DR.

NICOLAS LEBONVALLET (Orcid ID : 0000-0001-7263-1408)

Accepted Article
Article type : Regular Article

A new tool to test active ingredient using acid lactic in vitro, a help to understand cellular mechanism
involved in stinging test: an example using a bacterial polysaccharide (Fucogel®).

Mehdi Sakka1, Raphael Leschiera1, Christelle Le Gall1, Olivier Gouin1, Killian L’herondelle1,

Paul Buscaglia2, Olivier Mignen2, Jean-Luc Philbé3, Thibaut Saguet3, Jean-Luc Carré1,

Laurent Misery1, Nicolas Lebonvallet1

1. Laboratory Interactions Neurons-Keratinocytes, University of Western Brittany, Brest,

France.
2. INSERM U1227 «Lymphocyte B et Auto-Immunité», Brest, France
3. BioEurope – Groupe Solabia, Anet, France

Corresponding author: Prof Laurent Misery, Department of Dermatology, University Hospital, 5

Avenue Foch, 29200 Brest, France

Tel: + 33 298 22 33 15 Fax: + 33 298 22 33 82 E-mail: laurent.misery@chu-brest.fr.

Abstract

The stinging test is an in vivo protocol that evaluates sensitive skin using lactic acid (LA). A

soothing sensation of cosmetics or ingredients can be also appreciated through a decrease of

stinging score. To predict the soothing sensation of a product before in vivo testing, we

developed a model based on an LA test and substance P (SP) release using a co-culture of

human keratinocytes and NGF-differentiated PC12 cells. A bacterial fucose-rich

polysaccharide present in Fucogel® was evaluated as the soothing molecule in the in vivo

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/exd.13489
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 stinging test and our in vitro model. Excluding toxic concentrations, the release of SP was
Accepted Article significant from 0.2% of lactic acid for the PC12 cells and from 0.1% of lactic acid for the

keratinocytes. When the pH was adjusted to approximately 7.4, LA did not provoke SP

release. At these concentrations of LA, 0.1% of polysaccharide showed a significant decrease

in SP release from the two cellular types and in co-cultures without modifying the pH of the

medium. In vivo, a stinging test using the polysaccharide showed a 30% decrease of prickling

intensity versus the placebo in 19 women between the ages of 21 and 69. Our in vitro model

is ethically interesting and is adapted for cosmetic ingredients screening because it does not

use animal experimentation and limits human volunteers. Moreover, Fucogel® reduced

prickling sensation as revealed by the in vivo stinging test and inhibits the neurogenic

inflammation as showed by our new in vitro stinging test based on SP release.

Keywords

Lactic acid, keratinocyte, model, PC12 cells, polysaccharide

Introduction

The stinging test is an in vivo test to determine the severity of sensitive skin. Frosh et al.

described and recommended guidelines for using the stinging test (1–4). First, lactic acid

(LA) at 10 or 5 percent is applied on the skin of the individuals, precisely on the nasolabial

fold. LA is an alpha hydroxycarboxylic-acid which can potentially trigger a variety of

receptors, which may explain the effect on the skin by an acidic effect or by the specific

action of the ligand on a receptor. If the individuals have itching or unpleasant sensations, the

test is considered positive for sensitive skin. Therefore, cosmetic substances are applied on

“stingers” for testing an inhibition of sensation. This methodology is regularly used in the

cosmetic industry. Hence, it is considered as distinguishing sensitive and non-sensitive skins

by many authors and may distinguish biophysical parameters of the skin, including the

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 participation of neurons (5–7). However, the mechanism of LA acting as irritating substance
Accepted Article to induce unpleasant sensation is probably linked with a local cutaneous neurogenic

inflammation (CNI) on the skin, as recently shown in sensitive skins (8,9). In the stinging test

as well as in sensitive skins, keratinocytes and the nerve fibres of the sensory neurons of the

epidermis are the two major cells involved (10). With regard to CNI, proteases, cytokines and

neuropeptides are implicated in the dialogue between skin cells (11). Among all these

molecules, the neuropeptide substance P (SP), which is released after a cytosolic calcium

influx, is a good marker of CNI in vitro and in vivo (8,12–17). Consequently, it is probably

strongly associated with to the unpleasant sensation present in the stinging test.

Fucogel® is a hydrophilic polysaccharide with a repeating unit of acetylated charged linear

trisaccharide, fucose, galactose and galacturonic acid (18). Fucose-mannose receptors are

described in skin cells, keratinocytes and fibroblasts (19,20). Péterszegy et al. showed that in

a dose-dependent manner, this polysaccharide increased fibroblast proliferation and viability

and decreased ascorbate-induced cytotoxicity (21). Many studies in different models showed

a anti-inflammatory response of polysaccharides containing fucose or galactose (22–24).

