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.     

痤疮患者经异维A酸系统治疗后外周血CD14~+单核细胞TLR2的表达变化

目的:探讨Toll样受体2(Toll like receptor 2,TLR2)在寻常痤疮发病中的作用及异维A酸治疗痤疮的部分抗炎机制。方法:应用流式细胞术检测35例痤疮患者经异维A酸治疗前后外周血CD14+单核细胞TLR2的表达,并与对照组25例健康人进行比较。结果:未经治疗的痤疮患者外周血CD14+单核细胞TLR2表达明显高于对照组(t=10.63,P<0.01),治疗后TLR2的表达与对照组无明显差异(t=1.73,P>0.05)。结论:TLR2的表达与痤疮的发病相关;异维A酸治疗后可明显减少外周血CD14+单核细胞TLR2的表达,从而抑制炎症反应的发生。     

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.     

Clinical and microbiological comparisons of isotretinoin vs. tetracycline in acne vulgaris

The aim of this study was to compare the clinical and microbiological effect on Propionibacterium acnes of oral tetracycline plus topical adapalene vs. oral isotretinoin in moderate to severe acne vulgaris. Male and female acne patients with moderate or severe inflammatory disease were enrolled and assigned randomly to 6 months of treatment with oral tetracycline hydrochloride plus topical adapalene, or oral isotretinoin, in a controlled, open study. After cessation of oral treatment the antibiotic-treated group received topical adapalene for the 2-month follow-up period. Clinical and microbiological assessments were performed. Skin samples for microbial identification and quantification were taken at baseline, after 2, 4 and 6 months of treatment, and 2 months after cessation of treatment. Patients treated with isotretinoin showed prolonged significant remission compared with the other group. The density of resistant propionibacteria did not change significantly in any of the groups and there was no correlation between resistant P. acnes and the clinical response in any of the regions investigated. Antibiotic treatment was found to be a good alternative to isotretinoin, regardless of the presence of antibiotic-resistant P. acnes, although isotretinoin had a better effect, with prolonged remission after treatment.     

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.     

Topical aminolaevulinic acid-photodynamic therapy for the treatment of acne vulgaris: a study of clinical efficacy and mechanism of action

BACKGROUND: Acne affects 83-95% of 16-year-olds of both sexes, and many seek help from a clinician. Emerging problems with conventional acne treatments, specifically antibiotic resistance of Propionibacterium acnes and fears over the safety and tolerance of oral isotretinoin, create a demand for novel treatment modalities in acne. OBJECTIVES: To study the efficacy of aminolaevulinic acid-photodynamic therapy (ALA-PDT) in the treatment of acne and to identify the mode of action, looking specifically at the effects on surface numbers of P. acnes and on sebum excretion. METHODS: Ten patients (nine men and one woman, age range 16-40 years) with mild to moderate acne on their backs were recruited. Each patient's back was marked with four 30-cm2 areas of equal acne severity. Each site was then randomly allocated to either ALA-PDT treatment, light alone, ALA alone or an untreated control site. At baseline, numbers of inflammatory and noninflammatory acne lesions were counted, sebum excretion measured by Sebutapes (CuDerm, Dallas, TX, U.S.A.) and surface P. acnes swabs performed. ALA cream (20% in Unguentum Merck) was applied under occlusion to the ALA-PDT and ALA alone sites for 3 h. Red light from a diode laser was then delivered to the ALA-PDT and light alone sites (635 nm, 25 mW cm(-2), 15 J cm(-2)). Each patient was treated weekly for 3 weeks. At each visit acne lesion counts were performed and 3 weeks following the last treatment sebum excretion rates and P. acnes swabs were repeated. RESULTS: There was a statistically significant reduction in inflammatory acne lesion counts from baseline after the second treatment at the ALA-PDT site but not at any of the other sites. No statistically significant reduction in P. acnes numbers or sebum excretion was demonstrated at any sites including the ALA-PDT site. CONCLUSIONS: ALA-PDT is capable of clinically improving acne. An alternative mode of action for ALA-PDT other than direct damage to sebaceous glands or photodynamic killing of P. acnes is suggested from the results of this study.     

In vivo porphyrin production by P. acnes in untreated acne patients and its modulation by acne treatment

Propionibacterium acnes is often discussed as a contributing pathogenic factor in the aetiology of acne lesions. The aim of this study was to test which porphyrin patterns are synthesized by P. acnes in vivo in untreated acne patients and during standard acne regimens. These photosensitive compounds are potential targets for photo-dynamic therapy of acne and need to be better characterized in the skin. Using high-performance liquid chromatography coproporphyrin III was the main porphyrin identified in all patients. Coproporphyrin I and protoporphyrin were found at considerably lower concentrations. When the porphyrin concentration of individual patients receiving isotretinoin was analysed repeatedly over time, clinical improvement was associated with lowered levels of porphyrins. Statistical analysis demonstrated a significant reduction in the porphyrin fractions only in the isotretinoin group which was associated with clinical improvement 2 months after starting therapy.     

