-well Falcon plates for 24 h to reach 95-100% cell confluency. Scratches were done using a sterile 10 l-pipette tip across each well. After gentle washes to remove loose cells and debris, cells were incubated with supplemented DMEM without Red phenol, covered with mineral oil and placed in the incubator at 37 C. with 5% CO2 of BD Pathway 855 Bioimaging (Becton Dickinson, USA). Image captures every hour using the AttoVision Software (Becton Dickinson) were taken for a 16 h-period. The width of the scratch was measured at 3 different locations for each time point by using ImageJ Software. The width at each time point (tn) was subtracted from the width at time (t0) and normalized to the width at time 0 ((t0tn)/t0) and the values were expressed as percentage of scratch closure that reflects the FL and FNL migration capacity.

[0281] Statistical Analysis

[0282] Statistical significance was assessed using paired Student's t test (Prism5.0, Graph Pad Software). P values were considered significant with the following degrees: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

REFERENCES

[0283] 1. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65, 55-63 (1983).

[0284] 2. Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol 5, 99-118 (2010).

[0285] 3. Acosta, J. C. et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Bio115, 978-90 (2013).

[0286] 4. Waldera Lupa, D. M. et al. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin. J Invest Dermatol 135, 1954-68 (2015).

[0287] 5. Duval, C. et al. Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging. PLoS One 9, e114182 (2014).

[0288] Results

[0289] Upon aging, chronic exposure to ultraviolet radiations and pollution induces benign hyper-pigmented lesions, such as Solar Lentigo maculae (SL) (1). Well-defined histologically, SL is distinguishable from other hyper-pigmented diseases and can be classified relative to its evolution (2-4). Differential gene-profiling analyses comparing SL and normal skin biopsies revealed that SL tissues are mainly composed of epidermal activated melanocytes as well as hypo-proliferating and hypo-differentiated keratinocytes with a background of chronic inflammation. In absence of fibroblast markers, immuno-staining analyses for several growth factors and secreted proteins in the upper dermis of SL biopsies strongly suggest that dermal fibroblasts contribute functionally to dysregulation of epidermal cells (5). These observations are strengthened by recent studies using a pigmented reconstructed skin model that demonstrates the influence of dermal fibroblasts on skin pigmentation (6). However, data on the morphological and functional features of the SL primary fibroblasts that could explain their role in SL disease are not available.

[0290] In this study, we developed for the first time an in vitro model of SL primary fibroblasts obtained from 10 volunteers. Two biopsies, from a Solar Lentigo macula and from a non-pigmented skin respectively, were collected from the same patient. Fibroblastic explants from both biopsies (FL and its normal counterpart FNL; called FL/FLN couple) were cultured for two rounds before analysing and comparing their intra-individual morphological and functional features.

[0291] Global microscopic examination revealed that both primary fibroblasts with similar culture condition exhibit variable shape with the tendency of a more elongated appearance for the FL fibroblasts in all of the examined FL/FNL couples (data not shown). The Comparison of phalloidin stained FNL-versus FL showed that FL fibroblasts present a more intense labelling of F-actin (data not shown). This observation suggests that the two types of primary fibroblasts have different actin-based cytoskeletal frameworks. However, immunofluorescence analysis of -SMA revealed similar amounts of this myo-fibroblastic marker in FL and FNL (FIG. 4), in agreement with a possible reticular origin 7. Our data confirm the absence of difference between peri-lesional and SL skins in terms of -SMA-positive cells in the dermis 4 and proposes that the SL-fibroblast model shares functional features with the fibroblasts described in SL biopsies.

