ARTICLE
Auteur(s) : Audrey Gueniche1,
Jalil Benyacoub2, David Philippe2, Philippe
Bastien1, Nicole Kusy2, Lionel
Breton1, Stephanie Blum2, Isabelle
Castiel-Higounenc1
1L'Oréal Research, Charles Zviak Center, 90 Rue
du Général Roguet, 92 583 Clichy Cedex, France
2Nestle Research Center, Vers chez les blancs, PO Box
44, 1000 Lausanne 26, Switzerland
accepté le 23 Ao�t 2010
The term probiotic, popularized by Fuller [1], was recently
defined by an expert committee as “living microorganisms which,
when consumed in adequate amounts, confer a health effect on the
host” [2]. The most often used probiotic genera in humans and
animals are enterococci, lactobacilli and bifidobacteria, which are
natural inhabitants of the intestinal tract.
Beyond their capacity to positively influence the composition of
intestinal microbiota [3-6], several lines of evidence suggest that
some probiotic bacteria can modulate the immune system, both at
local and systemic levels [7-9], thereby improving immune defense
mechanisms and/or down-regulating immune disorders such as allergy
or intestinal inflammation [7, 10, 11]. Several strains
of lactic acid bacteria have been shown to modulate cytokines
and growth factor production in vitro and in vivo [12-14].
Indeed, results from different pre-clinical and clinical trials
have revealed the ability of various probiotic strains to enhance
non-specific and specific immunity [15-22]. Moreover, recent human
clinical trials widely suggest that probiotic supplementation might
be useful in the management of atopic dermatitis and dry skin [10,
11, 23-26].
About half of women and a third of men report having sensitive
skin [27, 28]. Subjects with sensitive skin primarily complain of
cutaneous discomfort. The main manifestations of this “cutaneous
discomfort” are neurosensory signs such as feelings of heat,
burning, stinging or itching [29-31]. Symptom onset is triggered by
several factors. The factors may be environmental (temperature
changes, heat, cold, wind, sun, air pollution, etc.) or consist of
the application of certain topical products such as “hard” water,
or internal factors (emotional factors, menstrual cycle, dietary
factors). The homeostatic hydration level of the epidermis is
related to the status of the skin barrier and the
interrelationships between the cell components and their lipid
environment. Lipids are involved in the rate of trans-epidermal
water loss (TEWL). Disruption of the skin barrier primarily gives
rise to an increase in TEWL. Impairment of the skin barrier most
frequently presents in the form of “dry” skin, or xerosis,
imparting a dull color to the skin, which appears fragile with
visible scaling.
The pathophysiology of reactive skin consists of an inflammatory
reaction resulting from the abnormal penetration in the skin of
potentially irritating substances, which occurs due to skin barrier
dysfunction and changes in the production of local neuromediators,
like substance P [32].
In addition to the development of soothing active ingredients
for topical application, the development of new approaches enabling
alleviation of symptoms related to reactive skin by the use of
active substances administered by the oral route appears highly
attractive. In that context, nutritional approaches, including
using live microorganisms such as probiotics has gained high
interest. Given the reported immune modulatory property of the
Lactobacillus paracasei ST11 probiotic strain both in vitro and
in vivo [33, 34], we hypothesized that this strain could
impact on the skin immune system and antagonize inflammatory
reactions underlying reactive skin conditions.
Using in vitro models, we demonstrate here that L. paracasei
ST11 modulates neurogenic inflammation mediated by substance P as
well as barrier function activity.
Material and methods
Probiotic cultures
Ready to use vials of freeze-dried powders of the probiotic strain
Lactobacillus paracasei CNCM I-2116 (ST11) were from Nestlé culture
collection (NCC2461). Fresh overnight cultures into deMan Rogosa
Sharpe (MRS) medium were used in the experiments. Bacterial counts
were determined by serial dilutions and plating onto MRS agar
plates. Bacteria were washed three times with PBS (Gibco BRL,
Basel, Switzerland) and diluted to obtain final cell densities of 1
× 107 CFU/mL in RPMI 1640 medium (Gibco BRL).
