ARTICLE
Auteur(s) : Édouard Begon1, Laurence
Michel2, Béatrice Flageul1, Isabelle
Beaudoin2, Francette Jean-Louis2, Hervé
Bachelez1, Louis Dubertret1, Philippe
Musette3
1INSERM U532, Institut de Recherche sur la Peau,
Centre Hospitalier Universitaire, Hôpital Saint Louis, 1 avenue
Claude Vellefaux, 75010 Paris, France
2Dermatology Unit, Centre Hospitalier Universitaire,
Hôpital Saint Louis, 1 avenue Claude Vellefaux, 75010 Paris,
France
3Dermatology Unit, Centre Hospitalier Universitaire
Charles Nicolle, 1 rue de Germont, 76031 Rouen Cedex, France
accepté le 30 Mai 2007
Mammalian Toll-like receptors (TLRs) are a recently recognized
family that play a critical role in innate immune defense against
diverse pathogens [1, 2]. In mammals, TLRs have been implicated in
both inflammatory responses and innate host defense against
pathogens. TLRs recognize conserved molecular patterns of microbial
pathogens termed pathogen-specific molecular patterns (PAMPs),
which promote responsiveness to a wide variety of pathogens. PAMPs
are produced by micro-organisms, but host cells are also able to
produce endogenous TLR ligands in stress conditions.So far, eleven
human-family members of TLRs have been discovered (TLR1 through
TLR11) [3-8]. Five have been shown to recognize a variety of
specific ligands: TLR 4 recognize lipopolysaccharide (LPS), a
bacterial membrane component of gram-negative bacteria [9];
Peptidoglycan (PGN) and lipoteichoic acid act as TLR2 ligands [10];
TLR3 has been shown to activate immune cells in response to
double-stranded RNA poly (I:C) [11]. Innate immunity receptors may
be activated not only by micro-organisms but also by endogenous
danger signals emitted by injured cells [12]. Such signals may be
generated when cells undergo pathological cell death (necrosis) or
by the release of extracellular matrix components occurring during
an inflammatory response [13-18]. In addition, genomic structures
such as non-methylated CpG and double-stranded RNA are
overexpressed in auto-immune diseases such as rheumatoid
polyarthritis and systemic lupus [19, 20].Activation of each TLR
triggers a signal transduction cascade culminating in nuclear
translocation of nuclear Nf-κB family members [21]. TLRs represent
a critical linkage between innate and adaptive immunity. After
stimulation by bacterial products, TLRs mediate a variety of
signals not only for inducing pro-inflammatory cytokines such as
TNF-α, IL-6, IFN-γ, and IL-12, but also for up-regulating
co-stimulatory molecules such as CD80 and CD86 to activate immune
responses [22, 23]. Expression of several TLRs is also specifically
up-regulated by cytokines such as TNF-α and IFN-γ [24, 25].TLRs are
expressed at higher levels in tissues exposed to the external
environment [26, 27]. One of the first lines of defense against
microbial invasion is the skin barrier. But little is known about
the role and expression of TLRs in the skin. Keratinocytes (KCs)
were found to constitutively express TLR2 and TLR4-LPS receptor
complex protein. In the present study, we therefore analyzed total
TLR expression in normal human keratinocytes and studied the
functionality of TLRs 2, 3 and 4 using specific PAMPs. We examined
how proinflammatory cytokines such as IFN-γ, TNF-α, and IL-8 could
enhance TLR expression and be released after TLR microbial
stimulation. We demonstrated that human KCs express all known TLRs
and that cytokines TNF-α and IFN-γ up-regulate intra-cytoplasmic
and surface expression of most TLRs, including TLRs 2, 3, and 4.
Subcellular localization of TLRs expressed was mainly
intra-cytoplasmic. Activation of TLR2 by PGN, TLR3 by Poly (I:C),
and TLR4 by LPS causes the nuclear translocation of NF-κB and
triggers the release of proinflammatory cytokines, providing
evidence for the functionality of TLRs 2, 3, and 4 in human
keratinocytes. The ligation of TLRs 2, 3, and 4 receptors, strongly
expressed by keratinocytes, induces the release of TNF-α and IL-8,
two pro-inflammatory cytokines largely involved in cutaneous
inflammation. We also observed that TLR2 is up-regulated in
psoriatic skin lesions. In conclusion, our results allow a better
understanding of the mechanisms of the immune response in skin
against invasive pathogens. In addition, TLRs may play a role in
the pathogenesis of several inflammatory diseases of the skin such
as psoriasis.
