ARTICLE
Auteur(s) : Josette Peguet-Navarro1, Colette Dezutter-Dambuyant1, Timo Buetler2, Jacques
Leclaire3, Hans Smola4, Stéphanie
Blum2, Philippe Bastien5, Lionel Breton3, Audrey Gueniche2
1Université de Lyon, EA 41-69, Hôpital Edouard
Herriot, Pavillon R, Département Dermatologie, Lyon, France
2Nestlé Research Center, Vers-chez-les-Blanc, Lausanne,
Switzerland
3L’Oréal Recherche, Centre Charles Zviak, Clichy,
France
4Dept. of Dermatology, University of Cologne, Cologne,
Germany
5L’Oréal Recherche, Aulnay-sous-Bois, France
accepté le 20 Avril 2008
Langerhans cells (LCs), the dendritic cells (DCs) from the
epidermis, constitute the first line of immune defence against
environmental attacks [1, 2]. Under steady state conditions, LC
turnover is very low [3, 4]. They reside in the epidermis in an
immature state and can be distinguished from other epidermal cells
by their surface expression of HLA-DR, CD1a and Langerin. Upon
stimulation by inflammatory mediators, LCs are activated and
acquire CCR7 expression, the chemokine receptor for CCL19 and CCL21
that mediate their migration to lymph nodes. Moreover, activated
LCs display a mature phenotype characterized by increased
expression of co-stimulatory molecules and acquisition of
maturation markers that facilitate their interaction with T-cells
and aid in elicitation of the immune response [2].
It has long been known that, in addition to being carcinogenic
via DNA damage and mutations, solar UV radiation induces local and
systemic immune suppression which represents a major risk for skin
cancer induction and progression in sun-exposed areas [5, 6]. The
process is partly related to direct LC damage through induction of
apoptosis and impairment of antigen-presenting function [7, 8].
Moreover, UV radiation elicits an inflammatory response and
subsequent recruitment of many immune cells, including
CD36+ monocytic cells. These cells colonize the
epidermis in the days following UV exposure and are the major
source of immunosuppressive cytokines such as IL-10 [9]. All these
mechanisms ultimately lead to impairment of cell-mediated immune
reactions and establishment of immune tolerance [10].
Nutritional intervention, particularly with dietary antioxidants
and vitamins, has been proposed to protect against UV-induced skin
damage and to a certain extent skin cancer occurrence [11]. In
recent years, there has been increasing interest in probiotics,
defined as live micro-organisms which, when consumed in adequate
amounts, confer a health benefit upon the host. Particular focus
has been on species of lactic acid bacteria, including Lactobacilli
and Bifidobacteria that are part of the natural human intestinal
microbiota. Indeed, it is well documented that the endogenous
intestinal microbiota plays a crucial role in immune maturation,
gut integrity and defence against pathogens [12, 13]. Recently, it
has been shown that some probiotic bacteria possess the ability to
modulate the immune system at both the local and systemic levels
and thereby improve immune defence mechanisms and/or down-regulate
immune disorders such as intestinal inflammations or allergies
[13-16].
Lactobacillus johnsonii NCC 533 (La1) has been isolated from
healthy adult microbiota and was shown to have strong
anti-pathogenic activity against a wide range of entero-pathogens
[17]. Furthermore, a recent study demonstrated that La1 can protect
against UV-induced LC depletion in murine epidermis [18].
Here we analyzed, in a randomized, double-blind,
placebo-controlled, clinical trial, whether the probiotic bacteria
La1 could modulate the cutaneous immune homeostasis after
solar-simulated UV exposure in humans. For this purpose, we
analyzed whether La1 could interfere with LC allostimulatory
function and the in situ activation/maturation phenotypic status of
skin DC after solar-simulated UV irradiation.
Materials and methods
Oral supplementation
The active ingredient was from Nestlé and one dose contained
1 × 1010 colony-forming-units (cfu) of
Lactobacillus johnsonii La1 (NCC533) while the excipient
(maltodextrin) served as placebo.
