Review of the innate immune response in acne vulgaris: activation of Toll-like receptor 2 in acne triggers inflammatory cytokine responses

Acne vulgaris is a common disorder that affects 40-50 million people in the USA alone. The pathogenesis of acne is multifactorial, including hormonal, microbiological and immunological mechanisms. One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). TLRs are pattern recognition receptors that recognize pathogen-associated molecular patterns conserved among microorganisms and elicit immune responses. We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Using transfectant cells we found that TLR2 was sufficient for NF-kappaB activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes.P. acnes induced activation of IL-12 and IL-8 production by primary human monocytes, and this cytokine production was inhibited by anti-TLR2-blocking antibody. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for the treatment of this common skin disease.     

Zinc salts inhibit in vitro Toll-like receptor 2 surface expression by keratinocytes

Propionibacterium acnes (P. acnes) plays an important role in the induction and maintenance of the inflammatory phase of acne. At the therapeutic level, it has been shown that zinc salts could have a beneficial effect on mild and moderate inflammatory acne lesions. However, their mechanisms of action are still only partially known. Immediate early immune response is a crucial route in the development of inflammatory reaction and, specifically, activation of Toll-like Receptors (TLRs) leading to nuclear factor (NF)-kappaB translocation and production of inflammatory cytokines such as interleukin-8 (IL-8). The aim of this work was to determine if cytokine secretion and innate immunity could be targets of zinc salts. Normal Human Epidermal Keratinocytes (NHEK) and skin explants were stimulated by P. acnes extracts and incubated (3 h) with zinc salts (1 microg/mL). Then we successively studied TLR2 expression by immunohistochemistry and IL-8 production by ELISA. After incubation with zinc salts, the increase of TLR2 surface expression in skin upon membrane fraction (FM) of P. acnes challenge was decreased as compared to that in control samples. However, this inhibition does not modify IL-8 secretion by keratinocytes. In conclusion the inhibition of TLR2 surface expression by keratinocytes could be one of the anti-inflammatory mechanisms of zinc salts in acne.     

Glucocorticoids enhance Toll-like receptor 2 expression in human keratinocytes stimulated with Propionibacterium acnes or proinflammatory cytokines

Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-alpha, and IL-1alpha. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis.     

All-trans retinoic acid shifts Propionibacterium acnes-induced matrix degradation expression profile toward matrix preservation in human monocytes

Propionibacterium acnes is a critical component in the pathogenesis of acne vulgaris, stimulating the production of various inflammatory mediators, such as cytokines and chemokines, important in the local inflammatory response found in acne. This study explored the role of P. acnes and its ability to induce matrix metalloproteinases (MMPs) in primary human monocytes and how this induction is regulated by retinoids. MMP-1- and MMP-9-expressing cells were present in perifollicular and dermal inflammatory infiltrates within acne lesions, suggesting their role in acne pathogenesis. In vitro, we found that P. acnes induced MMP-9 and MMP-1 mRNA, and the expression of MMP-9, but not of MMP-1, was found to be Toll-like receptor 2-dependent. P. acnes induced the mRNA expression of tissue inhibitors of metalloproteinase (TIMP)-1, the main regulator of MMP-9 and MMP-1. Treatment of monocytes with all-trans retinoic acid (ATRA) significantly decreased baseline MMP-9 expression. Furthermore, co-treatment of monocytes with ATRA and P. acnes inhibited MMP-9 and MMP-1 induction, while augmenting TIMP-1 expression. These data indicate that P. acnes-induced MMPs and TIMPs may be involved in acne pathogenesis and that retinoic acid modulates MMP and TIMP expression, shifting from a matrix-degradative phenotype to a matrix-preserving phenotype.     

Evidence for diversity within Propionibacterium acnes: a comparison of the T-cell stimulatory activity of isolates from inflammatory acne, endocarditis and the laboratory