Experimental procedures
Reagents and antibodies
Phosphate buffer saline solution (PBS), DMEM were purchased from
Gibco (In vitrogen, Paisley, UK). Lipopolysaccharide (LPS) derived
from Escherichia. coli K 235 (Sigma Aldrich, St Louis, MO),
peptidoglycan (PGN) (Biochemika, Buchs, Sw) derived from
Staphylococcus aureus, Poly (I:C) (Fluka, Sigma Adrich, Saint
Quentin-Fallavier, Fr) were used at respective concentrations of
100 ng/mL, 10 μg/mL, 25 μg/mL based on dose response studies in
published studies in several cell types. Before use, PGN was
re-suspended in PBS and sonicated at 20,000 Hz four times
10 s each, as has been reported elsewhere [28]. Antibodies
used to detect TLRS by flow cytometry were the following: anti-TLR1
Ab (goat polyclonal, sc-8687), anti-TLR2 Ab (goat polyclonal,
sc-8689), anti-TLR3 Ab (goat polyclonal IgG, sc-8692), anti-TLR4 Ab
(rabbit polyclonal IgG, sc-10741), anti-TLR5 Ab (rabbit polyclonal
IgG, sc-10742), anti-TLR6 Ab (goat polyclonal IgG, sc-5657) were
purchased from Santa Cruz (TEBU, Le Perray en Yvelines, Fr).
Anti-TLR2 Ab (Mouse IgG2a, clone TL2,1) and anti-TLR4 (Mouse IgG2a,
clone HTA 125) were from Hbt (HyCult biotechnology, TEBU, Le Perray
en Yvelines, Fr). Anti-TLR9 Ab (mouse polyclonal, clone 26c593) was
from IMGENEX (San Diego, Calif). Phycoerythrin-conjugated swine
anti-goat IgG (H+L) [G5004] and phycoerythrin-conjugated goat
anti-mouse IgG (H+L) [M30004] were obtained from Caltag
Laboratories (Burlingame, CA, USA). Human recombinant TNF-α and
IFN-γ were purchased from Peprotech (Peprotech, Rocky Hill, New
jersey, USA). FITC-conjugated anti HLA-dR and FITC-conjugated anti
CD54 (Beckmann Coulter, Immunotech, Marseille) were used to control
KC activation in flow cytometry assay.
Cell cultures and preparation of cultured epidermal human
keratinocytes
Fresh normal human KCs were isolated and cultured from skin
biopsies from healthy human adults (plastic surgery, mammoplasty)
as previously described [29]. Epidermal cells were maintained in
complete DMEM medium supplemented with 10% decomplemented fetal
calf serum and containing 1% glutamine, 100 Ui/mL
Penicilin-streptomycin, Insuline 5 microg/mL, 3,3,5
Triiodo-L-Thyronine (T3) 2.10-9M, Hydrocortisone 5 microg/mL,
Cholera-toxin 10–10 M and epidermal growth factor (EGF)
10 ng/mL added 48h latter. Cell culture medium was changed every
2-3 days and cells were harvested at subconfluency (15 to 20 days).
The tissue culture dishes, culture medium used in our experiments
were free of endotoxin contamination, according to the
manufacturer. NCTC 2544 cell line was obtained from Ree et al.
[30]. NCTC cells were cultured in DMEM medium containing
supplemented 10% fetal calf serum, 100 Ui per mL
penicillin-streptomycin, 10 mM hepes buffer, 2.5 UG/mL fungizone,
glutamate.
Determination of surface TLRs expression on human keratinocytes
by flow cytometry
To study surface expression, KCs were detached by trypsin and
washed twice in PBS containing 0.5% bovine serum albumin (PBS-BSA).
Cells were then incubated with the appropriate primary Ab on ice at
4 °C for 1 h. Cells were then washed twice in PBS BSA and
stained with the appropriate PE-labeled secondary Ab: PE-mouse
anti-rabbit mAb, PE-swine anti-goat mAb, PE-rabbit anti-mouse mAb.
