Propionibacterium acnes and inflammation in acne; P. acnes has T-cell mitogenic activity

BACKGROUND: Circumstantial evidence suggests that Propionibacterium acnes has a role in the inflammation of acne. This could be effected by antigenic or superantigenic or mitogenic reactions. OBJECTIVES: The purpose of this investigation was to determine whether P. acnes had only antigenic activity or additional superantigenic and mitogenic activity. METHODS: A lymphocyte transformation assay was used to detect responses to a mixture of eight P. acnes whole cell isolates, and their supernatant culture fluids. In order to determine the nature of T-cell reactions to P. acnes cells a mouse-antihuman major histocompatibility complex class II monoclonal antibody was used in the lymphocyte transformation assay to inhibit the antigenic stimulation of lymphocytes. An analysis of the T-cell receptor (TCR) variable region beta (BV) repertoire was undertaken using flow cytometry of the unstimulated and stimulated cells. RESULTS: Peripheral blood mononuclear cells (PBMNC) from adults with no history of acne responded strongly to stationary growth phase cells of P. acnes, less strongly to cells in the exponential growth phase. No response was detected to supernatant culture fluids. PBMNC from five cord blood samples (CBMNC) responded maximally after 3 and 7 days of incubation with stationary growth phase cells of P. acnes. The reaction of CBMNC to P. acnes cells was not suppressed completely by the blocking antibody. The analysis of the TCRBV repertoire indicated that P. acnes induced no deletion or over-representation of certain BV element-bearing T cells. The TCRBV analysis was repeated after preincubation with the blocking antibody. Deletion of T cells bearing certain BV components occurred and there was no over-representation of T cells carrying certain BV components. CONCLUSIONS: Two mechanisms of lymphocyte activation by P. acnes cells are proposed, antigen and mitogen driven. These results are consistent with the histological evidence of inflammation in acne lesions.     

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.     

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.     

Cyclosporin increases granulocyte/macrophage colony-stimulating factor (GM-CSF) activity and gene expression in murine keratinocytes.

Keratinocytes produce multiple cytokines in response to a variety of stimuli. The release of interleukin 1 (IL-1) from keratinocytes may be significant in initiation of cutaneous inflammation, and the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) is thought to be important in the regulation of antigen-presenting function by epidermal Langerhans cells. Because cyclosporin inhibits interleukin 2 release from T cells, it has been suggested that cyclosporin may function as an anti-inflammatory agent within the epidermis through inhibition of keratinocyte cytokine release. This investigation examined the direct effect of cyclosporin on the production of GM-CSF by murine keratinocytes and the keratinocyte cell line PAM 212. GM-CSF bioactivity increased in cell supernatants from keratinocytes exposed in vitro to 1 microgram/ml cyclosporin for up to 24 h. GM-CSF and IL-1 mRNA levels in keratinocytes cultured under similar conditions or in the presence of lipopolysaccharide also increased. The lack of inhibition of GM-CSF expression following cyclosporin treatment is consistent with recent observations in T cells and is opposite to the effect of cyclosporin on interleukin 2.     

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.     

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.     

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.     

Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin

Using the plasmid DNA injection method, we introduced cytokine genes into skin to determine whether systemic expression of cytokine genes is possible. Eight human cytokine [interleukin-4 (IL-4), IL-6, IL-10, transforming growth factor beta1 (TGF-beta1), monocyte chemotactic and activating factor (MCAF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)] gene expression vectors were constructed and injected into rat skin. Transgenic cytokines in local keratinocytes and in the sera were assayed with ELISA. Our results showed that transgenic cytokines were markedly increased in keratinocytes at the injection site. The serum concentrations of IL-4, 6, 10 and TauGF-beta1 reached levels high enough to have systemic biologic effects. However, other cytokines used in this study could not be detected in the sera. Moreover, the serum transgenic IL-10 level after subcutaneous injection was significantly higher than after intramuscular injection. We suggest that keratinocytes can be used as a bioreactor to achieve systemic expression of cytokine genes by DNA injection, but the transgenic protein level in circulation depends on different kinds of cytokine. This level also depends on different target cells used for gene transfer.     