[0292] To explore functionally both types of fibroblasts, we first analysed their proliferation rate. As shown in the FIG. 2a, cell number was significantly lower in FL relative to FNL after 96 hours of culture. Moreover, FL cells showed lower metabolic activity as compared to FNL cells (FIG. 2b). Since an arrest of cell proliferation with persistency of metabolic activity is a hallmark of senescence (8), we quantified the senescence-associated -galactosidase activity in FNL versus FL cells. As shown in FIG. 5, the percentage of -galactosidase positive cells was significantly higher in FL compared to FNL but lower than UV-treated primary fibroblasts used as control. Because senescent cells develop altered activities that may induce changes in the tissue microenvironment (8), we characterized the secretion capacity of both types of fibroblasts by quantifying cytokines and growth factors in their respective conditioned media (CM) by ELISA and V-plex assays. As shown in FIG. 3, HGF, KGF and SCF were detected in both FNL and FL CM with significant higher levels in FL CM. These results support data revealing higher levels of these 3 factors in the dermis of the SL skin (9). The analysis of 31 additional secreted factors involved in inflammation revealed that IL-13 cytokine and growth factor TGF1 as significantly increased in the FL media as compared to FNL; the concentration of 29 other soluble factors was either not significantly different or undetectable. Comparison of the FL secretory profile to secretomes of senescent cells (8), fibroblasts isolated from aged skin (10) and photo-aged vs unexposed fibroblasts in an in vitro skin model (6) confirmed the senescent-like phenotype of the FL (Table 1).

[0293] Since wound healing is compromised in aging adults, we examined the migration capacity of both fibroblastic cell types by a scratch assay and calculated the percentage of the scratch closure at each time point. The results presented in FIG. 2c show that migration kinetics of FL were significantly lower than those of FNL. The FL different actin-based cytoskeletal framework (data not shown) and/or its specific pattern of secreted proteins (FIG. 3) may explain the lower FL mobility.

[0294] In conclusion, isolation of fibroblasts from adjacent- and SL-skin biopsies allowed establishing primary dermal cell models with some specific morphological and functional features that are similar to those described in SL biopsies. New investigations are now required to clarify the autocrine/paracrine roles of the differential concentrations of soluble factors on melanocyte and keratinocyte functions. It is tempting to speculate that our primary fibroblast model constitutes a valid cellular tool to test compounds for the development of whitening and anti-aging agents in topical treatments.

