CHAPITRE V : Étude du rôle de l’IL dans l’inflammation cutanée induite par l’IMQ
1. Présentation de l’article: « Role of Interleukin-1 and in imiquimod-induced
psoriasis-like skin inflammation ».
La pathogenèse de maladies inflammatoires tel que le psoriasis est associé à une
production importante de cytokines dont les membres de la famille de l’Interleukin-1. Afin d’étudier les rôles respectifs de l’IL-1α et de l’IL-1 dans le développement d’une
inflammation cutanée de type « psoriasique », nous avons étudié l’expression des membres de
cette famille dans le modèle d’inflammation cutanée induit par l’IMQ. Nous avons montré que l’application topique et quotidienne d’IMQ pendant 6 jours consécutifs sur le dos
rasé/épilé de souris C57BL/6 (B6) induit une surexpression de l’ARNm codant l’IL-1α, l’IL-
1 ainsi que de leur récepteur antagoniste, l’IL-1Ra, suggérant un contrôle de la réponse inflammatoire induite par l’IMQ (Figure 1). En parallèle de cette induction, est associée une
surexpression des cytokines de l’axe Il-23/IL-17/IL-22, ainsi que des gènes cibles tels que les
peptides antimicrobiens BD3 et S100A9 ; et de la chimiokine CXCL3 (Figure 1), regroupant ainsi un profil observé dans les lésions psoriasiques. Par la suite, nous avons montré que
l’absence de l’IL-1α ou de l’IL-1 chez des souris transgéniques n’a pas protégé les animaux de l’inflammation cutanée induite par l’IMQ en présentant une hyperplasie de l’épiderme (Figure β) et une expression des cytokines de l’axe IL-23/IL-17 similaire aux souris B6 traitées avec l’IMQ (Figure γ). De façon intéressante, les souris déficientes en IL-1α reprennent rapidement leur poids initial dès le troisième jour d’application en comparaison
avec les souris déficientes en IL-1 et les souris B6 (Figure 7A. Supplémentaire). Nous
assimilons la perte de poids à un effet systémique dû au traitement. Chez les souris
doublement déficientes en l’IL-1α et en l’IL-1 ou en IL-1RI, l’inflammation est
partiellement réduite (Figure 2) et est caractérisée par une diminution de l’expression des
cytokines de l’axe IL-23/IL-17/IL-ββ (Figure γ) illustrant d’une part la redondance d’activité entre l’IL-1α et l’IL-1 , et d’autre part qu’elles agiraient en amont de l’axe IL-17/IL-22 dans
ce modèle. Ensuite nous avons montré que l’inflammasome NALPγ ne semble pas être
impliqué dans la mesure où l’absence de NALPγ ou de ASC n’a eu aucun effet sur l’inflammation cutanée induite par l’IMQ (Figure 4 et 5). Enfin nous avons confirmé la
dépendance totale de la voie MyD88 (protéine adaptatrice utilisée par l’IL-1RI et les TLR)
dans l’inflammation cutanée induite par l’IMQ, dans la mesure où les souris déficientes en cette molécule n’affichent ni lésion (Figure 6A,B et C), ni perte de poids (Figure 7D) et ni
induction de tous les marqueurs analysés (Figure 6D) confirmant que l’activité de l’IMQ est largement TLR dépendant.
Le travail présenté dans ce chapitre a fait l’objet d’une communication orale : « Role of
Interleukin-1 processing and signalling in imiquimod-induced psoriasis-like skin inflammation » (28 mai 2013, 16e Colloque Cytokines et Chimiokines, Presqu'Ile du
Croisic). De plus, il a été soumis pour publication dans la revue Mucosal Immunity. Le manuscrit est présenté ci-après.