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.     

Polymorphonuclear leukocyte lysosomal release in response to Propionibacterium acnes in vitro and its enhancement by sera from inflammatory acne patients

Propionibacterium acnes cells were tested for the ability to trigger lysosomal hydrolase release from human polymorphonuclear leukocytes. Representative strains of P. acnes serotype I and II failed to stimulate lysosomal release in the absence of serum. P. acnes growth culture supernatants failed to trigger release under any test condition. Addition of fresh or heat-inactivated human serum resulted in lysosomal hydrolase release directly proportional to the number of P. acnes/PMN. Pooled sera from acne patients, with a high anti-P. acnes titer stimulated release to P. acnes. Preabsorption of this reagent with P. acnes cells reduced the anti-P. acnes titer and produced 93.37 +/- 11.49% inhibition of lysosomal enzyme release compared to unabsorbed anti-serum. Electron microscopy indicated that P. acnes was readily phagocytosed by PMNs when fresh or heated serum was present.     

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.     

The evolving role of Propionibacterium acnes in acne

Propionibacterium acnes is a member of the resident cutaneous flora. Sebaceous follicles involved in acne are characterized by the accumulation of abnormally desquamated corneocytes and excess sebum-the microcomedo. This environment provides ideal growth conditions for P acnes. Several orders of magnitude level of P acnes are found in microcomedos. P acnes produces a variety of chemotactic factors and proinflammatory molecules that are responsible for the inflammatory phase of acne. Antibiotic therapy works by reducing the viable number of P acnes as well as by reducing the production of inflammatory stimuli. Antibiotic therapy has been a mainstay of treatment for more than 30 years. In the last decade, decreased sensitivity to antibiotics has developed and clinical resistance has been described. This development threatens the usefulness of antibiotic therapy in the future.     

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.     

痤疮丙酸杆菌的新命名及其与寻常痤疮的相关性

【摘要】 近来,有研究者建议将痤疮丙酸杆菌命名为Cutibacterium acnes（C. acnes）,以更好研究其分型并与其他丙酸杆菌区分。C. acnes是人类皮肤重要的定植菌,参与维持皮肤健康,但也可能转变成为寻常痤疮的机会性病原菌。最新的研究显示,C. acnes不同型别间的平衡和与其他微生物的相互作用在寻常痤疮发生发展中发挥关键作用。本文综述C. acnes分型及其与寻常痤疮的相关性、药物敏感性和与其他微生物的相互作用。     

Control of the differentiation state and function of human epidermal Langerhans cells by cytokines in vitro

Langerhans cells can originate in vitro from immature precursors stimulated with granulocyte macrophage-colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF)-alpha and stem cell factor (SCF). We asked whether these cytokines also control the differentiation state of Langerhans cells within the epidermis and upon leaving this tissue. We harvested sheets of human epidermis by controlled dispase hydrolysis of keratomes, cultured them in RPMI and 10% fetal calf serum for 48 h and analysed the sheets and the cells migrated spontaneously into the medium, most of which were Langerhans cells containing Birbeck granules. By flow cytometry, the intensity of CD1a expression was reduced quite evenly among Langerhans cells migrated from sheets within 48 h. The cells in the sheets underwent loss of dendrites, with a significant reduction in the cell perimeter that was prevented by GM-CSF and TNF-alpha together. Either of these cytokines induced expression of CD18 by cells in the sheets and those in the medium. Moreover, TNF-alpha induced expression of CD54 by cells in the medium, but not by those retained in the sheets, whereas human SCF induced, dose dependently, expression of CD54 by cells in the sheets, but not from those in the medium. The proliferation of allogeneic lymphocytes was much higher when stimulating Langerhans cells were harvested from cultures with any cytokine, rather than from cultures without cytokines. We conclude the following: (i) GM-CSF and TNF-alpha help to maintain full differentiation of Langerhans cells within the epidermis; (ii) cytokine influence on Langerhans cells adhesiveness is in part context dependent; and (iii) pretreatment with cytokines influences positively the number or accessory activity of Langerhans cells on lymphocytes during subsequent mixed leucocyte reaction.     