Human volunteers and experimental design
Results from a previous study [19] suggested that a minimum of 25
subjects per group would be needed to detect a variation of 2000
cpm for LC function variable (as determined by the ability of the
cells to induce proliferation of allogenic T cells), using the
usual sample size estimation equation with a two-sided test at the
5% significance level and a power of 90%.
Therefore, fifty-four healthy male Caucasian volunteers with
skin-type II/III, aged 20 to 40 years, were included in the study.
According to the inclusion criteria, they were low consumers of
fermented milk products and were not allowed to consume any
products containing live bacteria during the study. Exclusion
criteria included abnormal skin pigmentation, history of active
photo-induced or photo-aggravated skin diseases, recent exposure to
excessive or chronic UV exposure within four weeks prior to
inclusion or during the study period, use of systemic medications
that could affect inflammatory responses within two weeks prior to
inclusion, use of systemic or topical medications suspected of
causing photobiological reactions within one month prior to
inclusion and known sensitivity to any of the study
supplementations used. Moreover, subjects with a history of
intestinal surgery, vegetarian diet, or having taken mineral
supplements or vitamins in the 3 months preceding the study
initiation were excluded.
The study was double blind, conducted in Dermscan laboratories
(Lyon, France) in accordance with the Declaration of Helsinki and
its amendment, the International Conference on Harmonization of
Technical Requirements for Registration of Pharmaceuticals for
Human Use (ICH) guidelines for Good Clinical Practice (GCP) and the
protocol was reviewed and approved by an independent ethics
committee. Informed written consent was obtained from all study
participants.
The period of recruitment and follow up was from middle of
January until the beginning of July. After a washout period of 6
weeks, volunteers were randomly divided into two groups of 27
individuals. At that time, termed “before treatment”, suction
blister roofs (2 × 6 mm diameter) and biopsies (3 mm
diameter) were taken from the right and left buttocks of each
volunteer and the minimal erythema dose (MED) was determined (figure 1). Then, the
subjects received daily oral supplementation with either L.
johnsonii La1 or placebo (maltodextrin) for 66 days. On day 56 of
La1 supplementation, volunteers were exposed to 1.5 MED twice
within 10 h on the right buttock, the maximal exposure area
being 10 × 10 cm. Suction blister roofs and biopsies were
collected from the right and left buttocks at day 1, 4 and 10
post-irradiation (figure
1).
The study was registered in ClinicalTrials.Gov with NCT00351689
as the identifying number.
Ultraviolet radiation source
A 1000 W xenon solar simulator (Oriel, Stratford, CT, USA) fitted
with Oriel filters No. 81017 (Shott, Clichy, France) and No. 81019
(Shott) was used. The exposure spectrum was compliant with the
standards for determining sun protection factors (COLIPA standards,
2003 and FDA Federal Register, 1999). Solar simulated UV irradiance
was: UVB = 0.91 mW/cm2 and UVA = 7.79 mW/cm2.
The illumination received at subject level was determined using a
spectrophotometer (Bentham Instruments Ltd., Reading, United
Kingdom).
MED was determined using the same solar simulator. Increasing
total UV doses from 2437.9128 to 6673.1868 mJ/cm2 (from
255 to 698 mJ/cm2 in UVB) were applied to the right
buttock on day 0 (figure
1). MED was assessed one day later, as the lowest light
energy inducing perceptible and homogeneous erythema with a
clear-cut periphery. The mean MED were not statistically different
in the placebo and La1 groups: 4,739 ± 89 and 4,474 ±
97 mJ/cm2, respectively.
Suction blister roofs and biopsies
Suction blister roofs and biopsies were recovered at different
times of the study from both the right and left buttock of each
subject as illustrated in figure 1.
Immunohistochemical analyses
Skin biopsies were cryofixed and stored at –80 °C. Frozen
biopsies were embedded in OCT Tissue-Tek (OCT Compound, Sakura
Finetek Europe BV, Zoeterwoude, Netherlands). Vertical sections
(4 μm) were cut at –25 °C, air-dried on SuperFrost slides
(Menzel GmbH & Co KG, Braunschweig, Germany), fixed with cold
acetone (10 min) and air-dried before storage at –20 °C.
