Introduction
Acne vulgaris is a chronic inflammatory disorder of pilosebaceous unit characterized by increased sebum production, altered keratinization,
Propionibacterium acnes (
P. acnes) colonization, and inflammation [
35].
Propionibacterium acnes, which is a facultative anaerobic gram-positive bacterium, is believed to play a critical role in the induction and maintenance of inflammation in acne by activating inflammatory cells, keratinocytes, and sebocytes to induce various inflammatory mediators [
3,
23]. It is now accepted that
P. acnes contributes to inflammation via activation of toll-like receptors (TLRs) [
17,
18]. It is also known that, besides TLRs activation,
P. acnes triggers acne inflammation by releasing various enzymes which lead to rupture of follicular walls and tissue injury such as lipases, proteases and hyaluronidases [
8,
10,
28]. A previous study showed that
P. acnes, laboratory strain P-37, produced extracellular proteinase, which was a heterogeneous mixture of three molecular species of enzyme including a neutral proteinase with a serine group and two kinds of alkaline proteinases [
10]. In addition, the genome sequence of
P. acnes demonstrated various extracellular peptidases, including homologs of an O-sialoglycoprotein endopeptidase, an extracellular subtilisin-like protease and a tripeptidyl aminopeptidase [
4]. Extracellular proteases secreted by
P. acnes may contribute to inflammation of acne by matrix breakdown and proteolytic detachment of follicular keratinocytes, thereby releasing inflammatory mediators, however, their precise role in the development of acne has not been fully elucidated.
Proteases play an important role in skin homeostasis and various disease conditions [
7]. A number of biological activities of proteases are mediated, at least in part, via the activation of its receptor, protease-activated receptors (PARs) [
7]. In the human skin, PAR-2 is abundantly expressed by keratinocytes and seems to regulate permeability barrier homeostasis, inflammation, pruritus, pigmentation, and wound healing in response to various endogenous and exogenous serine proteases [
7]. Functional PAR-2 is also expressed by keratinocytes of hair follicles and sebaceous glands, fibroblasts, endothelium, afferent neuron, as well as inflammatory cells [
29]. During cutaneous inflammation, PAR-2 is activated by endogenous activators such as leukocyte elastase and mast cell tryptase, thereby amplifying inflammation via upregulation of inflammatory mediators [
30]. PAR-2 is also known to be activated by various pathogenic organisms with protease activity such as house dust mites, cockroaches, certain bacteria, or parasites [
12,
32].
As P. acnes has been reported to produce various proteases, exogenous proteases from P. acnes also can react with PAR-2 on keratinocytes to induce and amplify inflammation in acne.
In this study, we investigated whether the culture supernatant of P. acnes can activate PAR-2 on keratinocytes and then induce the gene expression for pro-inflammatory cytokines, antimicrobial peptides (AMPs), and matrix metalloproteinases (MMPs). In addition, we tested whether protease activity of P. acnes supernatant and PAR-2 are involved in the induction of these inflammatory mediators.
Materials and methods
Samples from patients
A total of six patients were enrolled in this study, with four patients diagnosed with acne vulgaris and two patients with nevus comedonicus. Skin of comedonal lesions were obtained from the face of patients with acne vulgaris and nevus comedonicus by 3-mm punch biopsy. The tissues from acne patients were divided into two groups; one group was fixed in 10% buffered formalin for paraffin embedding, and the other was embedded in optimal cutting temperature compound and immediately frozen in liquid nitrogen, and stored at −80°C for in situ zymography. All biopsies were taken after obtaining informed consents from the patients, and this study was approved by the institutional review board of Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.
Measurement of protease activity by in situ zymography
Frozen sections (5 μm thickness) were rinsed with washing solution (1% Tween 20 in deionized water) and incubated at 37°C for 1 h with 250 μl of BODIPY-Fl-casein substrate (1 μg/μl; EnZCheck® Protease Assay Kits, Molecular Probes, Eugene, OR, USA) in deionized water (2 μl/ml). After removal of excess of substrate solution, nuclei were stained with propidium iodide (PI; Sigma, MO, USA) and the sections were washed with 1% Tween 20 in deionized water. After incubation, the sections were rinsed with washing solution and visualized immediately under confocal microscope C1 Plus (Nikon, Japan).
Immunofluorescence study
Paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in an ethanol series. Sections were then incubated for 30 min in blocking buffer [1% BSA, 0.1% cold-water fish gelatin in phosphate-buffered saline (PBS)]. For PAR-2 staining, sections were incubated with a 1:250 dilution of a rabbit polyclonal antibody against PAR-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted in blocking buffer for 2 h at room temperature. After washing with PBS, the sections were incubated with secondary antibodies consisting of 1:100 dilution of FITC-labeled goat anti-rabbit antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h at room temperature and diluted in blocking buffer. Tissue sections then were washed with PBS, and visualized under a confocal microscope C1 Plus (Nikon, Japan).