The Abs were all diluted at 1/50. To study intracytoplasmic
expression, cells were re-suspended at
106 cells/mL, incubated with paraformaldehyde 0.45%
for 1 h on ice, then stained in PBS containing 0.2% Tween 20
for 15 min at 37 °C. Cells were then washed twice in PBS
containing 2% foetal serum bovine (PBS-SVF) and incubated with
primary Ab for 1h on ice. Cells were then washed twice in PBS SVF,
and then stained with the appropriate PE-labeled secondary Abs
described above. Ab dilutions were 1/50 for each Ab used. Flow
cytometry was performed using a dual laser Facs calibur (BD
Biosciences). All of the experiments were performed at least twice.
Determination of TLRs expression in cultured human
keratinocytes by reverse transcription polymerase chain reaction
(RT-PCR)
RNA was isolated from cells using an mRNA isolation kit (Qiagen
Sciences, Maryland, USA) according to the manufacturer’s
instruction. First strand RNA was synthesised from total RNA using
RT kit instruction (First strand cDNA synthesis kit Roche,
Indianapolis, USA) and was subjected to PCR amplification with Taq
polymerase (Promega, Charbonnières Les Bains, Fr). PCR was
performed in a final volume of 50 μL in the presence of dNTP, Mg
Cl2, Taq polymerase. Oligonucleotide primers used to amplify were
prepared according to the published sequences.
Primers used for human TLRs and β-actin control are as follows
(Eurogentec, Fr):
TLR1,5’-CGTAAAACTGGAAGCTTTGCAAGA-3’/3′-CCTTGGGCCATTCCAAATAAGTCC-5′,
TLR2,5’-GGCCAGCAAATTACCTGTGTG-3’/3′-CCAGGTAGGTCTTGGTGTTCA-5′,
TLR3,5’-ATTGGGTCTGGGAACATTTCTCTTC-3’ /
3′-GTGAGATTTAAACATTCCTCTTCGC-5′,
TLR4,5’-CTGCAATGGATCAAGGACCA-3’/3′-TCCCACTCCAGGTAAGTGTT-5′,
TLR5,5’-CATTGTATGCACTGTCACTC-3’/3′-CCACCACCATGATGAGAGCA-5′,
TLR6,5’-TAGGTCTCATGACGAAGGAT-3’/3′-GGCCACTGCAAATAACTCCG-5′,
TLR7,5’-AGTGTCTAAAGAACCTGG-3′/3′-CTTGGCCTTACAGAAATG-5’,
TLR8, 5’-CAGAATAGCAGGCGTAACACATCA-3′ /
3′-AATGTCACAGGTGCATTCAAAGGG-5′,
TLR9,5’-TTATGGACTTCCTGCTGGAGGTGC-3′/3’-CTGCGTTTTGTCGAAGACCA-5′,
TLR10, 5’-CAATCTAGAGAAGGAAGATGGTTC-3′ /
3′-GCCCTTATAAACTTGTGAAGGTGT-5′
ßactin,5’-ATCTGGGACCACACCTTCTACAATGAGCTGCG-3′/3′-
CGTCATACTCCTGCTTGCTGATCCACATCTGC-5′.
A PCR system was used with an initial denaturation step of
94 °C for 5 min, followed by 35 cycles of 94 °C for
30 s, 55 °C for 30s, 72 °C for 1 min and a
final elongation step of 72 °C for 7 min. DNA bands were
visualized on ImagerTM (Appligen-Oncor, Ilkirch,
Fr).
Immunohistochemistry
Four millimeter punch biopsies were obtained from psoriatic
patients on chronic plaques (n = 5) or healthy patients on normal
skin (n = 5). Serial cryostat sections were dehydrated in acetone
at 4 °C, stored at 4 °C, and hydrated in PBS for 10 min at a
room temperature. After rehydratation and inhibition of endogenous
peroxydases, cryosections were incubated with primary specific or
isotype control irrelevant antibodies for 30 min, then
incubated with a second multi-species biotinylated antibody for
10 min before the addition of streptavidin- peroxydase agent
for 10 min (Ultratech HRP Streptavidin Universal Detection
system). The amino-ethyl-carbazol (AEC) substrate-chromogen
(Beckmann-Immunotech, Marseille) was finally used to reveal
antibody fixation. The cells were counterstained with hematoxylin
for 1.5 min. The immunostaining was observed on a light
microscope. To assess the cell infiltrating area on the sections, a
larborlux S leitz (Leica) microscope was used at the 25× objective.