Frozen sections were thawed, air-dried, re-hydrated with PBS and
blocked in Antibody Diluent (LSAB2 System-HRP kit, Dako, Trappes,
France). Indirect immunohistochemistry staining was performed with
monoclonal antibodies: anti-CD1a (clone NA1/34, IgG2a, 1:200
dilution, Dako), anti-CD36 (clone FA6.152, IgG1, 1:60 dilution,
Dako), anti-HLA-DR (clone B8.12.2, IgG2b, 1:200 dilution,
Beckman/Coulter, Willepinte, Roissy CDG, France), anti-CCR7 (clone
150503, IgM, 1:100 dilution, R&D Systems Europe, Lille,
France), anti-CD86 (clone BU63, IgG1, 1:50 dilution, Dako) and,
anti-DC-Lamp/CD208 (clone 104.G4, IgG1, 1:30 dilution,
Beckman/Coulter), all revealed by streptavidin-biotin-peroxidase
labelling assay using a colorimetric substrate (LSAB2 System-HRP
kit). Each primary antibody was in contact with skin sections for
30 min at room temperature. Negative controls were done with
unrelated isotype-matched mouse immunoglobulins (IgM, IgG1, IgG2a
and, IgG2b, 1:25-1:50 dilution range).
Immunohistochemical analysis was carried out in double blind by
a single examiner. For every immuno-labelled skin section, a score
level (ranging for 0 to 3) was established for the epidermal and
dermal compartments. This score was based on immuno-labelling
intensity combined with a semi-quantitative estimation of
immunolabeled cell density. It was fixed according to the basal
staining level before supplementation and irradiation: i) score 2,
for CD1a and HLA-DR expressed on DC in epidermal and dermal
compartments; ii) score 0, for no expression of CCR7, CD86, DC-Lamp
and CD36 in epidermis and iii) score 0-2, for the latter antigens
in the dermis. The mean scores at irradiated and non-irradiated
sites were then calculated in the placebo and La1 group.
Preparation of epidermal cells from blister roofs
Epidermal cell (EC) suspensions were obtained by incubating the
suction blister roofs in a 0.05% trypsin solution (Difco
Laboratory, Detroit, MI) for one hour at 37 °C. The epidermis was
then freed of residual dermis with fine forceps and cut in very
small pieces. EC suspensions were obtained by repeated passing
through a syringe with a 0.5 mm diameter needle, which allowed
elimination of cell aggregates. The cells were then washed in
Hank’s buffer (Gibco Laboratories, Grand Island, NY) and
enumerated. Viability of the cell suspension was assessed by trypan
blue exclusion and cells were used directly in the MECLR.
Preparation of allogeneic T cells
Mononuclear cells were obtained from the peripheral blood of three
unrelated allogeneic donors and T cells were purified as described
[20]. The resulting suspensions contained more than 95%
CD3+ cells, as assessed by flow cytometry and cell
viability always exceeded 95%. Preliminary experiments showed that
T cell preparations mount similar responses towards allogeneic EC
suspensions. T cells were frozen in liquid nitrogen and viability
of thawed cells was again assessed before each MECLR. For each
subject, T cell preparation from the same donor was used at the
different times of the study.
Mixed epidermal cell-lymphocyte reactions (MECLR)
MECLR were carried out by using fresh ECs from irradiated and
non-irradiated skin samples. 5 × 104 ECs were added to
105 allogeneic T cells in U-bottom microtiter plates.
Controls with EC or T cells alone were included in each experiment.
Culture medium was RPMI-1640 (Gibco Laboratories) supplemented with
10% human AB serum (EFS, Lyon, France), 1μg/mL indomethacin and
antibiotics. Triplicate cultures were maintained for 5 days at 37
°C. T cell proliferation was then assessed by adding 2 μCi of
[3H]methyl-thymidine (2 Ci/mM, Amersham, Les Ulis,
France) for the final 18 h of culture, as previously described
[20]. Results are expressed as the ratio between exposed and
non-exposed skin of mean cpms from triplicate cultures.