Role of interleukin-1 and in imiquimod-induced psoriasis-like
skin inflammation
H. Rabeony1, P. Vasseur1,2, I. Petit-Paris 1,2, J.F. Jégou1, L. Favot1, D. Togbe3, B. Ryffel3, F.X. Bernard1,4, J.C. Lecron1,2, F. Morel1
1 Laboratoire Inflammation, Tissus Epithéliaux et Cytokines, UPRES EA4331, Pôle Biologie Santé, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073 POITIERS Cedex 9, France
2 CHU de Poitiers, 86021 Poitiers, France
3 INEM UMR 7355 CNRS and Université d’Orléans, France and IIDMM, University of Cape Town, RSA 4 BIOAlternatives, 1 bis rue des Plantes, 86160 Gençay, France
Corresponding author: Franck Morel, Address: Laboratoire Inflammation, Tissus Epithéliaux et Cytokines, UPRES EA 4331, Pôle Biologie Santé, Université de Poitiers, 1 Rue Georges Bonnet, TSA 51106, 86073 POITIERS Cedex 9, France; Phone: +33.5.49.45.40.54. ; Fax:
+33.5.49.45.37.03, E-mail address: f.morel@univ-poitiers.fr
Abstract
The pathogenesis of inflammatory skin diseases such as psoriasis involves the release of numerous proinflammatory cytokines, including members of the IL-1 family. To highlight the
role of IL-1α and IL-1 in the induction of psoriasis-like skin, we assessed the expression of
these IL-1 family members after topical application of the TLR7 agonist imiquimod (IMQ) in
C57BL/6J (B6) mice. Daily skin application of IMQ in B6 mice induced overexpression of
IL-1α, IL-1 , and IL-1 receptor antagonist (IL-1Ra) mRNA, associated to expression of IL-
23p19, IL-17A, IL-22, antimicrobial peptides such as -defensin3 and S100A9, and
chemokine CXCL3. IMQ-treated IL-1α- or IL-1 -deficient mice displayed inflammation,
epidermal hyperplasia and IL-23/IL-17 cytokines expression similar to IMQ-treated B6 mice. However, IMQ-induced skin inflammation is partially reduced in mice deficient for both IL-
1α and IL-1 or for IL-1R1. The inflammasome NALP3 appears not to be involved since
mice deficient for NALP3 or ASC have no significant reduction of inflammation. Finally,
IMQ-induced skin inflammation was totally abolished in mice deficient for MyD88, the
adaptor protein shared by IL-1R and TLR signaling pathways, confirming that IMQ induced
inflammation is TLR-signaling dependent. Therefore, both IL-1α and IL-1 contribute to the
Introduction
Psoriasis is a chronic inflammatory skin disease estimated to affect 2-3% of the
general population.1 Clinical presentation usually associates red, scaly and raised plaques.
These are the consequences of a marked thickening of epidermis due to an increased proliferation of keratinocytes (acanthosis), a retention of nuclei in the stratum corneum (parakeratosis) caused by alterations of keratinocyte differentiation leading to reduced or loss of the granular layer, and the presence of inflammatory cell infiltrates in the epidermis and
dermis.2 The inflammatory infiltrate is composed mainly of dendritic cells, macrophages and
T cells in the dermis and neutrophils, with some T cells in the epidermis.3 The combination of
this infiltration and acanthosis contribute to the overall thickness of lesions. Therefore, the pathogenesis of psoriasis clearly results from a cross-talk between immune cells, keratinocytes, endothelial cells and fibroblasts, together with the release of growth factors,
chemokines, and cytokines for the induction and the maintenance of this disease.3,4
An important role of the IL-23/IL-17/IL-22 axis in the induction of psoriasis has become evident in which IL-23 secreted by some DC is responsible for the production of
Th17-related proinflammatory cytokines IL-17A and IL-22.2 IL-1 family members also play
an important role in the regulation of the immune response and dysregulation of their expressions leads to tissue destruction and severe pathological effects, including skin diseases
such as psoriasis.5 Clearly, IL-1 is considered as a key player in the initiation and the
maintenance of psoriasis by inducing Th17 cell maturation and downstream cytokine
production.6,7 IL-1α and IL-1 , the original members of IL-1 family ligands, identically
recognize IL-1 receptor type I (IL-1RI) which leads to the recruitment of the adaptor molecule MyD88 (adaptor protein shared by IL-1R and TLR signaling) and activate transcription
factors NF-κB and AP1, whereas the binding of IL-1 receptor antagonist (IL-1Ra) to IL-1RI
The mature secreted form of IL-1 requires engagement of the protein complex termed inflammasome. NALP3 inflammasome includes the adaptor protein ASC (Apoptosis- associated Speck-like protein containing a Caspase recruitment domain) necessary for the
activation of the cysteine protease caspase-1, responsible for cleaving pro-IL-1 to mature IL-
