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.     

Systemic Isotretinoin Therapy Normalizes Exaggerated TLR-2-Mediated Innate Immune Responses in Acne Patients

Retinoids are used in the treatment of inflammatory skin diseases and malignancies, but studies characterizing the in vivo actions of these drugs in humans are lacking. Isotretinoin is a pro-drug for all-trans retinoic acid, which can induce long-term remissions of acne; however, its complete mechanism of action is unknown. We hypothesized that isotretinoin induces remission of acne by normalizing the innate immune response to the commensal bacterium Propionibacterium acnes. Compared with normal subjects, peripheral blood monocytes from acne patients expressed significantly higher levels of Toll-like receptor 2 (TLR-2) and exhibited significantly greater induction of TLR-2 expression following P. acnes stimulation. Treatment of patients with isotretinoin significantly decreased monocyte TLR-2 expression and subsequent inflammatory cytokine response to P. acnes after 1 week of therapy. This effect was sustained 6 months following cessation of therapy, indicating that TLR-2 modulation may be involved in the durable therapeutic response to isotretinoin. This study demonstrates that isotretinoin exerts immunomodulatory effects in patients and sheds light on a potential mechanism for its long-term effects on acne. The modulation of TLR-2 expression on monocytes has important implications in other inflammatory disorders characterized by TLR-2 dysregulation.     

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.     

Matrix metalloproteinases of epithelial origin in facial sebum of patients with acne and their regulation by isotretinoin

Acne vulgaris is a skin disorder of the sebaceous follicles, involving hyperkeratinization and perifollicular inflammation. Matrix metalloproteinases (MMP) have a predominant role in inflammatory matrix remodeling and hyperproliferative skin disorders. We investigated the expression of MMP and tissue inhibitors of MMP (TIMP) in facial sebum specimens from acne patients, before and after treatment with isotretinoin. Gelatin zymography and Western-blot analysis revealed that sebum contains proMMP-9, which was decreased following per os or topical treatment with isotretinoin and in parallel to the clinical improvement of acne. Sebum also contains MMP-1, MMP-13, TIMP-1, and TIMP-2, as assessed by ELISA and western blot, but only MMP-13 was decreased following treatment with isotretinoin. The origin of MMP and TIMP in sebum is attributed to keratinocytes and sebocytes, since we found that HaCaT keratinocytes in culture secrete proMMP-2, proMMP-9, MMP-1, MMP-13, TIMP-1, and TIMP-2. SZ95 sebocytes in culture secreted proMMP-2 and proMMP-9, which was also confirmed by microarray analysis. Isotretinoin inhibited the arachidonic acid-induced secretion and mRNA expression of proMMP-2 and -9 in both cell types and of MMP-13 in HaCaT keratinocytes. These data indicate that MMP and TIMP of epithelial origin may be involved in acne pathogenesis, and that isotretinoin-induced reduction in MMP-9 and -13 may contribute to the therapeutic effects of the agent in acne.     

Cutis laxa: analysis of metalloproteinases and extracellular matrix expression by immunohistochemistry and histochemistry

Upregulation of matrix metalloproteinases (MMPs) and downregulation of tissue inhibitors of metalloproteinases (TIMP) have been reported in cultured fibroblasts from patients with congenital cutis laxa (CL) or anetoderma. We determined the protein expressions of MMP-1, MMP-2, MMP-3, MMP-9, MMP-12, TIMP-1 and collagen I, collagen III in vivo, to confirm their roles in the pathogenesis of cutis laxa. The protein expression of the MMPs and collagens from skin lesions of CL were detected by immunohistochemistry and analyzed by image analysis software. Markedly increased MMP-1, MMP-2, MMP-3, MMP-9, MMP-12, TIMP-1 associated with alteration of elastic and collagen fibers were found in two cases of CL, whereas increased MMP-3, MMP-9, MMP-12 accompanying a degradation of elastic fibers were detected in the third case. These results suggest an elevated expression of MMPs may play a role in the evolution or genesis of CL.     

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.     

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.     

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.     

The P450 system and mTORC1 signalling in acne

In this issue, Hellmann‐Regen et al. suggested that anti‐acne effects of erythromycin and tetracyclines may be related to their inhibitory effect of cytochrome P450‐mediated degradation of all‐trans‐retinoic acid (ATRA). We have recently proposed that all anti‐acne agents function by attenuation of increased mTORC1 signalling. This commentary links the P450 system to mTORC1 regulation in acne. Drug‐mediated induction of P450 activity or P450 mutants with increased catabolic activity may reduce cellular ATRA levels and FoxO1 expression, thus reducing FoxO‐mediated mTORC1 inhibition. In contrast, agents blocking ATRA degradation such as erythromycin and tetracyclines may improve acne by increasing FoxO1 expression with consecutive inhibition of mTORC1 signalling.     

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.     

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.     