Isolation of nuclear extracts and electrophoretic mobility
shift assay
Cells were cultured with serum free culture medium alone for
1 h before being stimulated separately with LPS (10 ng/mL), or
Poly I:C (25 μg/mL) or PGN (10 μg/mL) for 4h. After pelleting at
10,000 rpm for 20 s, cells were re-suspended in one cell
pellet volume of lysis buffer (Hepes KOH 10 mM, MgCL2 1.5 mM,
Kcl 10 mM, DTT 0.5 mM, PMSF 0.2 mM) and incubated on ice
for 10 min with intermittent vortexing. Nuclei were collected
from the cell lysate by centrifugation at 10,000 rpm for
2 min, and then re-suspended in one cell pellet volume of
extraction buffer. After incubation on ice for 20 min with
intermittent vortexing , nuclear proteins were collected by
centrifugation at 10,000 rpm for 2 min. Supernatants also were
collected to ensure the elimination of nuclear debris. Nuclear
extracts were stored at – 70 °C until further use.
Nuclear extracts (10 μg) were incubated with a 32P-end labeled
oligonucleotide probe (Promega, oligonucleotides: 5’ AGT TGA GGG
GAC TTT CCC AGG C 3’ and 3’ TCA ATC CCC CTG AAA GGG TCC G 5’)
containing a binding site for Nf-κB, during 20 min at
4 °C. Samples were analyzed by electrophoresis in a 4%
polyacrylamlide gel. Positive and negative probes for competition
assay were used in order to verify the specificity of binding. For
supershift assays, nuclear extracts were preincubated for
15 min with 2 μL of rabbit polyclonal antibodies raised
against the NF-κB family subunits p65 or p50 (Santa Cruz
Biotechnology, Inc. Santa cruz, CA) prior to the addition of the
DNA probe. DNA complexes were separated on a 5% nondenaturating
polyacrylamide gel.
Elisa
To quantify cytokine secretion, cultured human keratinocytes were
plated in tissue culture flasks at sub confluence were incubated
with 100 ng/mL LPS, Poly I:C 25 μg/mL, 10 μg/mL PGN. After
18 h, the culture supernatants were collected. Then, the
quantity of TNF-α, Il-8 proteins in the supernatant was determined
by Elisa kit assays according to the manufacturer’s protocol
(Immunotech, Beckmann Coulter, Marseille, France).
NF-κB reporter assay
To assess NF-κB activity, a luciferase reporter plasmid assay was
used. Briefly, KCs were seeded in 6 well plates and cultured. At
approximately 60% of confluence, cells were co-transfected with
various plasmids including NF-κB luciferase and β-galactosidase by
using Fugene 6 reagent (Roche, Indianapolis, USA) according to
manufacturer’s instructions. A plasmid expressing constitutively
green fluorescent protein (GFP) was transfected concomitantly. The
mean efficacy for transfections was reached 15 to 20%. After 36
hours of co-transfection the cells were treated with 100 ng/mL LPS,
Poly I:C 25 μg/mL, 10 μg/ml PGN for up to 6 hours. Cell lysates
were prepared using a 1X passive lysis buffer provided by the
manufacturer and luciferase activity was measured with the dual
luciferase Reporter Assay System (Promega corp, Madison, USA). A
luminometer (1450 microbeta Trilux, Perkin-Elmer, Courtaboeuf,
France) was used for detecting the light intensity. βgalactosidase
activity was depisted by using β-gal reporter gene assay (Roche,
Indianapolis, USA). The results were expressed as the fold
increased compared with the luciferase activity of untreated cells.
Results
TLRs are expressed in normal skin
The immunohistochemical expression was first analyzed in normal
human skin and in in vitro cultured keratinocytes. Cryostat
sections of skin biopsies were immunostained to detect the presence
and location of TLRs 1, 2, 3, 4, 5, 6, and 9. We obtained cryostats
from normal human skin donors (figure 1). Different
dilutions were used for each anti-TLR mAb. TLRs 2, 3, 4, 5, and 6
were all expressed in the epidermis. TLRs 2 and 4 are consistently
expressed in epidermal layers of KCs, predominantly in lower
layers. TLRs 3 and 5 were principally detected in the basal layer.
