Reprint from Volume 2 - Number 3/2011

Innate immunity and Toll-likeReceptors modulation in acne

Marius-Anton IonescuLuc Lefeuvre

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Editorial StaffDirettore Responsabile: Pietro Cazzola Consulenza grafica: Piero Merlini

Direttore Generale: Armando Mazzù Impaginazione: Stefania Cacciaglia

Editorial BoardEditor

Stefano Veraldi (Milano)

Co-EditorMauro Barbareschi (Milano)

Scientific BoardVincenzo Bettoli (Ferrara), Stefano Calvieri (Roma), Gabriella Fabbrocini (Napoli), Giuseppe Micali (Catania)

Giuseppe Monfrecola (Napoli), Nevena Skroza (Roma), Annarosa Virgili (Ferrara)

Managing EditorAntonio Di Maio (Milano)

International Acne BoardTam El Ouazzani (Casablanca, Morocco), Marius Anton Ionescu (Paris, France), Monika Kapinska Mrowiecka (Cracow, Poland),

Noppakun Noppadon (Bangkok, Thailand), Gerd Plewig (Monaco, Germany), Robert Allen Schwartz (Newark, USA),Jacek Szepietowski (Wrocław, Poland), Shyam Verma (Vadodra, India).

Italian Acne ClubMario Bellosta (Pavia), Carlo Bertana (Roma), Alessandro Borghi (Ferrara), Francesco Bruno (Palermo), Maria Pia De Padova (Bologna),

Paolo Fabbri (Firenze), Carlo Pelfini (Pavia), Mauro Picardo (Roma), Maria Concetta Potenza (Roma), Alfredo Rossi (Roma),Patrizio Sedona (Venezia), Aurora Tedeschi (Catania), Antonella Tosti (Bologna/Miami), Matteo Tretti Clementoni (Milano)

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Introduction

The particular field that was subject of thehighest award in medical research in 2011 under-lined once again the importance of innate immuni-ty and of Toll-Like Receptors (TLRs). This domainof medical research was opened more than 15years ago by Nobel Prize recipients Jules Hoffmanwho discovered TLRs in Drosophila flyes1 andBruce Beutler that focused on the role of TRLs inmammals 2.With the discovery of TLRs, the understanding ofthe innate immunity role was widened from thepreviously described “nonspecific” mechanisms -

as the activation of complement and the phagocy-tosis - to the complex pathways of the TLRs acti-vation and the subsequent expression of pro-inflammatory molecules and of anti-microbial pep-tides, subject of several recent studies.This article presents in the first part ex vivo studieson TLR2 activation by P. acnes in human skin andits topical regulation by an association of a vegetalextract (Ombeliferae) with a long-chain lipid(TLR2-Regul®), then in the second part the resultsof a double blind, pilot clinical trial, using sametopical formulation in a series of acne patients.

European Journal of Acne and Related Diseases

Volume 2, n. 3, 2011

Innate immunity and Toll-like Receptors modulationin acne

Marius-Anton Ionescu1, Luc Lefeuvre2

1 Dermatology Polyclinic, Saint-Louis Hospital, Paris, France2 Laboratoires Dermatologiques d’Uriage, Courbevoie, France

SUMMARYBACKGROUND. Toll-like recep-tors (TLR) are trans-membranereceptors part of the innate immunesystem. Defects in TLR pathway

have the potential to lead to increased susceptibility todysregulation and play a role in the pathogenesis ofnumerous in!ammatory skin diseases (as seborrheic der-matitis, acne, atopic dermatitis). Toll-like receptor 2(TLR2) can be activated by peptidoglycans from P.acnesstructure, with a subsequent in!ammatory cascadeinvolving pro-in!ammatory interleukins (IL-8) andinducing human beta-defensine 2 (hBD2) as one of theinnate immune mechanisms of defense against microbes.PURPOSE. We sought to assess the effects of a vegetalnatural extract (Ombelliferae) associated with a longchain lipid (TLR2-Regul®) in ex vivo human skin in con-tact with P. acnes, focusing on the markers of TLR2 acti-vation: expressions of IL-8 and hBD2. In a second stepwe assessed the effects of the same emulsion in a doubleblind clinical trial in a series of patients with lightin!ammatory acne.METHODS. Normal human skin explants were treatedby an emulsion O/W formulated with the complex TLR2-RegulTM (20 μl per explant) or by its vehicle (control).Extracts of P. acnes were added.The dosages of IL-8 (ELISA) and of hBD2 were per-

formed at 24h. In a second part of this study was made adouble blind clinical trial including acne patients withlight in!ammatory acne (2-3 IGA scale) using same O/Wemulsion formulated with TLR2-Regul® (compared to itsvehicle): 2 applications/day during 12 weeks. Wereexcluded patients having systemic or topical treatmentfor acne.RESULTS. P. acnes induced signi#cant increase of IL-8expression in all untreated skin explants. Skin explantspreviously treated by TLR2-Regul® in contact with P.acnes had a signi#cant less important expression of IL-8compared to control (p<0.01) and a signi#cant increaseof hBD2 expression (p<0.01).CONCLUSIONS. In normal human skin explants incontact with microbial extracts, the expression of IL-8(as main cytokine linked to TLR2 activation) wasincreased. In all skin explants treated by the complexTLR2-Regul® and exposed to extract of P.acnes, theexpression of IL-8 was signi#cantly down-regulated andhBD2 expression was signi#cantly up-regulated. Resultsof the clinical trial showed in a series of 34 acne patientsa signi#cant decrease of IGA mean score (by 40%) in thegroup verum compared to the vehicle group(-24%)(p<0.05). Larger clinical trials are ongoing inorder to con#rm the results of this pilot study focusingon the modulation of TLR2 in acne patients.

