ARTICLE
Auteur(s) : Véronique Jarrousse1, Nathalie
Castex-Rizzi3, Amir Khammari1,2, Marie
Charveron3, Brigitte
Dréno1,2
1INSERM U601, 9, quai Moncousu, 44093 Nantes cedex
01, FranceFax: (+33) 2400831173
2Clinique dermatologique, CHU Hôtel-dieu, 1, place A.
Ricordeau, 44035 Nantes, France
3Institut de recherche Pierre Fabre, Laboratoire de
biologie cellulaire cutanée, Toulouse, France
accepté le 30 Mai 2007
Acne vulgaris is a skin disorder of the pilosebaceous follicles
that commonly occurs in adolescence and in young adulthood.
Although the precise mechanisms that induce the development and
progression of acne still remain unclear, three main factors have
been identified: an androgen-stimulated increase in the production
of sebum, obstruction of the pilo-sebaceous follicles resulting
from abnormal proliferation and differentiation of follicular
epithelium, and finally a bacterial factor with Propionibacterium
acnes which plays a central role in the initiation and the
maintenance of inflammatory reactions in and around the
pilo-sebaceous follicles.Zinc was recognized to be essential for
human health in 1963 [1]. Several studies [1, 2] have shown a
beneficial effect of zinc salts on inflammatory lesions in mild and
moderate acne. However, the mechanisms by which zinc salts act on
skin inflammation are still only partially known [3]. Indeed, zinc
inhibits polymorphonuclear cell chemotaxis [4], inhibits the growth
of P. acnes [5], activates natural killer (NK) cells and the
phagocytosic capacity of granulocytes [6, 7]. Its anti-inflammatory
activity in acne could also be related to a decrease in tumor
necrosis factor (TNF)-α and IL-6 production [8] and modulation of
the expression of integrins [9, 10], mainly intracellular adhesion
molecule (ICAM)-1 and leucocyte function associated antigen (LFA)-3
[11]. Zinc salts also have a specific action on type 5α-reductase,
shown in vitro [12].Among the different actors of immediate
immunity, Toll-like receptors have been described, these are
receptors specifically activated by microbial components [13, 14].
This activation of TLR by microbial antigens induces a strong
production of different inflammatory cytokines by the cell, and
among them IL-8 which strongly attracts and activates neutrophils
and T lymphocytes. Kim et al., [15] demonstrated that P. acnes
induces monocyte cytokine (IL-12 and IL-8) production through a
TLR2-dependent pathway and that the expression of TLR2 in acne
lesions indicates that activation of TLR2 can contribute to
inflammation at the site of disease activity. Furthermore Nagy et
al. [16] have shown that distinct strains of P. acnes
upregulate human beta-defensins (hBD2) and IL-8 mRNA and protein
expression in cultured keratinocytes.The aim of this work was to
determine if the anti-inflammatory activity of zinc salts in acne
could be linked to a modulation of TLR2 surface expression and
interleukin (IL)-8 production by keratinocytes.
Materials and methods
Materials
Bacterial extracts
We used three extracts of Propionibacterium acnes (IP 53113)
provided by Pierre Fabre Laboratories (Toulouse, France). This
reference strain was first described in 1968.
Supernatant A (SA) and pellet C were obtained after bacterial
culture centrifugation, several freeze/thaw cycles and a last
centrifugation at 4000 rpm for 15 min. Supernatant B (SB)
was obtained after a second centrifugation (4000 rpm,
15 min) of pellet C. The membrane fraction (FM) extract was
obtained after reconstitution of pellets re-suspended in
keratinocyte basal medium (KBM) without HC (hydrocortisone)
(Promocell, Heidelberg, Allemagne).
The distribution of cellular compartments in the three extracts
(FM, SA and SB) was partially known: the wall and cytosolic
membrane (peptidoglycane and lipoteichoic acid) was present in the
FM extract and a low proportion of clever wall elements were
present in the SB extract. The SA extract contained cytosolic
proteins.
Preliminary experiments were carried out in our laboratory to
determine the effect of different dilutions of P. acnes extracts on
keratinocyte viability (MTT test). The following extract dilutions:
FM diluted at 1/2, SA and SB diluted at 1/5, were considered to be
the most appropriate [17].
Trace elements
15 mg zinc salts (Labcatal, Montrouge, France), were
re-suspended in KBM without HC and incubated with NHEK or skin
explants.