However, there is no data on the fucose-rich anti-inflammatory effect on the skin, especially

concerning CNI. We propose that this polysaccharide, due to its composition, could be a

good candidate to have an effect act on the CNI process and a soothing activity in the

stinging test.

To mimic in vitro the in vivo stinging test and understand the effect of LA on the skin, we

developed a model that included neuronal-like cells and human keratinocytes exposed to LA

based on SP release. This model is based on previously published models studying CNI,

using primary animal sensory neurons or cell lines (13,25–30). To facilitate the study or the

screening of industrial or natural compounds the NGF-differentiated PC12 cell line, which

possess sensory-like characteristics, were used. Equally, we would like to propose a pre-

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 clinical screening model applicable for cosmetics to limit the time, cost and human resources
Accepted Article consumed by the in vivo stinging test. By using this model, the first objective was to

determine the effects of LA on CNI by analyzing the SP release and the cytosolic calcium

entry in keratinocytes or in NGF-differentiated PC12 cells or both (coculture) at non-toxic

concentrations. The second objective was to evaluate the effects of an anti-inflammatory

polysaccharide containded in Fucogel® on the LA induced-SP release in vitro and in

parallel, to define if Fucogel® could decrease prickling sensations induced by LA on

sensitive skin volunteers in vivo.

Methods

Cell culture

Normal Human Epidermal keratinocytes (NHEK) were obtained from patient

abdominoplasties after consent as previously published (25). The keratinocytes were

suspended in KSFM and were cultured at 37°C with 5% CO2. The PC12 cells (DSMZ,

ACC159 lot12) were cultured in DMEM/F12 10% FBS (Foetal Bovine Serum). The medium

was changed every 3 days.

Before the LA assay in monoculture, the keratinocytes were trypsinized, centrifugated and

plated at 29 000 cell/cm2 in KSFM on poly-L-Lysine. The keratinocytes were used the next

day for the assay. After EDTA (0.7mM in PBS, 5 minutes), PC12 cells were centrifugated

and plated at 23000 cells/cm2 on collagen I in a 96-well plate in their proliferation medium.

For LA and polysaccharide assay the PC12 cells were differentiated using NGF. After 24 h,

the supernatants were removed and replaced by DMEM with 50 ng/ml of NGF for 3 days.

For the co-culture, PC12 cells were prepared as previously described, and at the end of 3

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 days, the supernatants were removed, and the keratinocytes were placed in KSFM medium
Accepted Article with PC12 cells for 24 h.

For the assay, LA or Fucogel® were used. Fucogel® was supplied as a solution of purified

biotechnological anionic linear polysaccharide (medium mw of 106 Da) obtained by bacterial

fermentation and is composed of repeated L-fucose, D-galactose and galacturonic acid. The

supernatants from the PC12 cells and the keratinocytes were replaced by a solution of

DMEM or KSFM containing LA at various concentrations in water (0%, 0.04%, 0.07%,

0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 5%, and 10%) and/or the polysaccharide at 0.01%, 0.1%

or 1%. After 15 minutes, the supernatants were kept and stocked at -80°C until use for the SP

release analysis by ELISA test (Interchim Montluçon, France). The remaining seeded cells

were assessed for viability using MTS according to the manufacture’s protocol.

Intracellular Ca2+ measurement

The NHEK were loaded with 4 µM Fura 2-AM (Molecular Devices, Sunnyvale, CA, USA)

and 2 µM pluronic acid (Thermo Scientific, Waltham, MA, USA) in the dark for 45 min at

37°C and 5% CO2. The cells were washed to remove the excess dye, and intracellular Ca2+

measurements were done according to the protocol described in (31). For the data analysis,

the F340/F380 intensity ratio was obtained for each time point after having defined the region

of interest and subtracted the background. The amplitude of the Ca2+ responses was measured

by calculating the difference between the basal and maximal ratio.

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 Reverse transcription and real-time quantitative PCR
Accepted Article
The RNA extraction was performed according to the TRI-reagent protocol (Sigma-Aldrich,

Saint Louis, MO, USA). The RNA was reverse transcribed to cDNA with the High Capacity

cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). The

quantitative PCR for the analysis of TRPV1, HCA1, 2 and 3, and ASIC1, 2, 3 and 4 was

performed by using specific probes for human (keratinocytes) and rattus (NGF-Differentiated

PC12 cells) and the Power SYBR® Green PCR Master Mix (Applied Biosystems, Waltham,

MA, USA) on the Step One Real-Time PCR System (Applied Biosystems, Waltham, MA,

USA). The sequences for each probe are indicated in Table S1 and were purchased from

Eurogentec (Seraing, Liège, BEL). Results were expressed in Ct (threshold cycle). A control

without DNA and melting curve was realized.