Key words: Toll-like receptor 2, IL-8, betadefensin hBD2, in!ammatory acne.

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66

Toll-Like Receptors

The innate immune system is activated bycomplex mechanisms, among them TLRs aretrans- membrane cellular receptors of human main“interface” tissues as skin, gastrointestinal tractand lungs. Skin TLRs are expressed by ker-atinocytes, Langerhans’ cells, macrophages, mo-nocytes and granulocytes. TLRs are activated bytheir contact with ligands from pathogen-associat-ed molecular patterns (PAMPs) of microbes (bac-teria, yeasts or viruses). Up today more than 11types of TLRs were described 3, 4 (Figure 1).TLRs have an extracellular domain (leucine) that isable to recognize ligands from microbes’ struc-tures, and an intracellular domain of the receptorfor IL-1 and protein MyD88 Toll-IL-1R homology(TIR) from cytoplasm (Figure 2). The TIR domainparticipates to the induction of the cellular

response (pro-inflammatory cytokines and antimi-crobial peptides up- regulation). The bindingprocess between microbe structures and TLRs’extracellular domain triggers a signal that initiatesa succession of steps involving specific inflamma-tion messengers, resulting in the release of variouscytokines and defensines. Toll-like receptor 2(TLR-2) is activated by 3 compounds of microbesas P. acnes. This will trig an inflammatory cascadeinvolving mainly IL-8 (but also other cytokines asIL-1, IL-6) 3, 6.Inflammatory response triggered by microbes as P.acnes, S. aureus, Malassezias, human papilomaviruses - HPV is linked to the activation of TLRs –especially in inflammatory diseases with microbialaggravating factors as acne, seborrheic dermatitis,atopic dermatitis 3, 5.P. acnes-induced inflammation in acne is complex,a part of inflammatory pathway is due to TLR2

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Figure 1. Structures recognized by TLRs (microbial and host).

Figure 2. Toll Like Receptors – mechanisms of activation.

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Volume 2, n. 3, 2011

activation3, 4. Same receptor is activated in atopicdermatitis by S. aureus 3, 5, in leprosy 3 borreliosis/ Lyme disease (ligands from Borrelia burgdor-feri) 3, 5. Yeasts-induced inflammation can befound in seborrheic dermatitis (linked toMalassezias’ zymosan activating TLR2) and can-didiasis (Candida species activate same TLR).Papilloma viruses from warts activate TLR7, andTLR8 3,4.In psoriasis TLR2, TRL7, TLR8, TLR9 are activat-ed 4, 7. In acne TLR participate to innate responseto microbial presence: wall structure of P. acnes(mainly glyco-peptides) are acting as ligands andactivate TLR2 (and TLR4) 8-14. P. acnes activatesTLR-2 and consequently induces the expression ofIL-8 (and IL-6) within follicular keratinocytes, andof IL-8 and IL-12 within macrophages 13.Sebocytes’ and keratinocytes’ antimicrobial peptidesexpression can be up-regulated, this being an impor-tant contribution to the innate immunity responsewithin the pilo- sebaceous follicle. Changes ofsebum lipids - as a modified ratio between differentlipid fractions - induce alterations of keratinocyte interms of differentiation and IL-1 secretion, leading toinfra- infundibular hyperkeratosis and initiating firststeps of micro-comedo induction. It has been shownthat keratinocytes can respond to bacterial, fungal,and viral pathogens that have breached the stratumcorneum by producing 2 important classes ofendogenous peptides, beta-defensins and catheli-cidins 10, 14. Beta-defensin 1 is constitutivelyexpressed, beta-defensin 2 (hBD2) is strongly up-regulated upon antigenic stimulation 10, 15. In acneother factors can also increase hBD2 expression asfree fatty acids from sebum 16.

TLR modulation in human skin 4

We previously showed that in human skinin contact with microbial extracts the expression ofIL-8 significantly linked with the activation ofTLR2 17,18.We present here ex vivo tests on human skinassessing the modulation of TLR2 in presence ofP. acnes with the focus on IL-8 and also on antimi-crobial peptide expression hBD2.