BACKGROUND: Propionibacterium acnes is primarily associated with the pathogenesis of acne vulgaris but reports are increasing in number implicating P. acnes in other diseases such as abscess formation, meningitis and endocarditis. The pathogenicity of P. acnes is thought to be partly due to the interaction of the bacterium with the immune system. Historically, investigations have focused on humoral and cell-mediated immune responses to P. acnes antigens without attention to the possibility that different antigens may be expressed by different isolates. OBJECTIVE: Investigations were performed to determine whether there were differences between a laboratory strain of P. acnes (P-37) and fresh clinical isolates in their ability to stimulate naive and adult lymphocytes. MATERIAL AND METHODS: The fresh isolates were collected from a patient with inflammatory acne and a patient with P. acnes-induced prosthetic valve endocarditis. The lymphocyte transformation assay was used to detect responses to whole-cell suspensions of stationary phase P. acnes isolates during 7 days of incubation. RESULTS: The acne isolate was significantly more stimulatory for cord blood mononuclear cells (CBMNCs) than the laboratory isolate (P. acnes P-37) at day 4 of incubation. There were no significant differences between the three strains at any other time points. However, the isolate cultivated from inflammatory acne was significantly more stimulatory for peripheral blood mononuclear cells (PBMNCs) from acne donors than the endocarditis isolate or the laboratory strain at most time points. There were no significant differences between the endocarditis strain and the laboratory strain. CONCLUSION: It can be hypothesized that in case of P. acnes-induced endocarditis lymphocyte stimulation is a disadvantage for the microorganism and therefore a lack of lymphocyte stimulation may be relevant to the pathogenesis of endocarditis.     

Nadifloxacin, an antiacne quinolone antimicrobial, inhibits the production of proinflammatory cytokines by human peripheral blood mononuclear cells and normal human keratinocytes

BACKGROUND: Acne vulgaris is a chronic inflammatory disease involving colonization of Propionibacterium acnes (P. acnes), activation of neutrophils and lymphocytes. Circumstantial evidence suggests that antigen-independent and -dependent immune responses against P. acnes are involved in the pathogenesis of inflammatory acne. Epidermal keratinocytes are also suggested to be involved in initiation and progression of cutaneous inflammation. Nadifloxacin, a fluorinated quinolone, has potent antimicrobial activities against Gram-negative and -positive microbes and is used to treat multiple inflamed acne lesions. However, its effect on immune conferring cells such as mononuclear cells and keratinocytes has not been examined. OBJECTIVE: To evaluate the possible involvement of potential anti-inflammatory activity of nadifloxacin in its therapeutic effect on inflammatory acne, we examined the effects of nadifloxacin, in comparison with other antibiotics used to treat acne vulgaris, on cytokine production by human peripheral blood mononuclear cells (PBMC) and keratinocytes. METHODS: Cytokine production by PBMC was determined after treatment with heat-killed P. acnes in the presence or absence of antimicrobials using a real-time PCR and ELISA. Cultured human epidermal keratinocytes were stimulated by IFN-gamma plus IL-1beta and the effects of antimicrobials were examined by using ELISA. RESULTS: Nadifloxacin as well as macrolide antibiotics and clindamycin inhibited IL-12 and IFN-gamma production by PBMC stimulated by heat-killed P. acnes. The drug also inhibited the IL-1alpha, Il-6, IL-8 and GM-CMS production by keratinocytes treated with IFN-gamma plus IL-1beta. CONCLUSIONS: Inhibitory effects of nadifloxacin to activate T cells and keratinocytes may be involved at least in part in the mechanism of its therapeutic effect against inflammatory acne.     

Induction of toll-like receptors by Propionibacterium acnes

BACKGROUND: The bacterium Propionibacterium acnes is involved in the induction and maintenance of the inflammatory phase of acne. Recent studies have found that keratinocytes express toll-like receptors (TLRs) implicated in immediate immunity. No studies have, to date, been carried out on the action of P. acnes upon TLR activation in keratinocytes. OBJECTIVES: Focusing on the inflammatory phase of acne, to clarify the role of P. acnes in immediate immunity by inducing expression of TLR-2 and TLR-4 by keratinocytes. We also studied how the secretion and expression of matrix metalloproteinase (MMP)-9 is induced by P. acnes. METHODS: The work was carried out on two levels: in vivo with the study of the expression of TLR-2 and TLR-4 proteins in biopsies of acne lesions and in vitro on cultured keratinocyte monolayers to study the modulating effects of P. acnes on the expression of TLR-2 and TLR-4 and also on the expression and secretion of MMP-9. RESULTS: Our findings reveal that in vivo TLR-2 and TLR-4 expression is increased in the epidermis of acne lesions. In vitro, an increase in TLR-2 and TLR-4 expression by human keratinocytes occurred in the first hours of incubation with bacterial fractions as well as an increase of the expression and secretion by the keratinocytes of MMP-9, which plays a role in inflammation. CONCLUSIONS: This work demonstrates that P. acnes induces TLR expression and that this mechanism could play an essential role in acne-linked inflammation. These receptors could be involved notably in acute acne.     