In vivo analysis of the efficiency of Fucogel® by stinging test

For the study, a group of 19 healthy volunteer women (from 18 to 70 years old) were

included after consent. All the volunteers presented with sensitive skin, including five with a

history of an atopy but no current atopic dermatitis. The stinging test was performed as

previously described (2,3), with the application of 10% LA on naso-labial folds. After the

stinging sensation appeared, the volunteers were auto-evaluated by using a scale from 0 to 9

based on the intensity of the sting. Afterwards, a solution of 2 mg/cm2 on 15 cm2 of

polysaccharide 3% (polysaccharide, water and preservatives) or placebo (water and

preservatives) was applied on one of the two naso-labial folds (right or left). On the other

naso-labial fold (left or right) the other product (placebo or polysaccharide) was applied at the

same time. The determination of the sides was randomized. After five minutes, a new auto-

evaluation for each side was performed. The study was double blinded.

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 Statistical analysis
Accepted Article
Cytotoxicity and SP release results were normalized and expressed in fold change compared

to the control. Graph represents the mean of “n” independent experiment +/- standard

deviation for each condition. Data were analysed using non-parametric Mann-Whitney test.

For calcium imaging analysis, the graph represents the mean of 20-40 cells +/- standard error

of the mean of a representative experiment. PCR results represent the mean of n independent

experiment +/- standard deviation for each condition. Graph of in vivo results represent the

mean of stinging test score of 19 volunteers +/- standard error of the mean. Data were

analysed using Wilcoxon two-tail test. All Results were considered as significant for a

p<0.05.

Results

Lactic acid at a non-toxic concentration induces the release of Substance P by

keratinocytes or PC12 cells

The levels of SP released were significantly increased in the NGF-differentiated PC12 cells

or keratinocytes treated by LA (10%, 5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.07%, and

0.04%) than without LA exposure (Figure 1A). The release of SP increased in a dose-

dependent manner until it levelled off as a function of the concentration of LA. The cytotoxic

effect of lactic acid was demonstrated in the NGF-differentiated PC12 cells and keratinocytes

(Figure 1B). For the keratinocytes, the results were significant for the concentrations 10%,

5%, 1%, 05%, 04% and 0.3%. A tendency for cytotoxicity was observed at 0.2% lactic acid.

For the NGF-differentiated PC12 cells, the results were significant for the concentrations

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 10%, 5%, 1% and 0.5%. A tendency for cytotoxicity was observed at 0.4% and 0.3% lactic
Accepted Article acid.

For the next manipulations, we decided to work at an optimal concentration of LA comprised

between the cytotoxicity and the intensity of the response of the cells. These concentrations

were of 0.1% lactic acid for keratinocytes, corresponding to an increased release that was 10-

fold compared to the basal level, with no toxicity and 0.2% or 0.1% for the NGF-

differentiated PC12 cells, corresponding to an increased release that was 5-fold compared to

the basal level, with no toxicity.

Lactic acid induces eCa and the release of Substance P by an acidic mechanism

In DMEM (the PC12 medium), we found a pH of 6.95 (+/- 0.16, n=5), 6.22 (+/- 0.17, n=3)

and 5.63 (+/- 0.05, n=3) for 0.1%, 0.2% and 0.3% LA, respectively. In KSFM medium (the

keratinocyte medium), we found a pH of 6.66 (+/-0.17, n=3) for 0.1% lactic acid. When

polysaccharide was added at 0.1%, we did not observe a difference (a difference of 0.11 for

DMEM with 0.1% lactic acid and 0.03 for KSFM with 0.1% lactic acid, n=2). When the pH

was neutralised (approximately 7.4) before the addition of lactic acid to the cells, no SP

release was observed neither in keratinocytes nor in NGF-differentiated PC12 cells (Figure

2).

In the calcium imagery, we showed that LA at 0.1% and 0.2% induced a cytosolic calcium

entry in the keratinocytes or NGF-differentiated PC12 cells, respectively (Figure 3A). This

entry was abolished for both when the pH was 7.4 (Figure 3B).