1 , whereas IL-1α is already biologically active and its processing requires the protease
calpain.9 In vitro studies showed that IL-1α or IL-1 induce the production of CCL5 and
CCL20 by keratinocytes, and in synergy with IFN and TNFα, increase the production of
CCL5, CCL20, CXCL9, CXCL10 and CXCL11.10 Several studies showed that IL-1α is also
involved in the modulation of numerous genes within the epidermal differentiation complex
and transgenic mice overexpressing IL-1α in keratinocyte exhibited a spontaneous skin
disease characterized by scaling, hyperkeratosis and parakeratosis, consequences of an altered
keratinocyte differentiation.11,12 Moreover, we previously demonstrated that the association of
IL-1α, IL-17A, IL-ββ, OSM and TNFα exhibits a very strong synergistic effect on keratinocytes by increasing the expression of inflammatory effectors such as chemokines,
antimicrobial peptides that mimic some features of psoriasis in vitro, wherein IL-1α and IL-1
are functionally redundant.13 In addition, in mice, the mutation of nlrp3 gene resulting in
permanent activation of IL-1 associated to a Th17 cytokine-dominant response, or the lack
of IL-1Ra lead to a spontaneous skin inflammation resembling psoriasis.14,15 Finally,
Anakinra, an IL-1 receptor antagonist, has been used as an effective treatment against
psoriasis in several patients.16
Knowing these involvements of IL-1 in cutaneous inflammation, we used knockout mice to estimate the relative individual role of IL-1 ligands and the inflammasome contribution in the imiquimod (IMQ)-induced psoriasis-like skin inflammation described by
van der Fits et al.17 IMQ is a toll-like receptor (TLR)-7/8 ligand and a potent immune
side effect, it can induce psoriasis-like skin flares in predisposed humans.18 Daily topical application of IMQ on shaved back skin of mice leads to a psoriasis-like dermatitis with many hallmarks of human psoriasis, such as erythema, scaling, skin thickening associated with histopathological changes (acanthosis, parakeratosis neoangeogenesis), including the
recruitment of immune cells consisting of CD4+ T cells, CD11+ DCs and pDCs.17,19 This
model was shown to be critically dependent of the IL-23/IL-17 axis.17 However, limited data
are available on the role of the members of the IL-1 family in IMQ-induced skin inflammation.
We found that IL-1α and IL-1 were overexpressed in IMQ-treated B6 mice. We
observed an attenuated response in the absence of both IL-1α and IL-1 or IL-1RI, but not in
IL-1α or IL-1 deficient mice, demonstrating the important contribution of IL-1 to this model, despite, as expected the functional redundancy of the two cytokines. No major role for NALP3/ASC inflammasome was detected as evidenced by the induction of psoriasis-like skin in absence of these components. By contrast, IMQ-induced skin inflammation was totally
abolished in mice deficient for MyD88 confirming that IMQ induced inflammation is mainly
Materials and Methods
Mice and treatments
C57BL/6J mice were purchased from Janvier (Le Genest, France). IL-1α-/-, IL-1 -/-, IL-1α/ -/-,
20
NALP3-/-, ASC-/-,21 IL-1R1-/- 22 and MyD88-/- 23 mice were provided by Dr. B. Ryffel
(University of Orléans, France). All mice were bred in the animal facility of Centre National de la Recherche Scientifique (Orléans, France) and were kept under specific pathogen-free
conditions and provided food and water ad libitum. Experimental procedures were approved
by French Government’s ethical and animal experiment regulations. Mice at 8-10 weeks of
age were used in those experiments. Back skin of the mice was shaved and remaining hairs were removed using a depilatory cream (Veet@, Reckitt Benckiser, France). Psoriasis-like
skin disease was induced as described previously by van der Fits et al.17 Shaved back skin and
right ear were daily treated with 62.5 mg Aldara@ cream (5% imiquimod, 3M pharmaceuticals) for 6 days. Control mice were treated similarly with Vaseline (VAS) (Vaseline Lanette cream, Fagron). Due to deshydratation and weight loss observed in IMQ-
treated mice, a daily volume substitution by intraperitoneal injection of γ00 μl sterile PBS
was performed from day 1 to 5 in all experimental groups.
Evaluation of skin inflammation severity
Severity of skin inflammation was evaluated using a clinical score based on Psoriasis Area
Severity Index as described.17 Briefly, erythema, scaling and thickening were scored on a
scale from 0 to 4 (0: none; 1: slight; 2: moderate; 3: marked and 4: severe) and addition of these three parameters gave a cumulative score from 0 to 12, used to measure the severity of inflammation.