No immunoreactivity was detected for TLRs 1, 6 and 9.
Immunoreactivity was strong and diffuse throughout the cytoplasm
for TLR 2, slight and diffuse for TLR 4, and weak to absent for
TLRs 1, 6 and 9. With both methods of immunostaining (completed
with immunocytochemistry), immunoreactivity was principally
intra-cytoplasmic and detection of the different TLRs was not
localized on the cell surface.
Normal and immortalized KCs express intracytoplasmic TLRs
contrasting with a low-level surface expression
To determine whether TLRs are constitutively expressed, we analyzed
normal cultured human and immortalized KCs using flow cytometry
(figure 2).
Cell-surface expression of TLRs 1, 2, 3, 5, 6, and 9 was detected
at a low levels. The percentage of positive cells typically did not
exceed 10-15%. The results were similar at the different
concentrations of polyclonal antibodies used (1/50 to 1/20). We
hypothesized that cell treatment with trypsin, which most likely
degrades cell surface expression of many proteins, could explain
the lack of TLR cell surface expression. But the same experiments
carried out with any or lower dilutions showed no differences. We
then hypothesized that TLR expression is mainly intra-cytoplasmic,
as already observed in immunostaining studies. To test this
hypothesis, we examined TLR expression after membrane
permeabilization of the same human-KC cultures. Cytoplasmic TLR
expression was found to be much more intense. We found high levels
of intra-cytoplasmic expression for all TLRs, particularly TLRs 2
and 4. The percentage of positive cells detected reached 50-80% for
TLRs 2, 3, 4, 5, 6, and even TLRs 6 and 9, absent in histochemistry
analysis of skin sections.
The repertoire of TLR mRNA expressed in normal skin is
variable
Cultured human KCs obtained from five different healthy donors were
evaluated for expression of transcripts encoding TLRs by RT-PCR.
Using RT-PCR, we found that all known TLRs [1-10] can be detected
in human KCs. However, mRNA expression was variable among the
different samples studied. Bands of TLRs 3, 4, and 5 were
consistently detected at high intensity in the KC cultures (5/5).
Signals for TLRs 1, 2, 6, 8, and 10 were lower and found
inconsistently in the different cultures (3/5). Barely detectable
signals for TLRs 7 and 9 were found in only one sample. Because the
expression of each TLR varies in the different layers of the
epidermis, as observed by immunostaining, we hypothesized that the
variety of mRNA expression among donors could depend on the states
of confluence of the cultured KCs studied. Next, we examined the
possible relationship between the variability of TLR expression and
confluence states. We analyzed mRNA expression in cultured KCs with
different states of cell confluence. However, we found no strict
linkage between this variety and the confluence states of the
culture.
Intra-cytoplasmic and cell-surface TLR expression is increased
after stimulation by cytokines TNF-α and IFN-γ in normal human
keratinocytes
We examined, by flow cytometry, the enhancement of TLR expression
after addition of IFN-γ and TNF-α in normal human KCs and
immortalized cultured KCs (figure 3). KC cultures
were stimulated by increasing concentrations of IFN-γ and TNF-α
from 10 IU/mL to 1000 IU/mL over periods of 24 and 72
hours. The KC activation state was confirmed by detecting CD54 at
the KC surface. In this study, we demonstrated that addition of
IFN-γ and TNF-α increases intra-cytoplasmic TLR expression, as
determined by flow cytometry. Both of these cytokines led to the
activation of keratinocytes, as demonstrated by CD54
overexpression. Unstimulated cultured KCs were used as a control
and showed no CD54 up-regulation. By using several concentrations
and stimulation times, we demonstrated that kinetics and
dose-relation were different for each TLR. Addition of IFN-γ
increases intracytoplasmic expression of TLRs 2, 3, 4, 5, and 9
with a maximum observed after 72 hours of stimulation. TNF-α
enhances the intra-cytoplasmic expression of TLRs 2, 3, 4, 5, 6,
and 9, most visibly after 72 hours of stimulation. TLR1 expression
is not influenced by addition of either cytokine. In parallel to
intra-cytoplasmic expression increase, stimulation by cytokines led
to membrane translocation and increased cell-surface expression of
most TLRs (10-30% positive cells with high antigenic density).