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 Keratinocytes and PC12 cells express receptors for the response to lactic acid
Accepted Article In the keratinocytes, using quantitative PCR, we showed that TRPV1 (Ct 28.1 +/- 0.8), HCA1

(Ct 29.3 +/- 1.6), 2 (Ct 27.6 +/- 0.8) and 3 (Ct 27.9 +/- 0.8), ASIC1 (Ct 28.8 +/- 1.1), 2a (Ct

28.2 +/- 0.4), 3 (Ct 27.5 +/- 0.7) and 4 (Ct 33.3 +/- 2.9) were expressed (n=3). In the NGF-

differentiated PC12 cells, we showed that TRPV1 (Ct 27 +/- 0.8), HCA1 (Ct 30.8 +/-1.3), 2

(Ct 28.1 +/-1.7), ASIC1 (Ct 22 +/-2.4), 2a (Ct 30.8 +/-2.4), 3 (Ct 29.8+/-3.1) and 4 (Ct 24.3

+/- 4.3) were expressed (n=3).

The polysaccharide decreases the level of SP release induced by lactic acid in the PC12
cells and Keratinocytes and diminishes the prickling intensity in vivo without a pH
modification

To evaluate the potent effect of polysaccharide on SP release, we used the optimal

concentration of LA on the cells. With LA, SP release was induced on the keratinocytes or

NGF-differentiated PC12 cells. This release was considered as an induction control. In

parallel, Fucogel® was used at 0.1% and 0.01% and was added into the culture medium at

the same time as LA. The results showed a significant decrease of 50% in the release of SP

when polysaccharide was applied to the keratinocytes or PC12 cells (Figure 4A).

In the co-culture model, the release of SP was significantly decreased by 25% (n=3) when

polysaccharide was applied (Figure 4B left).

The polysaccharide Fucogel® decreases stinging scores

The polysaccharide was evaluated, in vivo, using classical stinging test on study of 19 women

volunteers. After the stinging appeared, the volunteers were auto-evaluated on a scale from 0

to 9 based on the intensity of the sting. Afterwards, polysaccharide or placebo was applied on

one of the two naso-labial folds in blind. After five minutes, a new auto evaluation for each

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 side was performed. The initial score of stinging test were of 4.79 (+/- 0.49) for placebo
Accepted Article group and of 4.58 (+/-0.48) for polysaccharide group. When placebo or polysaccharide was

applied the score after five minutes were of 3.68 (+/- 0.64) for placebo group and of 2.42 (+/-

0.56) for polysaccharide group. The results showed a significant decrease of 50% of prickling

intensity at five minutes for polysaccharide versus T0 and 30% at five minutes for

polysaccharide versus placebo in the 19 volunteers (Figure 4B right).

Discussion

To understand the implication of LA used in a stinging test and to screen molecules or active

compounds before in vivo testing, we developed an in vitro model based on the monoculture

and co-culture of human primary keratinocytes and NGF-differentiated PC12 cells that were

subjected to LA. The substance P dosed in the supernatant was used as a marker of LA–

induced CNI. The polysaccharide contained in Fucogel® was used as a soothing molecule,

and it was evaluated in our cellular model and in an in vivo stinging test.

On PC12 cells or keratinocytes, we showed that LA induced a significant release of SP and

an intra-cytosolic calcium entry, independently of a toxic effect. Then, we worked at non-

toxic concentrations of LA with the maximum release of SP, which corresponded to 0.1

(keratinocytes) or 0.1-0.2% (NGF-differentiated PC12 cells). On an in vitro reconstructed

model of differentiated human epidermis, an increased mortality with creams containing LA

at similar concentrations to our observed cytotoxic effect was previously shown (32). SP

release is correlated with CNI and is regularly used as a neuro-inflammatory marker (12,17).

We assumed that LA possesses a pro-inflammatory effect on the cells. Confirming our

results, other studies demonstrate that LA increases intra-cellular calcium and is an initiator

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 of SP release in DRG neurons (33). In vivo, stinging test induces a tendency to an increase of
Accepted Article the number of SP-positive nerve fibres (34).

LA uses a variety of a potent family of receptors, which may explain its effect on the skin by

acid function or by the specific action of a ligand on a receptor. The channels activated by an

acidic effect are numerous, but the two main families include the transient receptor potential

vanilloid 1 (TRPV1) and Asic-sensing ion channels (ASIC)(35). The Asic-sensing ion

channel family is composed of 6 members (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and

ASIC4), and they are proton receptors that are activated by LA (36). The activation of ASIC

with LA at 0.1% increases intracellular calcium in DRG neurons (33). TRPV1 is expressed

on keratinocytes and sensory neurons and is stimulated by many effectors, such as heat,

capsaicin, UV, and endovanilloids. Moreover, protons at a pH less than 5.9 activate TRPV1.