Epithelial-peripheral blood mononuclear cell co-cultures
The method was adapted from Haller et al. 2000 [35]. Briefly,
Human enterocyte-like Caco-2 cells were seeded on 10.5 mm inserts
(0.4 μm nucleopore size, Becton Dickinson, Basel, Switzerland) at
106 cells/well. The inserts were then cultured in a
12-well plate (Nunc) for 21 days at
37 °C/10%CO2 in DMEM supplemented with 10% FCS and
0.1% penicillin/streptomycin (10,000 IU/mL, Gibco BRL). Human
peripheral blood mononuclear cells (PBMC) from healthy donors were
purified from buffy coats (transfusion Centre, Lausanne,
Switzerland) by centrifugation through a Ficoll-Hypaque 1077 column
(Pharmacia, Dübendorf, Switzerland). PBMCs were suspended in a
complete RPMI medium supplemented with human AB serum (Gibco BRL)
at a final concentration of 2 × 106 cell/mL. When Caco-2
cells established a confluent polarized monolayer, the inserts were
washed twice and the PBMC (1ml) were added to the basolateral
compartment of transwell cultures. The co-cultures thus established
were stimulated by adding 1 × 107 CFU/mL of probiotics
at the apical surface of the monolayer of epithelial cells. The
system was then incubated for 16 hours at
37 °C/5%CO2. To avoid the growth of bacteria,
150 μg/mL of gentamicine was added to the medium after
4 hours of incubation (shown in figure 1). At the end
of the optimal incubation period (16 h) the ST11-conditioned
medium (ST11-CM) from the basolateral compartment was collected for
testing in a model of neurogenic skin inflammation and skin barrier
function (figure 1).
Organ culture of human skin
Human skin samples (n = 8) were obtained from plastic surgery on
patients (Caucasian women 35 to 45 years old). Formal
patient consent was obtained. They were washed three times with
antibiotic solution and then cut into 1 cm2
full-thickness pieces. Subcutaneous fat and lower dermis were
mechanically removed under a stereomicroscope using a surgical
scalpel.
Skin samples were then placed on culture inserts (filter pore
size 12 μm; Costar, Poly-Labo Paul Block, France) with the
epithelium uppermost at an air/liquid interface. The inserts were
set on 12 well plates (Costar) and culture medium was added to
the wells so that the surface of the medium reached the filter
level. Organ cultures were performed using Dulbecco's minimal
essential medium (Gibco BRL) containing antibiotics (100 U/mL
penicillin and 100 μg/mL streptomycin; Gibco BRL, USA), 200 μg/mL
L-glutamine (Gibco BRL), bovine pituitary extract (to promote the
survival of skin explants), epidermal growth factors and fetal calf
serum (DAP, France). Cohesion between skin and insert was obtained
with a polysiloxane vinyl seal in such a way that neither skin
retraction nor lateral passage of any applied topical product
towards the dermis was possible. Before the start of the
experiment, skin samples were kept under these survival conditions
for 5 hours at 37 °C in a humidified incubator with 5%
CO2. In order to test the effect of conditioned medium
in substance-P (SP) treated skin samples, skin culture medium was
replaced with a medium containing 30% (maximum amount of ST11-CM
with no cytotoxic activity) of CM obtained from co-cultures
stimulated or not with ST11. Ten μM of SP (Bachem, Switzerland)
suspended in DMEM were added to the culture medium and skin samples
were maintained in culture for 24 additional hours. Skin
inflammatory status was then assessed by histological evaluation
and measurement of histamine and TNF-alpha release.
Histological evaluation
After 24 hours, skin samples were removed from the inserts,
set in Bouin's liquid and embedded in paraffin. Thick sections of 5
μm were stained with hematoxylin and eosin to evaluate
vasodilation, edema and mast cells. Toluidine blue staining (0.1%
in 50% ethanol; pH 3.5) was used for mast cell counts. Sections
were evaluated under Olympus light-photomicroscope and photographed
with Ektachrome 64T film.