The optimal amount of zinc salts was determined in preliminary
experiments (dose-response studies, figure 1).
Keratinocyte culture
NHEK from healthy prepuces were obtained from the pediatric surgery
department of Nantes Hospital and were grown at 37 °C in a
humid 5% CO2 atmosphere, in serum free keratinocyte
growth medium (KSFM) (Invitrogen, Cergy-Pontoise, France)
supplemented with 25 μg/mL bovine pituitary extract (BPE),
0.5 ng/mL of recombinant epidermal growth factor (EGF),
2.5 μg/mL fungizone (Bristol-Myers Squibb, Paris, France) and
penicillin 100 UI/mL-streptomycine 100 μg/mL. Cells were
used after a limited number (n = 2) of subcultures. NHEK were
seeded at the density of 20000 per well in 4-wells chamber slides
(Dutcher, Brumath, France) in low calcium (CaCL2 0.09 mM)
defined medium KBM with HC. The medium was replaced by KBM without
HC 24 h before starting the experiment.
Skin explants
Explants were prepared from skin abdominal plasties received after
plastic surgery. After fat removing, explants (4 mm diameter)
were incubated at 37 °C in a moist atmosphere in the presence
of 5% CO2 in solution in order to cause stimulation, or in control
medium (KBM without HC). After incubation, explants were removed
from the culture medium and frozen in liquid nitrogen before
storage at – 80 °C.
NHEK and skin explants stimulation by LPS or by P. acnes
extracts
NHEK and explants were stimulated for six hours at 37 °C with
1 μg/mL lipopolysaccharide (LPS) purified from
Echerichia coli, 0111:B4 clone (Sigma) and prepared with fetal
calf serum (10%) as described [18].
The different P. acnes extracts (FM 1/2, SA 1/5 and SB 1/5)
were deposited on skin explants for 3 h (immunohistochemistry)
or 24 h (cytokine assays).
Incubation with zinc salts
After stimulation and two washes with PBS, zinc salts were added to
NHEK and skin explants for 3 h at 1 μg/mL. Then, culture
supernatants were collected and frozen at – 80 °C for
subsequent IL-8 secretion assay. NHEK and explants were washed
twice by PBS, then NHEK were fixed in acetone for 10 minutes and
frozen at – 20 °C. Explants were frozen in liquid
nitrogen before storage at – 80 °C.
Five independent experimental series were performed for each
condition.
Methods
Labelling of smears and skin explants
(immunohistochemistry)
Sections of deep-frozen skin explants were fixed in acetone for
10 min. The non-specific sites were saturated for 30 min
with TBS (Tris-Buffered Saline) 0.05% Tween20 w/v (Sigma), 0.1% BSA
w/v (Bovine Serum Albumin) (Sigma). The polyclonal rabbit antibody
anti-TLR-2 H-175 (TEBU, Le Perray-en-Yvelines, France) was
deposited on the slides for 30 min in a humid environment at
room temperature. Rinses of 10 min were made between each
stage using TBS, 0.1% BSA, 0.05% Tween20. The slides were incubated
successively with a secondary biotinylated antibody (DAKO ChemTek
detection kit peroxidase/AEC, Rabbit/Mouse) (30 min) and
streptavidin coupled with peroxidase (30 min) before deposing
AEC (a peroxidase substrate) for 5 min. The reaction was
stopped with distilled water (10 min) and counter-staining was
done with Mayer haemalun (VWR International) for about 1 min.
The slides were rinsed with distilled water, mounted in an aqueous
medium and observed with a Leitz microscope (X20 objective). The
control section included omission of the primary antibody [16]. Two
different examiners read the slides. The intensity of the labelling
was quantified according to semi-quantitative scale: none (0), weak
(1), moderate (2) and strong (3).
Production of IL-8 (cytokine assays)
Explants and NHEK were stimulated in the presence of P. acnes
extracts (FM 1/2, SA 1/5, SB 1/5). To determine the IL-8
concentration, the culture media were harvested after 24h of
culture and stored at – 80 °C until assay. The
concentration of IL-8 in the explant supernatants was measured by a
commercially available enzyme-linked immunoabsorbent assay kit (BD
Biosciences, le Pont de Claix, France), used in accordance with the
manufacters instructions. All samples were assayed in duplicate.