Addition of IFN-γ and TNF-α enhances the cell-surface expression of
TLRs 2, 4, and 5 but not 3 and 6. TLR9 cell-surface expression is
strongly induced by TNF-α (51% positive cells) but not by IFN-γ,
whereas TLR9 is not expressed at the surface of unstimulated KCs.
Stimulation of keratinocytes by peptidoglycan, Poly (I:C), and
lipolysaccharide led to the nuclear translocation of NF-κB as
demonstrated by EMSA and luciferase assay
TLRs 2, 3, and 4 are strongly expressed in human KCs. We tested the
hypothesis that these TLRs are functional receptors of KCs by using
specific PAMPs. Peptidoglycan (PGN), a component of outer membranes
of gram-positive bacteria, is a microbial ligand of TLR2. LPS, is
the first discovered microbial ligand of TLR4. Poly I:C
(double-stranded RNA) is the recognized viral TLR3 ligand. Previous
studies showed that stimulation of different cell types by PGN,
Poly I: C or LPS led to increased cell-surface expression of TLRs
2, 3, and 4, as well as to the release of pro-inflammatory
cytokines and NF-κB translocation. To further understand TLR
expression in KCs, we examined whether PGN, Poly I:C and LPS could
induce NF-κB activity in normal KCs (figure 5). PGN
(10 μg/mL), Poly I: C (25 μg/mL), and LPS (10 ng/mL) were
added to normal human cultured KCs for different stimulation times
(1 h, 4 h, 18 h). Addition of TNF-α (100 IU/mL) to
the culture served as positive control for NF-κB activation. We
used an electrophoretic mobility shift assay (EMSA) to detect NF-κB
complexes. The presence of a band corresponding to active Nf-κB
complex (p50/p65) was found in KCs stimulated by PGN, Poly I:C,
LPS, and TNF-α but not in unstimulated KCs (weak detection
corresponding to constitutive basal level consistent with previous
published data). These results demonstrated that PGN, Poly I: C,
and LPS strongly increased the nuclear translocation of Nf-κB as
measured by EMSA (figure
4). Interestingly, adding IFN-γ to LPS caused stronger
NF-κB activation than LPS alone (data not shown), consistently with
our finding that IFN-γ enhances TLR4 cell-surface expression. We
next confirmed those previous results by Nf-κB transfection assay
in cultured KCs. Using a cultured luciferase-based reporter assay,
we examined KCs before and after a 4-h exposure to PGN, Poly I:C or
LPS. Addition of TNF-α (100 IU/mL) served as a positive control for
Nf-κB activation. These tests showed a weak but significant
increase of NF-κB activation after stimulation by Poly I:C and LPS
and a strong increase after stimulation by PGN and TNF (figure 4). The
results provide evidence that normal epidermal KCs are capable of
expressing functional active TLRs 2, 3, and 4.
Stimulation of keratinocytes by specific microbial ligands of
TLRs 2, 3, and 4 led to the release of proinflammatory cytokines
TNF-α and IL-8
We measured pro-inflammatory cytokine production in response to
PGN, Poly I:C, and LPS. Based on the results obtained by EMSA and
luciferase activity, we sought to characterize the presence of two
cytokines, TNF-α and IL-8, after TLR stimulation. Immortalized
cultured KCs were stimulated by addition of PGN (10 μg/mL), Poly
I:C (25 μg/mL), and LPS (10 μg/mL) for three stimulation times
(4 h, 8 h, and 24 h). We found that addition of PGN,
Poly I:C or LPS led to the release of pro-inflammatory cytokines
such as TNF-α and IL-8 as determined by ELISA (figure 5).
TLR2 is up-regulated in psoriatic skin: comparison of
immunostaining between normal and psoriatic skin
Our goal was to determine whether there was any evidence for TLR
implication in a pathologic model of skin. We studied psoriatic
skin because it is one the most frequent inflammatory skin
disorders and its pathogenesis remains unclear. Mechanisms leading
to T-cell activation in psoriasis are yet undetermined. In
addition, infectious agents such as streptococcus are suspected of
involvement in its pathogenesis. We compared TLR expression in
psoriasis and normal skin by immunostaining. Samples of
pathological skin were obtained from five patients suffering from
the common form of psoriasis (plaque psoriasis). We performed skin
biopsies on erythematous and squamous plaques for each patient. We
compared the result to the TLR expression in normal skin samples
obtained from healthy donors, as described in the first part of the
results. We found a strong TLR2 overexpression in the epidermis of
all five psoriatic lesional skin samples as compared with the five
biopsies from normal skin from healthy subjects (mammary plastic
surgery) (figure
6).