Histological evaluation of the inflammatory reaction was
performed on papillary dermis and on the upper part of reticular
dermis. Double blind assessment was carried out by two
investigators through visual scoring. Four slides per skin sample
were evaluated for each treatment.
a) Microvascular vasodilation: After staining with hematoxylin
& eosin, vascular dilatation was evaluated by counting the
number of vessels dilated over the histological section (16 fields
at 40× magnification). This number was related to the total number
of vessels to calculate the percentage of dilated vessels.
b) Edema: Evaluation was performed using scores: 0- no edema, 1-
slight edema, 2- moderate edema, 3- marked edema and 4- very
important edema
c) Mast cells: (characterized by toluidine blue staining) was
scored as follow: The mast cells present in the dermis are revealed
in blue-violet by the toluidine blue stain. Histologically, a more
or less intense blue-violet and granular appearance of the mast
cells was observed, in combination with the more or less
significant presence in their cytoplasm of basophilic and
metachromatic granulations, notably containing histamine. The
degranulated mast cells were then counted in an optical microscope
(15 fields at a 40× enlargement on 3 sectional planes).
The degranulation obtained after application of substance P is
responsible for a reduction or absence of toluidine blue stain in
the mast cells. This reduction in stain is related to the reduction
or more or less complete disappearance of the number of
granulations initially present in the cytoplasm of the mast cells
(mast cells containing a small number of basophilic granulations).
The results are expressed in the following manner: for each
subject, the percentage of mast cells for each score was calculated
relative to the total number of mast cells.
Measurement of TNF-alpha
The TNF-alpha level was measured in skin culture medium using
commercially available enzyme immunoassay kits (Chemicon
International, USA). The results were expressed in pg per ml of
culture medium.
Skin barrier function recovery after SLS treatment using organ
culture of human skin
Human abdominal plastic surgery skin sample from 4 donors,
randomly selected, was set on Franz cell in an ambient atmosphere
(figure 2).
The skin sample was pre-treated with 30% of conditioned media
(3 h after setting of the skin) for 24 h and maintained
throughout the test, then the surface of the skin sample was
treated with SLS 10% for 3 h to alter the barrier function
(reversible alteration). Skin barrier function recovery was
assessed over 4 days post challenge by measuring the
trans-epidermal water loss using Servomed® evaporimeter
[36-38].
The TEWL were measured and the % were calculated based on a
ratio between TEWL before (considered as 100%) and after SLS
treatment (considered as X%). For example in volunteer 1, before
SLS, TEWL was 2.9 g/m2/h and after SLS was 17.4 g/m2/h : in this
case the variation % is 600% of TEWL after SLS (see figure 5 high
responder).
Statistical analysis
The statistical analysis was performed using the Student
reduced-scale test or a pairwise test, with a risk of 5%.
Results
Part 1. Effect of ST11 on neurogenic inflammation
induced by substance P
In order to test for the effect of ST11 on the SP-induced skin
inflammation, normal human skin from 8 donors was treated with
10 μM SP diluted in medium containing 30% of conditioned
medium obtained from co-cultures (Caco-2/PBMC) stimulated or not
with ST11 for 24 hours, as shown in figures 1 and 2.
Histological changes (edema, vasodilatation and mast cell numbers)
and measurement of TNFα in the culture supernatant of SP-treated
versus untreated skin were carried out at 24 hours.
Histological changes in edema and vasodilation
When applied directly to the culture medium, SP induced
vasodilatation in the superficial dermis: the global % of capillary
dilatation was significantly increased by approximately 30%,
compared to control. The capillary surface was also markedly
increased, as indicated in figure 3 A. These
effects were still observed using non-stimulated CM. Interestingly,
the application of the ST11-CM prevented the increase in vessel
dilatation (figures 3A, B).