Preparation of culture supernatant of P. acnes
Propionibacterium acnes (ATCC 6919, Manassas, VA, USA) was grown in brain heart infusion broth (Difco, Sparks, MD, USA) at 37°C for 24 h under anaerobic condition. The log phase culture was centrifuged at 5,000×g for 15 min. The supernatants were harvested, filtered through a 0.2 μm-pore size filter and then stored at −20°C until used.
Protease activity measurement
To detect the secreted protease activity of P. acnes, the protease activity of the P. acnes culture supernatant was assayed using an EnzChek Protease Assay Kit (Molecular Probes Inc., Eugene, OR, USA). The pH-insensitive green fluorescent BODIPY-FL-conjugated casein was used as a substrate. Trypsin type IX-S (Sigma Co., St Louis, MO, USA), was used as a positive control. BODIPY-FL-conjugated casein solution (10 μg/ml) was prepared with 10 mM Tris–HCl buffer solution (pH 7.8) containing 0.1 mM sodium azide. The exponential phase culture supernatant of P. acnes strain ATCC 6919 was incubated with the substrate in a 96-well plate (OptiPlate 96F; Perkin Elmer, Boston, MA, USA) according to the manufacturer’s protocol for 1 h at 37°C and the fluorescence was measured by the HTS Multilabel Reader (Perkin Elmer, Boston, MA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Reagents
PAR-2 agonist peptide (AP) (SLIGKV-NH2) was purchased from Peptron (Daejeon, Korea) and dissolved in PBS at a concentration ranged from 0.3125 to 2.5 μM. Serine protease inhibitor, PMSF (phenylmethyl sulfonyl fluoride) was purchased from Sigma Chemicals (St Louis, MO, USA) and dissolved in ethanol, to a final concentration of 1.5 mM. A novel and selective PAR2 antagonist, ENMD-1068, was dissolved in PBS at a concentration ranged from 0.2 to 1.5 mM. The selectivity for PAR-2 in vivo in mice and in vitro in both murine and human cell lines of this PAR-2 antagonist was confirmed previously [
16].
Culture and stimulation of keratinocytes
The human keratinocyte cell line HaCaT grown to 80% confluency were maintained in EPILIFE Medium (Gibco, Invitrogen Ltd., Paisley, UK) with human keratinocyte growth supplement (Gibco, Invitrogen Ltd., Paisley, UK) for 24 h before stimulation. HaCaT cells were treated with the culture supernatant of P. acnes (2.5%) or AP (2.5 μM) at the optimal concentration to induce the maximal calcium influx for 3, 6, 12, 18 h at 37°C/5% CO2 in culture medium. In the inhibition study, cells were pretreated with PMSF (1.5 mM) or ENMD-1068 (1.5 mM) for 30 min and 6 h, respectively, and then treated with P. acnes culture supernatant (2.5%) or AP (2.5 μM) under the same condition.
Measurement of Ca2+ mobilization
We assessed PAR-2-mediated intracellular calcium mobilization in keratinocytes using a fluorometric imaging plate reader (FLIPR) calcium assay kit (Molecular Devices, Sunnyvale, CA, USA). Keratinocytes were plated in 96-well plates 24 h prior to the assay and grown to reach confluence at 37°C, 5% CO2. A calcium indicator dye (100 μl per well; FLIPR Calcium 4 assay kit; Molecular Devices, Sunnyvale, CA, USA) was added, and then plates were incubated for 1 h at 37°C. Test compound solutions were prepared in vehicle, consisting of 20 mM HEPES in Hank’s balanced salt solution (HBSS) with 0.1% BSA at pH 7.4. Test solutions were added at 25 μl to each well in duplicate for each experiment. Test compounds were added 30 min prior to the agonist stimulation. Then cells were stimulated with various concentrations of AP or P. acnes supernatant and the fluorescence change measured at 25°C (excitation 485 nm and emission 525 nm). ENMD-1068 was added 30 min prior to the stimulation of cells. Inhibitory effects of ENMD-1068 on calcium responses were expressed as percentage of the reference calcium response induced by either AP (2.5 μM) or P. acnes supernatant (2.5%) alone.