An association of a natural vegetal extract (fromthe family of Ombelliferae) with a synthetic long-chain lipid (TLR2-Regul®) was tested on ex vivohuman normal skin in contact with purified micro-bial extract of P. acnes.The objective of this study was to assess the effectsof the TLR2-Regul® on the expression of IL-8 –considering that this interleukin is not specific forTLR2 activation but is one of the main cytokinesexpressed after the activation of TLR2 in ker-atinocytes in contact with microbial extracts.3,4This first test was followed by the assessment ofantimicrobial peptide hBD2 expression in humanskin in contact with P. acnes in the presence ofsame complex TLR2-Regul®.Methods: skin explants A. were incubated (1 h at37°C) in absence (control) or in presence of an emul-sion O/W formulated with the complex TLR2-Regul®

(20 μl per explant); B. at 1 h were added inactivatedextracts of P. acnes (20 μl per explant); C. after 24 hincubation was performed the dosage of IL-8(ELISA). Same steps A and B were used in order toassess antimicrobial peptides expression in humanskin explants, in last step C. after 24 h incubation waspreformed the dosage of human beta-defensin 2(hBD2) (ELISA). Comparisons for each type of testwere made between skin explants coming from samedonor. A difference of IL-8 or hBD2 expression lessthat 0.01 was considered as significant.Results: microbial extract of P. acnes induced asignificant increase of IL-8 expression in alluntreated skin explants. Skin explants treated byTLR2-Regul® in contact with microbial extractshad a significant less important expression of IL-8compared to control (p<0.01) (Figure 3).Skin explants in contact with P. acnes pre-treatedwith TLR2-Regul® had a significant up-regulatedexpression of hBD2 compared to untreatedexplants in contact with the same microbial extract(p<0.01) (Figure 4).In these studies on human skin explants previouslytreated by the TLR2-Regul® and exposed to P. acnesextract, the expression of IL-8 was significantlydecreased compared to untreated skin (p<0.01),antimicrobial peptide hBD2 expression was signifi-cantly up-regulated in skin treated with TLR2-Regul®

in contact with P. acnes compared to untreated skin.

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Clinical application of TLR2modulation in inflammatory acne

The purpose of this in vivo pilot study wasto assess the effects of TLR2-Regul® in a doubleblind clinical trial study in patients with light inflam-matory acne.Methods: TLR2-Regul® was included in a formula ofan O/W emulsion (at 1%) and tested in monotherapyin acne patients with light inflammatory forms. Wereincluded adult acne patients (above 18 years old)with light inflammatory acne on IGA scale19, rated2 to 3 on the 5-point investigator global assessmentof acne severity scale.Included patients were randomized and treated withverum or by its excipient. Were excluded patientswith IGA scores less than 2 or over 3, patients under

topical and/or systemic treatment for acne or havingstopped their treatment less than 4 weeks beforebaseline. Daily cleansers used by the patients duringthe trial were “neutral” (no anti-acne effect).Results: were included 34 adult acne patients, 16 inthe group A (verum) and 18 in group b (excipient),with a mean score of acne IGA score of 2.3 in groupA vs 2.2 in group B.Inflammatory lesions decreased significantly at 3months in the group verum (mean IGA decreased by40% compared to baseline) compared to its excipi-ent (mean IGA decreased by 24% compared tobaseline) (p>0.05) (Figure 5). Skin tolerance wasvery good in both groups.

European Journal of Acne and Related Diseases

Volume 2, n. 3, 2011

Figure 5. O/W emulsion (with 1% TLR2-RegulTM ) applied 2X/day inmonotherapy in patients with light inflammatory acne (baseline and week 12).

Figure 3. IL-8 release in human skin explantsin contact with P. acnes extract.

Figure 4. Human beta-defensin 2 (hBD2) expressionin human skin explants in contact with P. acnes extract.

baseline week 12

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Conclusions

Human skin explants in contact with P.acnes express increased levels of IL-8, in skinexplants pre- treated with TLR2-Regul® emulsionthe expression of IL-8 was less important comparedto control (p<0.01). In pre-treated skin with TLR2-Regul® in contact with P. acnes the expression ofanti- microbial peptide hBD2 was up-regulated. Inthis double blind, pilot clinical trial in a series of 34patients with light inflammatory acne (IGA score 2to 3), the group treated with the emulsion based onTLR2-Regul® showed at week 12 a significantdecrease of inflammatory lesions compared to excip-ient group (p<0.05). As a recently developed area ofresearch, the interest for the innate immunity is con-tinuously growing in dermatology, especially with

the opening of new and therapeutic prospective tar-geting TLRs. The development of active ingredientsthat are able to modulate the innate response canopen new ways of treatment in the field of inflamma-tory skin diseases aggravated by microorganisms asacne. Larger clinical trials are ongoing in order toconfirm the results of the pilot trial presented in thisarticle. Most recently, the topical calcineurin antago-nists such as tacrolimus 16 and pimecrolimus 17, 18

have been successfully used. Thus, they should beconsidered as alternative or adjuvant therapies forpatients who do not respond to traditional treatment.Otherwise, prevention becomes a priority in the man-agement of SIRD. It may be a recalcitrant problem,imposing a great psychological stress on the patients.

European Journal of Acne and Related Diseases

Volume 2, n. 3, 2011

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Disclosures:Studies were supported by Laboratoires Dermatologiques d’Uriage®