Discussion
Our findings show that KCs express a large repertoire of TLRs. In
addition, we demonstrated that TLRs 2, 3, and 4 are functional
ligands of KCs: stimulation of normal KCs by microbial ligands
leads to the translocation of Nf-κB and over-expression of
pro-inflammatory cytokines. All adult tissue is capable of
expressing at least one member of the TLR family, but the broadest
TLR repertoire is found in tissues exposed to the external
environment, such as lungs and the gastrointestinal tract [27].
Since the primary function of skin is to provide an effective
barrier against outside aggression, the fact that KCs are capable
of expressing a wide variety of TLRs, and most likely play a role
in a rapid and efficient host defense system, is not surprising. A
link between a TLR deficient response and skin infections such as
human leprosy or pyogenic infections has already been established
[31-33]. Others authors have already shown that normal human KCs
display a large variety of TLRs although they did not observe any
evidence of the expression of TLRs 7 and 8 [34, 35]. This
discrepancy between our results and those already reported may be
due to the site and type of skin examined from which primary KCs
were obtained (e.g. samples of skin biopsies obtained from plastic
surgery versus neonatal foreskins).
TLRs were expressed at very low levels on KC cell surfaces, as
determined by flow cytometry and immunohistochemistry. This
preferential intra-cytoplasmic localization was confirmed by
immunocytochemistry that revealed strictly cytoplasmic paranuclear
distribution of TLR staining (data not shown). TLR expression is
already known to be very low on cell surfaces [36]. A preferential
intracellular expression of this kind has also been observed in
intestinal epithelial cells [37, 38]. Most of the TLRs are
displayed on the cell surface. By contrast, TLRs 3, 7 and 8 are
localized intra-cellularly. In tissues constantly exposed to a
bacterial environment, like intestinal and lung epithelial cells,
TLRs presents an intracellular localization and epithelial cells
appear to be generally unresponsive to several different PAMPs
[39]. This hyporesponsiveness can be explained by several
strategies that are likely to limit the potential encounters
between TLRs and luminal flora. Among them, reduced cell surface
expression and distinct subcellular distribution mean the constant
recognition of PAMPs can be avoided. We therefore hypothesize that
this down regulation of TLR cell surface expression may preserve
skin from chronic inflammation by various infectious ligands, given
the constant exposure to various infectious and environmental
stimuli. The preferential intra-cytoplasmic localization observed
in KCs might contribute to the hyporesponsiveness of these cells to
microbial stimulation.
The mechanisms of regulation of TLR traffic from inside the cell
to the surface are poorly known. One particularity of TLRs is that
their surface expression may be increased after stimulation by
microbial ligands or pro-inflammatory cytokines, as has been
reported by several authors [24, 25]. Moreover, TH1
cytokines sensitize cells to microbial recognition by up-regulating
the expression of several TLRs. Tohyama M et al. demonstrated that
IFN-α enhanced the TLR3 expression and reinforced the response of
poly(I:C) in KCs [41]. In epithelial and endothelial cells, an
enhanced TLR3 expression by IFN-alpha stimulation conferred poly
(I:C) responsiveness [42]. Consistently with these previous
studies, we found that stimulation by IFN-γ and TNF-α not only
increases the intra-cytoplasmic expression of most TLRs, but also
leads to their membrane translocation. So TH1 cytokines
may act as regulators of traffic and cell surface expression of
KCs. KC cytoplasmic domains may function as reserves of TLRs, which
are released in surface cells after stimulation by microbial
ligands and/or proinflammatory cytokines. These results raise the
possibility that stimulation by bacterial products may be
facilitated in a proinflammatory environment.
Given the essential role of the innate immune system in
regulating all aspects of immunity, the dysfunction of
innate-immunity components may conceivably contribute to infectious
or inflammatory diseases. Deficient TLR activation has also been
implicated in severe infectious skin diseases [31, 32, 43, 44].