As expected, adding SP to culture medium significantly enhanced
the edema score by a factor of 2 compared to control. The same
observation was made when using non-stimulated CM. ST11-CM
prevented the SP-induced increase in edema scores and maintained
them at a level similar to non-challenged control skin (figure 3C).
Mast cell degranulation
SP induced a statistically significant increase in mast cell
degranulation as reflected by a significant decrease in highly
toluidine blue stained granulated mast cells (figures 4A and B).
Mast cell degranulation induced by SP was not affected by adding
non-stimulated conditioned medium but was prevented by the
application of the ST11 conditioned media.
TNF-alpha levels after SP stimulation
When SP was added to culture medium of ex vivo surviving skin, we
observed a significant increase in TNF-alpha level by a factor of
3 compared to non-challenged control skin. Using
non-stimulated conditioned medium led to a lesser, but still
significant, increase in TNF-alpha (figure 4C). Applying
ST11 conditioned media prevented the release of TNF-alpha. Levels
were maintained unchanged as compared to non-challenged control
skin.
Part 2: Skin barrier function recovery
To test for the effect of ST11 on the skin barrier function
recovery, normal human skin from 4 donors was pre-treated with
conditioned medium from Caco-2 cells/PBMC/ST11 co-cultures for
24 hours, as shown in figure 5, and then
exposed to SLS 10% for 3 hours. Skin barrier function was
repeatedly measured by TEWL during 4 days.
Samples from 2 of 4 donors were highly reactive to SLS
application. These samples presented a strong 5-6 fold increase in
TEWL 1h after SLS application and a mean recovery time of 45h. As
shown in figure 5A, for a
representative high responder to SLS, the addition of ST11
conditioned medium protected strongly from SLS-induced increase in
TEWL, since after SLS only a 100% increase of TEWL was observed
instead of the 600% increase obtained with non-stimulated
conditioned medium.
The two other donors behave as moderate responders to
SLS-induced alteration of skin barrier since these samples
presented a mild 1.5 increase in TEWL 1 h after SLS painting
and a mean recovery time of 21 h (figure 5B). The
addition of ST11 conditioned medium strongly inhibited the
alteration generated by SLS and allowed a faster recovery of the
barrier function compared to non-stimulated conditioned medium or
control.
Discussion
A number of authors have suggested a role for impairment of barrier
function in the onset of sensitive skin [28, 39 and, 40].
Failure in barrier function is reported to be responsible for
enhanced penetration of potentially irritant substances which are
considered as the primary triggering factor. Impairment of the skin
barrier most frequently presents in the form of “dry” skin, or
xerosis, imparting a dull color to the skin, which appears fragile
with visible scaling [41, 42]. These features are reflected by a
strong increase in the rate of TEWL. This is commonly assessed by a
repeated tape-stripping test in the clinic. In our study we
developed a new system of ex vivo skin organ culture with a topical
SLS challenge that mimics the TEWL phenomenon associated with
alterations of the skin barrier function usually observed in
reactive skin conditions.
In addition to skin barrier function alteration, skin
sensitivity is generally characterized by an exacerbated reactivity
of sensory nerves associated with neurogenic inflammation [43-45].
Neurogenic inflammation is triggered when a massive release of
mediators, particularly SP derived from sensory fibers, occurs and
the phenomenon extends outwards via the axonal reflex. Thus, the
axonal reflex induces spatial and temporal amplification of the
inflammatory process [46-48]. A number of events occur
subsequently, including mast-cell degranulation and changes in the
contractility of smooth muscle fibers in the vessels
(vasodilatation, extravasation).
Topical application of an irritant substance or environmental
stimulation induces vascular changes and the production of
inflammatory mediators. As indicated above, substance P is one of
the main neuropeptides that trigger skin inflammatory responses. In
our in vitro system, even in the absence of systemic blood
circulation, SP addition to culture medium of surviving skin
induced a dose dependent edema, vasodilation and extravasation of
lymphocytes through microvascular walls and mast cell
degranulation. Moreover, the inflammatory response was associated
with the release of pro-inflammatory mediators, such as
TNF-alpha.