The activation of TRPV1 increases intracellular calcium and participates in SP release and

CNI (8,17). The receptors activated by the ligand lactic acid are hydroxycarboxylic acid

receptors (HCA). HCA are a family of three G protein coupled receptors, including HCA1,

HCA2 and HCA3. LA is their main agonist. Most of these receptors are expressed in skin

cells and are implicated at different levels in CNI, pain, itching or other affiliated unpleasant

sensations (8,15–17,37).

To identify the dependent or independent acidic effects of LA, we used the same conditions

of LA, but we modified the pH, which was initially between 6 and 7, to 7.4. In these

conditions, no SP release or calcium entry was noted, leading to a pH-dependent mechanism.

These data excluded, in our conditions, the participation of the HCA receptor in SP release

and calcium entry. Interestingly, in an in vitro model of sciatic nerve acidification, the

authors showed a release of CGRP, which is often correlated with SP release (38). Peng et al.

showed the participation of TRPV1 at a pH of 5.2 but not at 6.1. Treatment with acetic acid

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 in mice skin provokes itching, a CNI related mechanism; moreover, the itching decreased in
Accepted Article KO ASIC3 mice (39). Another study demonstrated the participation of ASIC1 but not ASIC2

using extracellular acidosis in central neurons (40). The pH determinated in our culture

conditions is compatible with the activation of the ASICs receptors (35), which were also

expressed in our cells. However, the pH was too basic for an activation of TRPV1 (pH 5.9),

but sensitisation was possible, but was not studied here.

Fucose-rich polysaccharides possess various anti-inflammatory effects, but no study was

performed on CNI. Interestingly, the fucose-manose receptors exist in the skin, and fucose

binds on keratinocytes and fibroblasts (Figure S1)(21). To identify the soothing effect of this

polysaccharide and its action on CNI, we evaluated a classical stinging test in vivo. Our data

showed a significant decrease in the prickling sensation by 50% when the polysaccharide was

applied versus T0, and the decreased sensation was 30% versus the placebo. Interestingly, in

our in vitro model, we demonstrated that polysaccharide significantly decreased the release of

SP by 25% in co-culture (NGF-differentiated PC12 cells with keratinocytes) and until 50% in

monoculture (Keratinocytes or NGF-differentiated PC12 cells). This surprising but

interesting result may be explained by the interaction/communication between keratinocytes

and PC12 in co-culture, that is absent in monoculture, probably through other inflammatory

molecules that will stimulate the cells to produce SP by another way that is not blocked by

the polysaccharide. The decreasing effect of the polysaccharide was independent of pH

because the polysaccharide does not provoke a significant modification of the pH of the

medium. In a different model, the polysaccharide demonstrates an anti-inflammatory effect,

which we confirmed here by the decreased SP release (an inflammatory mediator) in vitro

and which was correlated by the diminution of a soothing sensation in the 19 volunteer

women by a mechanism dependant or independent of SP release. Consequently the

polysaccharide contained in Fucogel® possessed, by partially inhibition of the effect of acid

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 lactic probably via ASIC signalling, an anti-inflammatory and soothing effect independently
Accepted Article of the pH.

Because our in vivo and in vitro results went in the same direction, this in vitro model appears

as a valuable tool to predict or maybe replace the stinging test. Moreover, it is ethically

interesting and is adapted for cosmetic ingredients screening because it does not use animal

experimentation and limits human volunteers.

In conclusion, we developed a co-culture model to predict the soothing effect of a compound

(here a polysaccharide) by the inhibition of SP release induced by LA in keratinocytes and

PC12 cells. Control of the pH and mechanistic evaluation are easier in this model than in

vivo, and hence, this model permits the investigation of lactic acid on skin cells in vitro.

Acknowledgements

We thank Dr Etienne Camel of the Institut d’Exprertise Clinique for providing the stinging

test expertise. We thank Dr. S. Valentin and Pr Weigo Hu for providing skin samples.

MS, RL, NL, PB, KL performed the research. NL, CLG designed the research study. OG,

KL, OM, PB, JLP, TB contributed essential reagents or tools. NL, MS, JLP, TS analysed the

data. NL, MS wrote the paper. MS, NL, LM, TS, JLC, CLG revised the paper critically and

corrected it. All approved the submitted and final versions.

The experiments were realized in conformity with ethical consideration, in accordance with

declaration of Helsinski, European Union rules and French law. Sample of skin and stinging

test was realized after obtain the consent of patient and volunteers.