Effects of Propionibacterium acnes on various mRNA expression levels in normal human epidermal keratinocytes in vitro

Propionibacterium acnes is one of the most significant pathogenic factors of acne vulgaris. This bacteria relates to acne by various pathways. It has also been reported that P. acnes influences pro-inflammatory cytokine production in keratinocytes in vitro . However, the influence on the differentiation of keratinocytes by P. acnes has not been studied extensively. We analyzed the expression of keratinocyte differentiation-specific markers, keratins, and pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEK) exposed to P. acnes in vitro . All P. acnes strains used in this study increased transglutaminase (TGase), keratin 17 (K17) and interleukin (IL) mRNA expression levels in NHEK, and decreased K1 and K10 expression levels. Some P. acnes strains increased involucrin and K6 mRNA expression levels in NHEK and decreased filaggrin, K6 and K16 expression levels in vitro . This experiment clarified that P. acnes influences the differentiation of NHEK in vitro . As a result, P. acnes influenced the expression of not only pro-inflammatory cytokines but also some keratinocyte differentiation-specific markers and keratins in NHEK. Our results suggest that P. acnes relates to acne pathogenesis by not only the induction of inflammation but also in the differentiation of keratinocytes. Moreover, it was considered that the reaction of NHEK to P. acnes may be different depending on the type of bacteria.     

Toll样受体2与痤疮相关性研究进展

痤疮是一种累及毛囊皮脂腺的慢性炎症性皮肤疾病。尽管痤疮的确切病因及发病机制尚不明确,但炎症反应是其发病的重要因素之一,其中痤疮丙酸杆菌是主要致病菌。痤疮丙酸杆菌可通过激活Toll样受体2(Toll-like receptors 2,TLR2)介导的信号传导通路诱导炎症级联反应,从而形成炎症性痤疮。一些药物可以通过调节TLR2受体的功能与表达,发挥抗炎及治疗痤疮的作用。该文就TLR2在痤疮发病中的作用及与之相关的治疗方法做一综述。     

Polyphenon-60 displays a therapeutic effect on acne by suppression of TLR2 and IL-8 expression via down-regulating the ERK1/2 pathway

Propionibacterium acnes (P. acnes) is a well-known acne-inducing factor which causes inflammatory skin lesions by enhancing cytokine production through toll-like receptor 2 (TLR2). Green tea extract catechin has been documented to possess anti-inflammatory effects. However, little is known about the mechanisms involved or any direct effect of green tea catechin on acne. The present study investigated the therapeutic effects and mechanism of polyphenon-60, also known as green tea catechin compound, on acne in vitro and in vivo. In a clinical study using topical polyphenon-60 treatment, acne patients showed symptomatic improvement with decrease in the number of comedos and pustules. To investigate the mechanism underlying the activity of polyphenon-60 in acne therapy, an in vitro study was performed. We found that polyphenon-60 reduced the levels of P. acnes-enhanced TLR2 and interleukin-8 (IL-8) in THP-1 cells, human monocyte cell line and human primary monocytes. Taken together, these data demonstrate that polyphenon-60 has a therapeutic effect on acne by suppressing inflammation, specifically by inhibiting TLR2 expression and IL-8 secretion via down-regulation of extracellular signal-regulated kinases 1/2 (ERK1/2) pathway and activator protein-1 (AP-1) pathway.     

Antibodies elicited by inactivated propionibacterium acnes-based vaccines exert protective immunity and attenuate the IL-8 production in human sebocytes: relevance to therapy for acne vulgaris

Propionibacterium acnes is a key pathogen involved in the progression of inflammation in acne vulgaris. We examined whether vaccination against P. acnes suppressed P. acnes-induced skin inflammation. Inactivation of P. acnes with heat was employed to create a P. acnes-based vaccine. Intranasal immunization in mice with this inactivated vaccine provoked specific antibodies against P. acnes. Most notably, immunization with inactivated vaccines generated in vivo protective immunity against P. acnes challenge and facilitated the resolution of ear inflammation in mice. In addition, antibodies elicited by inactivated vaccines effectively neutralized the cytotoxicity of P. acnes and attenuated the production of proinflammatory cytokine IL-8 in human sebocyte SZ95 cells. Intranasal immunization using heat-inactivated P. acnes-based vaccines provided a simple modality to develop acne vaccines. These observations highlight the concept that development of vaccines targeting microbial products may represent an alternative strategy to conventional antibiotic therapy.     