Real-time PCR
Tissues were homogenized in lysis buffer using gentleMACSTM Dissociator (Miltenyi Biotec,
France). Total RNA was isolated using Nucleospin® RNAII (Marchery-Nagel) and quantified
using NanoDrop® β000c (Thermo Scientific, Fisher). cDNA was obtained from 1μg of total
RNA by reverse transcription using Superscript II reverse transcriptase (Invitrogen Life
technologies) and random hexamer (Invitrogen), according to the manufacturer’s instructions
(Invitrogen Life technologies).
Quantitative PCR was conducted using LightCycler 480 SYBR Green I Master (Roche
Applied Science) on a LightCycler 480 System (Roche) with 0.5 μM of forward and reverse
primers, designed using Primer3 software. The PCR program consisted of 5 minutes at 95°C for polymerase activation, followed by 40 cycles at 95°C for 20 s, 60°C or 64°C for 15 s, and 72°C for 20 s. The melting curves were established by increasing temperature from 60°C to 95°C with a continuous fluorescence measurement. Samples were normalized to independent
control housekeeping genes GAPDH and B2M and reported according to the ∆∆CT method
as RNA fold increase over normal untreated skin.
Histological evaluation of the tissue sections
Freshly obtained ear skin was snap frozen in liquid nitrogen and stored at -80°C until use. We next embedded the tissue in a cryogenic medium (Cryomatrix Shandon, Thermo Scientific, France) and eight-micrometer cryosections were prepared using a cryotome (Leica CM 3050S) and stained with hematoxylin and eosin (Sigma Aldrich, France). Tissues were observed under light microscope (Olympus CKX41, Olympus Optical, Japan) and were photographed (Olympus DP72). Epidermal thickness, considered as the distance from the basal layer to the stratum corneum, was measured every 50 micrometers using cellSens standard software (Olympus) on all part of the sectioned tissue.
Statistical analysis
The results are presented as mean ± S.E.M for n= 4 mice/group, and Mann-Whitney test was
used to compare groups using GrapdhPad Prism 5 (GrapdhPad Software, Inc, San Diego,
Results
IMQ-induced psoriasis-like skin inflammation leads to increased expression of proinflammatory cytokines including interleukin-1α and β
We first assessed in IMQ-induced skin inflammation the expression of several proinflammatory cytokines and receptor belonging to the IL-17/IL-23 axis and interleukin-1
family, including the agonists IL-1α and IL-1 , the IL-1Ra and the IL-1R1. Shaved back skin
and right ear of B6 mice were treated daily with IMQ or Vaseline as control and cytokine mRNA expressions were analyzed by RT-quantitative PCR. Consistent with the original report (van der Fits et al., 2009), a rapid and significant overexpression of IL-23p19 was observed after IMQ treatment, compared to VAS-control mice (figure 1). This was followed by an overexpression of IL-17A and IL-22, which were strongly induced at day 6 (figure 1).
IMQ also induced the expression of IL-1α and IL-1 after β days, with a maximum after 6
days, accompanied by an overexpression of IL-1Ra which was significantly expressed after 6 days. Finally we have demonstrated that IL-1RI was constitutively expressed and not influenced by IMQ application (figure 1). As a control for inflammation, S100A9 mRNA expression was strongly induced after IMQ-treatment.
IL-1α and IL-1β display redundant activity in imiquimod-induced psoriasis-like skin inflammation
To investigate the role of IL-1 in IMQ model, we daily treated IL-1α-, IL-1 -, IL-
1α/ - or IL-1R1-deficient mice with IMQ for 6 consecutive days. None of the mice treated
with Vaseline control cream had any clinical sign of inflammation (figure 2A). After 2 days
of IMQ application, the back skin of IL-1α- or IL-1 -deficient mice displayed skin
signs increased in severity up to 6 days as illustrated by the cumulative disease severity index (figure 2A and 2B). By contrast, IMQ-induced skin lesions were partially reduced in absence
of IL-1α/ or IL-1R1 (figure 2A) with decreased scores for the independent parameters
leading to a lower cumulative disease index from day 3 to day 6 (figure 2B). Consistent with macroscopic features, histological analysis showed increased epidermal thickness in ear of
IMQ-treated IL-1α or IL-1 -deficient mice comparable to IMQ-treated WT mice (figure 2C
and 2D), whereas a significant decrease of epidermal thickening was observed between WT
mice and IL-1α/ or IL-1R1 deficient mice after IMQ treatment (figure 2C and D).
Nevertheless, an increase in epidermal thickness was observed in all IMQ-treated mice compared to Vaseline-treated groups (figure 2C and 2D).