痤疮患者经异维A酸系统治疗后外周血CDl4＋单核细胞TLR2的表达变化

目的：探讨Toll样受体2（Tolllikereceptor2,TLR2）在寻常痤疮发病中的作用及异维A酸治疗痤疮的部分抗炎机制。方法：应用流式细胞术检测35例痤疮患者经异维A酸治疗前后外周血CDl4＋单核细胞TLR2的表达,并与对照组25例健康人进行比较。结果：未经治疗的痤疮患者外周血CDl4＋单核细胞TLR2表达明显高于对照组（t=10．63,P〈0．01）,治疗后TLR2的表达与对照组无明显差异（t=1．73,P〉0．05）。结论：TLR2的表达与痤疮的发病相关;异维A酸治疗后可明显减少外周血CDl4’单核细胞TLR2的表达,从而抑制炎症反应的发生。     

Effects of topical erythromycin on aerobic and anaerobic surface flora

The contents of acne lesions from 30 men with inflammatory acne vulgaris, obtained before and at the conclusion of 4 weeks of topical 2% erythromycin therapy, were cultured on aerobic and anaerobic plates. Sensitivity testing of cultures was performed with discs containing 50 mg erythromycin, in order to ascertain the prevalence of erythromycin-resistant strains of Propionibacterium acnes and aerobic micrococci. The percentages of patients in whom erythromycin-resistant micrococci could be isolated increased from 3 to 60% during topical erythromycin therapy, but no resistant P. acnes strains were isolated either before or after therapy. Considering the widespread use of topical antibiotics, long-term studies on their ecological effects are desirable.     

Resolution of inflammatory acne vulgaris may involve regulation of CD4+ T-cell responses to Propionibacterium acnes

BACKGROUND: Propionibacterium acnes has been strongly implicated in inflammatory acne. However, its role in the disease is unclear. It has been hypothesized that an immune response to P. acnes and/or P. acnes heat shock proteins (HSPs) may play a role in the pathogenesis of inflammatory acne. OBJECTIVES: To compare the cell-mediated immune response to P. acnes and HSPs in acne patients, nonacne controls and individuals with resolved acne. METHODS: The proliferative response of peripheral blood mononuclear cells (PBMC) from acne patients, resolved acne donors and healthy controls to P. acnes, P. acnes HSP60 and HSP70, and mycobacterial HSPs was assessed by lymphocyte transformation assay (LTA). The proliferative response of purified CD4+ T cells was further analysed by limiting dilution analysis (LDA). Contingency tables (G-test) were used to analyse the proportion of individuals in each group showing a positive proliferative response for LTA or data fitting single-hit kinetics for LDA. RESULTS: Analysis of stimulation of PBMC with P. acnes, P. acnes HSP60 and HSP70 in the LTA showed the proportion of positive responders to be independent of subject group. However, the proportion of acne patients with a positive response to mycobacterial HSPs was significantly higher than those for the other subject groups. Analysis of LDA data showed the proportion of resolved donors with responses to P. acnes fitting the single-hit kinetics model to be significantly lower than those of the other groups. There were no significant differences in responses to other antigens. CONCLUSIONS: The significantly lower proportion of resolved donors demonstrating a single-hit kinetics response to P. acnes by LDA may represent negative regulation of the CD4+ T-cell response to P. acnes in these subjects.     

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.     

Distinct strains of Propionibacterium acnes induce selective human beta-defensin-2 and interleukin-8 expression in human keratinocytes through toll-like receptors

Acne is a chronic inflammatory disease of the pilosebaceous follicle. One of the main pathogenetic factors in acne is the increased proliferation of Propionibacterium acnes (P. acnes) in the pilosebaceous unit. We investigated whether direct interaction of P. acnes with keratinocytes might be involved in the inflammation and ductal hypercornification in acne. The capacities of different P. acnes strains to activate the innate immune response and the growth of cultured keratinocytes were investigated. We have found that two clinical isolates of P. acnes significantly induced human beta-defensin-2 (hBD2) messenger RNA (mRNA) expression; in contrast a third clinical isolate and the reference strain (ATCC11828) had no effect on hBD2 mRNA expression. In contrast, all four strains significantly induced the interleukin-8 (IL-8) mRNA expression. The P. acnes-induced increase in hBD2 and IL-8 gene expression could be inhibited by anti-Toll-like receptor 2 (TLR2) and anti-TLR4 neutralizing antibodies, suggesting that P. acnes-induced secretion of soluble factors in keratinocytes are both TLR2 and TLR4 dependent. In addition, the clinical isolate P. acnes (889) was capable of inducing keratinocyte cell growth in vitro. Our findings suggest that P. acnes modulates the antimicrobial peptide and chemokine expression of keratinocytes and thereby contributes to the recruitment of inflammatory cells to the sites of infections.     