Statistical analysis
Results were expressed as the mean ± standard deviation
(SD). Data were compared to controls, which consisted of
keratinocytes or explants treated in the same manner as the test
samples but without trace elements or LPS or P. acnes
extracts. The statistical significance of the data was determined
by T test. A P < 0.05 was taken as significant.
Results
TLR2 surface expression on keratinocytes
Zinc salts decreased TLR2 expression by NHEK and skin explants
previously stimulated by LPS (1 μg/mL).
TLR2 expression on NHEK stimulated by LPS (figure 1A) was
significantly (p = 0.047) stronger (2.67 ± 0.58) compared to
control (1.33 ± 0.58).
With zinc salts, TLR2 expression was significantly decreased in
a dose-dependent manner from 2.33 (± 0.58) with 0.1 μg/ml of zinc
salts to 1 (± 0.87) with 1.5 μg/mL.
TLR2 expression on skin explants stimulated by LPS (figure 1B) was increased
(2.40 ± 0.65) (not significantly) compared to control
(1.80 ± 0.65).
In the presence of the three concentrations of zinc salts (0.5
μg/mL, 1μg/mL and 1.5 μg/mL), the level of TLR2 expression was
decreased (not significantly) and was similar to that observed in
control.
According to the results, the concentration of 1 μg/mL of
zinc salts with 3h of incubation was used in further
experiments.
Zinc salts decreased TLR2 expression by keratinocytes in skin
explants previously stimulated by FM P. acnes extracts.
TLR2 protein was express through the epidermis except by basal
layer.
When skin explants were stimulated with P. acnes extracts
for 3 h (figure
2), the expression of TLR2 was induced: 2.20
(± 0.76) on skin explants incubated with FM1/2 extract
compared to control: 1.70 (± 0.55) but not with the SA1/5
(1.80 ± 0.67) and SB1/5 (1.90 ± 0.76) extracts. After
incubation of skin explants for 3h with zinc salts prior to FM1/2
extract, the expression of TLR2 was lower (at the limit of
significance: p = 0.08) 1.20 (± 0.65) than control
expression. The expression of TLR2 on skin explants incubated with
SA: 1.80 (± 0.71) or SB: 1.90 (± 0.82) and then with
zinc salts was not modified.
IL-8 secretion in NHEK or skin explant supernatants
Zinc salts had no effects on IL-8 secretion on NHEK nor on skin
explants stimulated by P. acnes extracts.
IL-8 secretion was significantly (p < 0.001) increased by FM:
515 pg/ml (± 30) in NHEK (figure 3A)) compared to
control medium: 20 pg/mL (± 9). However, interleukin-8
secretion of keratinocytes after incubation with zinc salts
(1 μg/mL for 3 h) was not modified.
Regarding skin explants (figure 3B), we observed
that IL-8 secretion was not stimulated by P. acnes extracts
compared to control. IL-8 secretion pattern obtained with P acnes
extracts was not modulated by zinc salts (1 μg/mL, for
3 h).
Discussion
Our study shown that the expression of TLR2 by keratinocytes
induced either by LPS or by FM P. acnes extract is decreased
by zinc salts. This modulation seems to be obtained both with NHEK
and skin explants. Explants have the advantage of being a model
close to in vivo conditions. Thus, it permits study of the role of
differentiation on the expression of an antigen by the
keratinocytes and, moreover, localization of this marker in the
epidermis. The model of NHEK has the advantage of studying
keratinocytes alone and thus of ensuring a direct modulation on
these cells.
Recently, we have shown that extracts of P. acnes are able
to increase TLR2, TLR4 and matrix metaloproteinase (MMP9)
expression by keratinocytes [17]. In particular, we have shown on
acne biopsies that TLR2 was clearly over expressed in comparison to
TLR4. These facts suggested that P. acnes plays a central role
in the induction and maintenance of the inflammatory phase of acne,
through TLR2 expression. This present study focused on
P. acnes (GRAM+) extract effects on TLR2 expression by
keratinocytes. Indeed, TLR2 specifically recognizes a GRAM+ pattern
(e.g. peptidoglycan) whereas TLR4 recognize a GRAM- pattern (LPS).
However, it has been shown that commercial LPS preparations could
stimulate epidermal keratinocytes to produce β-defensins 2 and IL-8
and that the LPS response was inhibited with mAB specific for TLR2,
but not for CD14 and TLR4 [18].