Real-time reverse transcription-polymerase chain reaction (RT-PCR)
After treatment, the cells were harvested using 0.25% trypsin/ethylene diamine tetra acetic acid (EDTA) and resuspended in PBS. Total RNA was extracted from the cells using Trizol reagent (Invitrogen). The complementary DNA was synthesized with 500 ng of total RNA using a TaKaRa RNA PCR kit (AMV) Ver. 3.0 (Takara, Shuzo Shiga, Japan). The primer sets were designed using NCBI Primer-Blast. Real-time PCR was performed using the SYBR Green PCR master mix and the ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the following parameters: 50°C for 2 min, 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 s; and primer extension at 60°C for 1 min. The expression of β-actin was used as reference. The PCR conditions for human β-actin were denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 1 min for 30 cycles. All reactions were done in triplicate. Melting-curve analysis of the PCR products was performed at the end of each assay to confirm the specificity of the amplification and absence of primer dimers. The relative amount of all mRNAs was calculated using the comparative threshold (CT) method. Primers in the present study are shown in Table
1.
Table 1
Primers for RT-PCR used in the present study
Pro-inflammatory cytokines | IL-1α | 5′-GACGCACTTGTAGCCACGTA-3′ |
5′-ACCGCCAATGAAATGACTCC-3′ |
IL-6 | 5′-GAAAGCAGCAAAGAGGCACT-3′ |
5′-TTTCACCAGGCAAGTCTCCT-3′ |
IL-8 | 5′-TCTGGCAACCCTAGTCTGCT-3′ |
5′-GCTTCCACATGTCCTCACAA-3′ |
TNF-α | 5′-ATGTTCGTCCTCCTCACAGG-3′ |
5′-CTATCTGGGAGGGGTCTTCC-3′ |
Antimicrobial peptides | hBD-2 | 5′-CAGCCCATTGAAACCAACTT-3′ |
5′-CTCTGGTGCCTCTCAGAACC-3′ |
LL-37 | 5′-GACATGGGGACCATGAAGAC-3′ |
5′-AGGAGGCGGTAGAGGTTAGC-3′ |
Matrix metalloproteinases | MMP-1 | 5′-CTGGCCACAACTGCCAAATG-3′ |
5′-CTGTCCCTGAACAGCCCAGTACTTA-3′ |
MMP-2 | 5′-TTGACGGTAAGGACGGACTC-3′ |
5′-ACTTGCAGTACTCCCCATCG-3′ |
MMP-3 | 5′-TGATCCTGCTTTGTCCTTTG-3′ |
5′-TTCAAGCTTCCTGAGGGATT-3′ |
MMP-9 | 5′-TTGACAGCGACAAGAAGTGG-3′ |
5′-GCCATTCACGTCGTCCTTAT-3′ |
MMP-13 | 5′-AACATCCAAAAACGCCAGAC-3′ |
5′-GGAAGTTCTGGCCAAAATGA-3′ |
TIMP-1 | 5′-AAGGCTCTGAAAAGGGCTTC-3′ |
5′-GAAAGATGGGAGTGGGAACA-3′ |
TIMP-2 | 5′-CCAAGCAGGAGTTTCTCGAC-3′ |
5′-GACCCATGGGATGAGTGTTT-3′ |
Housekeeping gene | β-actin | 5′-TGAAGGTCGGAGTCAACGGATTTGT-3′ |
5′-CATGTGGGCCATGAGGTCCACCAC-3′ |
Statistical analysis
All experiments were carried out in triplicate, and results are expressed as mean ± standard deviation. Comparisons between tests were done by the Student’s t test.
Discussion
PAR-2 is known to be expressed in the adnexal structures of the skin. As a sensor for exogenous or endogenous danger molecules, PAR-2 activation is tightly regulated by endogenous serine proteases as well as by secreted proteases from various microbes [
29,
30]. In previous studies,
P. acnes was found to produce various proteases and contribute to tissue injury [
8,
10,
28]. In addition to this direct proteolytic effect, proteases derived from
P. acnes could be potential activators of PAR-2 and activate innate immune response and inflammation. However, the possible role of PAR-2 in the pathogenesis of acne has not been investigated. Consistent with previous studies, we demonstrated that the culture supernatant of
P. acnes strain ATCC 6919 in the exponential phase of growth had proteolytic activity with a casein substrate (data not shown). We also showed that the protease activity was increased in follicular epithelium of acne lesion compared to that of non-lesional normal epidermis by in situ zymography, suggesting that the proteases secreted by
P. acnes might be attributed to the higher protease activity in acne lesions. Then we demonstrated that the culture supernatant of
P. acnes induced intracellular calcium influx and desensitized PAR-2 activation, suggesting that the proteases derived from
P. acnes can proteolytically activate PAR-2 on keratinocytes. To further confirm the involvement of
P. acnes proteases in the PAR-2 expression in comedonal lesions of acne, we compared the expression of PAR-2 immunoreactivity in the comedo of acne vulgaris and nevus comedonicus. As a negative control, we used the lesion of nevus comedonicus, because it is characterized by the abnormal keratinization and comedo formation similar to acne vulgaris without colonization of
P. acnes [
19]. PAR-2 immunoreactivity was upregulated in the follicular wall of comedones of acne vulgaris in compared with that of nevus comedonicus. Taken together we hypothesized that protease/PAR-2 signaling might have an important role in acne pathogenesis. Based on this hypothesis, we investigated whether
P. acnes-derived protease activity is able to stimulate keratinocytes to induce inflammatory mediators via PAR-2 activation.