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 Conflicts of interest
Accepted Article
If yes, please state:

• Mehdi Sakka: Solabia

• Raphael Leschiera: none

• Christelle Le Gall: Beiersdorf, Clarins, Nestlé, Pierre Fabre

• Olivier Gouin: Uriage

• Killian L’herondelle: Uriage

• Paul Buscaglia: none

• Olivier Mignen: none

• Jean-Luc Philbé is an employee of Solabia

• Thibaut Saguet is an employee of Solabia

• Jean-Luc Carré: none

• Laurent Misery: BASF, Bayer, Beiersdorf, Bioderma, Clarins, Expanscience,

Johnson&Johnson, La Roche-Posay, Nestlé, Pierre Fabre, Sofibel, Solabia and

Uriage.

• Nicolas Lebonvallet: BASF, Johnson&Johnson, Sofibel, Solabia and Uriage.

This article is protected by copyright. All rights reserved.

 Reference
Accepted Article
1. Issachar N, Gall Y, Borell MT, Poelman MC. pH measurements during lactic acid stinging test
in normal and sensitive skin. Contact Dermatitis. 1997;36(3):152‑5.

2. Frosch PJ, Kligman AM. A method for appraising the stinging capacity of topically applied
substances. J Soc Cosmet Chem. 1977; 28(5):197-209.

3. Christensen M, Kligman AM. An improved procedure for conducting lactic acid stinging tests
on facial skin. J Soc Cosmet Chem. 1996;47(1):1‑11.

4. Seidenari S, Francomano M, Mantovani L. Baseline biophysical parameters in subjects with

sensitive skin. Contact Dermatitis. 1998;38(6):311‑5.

5. Roussaki-Schulze AV, Zafiriou E, Nikoulis D, Klimi E, Rallis E, Zintzaras E. Objective Biophysical

Findings in Patients With Sensitive Skin. Drugs Exp Clin Res. 2005;31:17-24.

6. Ham H, An SM, Lee EJ, Lee E, Kim HO, Koh JS. Itching sensation and neuronal sensitivity of
the skin. Skin Res Technol. 2016;22(1):104‑7.

7. Misery L, Loser K, Ständer S. Sensitive skin. J Eur Acad Dermatol Venereol JEADV.
2016;30:2‑8.

8. Gouin O, L’Herondelle K, Lebonvallet N, Le Gall-Ianotto C, Sakka M, Buhé V, et al. TRPV1 and

TRPA1 in cutaneous neurogenic and chronic inflammation: pro-inflammatory response induced by
their activation and their sensitization. Protein Cell. 2017;8(9):644-661

9. Costa A, Eberlin S, Polettini AJ, da Costa Pereira AF, Pereira CS, Ferreira NMC, et al.
Neuromodulatory and anti-inflammatory ingredient for sensitive skin: in vitro assessment. Inflamm
Allergy Drug Targets. 2014;13(3):191‑8.

10. Misery L. Neuropsychiatric factors in sensitive skin. Clin Dermatol. 2017;35(3):281‑4.

11. Ständer S, Schneider SW, Weishaupt C, Luger TA, Misery L. Putative neuronal mechanisms of
sensitive skin. Exp Dermatol. 2009;18(5):417‑23.

12. Pereira U, Boulais N, Lebonvallet N, Lefeuvre L, Gougerot A, Misery L. Development of an in

vitro coculture of primary sensitive pig neurons and keratinocytes for the study of cutaneous
neurogenic inflammation. Exp Dermatol. 2010;19(10):931‑5.

13. Pereira U, Boulais N, Lebonvallet N, Pennec JP, Dorange G, Misery L. Mechanisms of the
sensory effects of tacrolimus on the skin. Br J Dermatol. 2010;163(1):70‑7.

14. Pereira U, Garcia-Le Gal C, Le Gal G, Boulais N, Lebonvallet N, Dorange G, et al. Effects of
sangre de drago in an in vitro model of cutaneous neurogenic inflammation. Exp Dermatol.
2010;19(9):796‑9.

15. Roosterman D, Goerge T, Schneider SW, Bunnett NW, Steinhoff M. Neuronal control of skin
function: the skin as a neuroimmunoendocrine organ. Physiol Rev. 2006;86(4):1309‑79.

This article is protected by copyright. All rights reserved.

 16. Misery L. Skin, immunity and the nervous system. Br J Dermatol. 1997;137(6):843‑50.
Accepted Article 17. Gouin O, Lebonvallet N, L’Herondelle K, Le Gall-Ianotto C, Buhé V, Plée-Gautier E, et al. Self-
maintenance of neurogenic inflammation contributes to a vicious cycle in skin. Exp Dermatol. 2015;
24(10):723-6.