Our study confirms that P. acnes is indeed able to induce
the expression of TLR2. Interestingly, only the membrane fraction
(FM) which contains peptidoglycan (PGN) and lipoteichoic acid, but
neither supernatant A (SA) which contains cytosolic proteins nor
supernatant B (SB) which is rich in membrane proteins are able to
activate TLR2. Therefore, FM extracts of P. acnes contain one
or several patterns which specifically activate TLR2 expression by
keratinocytes.
Zinc salts act mainly on inflammatory lesions in acne. It has
been previously shown that its anti-inflammatory activity has
different targets. Zinc is known to participate in the activation
of nearly 300 enzymes. Some of these zinc-dependent enzymes are
involved in DNA synthesis, cell division, and protein synthesis.
Zinc is also required to stabilize three-dimensional structures of
nearly 1000 transcription factors, such as “the zinc-finger”
proteins. Zinc also plays important roles in immune functions. Zinc
regulates several functions of lymphocytes, such as mitogenesis,
antibody synthesis, the activation of T-cells and natural killer
cells, and more specifically cellular immunity [19, 20]. Zinc
deficiency has been reported to impair cellular immune
functions.
Our results add another anti-inflammatory target of zinc salts
with TLR2. Indeed, we have shown that zinc salts decrease TLR2
surface expression by keratinocytes induced by FM extracts of
P. acnes. Interestingly, recently, Kitamura et al., [21]
showed a link between zinc and TLR on dendritic cells (DC), which
are also present in the epidermis. Indeed, they have demonstrated
that some TLR stimuli decrease intracellular free zinc in DCs,
which is critical for DC maturation and, moreover, that activation
of Toll/Il-1R gene homology (TIR)-containing adaptor-inducing
interferon (IFN) (TRIF)-dependent signalling, alters the expression
of zinc transporters and results in decreased intracellular free
zinc. These results, added to ours concerning keratinocytes,
demonstrate that innate immunity with TLR is an important target of
zinc salts both on keratinocytes and DC.
Concerning the activation of TLR2, the intracellular domain of
TLR2 may activate NF-κB which then modulates the expression of many
immune response genes [22]. By western-blot, we observed that NF-κB
expression on explants was not modulated by P. acnes extracts
or by zinc salts (1 μg/mL) (data not shown).By ELISA we
firstly confirmed that FM extracts of P. acnes induced IL-8
secretion by NHEK. Previously, Nagy et al. [16] demonstrated that
IL-8 secretion induced by P. acnes on keratinocytes are both
TLR2 and TLR4 dependent.
Our results shown that zinc salts (1 μg/mL) have no
modulatory effect on IL-8 secretion (by explants or by NHEK)
despite their inhibitory effect on TLR2 surface expression. Several
studies have shown that Il-8 is not only produced by keratinocytes
via TLRs stimulation but in response of pro-inflammatory cytokines
stimulation. In particular, NHEK have been shown to produce IL-8 in
response to IL-1 or TNF-α [23] and it has been shown that
P. acnes produce IL-1α like and TNF-α like [3]. In healthy
tissues, IL-8 is barely detectable, but is rapidly induced by ten-
to 100-fold in response to pro-inflammatory cytokines such as TNF,
IL-1, bacterial or viral products, and cellular stress [24].
Concerning the skin explants, we observed a high basal level of
IL-8 secretion in normal skin. One hypothesis could be that in
culture, cells present in explants as fibroblasts secrete cytokines
such as IL-1 and TNF-α, inducing the production of IL-8 by
keratinocytes. Thus it could explain the lack of modulation of IL-
8 by zinc salts, via inhibition of TLR.
In conclusion, our results demonstrate for the first time, that
zinc salts inhibit TLR2 expression by keratinocytes induced both by
P. acnes FM extracts and LPS. Interestingly, this inhibition
was not associated with an inhibition of interleukin-8
secretion.
Acknowledgements
Financial support and P. acnes extracts were provided by
Pierre Fabre Laboratories (Toulouse, France) and Labcatal
Laboratories (Montrouge, France). No conflicts of interest have
been declared. We thank surgeons from the plastic surgery
Department of Nantes Hospital for abdominal skin samples and the
pediatric surgery Department of Nantes Hospital for foreskin
samples.
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