Heterogeneity of cytokine production by human malignant melanoma cells

Recent investigations indicate that malignant melanoma cells can produce distinct cytokines. While differences in the production of single cytokines have been observed among different melanoma cell lines, the extent of variability in the production of single and multiple cytokines between individual melanoma cell lines has not been as thoroughly investigated. A heterogeneity in melanoma cell cytokine production could have important implications for the biology of this aggressive neoplasm since certain cytokines may act as autocrine growth factors or be potent modulators of host immune response to the developing tumor. The purpose of this study is to assess the cytokine production profile of two widely available human melanoma cell lines, A375 and G361. The A375 cell line constitutively expressed the mRNA for IL-1 alpha, IL-1 beta and PDGF-A, with increased expression of these cytokines after induction with PMA. GM-CSF mRNA was expressed by the A375 melanoma line only after induction with PMA. No IL-6 mRNA was detected in the A375 melanoma cell line. The cell culture supernatants from the A375 cells likewise contained a parallel increase in IL-1 activity as determined in the D10 bioassay and secreted GM-CSF and PDGF-AA as measured by ELISA. In contrast, the G361 cell line did not express IL-1, GM-CSF or PDGF-A mRNA (constitutively or after PMA induction) but expressed only IL-6 mRNA and secreted IL-6 activity after PMA induction. These results demonstrate a significant heterogeneity in the production of IL-1 alpha, IL-1 beta, IL-6, GM-CSF, and PDGF in two distinct melanoma cell lines. This study demonstrates that individual melanoma cell lines express and secrete multiple cytokines both constitutively and after stimulation with PMA. The immunodulating and mitogenic properties of these melanoma-derived cytokines may have implications in determining the biologic behavior of different malignant melanomas.     

The pathogenesis of acne

Acne vulgaris is a multifactorial disease of as yet incompletely elucidated etiology and pathogenesis. The following have been identified as the most significant factors: follicular hyperkeratosis, increased sebum secretion, Propionibacterium (P.) acnes, and inflammation. Increased sebum production and follicular hyperkeratosis result in the development of microcomedones, and changes in follicular milieu in intensive growth of P. acnes. P. acnes secretes several proinflammatory products, which play an important role in the development of inflammation. These include lipases, proteases, hyaluronidases, and chemotactic factors. Immune response to P. acnes includes humoral and cell-mediated immunity as well as complement activation. Recent results indicate that keratinocytes and sebocytes, as major components of pilosebaceous unit, may act as immune cells and may be activated by P. acnes via toll-like receptors (TLRs) and CD14, and through CD1 molecules may recognize altered lipid content in sebum, followed by the production of inflammatory cytokines.     

The production of inflammatory compounds by Propionibacterium acnes and other skin organisms

Culture supernatants from four species of skin microorganisms (P. acnes, P. avidum, P. granulosum and S. epidermidis) were assayed for smooth muscle contracting substances which are indicative of inflammatory activity. At least three types of smooth muscle contracting substances were detected. These were: first, a substance active on a rat fundic strip preparation and antagonized by N,N-DMT. Activity was enhanced when cultures were grown in the presence of tryptophan. This substance was probably tryptamine. Second, a substance active on a guinea-pig ileum preparation and antagonized by mepyramine. Activity was enhanced when cultures were grown in the presence of histidine. This substance was probably histamine. Third, a substance active on a rat fundic strip preparation but not antagonized by N,N-DMT, mepyramine, atropine or indomethacin. Activity was enhanced when cultures were grown in the presence of glucose. This activity was probably due to acetate, propionate or other short-chain fatty-acid salts. Chromatographic analysis confirmed the presence of histamine, tryptamine and short-chain fatty acids in the culture supernatants. These substances if produced in vivo may cause or contribute to inflammation and pain directly without the prior mediation of the immune system. Cell extracts of Propionibacterium species were analysed by bioassay for the presence of prostaglandin-like compounds. These could not be detected in any of the eight strains of organisms tested.