18. Guetta O, Mazeau K, Auzely R, Milas M, Rinaudo M. Structure and properties of a bacterial
polysaccharide named Fucogel. Biomacromolecules. 2003;4(5):1362‑71.

19. Szolnoky G, Bata-Csörgö Z, Kenderessy AS, Kiss M, Pivarcsi A, Novák Z, et al. A mannose-
binding receptor is expressed on human keratinocytes and mediates killing of Candida albicans. J
Invest Dermatol. 2001;117(2):205‑13.

20. Hespanhol RC, de Nazaré C Soeiro M, Meuser MB, de Nazareth S L Meirelles M, Côrte-Real S.
The expression of mannose receptors in skin fibroblast and their involvement in Leishmania (L.)
amazonensis invasion. J Histochem Cytochem Off J Histochem Soc. 2005;53(1):35‑44.

21. Péterszegi G, Isnard N, Robert AM, Robert L. Studies on skin aging. Preparation and
properties of fucose-rich oligo- and polysaccharides. Effect on fibroblast proliferation and survival.
Biomed Pharmacother. 2003;57(5):187‑94.

22. Lima ATM, Santos MN, de Souza LAR, Pinheiro TS, Paiva AAO, Dore CMPG, et al. Chemical
characteristics of a heteropolysaccharide from Tylopilus ballouii mushroom and its antioxidant and
anti-inflammatory activities. Carbohydr Polym. 2016;144:400‑9.

23. Wen Z-S, Xiang X-W, Jin H-X, Guo X-Y, Liu L-J, Huang Y-N, et al. Composition and anti-
inflammatory effect of polysaccharides from Sargassum horneri in RAW264.7 macrophages. Int J Biol
Macromol. 2016;88:403‑13.

24. Cheng J-J, Yang C-J, Cheng C-H, Wang Y-T, Huang N-K, Lu M-K. Characterization and
functional study of Antrodia camphorata lipopolysaccharide. J Agric Food Chem. 2005;53(2):469‑74.

25. Le Garrec R, L’herondelle K, Le Gall-Ianotto C, Lebonvallet N, Leschiera R, Buhe V, et al.

Release of neuropeptides from a neuro-cutaneous co-culture model: A novel in vitro model for
studying sensory effects of ciguatoxins. Toxicon Off J Int Soc Toxinology. 2016;116:4‑10.

26. Le Gall-Ianotto C, Andres E, Hurtado SP, Pereira U, Misery L. Characterization of the first
coculture between human primary keratinocytes and the dorsal root ganglion-derived neuronal cell
line F-11. Neuroscience. 2012;210:47‑57.

27. Lebonvallet N, Pennec J-P, Le Gall C, Pereira U, Boulais N, Cheret J, et al. Effect of human skin
explants on the neurite growth of the PC12 cell line. Exp Dermatol. 2013;22(3):224‑5.

28. Lebonvallet N, Boulais N, Le Gall C, Pereira U, Gauché D, Gobin E, et al. Effects of the re-
innervation of organotypic skin explants on the epidermis. Exp Dermatol. 2012;21(2):156‑8.

29. Lebonvallet N, Pennec J-P, Le Gall-Ianotto C, Chéret J, Jeanmaire C, Carré J-L, et al. Activation
of primary sensory neurons by the topical application of capsaicin on the epidermis of a re-
innervated organotypic human skin model. Exp Dermatol. 2014;23(1):73‑5.

This article is protected by copyright. All rights reserved.

 30. Sevrain D, Le Grand Y, Buhé V, Jeanmaire C, Pauly G, Carré J-L, et al. Two-photon microscopy
of dermal innervation in a human re-innervated model of skin. Exp Dermatol. 2013;22(4):290‑1.
Accepted Article
31. Philippe R, Antigny F, Buscaglia P, Norez C, Becq F, Frieden M, et al. SERCA and PMCA pumps
contribute to the deregulation of Ca2 + homeostasis in human CF epithelial cells. Biochim Biophys
Acta BBA - Mol Cell Res. 2015;1853(5):892‑903.

32. Rendl M, Mayer C, Weninger W, Tschachler E. Topically applied lactic acid increases
spontaneous secretion of vascular endothelial growth factor by human reconstructed epidermis. Br J
Dermatol. 2001;145(1):3‑9.

33. Light AR, Hughen RW, Zhang J, Rainier J, Liu Z, Lee J. Dorsal root ganglion neurons
innervating skeletal muscle respond to physiological combinations of protons, ATP, and lactate
mediated by ASIC, P2X, and TRPV1. J Neurophysiol. 2008;100(3):1184‑201.