外周血单一核细胞的体外活化对成纤维细胞增殖和基质金属蛋白酶产生的影响

目的 研究不同活化剂对外周血单一核细胞（PBMC）产生基质金属蛋白酶（MMP）的影响以及PBMC活化后培养上清液［称为条件培养基（CM）］对培养的皮肤成纤维细胞增殖活性、产生MMP的影响。方法 抽取、分离健康人PBMC,分为植物血凝素（PHA）组、双抗体组、对照组,分别用活化剂PHA、CD3和CD28双抗体、含10%胎牛血清的RPMI 1640培养基处理细胞,72 h后收集三组CM,并将获得的CM按不同比例稀释后作用于培养的成纤维细胞,以不加CM作为对照组,培养48或24 h。采用噻唑蓝法检测细胞增殖能力,半定量反转录（RT）-PCR法检测细胞MMP-1、MMP-3、MMP-9 mRNA表达水平,酶联免疫吸附实验（ELISA）检测上清液中白介素（IL）-6、MMP-1、MMP-3、MMP-9蛋白含量。采用单因素方差分析、Tukey HSD检验、Games-Howell检验等方法进行统计学分析。 结果 与对照组相比,PHA组PBMC增殖活性及分泌IL-6、MMP-3水平明显增加,差异均有统计学意义（P < 0.05）;3组活化后上清液中均未检测到MMP-1、MMP-9蛋白。对照组、双抗体组、PHA组PBMC中MMP-1 mRNA相对表达水平（以靶基因与β肌动蛋白mRNA之比表示）0.083 ± 0.016、0.188 ± 0.030、0.714 ± 0.104,差异有统计学意义（P < 0.05）,MMP-3、MMP-9 mRNA均无表达。不同稀释度CM刺激成纤维细胞后上清液中可检测到MMP-3蛋白,MMP-3含量以原液CM中最高,1/10 CM刺激时最低;在相同的稀释度刺激时,上清液中MMP-3含量以PHA刺激组最高,对照刺激组最低;不同CM刺激成纤维细胞后上清液中均未检测到MMP-1、MMP-9蛋白。不同处理组成纤维细胞的增殖能力及MMP-1、MMP-3 mRNA表达差异均无统计学意义（P > 0.05）,MMP-9 mRNA无表达。 结论 PBMC活化后可表达MMP-1 mRNA并分泌MMP-3蛋白,PBMC活化后上清液不能刺激成纤维细胞MMP-1、MMP-3、MMP-9 mRNA及蛋白表达,提示炎症细胞可能通过自身产生MMP而发挥作用。     

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.     

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.     

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.     

Gene array expression profiling in acne lesions reveals marked upregulation of genes involved in inflammation and matrix remodeling

The pathogenesis of acne has been linked to multiple factors such as increased sebum production, inflammation, follicular hyperkeratinization, and the action of Propionibacterium acnes within the follicle. In an attempt to understand the specific genes involved in inflammatory acne, we performed gene expression profiling in acne patients. Skin biopsies were obtained from an inflammatory papule and from normal skin in six patients with acne. Biopsies were also taken from normal skin of six subjects without acne. Gene array expression profiling was conducted using Affymetrix HG-U133A 2.0 arrays comparing lesional to nonlesional skin in acne patients and comparing nonlesional skin from acne patients to skin from normal subjects. Within the acne patients, 211 genes are upregulated in lesional skin compared to nonlesional skin. A significant proportion of these genes are involved in pathways that regulate inflammation and extracellular matrix remodeling, and they include matrix metalloproteinases 1 and 3, IL-8, human beta-defensin 4, and granzyme B. These data indicate a prominent role of matrix metalloproteinases, inflammatory cytokines, and antimicrobial peptides in acne lesions. These studies are the first describing the comprehensive changes in gene expression in inflammatory acne lesions and are valuable in identifying potential therapeutic targets in inflammatory acne.     

皮肤恶性黑素瘤中基质金属蛋白酶-9及其组织抑制物-1的表达及意义

目的:探讨基质金属蛋白酶-9(MMP-9)及其组织抑制物-1(TIMP-1)在皮肤恶性黑素瘤中的表达及其临床、病理学意义。方法:采用免疫组织化学S-P方法观察MMP-9和TIMP-1在51例皮肤黑素瘤及15例色素痣石蜡切片中的表达,使用图像分析技术对免疫组化结果进行定量分析。结果:与色素痣比较,皮肤恶性黑素瘤中MMP-9表达较强(P<0.05),MMP-9/TIMP-1比值较高(P<0.05)。MMP-9和TIMP-1的表达及MMP-9/TIMP-1比值与黑素瘤浸润深度有关(P<0.05)。有淋巴结转移者MMP-9表达较强(P<0.05),MMP-9/TIMP-1比值较高(P<0.05)。结论:MMP-9、TIMP-1蛋白的表达与皮肤恶性黑素瘤的侵袭性有关,MMP-9/TIMP-1比值增高提示肿瘤具有较高侵袭转移能力。     

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。