TABLE-US-00001 TABLE 1 Comparison of soluble secreted factors from fibroblasts isolated from Solar lentigo (FL) and non-lesional (FNL) skins (this study) with those from senescent cells (SASP) 2, 3, fibroblasts isolated from intrinsically aged skin (SAASP) 4 and from photo-aged vs young fibroblasts in an in vitro skin model5. FNL and FL-conditioned media (n = 10 couples) were subjected to quantification of 34 soluble factors using ELISA or V-PLEX assays (Figures, Table 1 and Materiel and Methods). Concentration of each factor was calculated by normalization to cell number. Means of concentration (column Mean), standard deviations (column ET) and p values were calculated using paired Student's t-test. Grey lines indicate quantified factors below detection (BD). means variation of the measured soluble factors ( = increased, = decreased, x = no change and nd = not determined) between FNL and FL. Comparison of the variations () of SL fibroblast associated secretory profile, SASP, SAASP and photo-aged fibroblasts reveals that the FL secretory profile behaves as SASP. Secreted Skin soluble factors Aging- from culture Solar Lentigo Fibroblast-associated secretory Senescence- Associated supernatants of profile (this study) associated Secreted fibroblasts from FNL FL phenotype proteins photo-aged vs young Soluble Mean Mean (SASP) (SAASP) unexposed skin factors (pg/ml) ET (pg/ml) ET p HGF 7.26E03 1.37E03 3.73E02 5.30E03 0.0003*** nd X IL-13 4.35E05 7.55E06 7.60E05 1.70E05 0.0294* X nd KGF 5.56E04 1.66E04 9.15E04 2.49E04 0.0388* X X SCF 1.28E03 3.39E04 2.20E03 4.41E04 0.0168* nd X TGF1 2.31E03 2.39E04 3.70E03 5.17E04 0.0076** nd nd Eotaxin 6.30E05 5.58E05 8.98E05 6.69E05 0.152 X X X nd ns Eotaxin-3 8.88E05 4.70E05 1.38E04 1.16E04 0.192 X nd nd ns GM- 4.90E06 3.93E06 1.14E05 1.42E05 0.167 X nd CSF ns IFN- 6.69E06 4.86E06 1.25E05 1.15E05 0.102 X X nd ns IL-10 1.49E06 9.92E07 3.41E06 3.93E06 0.102 X nd nd nd ns IL-12 BD BD nd nd nd IL- 3.99E06 2.12E06 6.83E06 5.93E06 0.089 X nd nd nd 12p70 ns IL-15 BD BD nd IL-16 2.45E05 1.27E05 3.60E05 2.20E05 0.116 X nd nd nd ns IL-17A 5.17E06 2.21E06 7.43E06 3.67E06 0.110 X nd nd nd ns IL-1 BD BD nd X IL-1 BD BD nd IL-2 BD BD nd nd nd IL-4 1.13E06 6.95E07 1.80E06 1.62E06 0.133 X nd nd ns IL-5 ND ND nd nd nd IL-6 7.54E04 5.29E04 1.06E03 1.20E03 0.258 X X X ns IL-7 2.79E05 2.24E05 5.45E05 5.15E05 0.135 X X nd ns IL-8 1.60E03 1.67E03 3.46E03 4.49E03 0.168 X nd ns IL-8 cck BD BD nd nd nd IP-10 1.20E05 1.02E05 2.12E05 2.42E05 0.094 X nd nd ns MCP-1 BD BD nd X MCP-4 2.84E05 2.48E05 4.54E05 7.11E05 0.430 X nd nd ns MDC 1.73E04 1.36E04 2.01E04 1.12E04 0.470 X nd nd nd ns MIP-1 1.73E04 1.36E04 2.01E04 1.12E04 0.470 X X nd ns MIP-1 1.60E05 1.03E05 3.13E05 4.02E05 0.260 X nd nd nd ns TARC 5.04E05 4.53E05 7.72E05 4.78E05 0.201 X nd nd nd ns TNF- 2.99E06 2.48E06 6.17E06 5.84E06 0.089 X nd nd ns TNF- BD BD nd nd nd VEGF-A 1.76E03 1.09E03 2.20E03 1.23E03 0.366 X X nd ns

REFERENCES

[0295] 1. Nakamura, M. et al. Environment-induced lentigines: formation of solar lentigines beyond ultraviolet radiation. Exp Dermatol 24, 407-11 (2015).

[0296] 2. Lin, C. B. et al. Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development. J Dermatol Sci 59, 91-7 (2010).

[0297] 3. Goorochurn, R. et al. Biological processes in solar lentigo: insights brought by experimental models. Exp Dermatol (2016).

[0298] 4. Cario-Andre, M. et al. Perilesional vs. lesional skin changes in senile lentigo. J Cutan Pathol 31, 441-7 (2004).

[0299] 5. Bastonini, E., Kovacs, D. & Picardo, M. Skin Pigmentation and Pigmentary Disorders: Focus on Epidermal/Dermal Cross-Talk. Ann Dermatol 28, 279-89 (2016).

[0300] 6. Duval, C. et al. Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging. PLoS One 9, e114182 (2014).

[0301] 7. Mine, S., Fortunel, N. O., Pageon, H. & Asselineau, D. Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging. PLoS One 3, e4066 (2008).

[0302] 8. Coppe, J. P., Desprez, P. Y., Krtolica, A. & Campisi, J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol 5, 99-118 (2010).

[0303] 9. Kovacs, D. et al. Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo. Br J Dermatol 163, 1020-7 (2010).

[0304] 10. Waldera Lupa, D. M. et al. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin. J Invest Dermatol 135, 1954-68 (2015).