Statistical analysis
The statistical analysis was conducted using the SAS©
software package, version 8.2 (SAS Institute Inc., Cary, NC, USA),
including the MIXED procedure to perform analysis of variance on
quantitative variables for the blisters and the GENMOD procedure to
perform analysis of qualitative variables for the biopsy specimens.
For both methods, contrasts were used to assess time evolution. The
normal distribution of the data was tested using the Shapiro-Wilk
test at the 0.01 level. Homogeneity of variances was tested using
Levine’s test.
The significance levels for all other tests were 0.05 except for
the Shapiro-Wilk normality test. The baseline comparability of the
groups was verified with respect to the efficacy parameters.
Statistical method applied to MECLR: Log linear analysis (with
the ratio for each subject, at each time point: cpm exposed
buttock/cpm unexposed buttock) was implemented. Analysis of
supplementation was done for each day, using mixed-effect analysis
of variance. The supplementation and day factors were considered
fixed and the subject factor was considered random.
Statistical methods applied to the results from
immunohistochemical analysis: Generalized Estimating Equation
models were implemented to estimate the supplementation effect. The
buttock, supplementation and day factors were considered fixed. The
subject factor was considered random.
Results
La1 does not prevent LC phenotypic activation/maturation on day
1 post-irradiation
Before supplementation, the distribution and density of epidermal
CD1a+ and HLA-DR+ LCs were similar in the
placebo and La1 group. On day 1 post-irradiation, a significant
increase in the expression of all the antigens tested was observed
in irradiated versus non-irradiated skin samples from placebo and
La1 groups (figures 2 and 4).
Indeed, the CD1a+ epidermal LCs were more dendritically
shaped and exhibited a significantly more intense membrane staining
pattern (figure
4, prints 2 and 5). However, we did not detect any changes
in LC distribution and density. Similar observations were noted for
HLA-DR expression (not shown). Furthermore, irradiation induced the
acquisition of activation (CD86, CCR7) and maturation (DC-Lamp)
markers on some resident epidermal DC (figure 4, prints 8 and 11).
All these observations demonstrate local phenotypic activation and
maturation of LCs upon UV radiation without notable differences
between the placebo and La1 group.
It should be noted that, in both groups, a significant
up-regulation of HLA-DR, CD86 and DC-Lamp expression was also
observed in the dermis, following UV irradiation (figure 3).
La1 limits the depletion of epidermal
CD1a+/HLA-DR+ cells and accelerates basal
epidermal staining pattern
On day 4 and only in the placebo group, the number of
CD1a+ cells was significantly decreased in irradiated
compared to non-irradiated epidermis, indicating either a loss of
antigen expression or a decrease in LC density (figures 2 and 4,
prints 3 and 6). This decrease may be due to cell migration
although, at this time, no significant increase in CD1a+
LC was observed in the dermis (figure 3). Some mature
DC-Lamp+ cells appear to persist in the irradiated
epidermis of the placebo group only (figure 2). By contrast,
the expression of CD1a, as well as DC-Lamp, was normalized in the
epidermis of La1 supplemented individuals, 4 days after UV
exposure.
On day 10 post-irradiation, most antigens recovered basal
expression in the epidermis and dermis of both groups of subjects
(figures 2 and
3).
CD36+ monocytic cells disappear more quickly from
irradiated epidermis in the La1 supplemented group
On day 1 and 4 post-irradiation, CD36+ monocytic cells
were significantly increased in irradiated epidermis from both
groups (figures 2
and 4, prints 14 and 17). In the dermis, CD36+
was significantly increased in both groups on day 4, only (figure 3). Most
interestingly, CD36+ cells remained increased in
irradiated epidermis from the placebo group at day 10 (figure 2) while in the La1
supplemented group residual CD36+ cells were rare and
distributed along the basal membrane (figure 4, prints 15 and
18).