34. Lonne-Rahm S, Berg M, Mårin P, Nordlind K. Atopic dermatitis, stinging, and effects of
chronic stress: A pathocausal study. J Am Acad Dermatol. 2004;51(6):899‑905.

35. Holzer P. Acid-sensitive ion channels and receptors. Handb Exp Pharmacol.
2009;(194):283‑332.

36. Sherwood TW, Frey EN, Askwith CC. Structure and activity of the acid-sensing ion channels.
Am J Physiol - Cell Physiol. 2012;303(7):C699‑710.

37. Boulais N, Pereira U, Lebonvallet N, Misery L. The whole epidermis as the forefront of the
sensory system. Exp Dermatol. 2007;16(8):634‑5.

38. Fischer MJM, Reeh PW, Sauer SK. Proton-induced calcitonin gene-related peptide release
from rat sciatic nerve axons, in vitro, involving TRPV1. Eur J Neurosci. 2003;18(4):803‑10.

39. Peng Z, Li W-G, Huang C, Jiang Y-M, Wang X, Zhu MX, et al. ASIC3 Mediates Itch Sensation in
Response to Coincident Stimulation by Acid and Nonproton Ligand. Cell Rep. 2015;13(2):387 ‑98.

40. Yermolaieva O, Leonard AS, Schnizler MK, Abboud FM, Welsh MJ. Extracellular acidosis
increases neuronal cell calcium by activating acid-sensing ion channel 1a. Proc Natl Acad Sci U S A.
2004;101(17):6752‑7.

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 Legend for illustrations
Accepted Article Figure 1: Substance P released and cytotoxicity induced by lactic acid treatment

Substance P released (A) and cytotoxicity (B) after lactic acid treatment on NGF-

differentiated PC12 cells (left) or keratinocytes (right). The results was normalised and

expressed in fold change +/- standard deviation compared to the control without lactic acid

(*, Mann-Whitney, p< 0.05, n=3).

Figure 2: Substance P released by lactic acid treatment after pH neutralization

Substance P released by lactic acid treatment at 0.2% and at 0.1% in the NGF-differentiated

PC12 cells or keratinocytes, respectively, after 15 minutes. The pH of the lactic acid solution

was neutralized by NaOH solution (1 M) to have a pH at 7.4. The results was normalised and

expressed in fold change +/- standard deviation compared to the control without lactic acid

(*, Mann-Whitney, p< 0.05, n=3).

Figure 3: Analysis of the calcium cytosolic entry induced by lactic acid

Calcium entry was analysed using calcium imaging. After record for 50ms of control signal,

the NGF-differentiated PC12 cells or keratinocytes were treated with lactic acid at 0.2%

(n=3) and at 0.1% (n=3), respectively or with neutralized lactic acid (ph7.4) at 0.2% (n=2)

and at 0.1% (n=3), for 200ms. The graphics represent the mean of 20-40 cells in the

representative experiment.

Figure 4: Fucogel® decreased substance P release in an in vitro mono and co-culture

model and stinging test score in in vivo conditions

A Substance P released by lactic acid treatment at 0.2% or at 0.1% in the NGF-differentiated

PC12 cells (left) or keratinocytes (right), respectively, after 15 minutes, with or without

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 Fucogel® at different concentrations of 0.01% and 0.1%. The results are normalised and
Accepted Article expressed in fold change +/- standard deviation compared to the control with lactic acid (*,

Mann-Whitney, p< 0.05, n=3).

B Substance P released by lactic acid treatment at 0.1% after 15 minutes in the co-cultured

model (left) containing together the NGF-differentiated PC12 cells and keratinocytes, with or

without Fucogel® at 0.1%. The results are normalised and expressed in fold change +/-

standard deviation compared to the control with lactic acid (*, Mann-Whitney, p< 0.05, n=3).

In in vivo conditions (right), a stinging test (with 10% lactic acid) was performed on 19

volunteer women with hypersensitivity of the skin. A solution, using 2 mg/cm² on 15 cm² of

Fucogel® 3% or placebo (water and preservatives), was applied after the lactic acid

application. An auto-evaluation on a scale of 0 to 9, where 9 is the maximal sting, at the

beginning (0 minutes) and at 5 minutes (*, Wilcoxon test, p< 0.05, n=19).

Figure S1: Fucosyl-BSA fluoresceine binding assay (green) on a slice of human skin

explants. The cell nuclei are labelled by a Hoechst solution. Scale bar: 50 µm.

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Accepted Article

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.