The culture supernatant of
P. acnes increased the gene expression of IL-1α, IL-6, IL-8, and TNF-α in keratinocytes. The induction of IL-1α, IL-8, and TNF-α mRNA expression was significantly inhibited by incubating
P. acnes culture supernatants with serine protease inhibitor or selective PAR-2 antagonist, suggesting that
P. acnes proteases contribute to the upregulation of IL-1α, IL-8, and TNF-α via PAR-2 activation, thereby attributes to the comedo formation [
9], neutrophil infiltration [
1], and sustained inflammation in the acne pathogenesis. Viable
P. acnes in the exponential and stationary phase of growth was found to stimulate the production of TNF-α and granulocyte macrophage-colony stimulating factor (GM-CSF) by human keratinocytes [
6]. Viable
P. acnes in the stationary phase of growth also reported to contribute to IL-1α production in keratinocytes [
6]. In addition, it is well established that
P. acnes induces keratinocyte IL-8 production through a TLR2-dependent pathway [
24]. In the present study we demonstrated that
P. acnes proteases induce the gene expression of proinflammatory cytokines in keratinocytes via activation of PAR-2. These results are agreement with previous studies which reported that several pathogen-derived serine proteases are able to activate PAR-2, thereby inducing inflammation [
14,
31]. Protease derived from
Aggregatibacter actinomycetemcomitans extracts was demonstrated to upregulate IL-8 and intercellular adhesion molecule-1 expression in human gingival cells via PAR-2 activation [
31]. Recent study also showed that serine proteases in mite culture extracts activated PAR-2 on keratinocytes leading to upregulation of IL-8 and GM-CSF [
14].
Next, we demonstrated that the P. acnes supernatant increased the gene expression of hBD-2 and LL-37 in keratinocytes, which was blocked by serine protease inhibitor as well as PAR-2 specific inhibitor, suggesting that P. acnes-derived protease contributes to these AMPs induction via PAR-2.
Our findings are consistent with previous studies which demonstrated that PAR-2 stimulation on gingival epithelial cells by
Porphyromonas gingivalis-secreted proteases led to an upregulation of hBD-2 expression [
5]. In addition, immunoglobulin A in human milk with protease activity has been demonstrated to activate PAR-2 on intestinal epithelium, inducing hBD-2 expression via PAR-2 signaling [
2]. Previous study showed that hBD-2 expression is upregulated in acne lesions, suggesting the role of hBD-2 in acne pathogenesis [
27]. Nagy et al. found that
P. acnes induced hBD-2 in keratinocytes through TLR-2 and -4 and reported that hBD-2 does not have any bacteriostatic or bactericidal effect against any of the four
P. acnes strains they used, suggesting that the primary contribution of hBD2 to acne pathogenesis is the regulation of adaptive immunity [
24]. In contrast, Nakatsuji et al. demonstrated that high concentrations of synthetic hBD-2 (5–20 μM) had bactericidal effect on
P. acnes (ATCC 6919), dose dependently [
25]. These observations suggest that hBD-2 has a dual role in the pathogenesis of acne: the protective role against
P. acnes colonization when its local concentration rises above the bactericidal level and the role of innate immune responses and cutaneous inflammation. Nakatsuji et al. demonstrated that sebum free fatty acids (FFAs) enhanced hBD-2 mRNA levels up to 45,260-fold in sebocytes and that the FFA-induced sebocyte culture supernatant showed antimicrobial activity against
P. acnes [
25]. Our results showed that
P. acnes culture supernatant or AP treatment of keratinocytes significantly induced the gene expression of hBD-2 by sevenfold or tenfold, respectively, but to a lesser extent than in a previous report [
25]. These observations may require further study to investigate the clinical significance of PAR-2-mediated hBD-2 induction in response to