Ex vivo demonstration of a synergistic effect of Adapalene and benzoyl peroxide on inflammatory acne lesions

Acne is a chronic inflammatory disease of the pilosebaceous follicle. Thanks to its ability to reduce both comedones and inflammatory lesions, the association of a retinoid and benzoyl peroxide (BPO) is now recommended for the treatment of acne. However, the mechanisms of action of this combined therapy on inflammatory acne lesions are not well understood. In an ex vivo immunohistochemistry study, we investigated the potential synergistic modulator effect of Adapalene associated with BPO on keratinocytes proliferation/differentiation and innate immunity in inflammatory acne lesions. We demonstrated that proliferation (Ki-67), adhesion/differentiation (integrin α(2), α(3) and α(6)) and innate immunity (TLR-2, β-defensin 4, IL-8) markers are overexpressed in inflammatory acne skin compared with uninvolved acne skin. Association of Adapalene and BPO significantly decreased expression of Ki67, α(2) and α(6) integrins, TLR-2, β-defensin 4 and IL-8 in inflammatory acne skin, whereas single treatments with Adapalene or BPO alone were less effective. These results contribute to explain the comedolytic and anti-inflammatory activities of this combined therapy observed in recent clinical trials.     

Lymphocyte transformation in subjects with nodulo-cystic acne

Patients with severe nodulo-cystic acne are known to have elevated serum antibody levels and increased immediate hypersensitivity reactions to Propionibacterium acnes. This organism is the predominant bacterium in normal pilosebaceous follicles of human skin, and can be consistently isolated from pustular lesions in acne. Previously it had been observed that delayed cutaneous hypersensitivity reactions to P. acnes were negative in patients with acne. The present study investigated the proliferative response of lymphocytes from patients with nodulo-cystic acne to phytohaemagglutinin (PHA) and P. acnes antigen stimulation. The response to PHA stimulation was within normal limits. The response to P. acnes antigen showed a significant increase over control values obtained by testing lymphocytes from acne-free subjects. Thus cell mediated immunity to P. acnes may be present in subjects with severe inflammatory acne. These findings raise the possibility that reactions to P. acnes may contribute to intensifying the inflammatory response in acne lesions.     

In vitro modulation of TLR-2, CD1d and IL-10 by adapalene on normal human skin and acne inflammatory lesions

The anti-inflammatory mechanisms of adapalene, a synthetic retinoid used for the treatment of acne patients, are partially understood. They seem particularly related to the modulation of the non-specific immunity. Recent studies have shown that Toll-like receptor (TLR)-2 expression, a receptor of the innate immune system, was increased in acne lesions and could play an essential role in acne-linked inflammation. The aim of our study was to investigate the new mechanisms of the anti-inflammatory activity of adapalene in vitro, and more specifically the modulatory effect of adapalene on the expression of TLR-2, CD1d, a cell surface glycoprotein that plays a role as antigen-presenting molecules and is responsible for the development of cutaneous inflammation, and also on the expression and the secretion of the anti-inflammatory interleukin (IL)-10 cytokine. Both explants of normal human skin and explants of acne patients were incubated with adapalene (10(-7) or 10(-6) M) or the control medium for 24 h. Evaluation of epidermal expression by immunohistochemistry showed a decreased expression of TLR-2 and IL-10 in explants of normal skin and explants of acne with adapalene. On the contrary, adapalene increased CD1d expression in explants of acne patients. Thus, adapalene can modulate the epidermal immune system by increasing the CD1d expression and by decreasing the IL-10 expression by keratinocytes. Moreover, these modulations could increase the interactions between dendritic cells and T lymphocytes and could strengthen the antimicrobial activity against Propionibacterium acnes. The decreased expression of TLR-2 by the keratinocytes can contribute to explain the anti-inflammatory activity of adapalene observed in clinical practice.     

Propionibacterium acnes stimulates pro-matrix metalloproteinase-2 expression through tumor necrosis factor-alpha in human dermal fibroblasts

Propionibacterium acnes (P. acnes) is a commensal microorganism found in sebum-rich skin and plays a role in acne inflammation by stimulating keratinocyte to produce a number of proinflammatory cytokines. However, the role of P. acnes in the dermis of acne lesions, where tissue remodeling after inflammation eventually takes place, is not known. In this study, we investigated whether P. acnes induces matrix metalloproteinase (MMP), a key enzyme involved in matrix remodeling in human dermal fibroblasts (hDF). We found that P. acnes increased expression of pro-matrix metalloproteinase (proMMP)-2 mRNA/protein in hDF, but not that of proMMP-9. Concomitantly, P. acnes induced tumor necrosis factor-alpha (TNF-alpha) mRNA/protein expression in hDF, which in turn increases both proMMP-2 mRNA and protein expression. P. acnes induced such changes through the activated NF-kappaB pathway. Doxycycline was found to inhibit the expression of proMMP-2 induced either by P. acnes or TNF-alpha. These results suggest that P. acnes stimulates hDF to produce TNF-alpha, which mediates the expression of proMMP-2 through the NF-kappaB pathway. The secretion of proMMP-2 from hDF upon P. acnes stimulation may contribute to the pathogenesis of tissue remodeling in acne skin.     