Our data shown that treatment with ST11 was able to promote the
recovery and maintenance of the skin barrier function, since
ST11-CM strongly reduced TEWL upon SLS challenge as compared to
control. Moreover, the study supports the ability of the probiotic
to antagonize SP-mediated skin inflammatory reactions. This was
reflected in a significant decrease in SP-induced vasodilatation,
edema scores and release of TNF-alpha. Of note, a similar trend was
observed for the release of histamine, although the effect did not
reach statistical significance (data not shown).
The mechanism underlying immune modulation by probiotics
involves, in part, regulation of the composition and/or metabolic
activity of the gut microbiota. However, a direct interaction of
probiotics (and/or bacterial components) with the immune system
underlying the gut mucosa has also been proposed. Indeed, effects
on immunocompetent cells and regulation of pro-inflammatory
cytokine production have been reported [10, 11]. Among the immune
cells, dendritic cells play a major role in sensing signals from
luminal bacteria and/or antigens (reviewed by Uhlig and Powrie 2003
[49]. Furthermore, it was demonstrated that mucosal dendritic cells
could express tight junction proteins and penetrate gut epithelial
monolayers to sample bacteria and antigens directly in the
intestinal lumen [50, 51].
It is postulated that following interaction of probiotics with
the intestinal epithelium, associated immune cells become activated
and consequently immune mediators such as cytokines are released
into the blood circulation. Cytokines activate immune cells and
possibly bacterial fractions might reach the skin through the
bloodstream where they potentially could exert immune modulation
effects. In that context, it has been suggested that the
immune-modulatory properties of certain strains of probiotics at
the skin level may modulate the inflammatory reactions generated by
the release of neuromediators involved in cutaneous neurosensory
functions [52, 53].
Our strategy was to evaluate such benefits in relevant in vitro
models. For that purpose the probiotic strain Lactobacillus
paracasei ST11 was used to stimulate enterocyte-leukocyte
co-culture systems that mimic the intestinal epithelium
and related bacteria-cell interactions [35]. A fraction
of the culture medium enriched with molecules and mediators
secreted upon these interactions (so called conditioned medium),
was collected and tested in in vitro models of skin inflammation
and barrier function, as described above.
It has previously been shown that ST11 was able to induce an
increase in the production of systemic regulatory cytokines such as
TGF-beta and IL-10 in mice [13]. Expression of IL-8, TGF-beta,
IL-10, IL-12, IFN-gamma, and TSLP was assessed in the ST11-CM
versus control CM. However no significant differences were observed
after 16h of incubation (data not shown). Besides, IL-4, IL-2 and
IP-10 were undetectable in both ST11-CM and CM. Nevertheless, the
consistent preventing effect against SP-mediated inflammatory
reaction, as well as against topical SLS-induced alteration of the
skin barrier observed in the skin culture systems, revealed that,
so far, non-identified bioactive compounds are effectively released
in the medium. Moreover, priming of immune cells and the
development of anti-inflammatory cell phenotypes is also a possible
mechanism and would deserve further investigation. For instance, it
was reported that commensal bacteria have the potential to commit
mucosal dendritic cells towards an anti-inflammatory phenotype in
similar in vitro experimental settings [54].
All together, our data demonstrates a beneficial role for ST11
in reinforcing skin barrier function recovery and antagonizing
neurogenic skin inflammation and therefore suggests that an oral
application of such probiotic strains may be an effective approach
to prevent or treat reactive skin conditions.
Disclosure
Conflict of interest: none. The authors have no financial support
or relationships that may pose conflict of interest by disclosing
at the time of submission any financial arrangements they have with
a company whose product figures prominently in the submitted
manuscript or with a company making a competing product. However,
Audrey Gueniche, Philippe Bastien, Lionel Breton and Isabelle
Castiel Higounec are employees of L'OREAL and Jalil Benyacoub,
David Philippe, Nicole Kusy and Stephanie Blum are employed by
Nestlé.
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