La1 supplementation induces earlier recovery of LC
allostimulatory function
The MECLR results are expressed as the ratio of cpm obtained with
ECs from the left (irradiated) versus the right (non-irradiated)
buttock. As shown in figure 5 and as expected,
the mean ratio averaged 1 in both placebo and La1 groups before
irradiation, showing a similar allostimulatory capacity of EC from
the right and left buttock of a given donor and, therefore,
validating the assay. In contrast, on day 1 post-irradiation the
mean ratio decreased to 0.7 in both groups of subjects, therefore,
demonstrating the UV-induced immunosuppressive effect. The decrease
was highly significant (p < 0.001) and persisted on days 4 and
10 post-irradiation in the placebo group. The interesting finding
was that in the La1 group the allostimulatory capacity of
irradiated ECs was completely restored on day 4 days
post-irradiation (figure
5A).
However, when considering the MECLR results at day 1
post-irradiation individually, a significant decrease in EC
allostimulatory function (i.e. non-overlapping mean ± SD of
triplicate data) was only observed in about half of the volunteers
(13 out 25 in the placebo group and 15 out 25 in the La1 group, not
shown), thereafter considered as UV-sensitive (UVS) and
UV-resistant (UVR) subjects. Therefore, MECLR results were
re-calculated separately for the UVS and UVR subjects. In UVR
donors, the mean cpm ratio from irradiated as compared to
non-irradiated skin sites averaged 1 and was not significantly
altered at any times after UV exposure, in both the placebo and La1
groups (figure
5B). In contrast, and as expected, the EC allostimulatory
function was significantly decreased at day 1 post irradiation in
the in UVS subjects and the decrease persisted at day 4
post-irradiation in the placebo group, only (figure 5B). Interestingly,
when restricting analysis to the UVS subjects, a complete recovery
of EC allostimulatory function was observed at day 4
post-irradiation, in the La1 supplemented group. Thus, the overall
significant recovery at day 4 post-UV can be attributed to the
strong effect of La1 in the UVS subjects. At day 10 after UV
exposure, the difference between placebo and La1 supplemented UVS
individuals was no longer significant.
Discussion
The present study analyzed for the first time the effect of oral
supplementation with the probiotic bacteria L. johnsonii La1 on
cutaneous immune status after acute solar-simulated UV exposure.
Results show that La1 intake was well tolerated and did not modify
erythema upon UV exposure (chromameter data, not shown). All
adverse events (miscellaneous pain, flu syndrome) were not related
to the study products.
Moreover, La1 intake did not prevent the early UV-induced
phenotypic activation of LCs. The results extend previous in vivo
studies by Laihia and Jansen [21], by showing that a large number
of irradiated LCs not only acquire expression activation markers
such as CD86, but also express DC-Lamp as early as day 1
post-irradiation and most probably reflect a population that
matured within the epidermis. Despite in vivo phenotypic
activation/maturation known to favour T cell priming [2], LCs
displayed reduced in vitro allostimulatory function on day 1 post
irradiation. This is in agreement with many reports [22, 23] and
might be due to rapid in situ LC death following phenotypic
maturation or, alternatively, to increased sensitivity to trypsin
treatment during isolation procedure and, subsequently, to
increased in vitro cell death. In agreement with this, we observed
significant higher mortality in ECs recovered from the irradiated
versus non-irradiated suction blisters in both the La1 and
untreated groups (not shown). The fate of UV-activated LCs in vivo
is questionable, however. Kolgen et al. [24] detected LCs in the
blister fluids from the UVB-exposed, but not the unexposed, skin
samples. Some LCs were positive for DNA damage, suggesting that
they originate from epidermis and migrate rather than die in situ
after irradiation.
Although UV-induced LC depletion has been widely reported,
little is known about the kinetics of reconstitution of skin immune
function. We show here that under normal conditions (placebo group)
a relatively minor skin injury, i.e. UV exposure to twice 1.5 MED,
still induces significant inhibition of EC allostimulatory function
4 days post-irradiation and this inhibition correlates with
significant decrease in CD1a+ in irradiated epidermis.