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.     

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.     

Propionibacterium acnes resistance to antibiotics in acne patients

The minimal inhibitory concentration (MIC) of Propionibacterium acnes in seventy-five acne patients receiving long-term antibiotic therapy demonstrated the emergence of resistant strains. The mean MIC in thirty-three patients receiving long-term tetracycline was four to five times higher than that found in control groups of acne patients not receiving antibiotic therapy and controls free of acne. The average MIC for erythromycin was more than 100 times higher in those receiving long-term antibiotic therapy. In a second group of sixty-two patients, the clinical course and number of P. acnes were correlated with the presence of "resistant strains" defined as P. acnes with a tenfold increase in MIC to tetracycline or erythromycin. Patients with resistant strains had higher counts of P. acnes and clinically were not doing as well as those with sensitive strains.     

异维A酸红霉素凝胶治疗寻常痤疮的多中心随机对照临床研究

目的 比较异维A酸红霉素凝胶与阿达帕林凝胶治疗轻中度寻常痤疮的疗效和安全性。方法 多中心、开放、随机、平行对照研究。按照中国痤疮治疗指南痤疮严重程度标准入选轻、中度（Ⅰ ~ Ⅲ级）寻常痤疮患者192例,纳入疗效分析169例,安全性分析190例。试验组86例,外用异维A酸红霉素凝胶;对照组83例,外用阿达帕林凝胶,均为每晚1次,共用药6周。在基线、治疗2、4和6周时分别记录患者白头粉刺、黑头粉刺、炎性丘疹、脓疱数,评价各时间点患者痤疮严重程度,同时记录皮肤局部耐受性以及其他不良反应。结果 随着治疗时间延长,两组的有效率逐渐提高,总体病情严重程度改善。在治疗6周时,试验组总有效率为51.16%,对照组为40.96%,两组差异无统计学意义（P > 0.05）;但在治疗4周及6周时,试验组患者脓疱及炎性皮损的疗效优于对照组（P < 0.05）,同时试验组痤疮分级较对照组低（P < 0.01）。两组不良反应类似,均表现为局部刺激,可耐受。结论 异维A酸红霉素凝胶治疗轻、中度寻常痤疮总有效率与阿达帕林凝胶类似,对于炎性损害疗效优于阿达帕林凝胶,并且对患者总体严重程度降低更快。     

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.     

Delayed skin test reactivity to Propionibacterium acnes correlates with severity of inflammation in acne vulgaris

Propiontibacterium acnes is the bacterial species most consistently isolated from acne lesions. Intradermal injection of a heat-killed suspension of P. acnes induced a delayed erythematous and often papular inflammatory reaction which was maximal after 24–48 h. This response was dose related and was probably mediated at least partly by immune mechanisms. In eighty-one subjects with acne of varying severity the intensity of the 48 h skin test reaction was significantly related to the severity of the acne. These findings indicate that the host response to P. acnes is an important variable in determining the severity of inflammatory acne.     

The use and long-term benefit of isotretinoin

Isotretinoin is effective in the treatment of acne, but at low doses (0·1 mg/kg) many patients relapse. Over the past 5 years we have treated 250 patients with either 0·5 mg/kg or 1·0 mg/kg of isotretinoin and have followed them up for up to 3 years to determine the relapse rate. All patients in each dose group responded well, with a 90% improvement in acne grade. The majority (86%) needed only 4 months'treatment, but 13% needed 6 months, and two patients required 9 months of continuous therapy. Patients with marked mucous membrane side-effects tended to respond better than those without such side-effects.
The relapse rate in the 0·5 mg/kg group was significantly greater (42%) than in the 1·0 mg/kg group (13%) (P < 0·01); relapse was defined as the need for further oral therapy—either antibiotics or isotretinoin. Truncal acne was more likely to relapse than facial acne (P < 0·05). Younger patients relapsed more quickly than older patients (P < 0·05). Patients treated with isotretinoin fell into one of five categories: (i) severe acne with isotretinoin as first-line therapy (18%); (2) failure to improve on conventional antibiotic therapy (55%); (3) rapid relapse after two courses of conventional antibiotic therapy (21%); (4) Gram-negative folliculitis (3%); and (5) severe psychological disturbance about minor degrees of acne that normally would not require isotretinoin (3%).
We thus recommend 1·0 mg/kg as the best dose of isotretinoin for long-term benefit in acne, and that isotretinoin should now be considered as part of the treatment for less severe types of acne.