P. acnes.
The regulation and role of cathelicidins, which are another group of AMPs, in acne are largely unknown. Cathelicidins are reported to be controlled at transcriptional and post-transcriptional level by infection, inflammation, and 1,25-dihydroxyvitamin D
3 [
20]. Our results demonstrated that
P. acnes-derived protease and PAR-2 signaling induced the upregulation of cathelicidin expression in keratinocytes. The importance of serine protease-mediated proteolysis in the regulation of the antimicrobial effects of cathelicidin has been well known. Recent study observed increased protease activity and cathelicidin expression in the lesions of rosacea patients and suggested that increased protease activity leads to abnormal cathelicidin processing, thereby promotes inflammation in rosacea [
34]. However, this protease-induced activation of cathelicidin was post-transcriptional regulation. Taken together, it remains to be determined whether the increased gene expression of LL-37 in response to
P. acnes supernatant in our study is a nonspecific reaction to inflammatory cytokines or a more specific response to protease/PAR-2 signaling.
Recently, an active role of MMPs in acne has become an interesting topic of research in acne pathogenesis [
11,
26]. Papakonstantinou et al. reported that facial sebum from acne patients contained MMP-1, MMP-13, TIMP-1, and TIMP-2, which are thought to originate in keratinocytes and sebocytes [
26]. A recent in vitro study by Jalian et al. demonstrated that
P. acnes induced MMP-9, MMP-1, and TIMP-1 transcript in human monocytes [
11]. We demonstrated here that keratinocytes are important source of MMPs in acne and that protease activity of
P. acnes could induce the expression of several MMPs, including MMP-1, -2, -3, -9, and -13 via PAR-2 activation. We also observed that MMP-1, -2, -3, and -9 were significantly upregulated in acne lesions by immunohistochemistry (data not shown) as previous study which showed that MMP-1, -3, and -9 were markedly elevated in inflammatory acne lesions [
15]. Although the mechanism underlying the elevation of MMPs in acne lesions has not been fully elucidated, one report suggested that the enhanced expression of activator protein-1 (AP-1) regulated MMPs in inflammatory acne lesions by demonstrating that AP-1 was activated in acne lesions [
15]. Retinoids, which are known to modulate AP-1 expression, have been demonstrated to reduce MMP-9 and -13 in sebum of acne patients [
26], whereas, upregulate TIMP-1 in vitro [
11], thereby inducing clinical improvement of acne. It was reported that PAR-2 induced c-Jun/AP-1 activation via Jun activation domain-binding protein 1 (Jab1)-mediated signaling pathway [
21]. These data led us to suggest that PAR-2-induced AP-1 activation may stimulate MMPs in keratinocytes when exposed to
P. acnes with protease activity. The upregulated MMPs might contribute to the acne pathogenesis by inducing inflammation and tissue destruction.
It has been known that there are two distinct types of
P. acnes (types I and II) based on serological agglutination tests and cell-wall sugar analysis [
13]. Recent studies have profiled these two phenotypes of
P. acnes using nucleotide sequencing of the recA housekeeping gene, demonstrating that types I and II represent phylogenetically distinct lineages and the type I strains could be split into two further clusters, types IA and IB [
22]. Because of the antigenic and biochemical differences between these phylogenetic types of
P. acnes, including the expression of putative Christie–Atkins–Munch–Peterson (CAMP) factor [
33], a number of recent studies have attempted to correlate the different phylogenetic types of
P. acnes with their different role in acne pathogenesis [
23,
24]. The
P. acnes strain ATCC 6919, which we used in our study, belongs to the
P. acnes type IA strain [
33]. A previous microbiological study has found that
P. acnes isolates from patients with acne were predominantly type IA [
22] and type IA isolates have been demonstrated to have a greater effect on AMPs and proinflammatory cytokines production by keratinocytes [
24] and sebocytes [
23]. However, the lesions of acne patients are likely to harbor different skin microbes and
P. acnes strains other than type IA. From these findings, our study has a limitation that should be taken into account in interpreting the results. It should be noted that our data obtained with only one isolate type of
P. acnes, do not necessarily translate into other phylogenetic types of
P. acnes and their role in acne pathogenesis. Moreover, in future study it might be worthwhile to investigate the possible involvement of PAR-2 in the pathogenesis of acne using the different other phylogenetic types of
P. acnes.
In conclusion, our results indicate that PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from P. acnes, and plays an important role in acne pathogenesis by inducing inflammation, innate immune responses, and acne scar formation. Together, these finding also suggest that specific protease activity or PAR-2 might be a future target for therapeutic intervention for the treatment of acne vulgaris.