To highlight the role of IL-1 in the development of psoriasis-like skin disease after IMQ treatment, we analyzed gene expression levels of different proinflammatory cytokines, antimicrobial peptides and chemokines. In B6 mice, IMQ treatment significantly induced the
expression of IL-23p19, IL-17A, IL-22, IL-1α and IL-1 , as well as downstream genes known
to be induced by these cytokines such as antimicrobial peptides (BD3 and S100A9), chemokine (CXCL3), and cytokeratin (KRT) 6, a cytokeratin associated with keratinocyte
hyperproliferation (figure 3). The expression of Gr1 was also increased as a marker of skin-
infiltrated granulocytes. IMQ-treated IL-1α-deficient mice expressed elevated levels of
proinflammatory cytokines mRNA encoding IL-23p19 and IL-22, similarly to IMQ-treated
B6 mice, while IL-17A and IL-1 expressions were significantly reduced when compared
with IMQ-treated B6 mice (figure 3). Although expression of BD3 and S100A9 was strongly
induced by IMQ in absence of IL-1α, we observed a decrease compared to B6 mice (figure 3).
Additionally, IMQ induced comparable expression of CXCL3, KRT6 and Gr1 in IL-1 α -
deficient mice and B6 mice (figure 3). In IMQ-treated IL-1 -deficient mice, a significant but
B6 mice while IL-1 α, IL-17A, IL-22, S100A9, KRT6, CXCL3 and Gr1 induction were
similar (figure 3). However, in the skin of IMQ-treated IL-1α/ -deficient mice, we observed a
significant reduction of the proinflammatory cytokines IL-23p19, IL-17A and IL-22, as well as for the chemokine CXCL3 and the granulocyte marker Gr1 when compared to IMQ-treated
B6 mice (figure 3). Interestingly, the absence of both IL-1α and IL-1 did not affect the
induction of antimicrobial peptides (BD3 and S1009) and KRT6 expression by IMQ treatment (figure 3). Finally, while IL-23p19, BD3 and KRT6 expression were unchanged in absence of IL-1R1, we found a profound decrease in the expression of all other proinflammatory cytokines and downstream genes (figure 3). Our data demonstrate the involvement and the
redundant roles of IL-1α and IL-1 in this model, as evidenced by the attenuation of IMQ-
induced skin inflammation in absence of both IL-1α and IL-1 or IL-1 receptor.
IMQ-induced skin inflammation is NALP3/ASC inflammasome independent
The role of IL-1α and IL-1 in the IMQ-induced skin inflammatory response led us to
investigate the involvement of NALP3/ASC inflammasome pathway. As shown in figure 4A, IMQ-treated NALP3- and ASC-deficient mice displayed inflamed, red and scaly skin lesions similar to IMQ-treated B6 mice. IMQ-induced skin inflammation was reflected by a strong cumulative clinical score in NALP3- and ASC-deficient mice, similar to IMQ-treated B6 mice (figure 4B). Histological analysis revealed an important epidermal thickening in IMQ- treated mice deficient for NALP3 or ASC as observed in IMQ-treated B6 mice (figure 4C and 4D).
We also analyzed gene expression in IMQ-treated NALP3- or ASC-deficient mice in comparison to B6 mice. As expected, the absence of NALP3 or ASC did not modify the IMQ- induced profile of proinflammatory gene expression, excepted an increased IL23p19 expression in NALP3-deficient mice and a small increased BD3 expression in ASC-deficient
mice (figure 5). These data indicate that IMQ-induced psoriasis-like skin inflammation is NALP3/ASC inflammasome independent as evidenced by the development of skin lesions as well as the high level of all inflammation markers.
MyD88-signaling is critical for IMQ-induced skin inflammation
Since IMQ is an agonist of TLR7, we targeted MyD88 signaling, the adaptor protein shared by IL-1R and TLR signaling pathways. IMQ-induced skin lesions were totally abolished in MyD88-deficient mice compared to IMQ-treated WT mice (figure 6A). Neither skin erythema nor scaling or thickening was observed in the absence of MyD88 after IMQ treatment, as evidenced by the cumulative clinical score (figure 6C). Histological analysis showed an absence of inflammation and epidermal thickening in IMQ-treated MyD88- deficient mice (figure 6B and 6C). At the molecular level, we observed a significant and profound decrease expression of all proinflammatory cytokines as well as downstream inflammatory markers (figure 6D). Therefore, the data confirmed that IMQ engagement of TLR7 fully involved MyD88. In this context, the downstream role of MyD88 in IL-1