Recently TLRs have attracted particular interest because of their
manifold functions in the regulation and linking of immune and
inflammatory processes. Several authors have raised the hypothesis
that an inadequate or poorly regulated natural anti microbial
response may lead to the development of long-lasting inflammatory
processes with the induction, perpetuation or severity of
inflammation. This involvement of TLR in inflammatory skin disease
has already been corroborated by Kim et al. [45] regarding TLR2
up-regulation in acne lesions and the role of Propionibacterium
acnes. Evidence has been recently accumulating about the potential
role of innate immunity receptors such as TLRs and NOD in
inflammatory diseases such as Crohn’s colitis [46-48], rheumatoid
arthritis [49], and atherogenesis [50].
Several studies demonstrated that KCs express a variety immune
functional receptors [35, 51-54] but TLR implication in human skin
diseases remains to be explored. We found that TLR2 is
over-expressed in KCs in patients presenting psoriasis. Our results
are consistent with previous findings about the variability of TLR
expression in psoriatic skin [55, 56]. Psoriasis is a frequent and
chronic inflammatory skin disease characterized by increased
keratinocyte proliferation and infiltration of inflammatory cells.
Recent reviews have stressed the central role of T-cells in its
pathogenesis. But other authors argue that KCs may be key cells in
the inflammation process by releasing pro-inflammatory cytokines
and recruiting T cells [57].
Innate immune receptors could promote persistent dermal
inflammation in psoriatic skin through the release of
pro-inflammatory cytokines. IL-8, IFN-γ TNF-α are known to play a
crucial role in promoting inflammation and recruiting immune cells
in psoriatic skin. They are expressed at increased levels in
psoriatic lesions [58]. Here we demonstrated that two of these
cytokines are released after KC stimulation through TLR infectious
ligands: TNF-α and IL-8.
The causes of TLR2 over-expression in psoriatic skin and the
mechanism of its possible involvement in the pathogenesis of
psoriasis remain to be explored. TLR2 over-expression could result
from inappropriate stimulation by TLR2 ligands expressed on KC
surfaces but, on the other hand may be the ultimate consequence of
the pro-inflammatory skin environment known in psoriatic skin.
In the light of previous results for antigenic stimulation in
psoriasis, it may be worth discussing the role of two TLR2 ligands:
streptococcus and heat-shock proteins (HSPs). Although many factors
are regarded as triggers for the onset or exacerbation of the
disease, streptococcal throat infection is the main accepted factor
involved in the disease. Many authors have hypothesized that
psoriasis is a disease precipitated by super antigens derived from
streptococci or nominal antigens that promote clonal expansion of
lymphocytes [59-63]. Streptococcus, as a gram-positive bacterium,
is a natural TLR2 ligand. We hypothesize that a facilitated TLR2
stimulation by streptococcus, in certain subjects, could lead to an
excessive inflammatory response. The role of endogenous TLR ligands
such as heat-shock proteins (HSPs) in the pathogenesis of many
inflammatory diseases is currently being debated [64]. One
endogenous TLR2 ligand is HSP70. Recent studies have found abundant
levels of several HSPs in psoriatic skin [65]. Perez-Lorenzo et al.
have observed higher levels of HSP antibodies in patients with
psoriasis than in healthy subjects. These HSPs proteins have
molecular masses coincidental with streptococcal HSPs [66]. In
particular subjects, TLR2 stimulation by endogenous HSPs, or
streptococcus HSPs, may prolong inflammatory response and
contribute to the pathogenesis of psoriasis. TLR2 over-expression
could be an indirect sign of this continual stimulation.
In conclusion, we have demonstrated that human keratinocytes
express a large repertoire of TLRs and functional TLRs 2, 3, and 4.
TLR2 over-expression in psoriatic skin provides new insights into
the involvement of these newly discovered innate-immunity receptors
in the pathogenesis of cutaneous inflammation [67, 68]. The
pathogenesis of psoriasis may be viewed as a deficiency in the down
regulation process and/or the persistence of unknown triggers
resulting in an exaggerated innate immune reponse [40].
Acknowledgments
This study was supported by SRD (Société de Recherche
Dermatologique) and SFD (Société Française de Dermatologie). The
authors would like to address special acknowledgments to Robin
Nancel, Elisabeth Savariau, and Bernard Boursin (Document
Reproduction Unit, Department of Hematology, Hôpital Saint Louis)
and Pascal Lorre, for their excellent technical assistance.
The authors affirm that the content of this paper has not been
influenced, directly or indirectly, by any actual or potential
conflict of interest.
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