In agreement with previous studies by Cooper et al. [22]
concomitant epidermal infiltration with CD36+
macrophages was observed in both groups, beginning on day 1 and
still visible on day 4 post-irradiation. The important result is
that La1 intake facilitated an earlier recovery of the EC
allostimulatory function, a process that correlated with recovery
of basal CD1a+ cell staining within the irradiated
epidermis. The origin of these CD1a+ cells remains an
open question. It is unlikely that La1 has protected LCs from
UV-induced DNA damage and thus facilitated the cell repair. Indeed,
numerous cells containing pyrimidine dimers were observed at day 1
that persisted until day 10 after UV, but no differences were
noticed between the La1 and placebo supplemented groups (not
shown). On the other hand, it is likely that activated LCs had
disappeared from epidermis either by apoptosis or migration, a
process that might be facilitated by La1 treatment. Accordingly,
the recovered EC allostimulatory function in the La1 supplemented
group might be related to repopulating cells, most probably to
CD1a+ cells derived from precursor cells. These might be
local proliferative precursor cells as previously described [3].
Alternatively, we show here that CD36+ monocytic cells
that colonize irradiated epidermis completely disappeared on day 10
in the La1 supplemented group, whereas CD36+ cells could
still be observed in biopsies from the placebo group. This might
suggest that the monocytic cells could have served as potential LC
precursors and that La1 intake favours their differentiation.
Indeed, there is now evidence in murine models that LCs arise from
monocytes following severe skin injury [25].
It is tempting to speculate that La1 is somehow able to modulate
local or systemic cytokine levels. Using ELISA assays, we found
increased levels of IL-8, TNFα and IL-10 in the suction blister
fluids from irradiated skin. However, the assays did not reveal a
significant effect of La1 relative to placebo at any time of the
study (not shown). Whether La1 favour the production of TGFβ, known
to promote LC differentiation [26], or some chemokines that favour
the homing of skin LC precursors requires further investigations,
however.
Susceptibility to UV-induced immunosuppression appears to be
genetically determined in mice [27] but whether this holds true for
humans is not clear. It has been reported that only 40% of the
normal population were susceptible to a high cumulative UVB dose
(four times 144 mJ/cm2) for UV-induced suppression of
CHS [28]. However, in a more recent study, a single solar-simulated
UV exposure to 3 MED was sufficient to suppress the CHS response to
DNCB in twelve out of twelve skin type I/II human volunteers [29].
In the present study, we show that twice 1.5 MED significantly
inhibited the MECLR in about half of the twenty-five subjects.
Interestingly, similar numbers of UV-S subjects were observed in
the placebo and La1 groups, suggesting that the probiotic intake
did not modify the individual susceptibility to UV radiations. In
addition, MED was similar in the UV-S or UV-R volunteers, showing
that UV-susceptibility was not related to the physical UV dose
received by the volunteers (not shown). La1 had no effect on the
allostimulatory activity in the UVR subjects, thus confirming that
in the absence of any challenge or in resistant individuals La1 has
no unwanted immunological effects. It should be noted that the
dichotomy between the UVS and UVR groups did not alter the
phenotypic analysis in either placebo or La1 groups (not
shown).
In conclusion, in this randomized double blind clinical study,
we show for the first time that the intake of L. johnsonii La1
contributes to reinforce cutaneous immune homeostasis following UV
exposure in humans and may thus represent a new strategy for
photoprotection.
Acknowledgments
We would like to acknowledge the help of C. Weiss and N. Blanc
(Nestlé PTC Konolfingen, Switzerland) for the preparation of the
probiotic bacteria; B. Ducarre and S. Maréchal (EA 37-32, Lyon,
France) for phenotypic and functional LC analysis; Drs. F.
Boudejma, C. Noize-Pin and S. Chartier (Dermscan, Lyon, France) for
the clinical part of the study and R. Marion-Gallois and F.
Makori (Effi-stats, Paris, France) for the statistical analyses.
There is no conflict of interest.
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