Anti-inflammatory properties of a new undecyl-rhamnoside (APRC11) against <Emphasis Type="Italic">P. acnes</Emphasis>

Acne vulgaris is a skin disease affecting pilosebaceous glands in which Propionibacterium acnes (P. acnes) induced inflammation plays a central role. In order to develop new therapies against the inflammatory events, we evaluated the modulating effect of a new undecyl-rhamnoside, APRC11, on different markers of the inflammation. For this purpose, normal human keratinocytes taken from five healthy donors were pre-incubated for 24 h with APRC11 or Zinc Gluconate (Zn) which was used as reference molecule for its anti-inflammatory properties. Then, keratinocytes were stimulated with P. acnes Membrane Fraction for 6 h, in the presence of either APRC11 or Zn. Different markers were evaluated at mRNA level using a Luminex-based Quantigene array system and at protein level using an ELISA test and a Luminex array system. Results showed that P. acnes significantly increased the expression of IL-1α, IL-1RA, IL-8 and MMP-9. A 24-h treatment with APRC11 prior to the P. acnes stimulation down-regulated the P. acnes-induced cytokines over expression (IL-1α, IL-8 and MMP-9) and up-regulated IL-1RA level in a similar manner than Zn. These regulations were noted at both protein and mRNA levels. In conclusion, the new undecyl-rhamnoside APRC11 is able to down-regulate the expression of molecules implicated in cutaneous inflammation and whose expression is induced by P. acnes, confirming its potential interest in inflammatory acne.     

Propionibacterium acnes activates the IGF-1/IGF-1R system in the epidermis and induces keratinocyte proliferation

Propionibacterium acnes has a major role in the development of acne lesions. IGF-1 stimulates the proliferation of keratinocytes via an activation of the IGF-1 receptor (IGF-1R). Zinc has been proven to work efficiently against inflammatory acne and to modulate the IGF-1 system. Our objectives were to study the modulation of IGF-1 and IGF-1R expression by P. acnes extracts and to determine their modulation by zinc gluconate. In vivo, we analyzed biopsies of acne lesions and healthy skin, and in vitro we used skin explants incubated with two P. acnes extracts--membrane fraction (MF) and cytosolic proteins--with or without zinc. IGF-1 and IGF-1R expression was evaluated using immunohistochemistry, and the IGF-1 production in supernatants was measured by ELISA. Then, IGF-1 and IGF-1R mRNA levels were analyzed using quantitative PCR on normal human epidermal keratinocytes (NHEKs). IGF-1 and IGF-1R were overexpressed in acne lesions. MF increased IGF-1 and IGF-1R expression in the epidermis of explants and was associated with an overexpression of both Ki-67 and filaggrin. Zinc had the effect of downregulating IGF-1 and IGF-1R levels. These observations were confirmed at the mRNA level for IGF-1R in NHEKs. These results demonstrate that P. acnes can induce the formation of comedones by stimulating the IGF/IGF-1R system. Moreover, zinc downregulates this pathway.     

Different strains of Propionibacterium acnes modulate differently the cutaneous innate immunity

Acne is a chronic inflammatory illness of the pilosebaceous follicle where innate immunity plays a central role. In acne, the density of Propionibacterium acnes is increased in the pilosebaceous unit. We hypothesized that the severity of acne is not only dependent on the proliferation of P. acnes but also dependent on the pro‐inflammatory potential of P. acnes strains and consequently constitutes potential triggering factor for acne scarring. We investigated pro‐inflammatory potential of five different strains of P. acnes and P. avidum in skin explants and the preventive effect of zinc gluconate. The expression of immune markers was studied by immunohistochemistry, RT‐qPCR and ELISA. P. acnes strains modulate differently the expression of immune markers both at gene and at protein levels. P. acnes type III had the highest pro‐inflammatory potential by up‐regulating the expression of PAR‐2, TNF‐alpha, MMP‐13 and TIMP‐2, whereas P. avidum had the weakest by up‐regulating only MMP‐13 and TIMP‐2. Preincubation of zinc gluconate, which is a modulator of innate immunity, down‐regulates the expression of most immune markers induced by P. acnes, PAR‐2, TIMP‐2, up‐regulates MMP‐1, TIMP‐1. Our results demonstrate that different P. acnes strains have different inflammatory potential targeting markers of cutaneous innate immunity, and that inflammatory potential can be down‐regulated by zinc gluconate. As such, the inflammatory potential of P. acnes strains on acne skin may influence the severity of inflammatory acne lesions and scars.