Propionibacterium acnes host cell tropism contributesto vimentin-mediated invasion and inductionof inflammation

Tim N. Mak,1 Natalie Fischer,1† Britta Laube,2

Volker Brinkmann,2 Matteo M. E. Metruccio,1‡

Karen S. Sfanos,3 Hans-Joachim Mollenkopf,4

Thomas F. Meyer1 and Holger Brüggemann1,5*1Department of Molecular Biology, Max Planck Institutefor Infection Biology, Berlin, Germany.2Core Facility Microscopy, Max Planck Institute forInfection Biology, Berlin, Germany.3Department of Pathology, Johns Hopkins UniversitySchool of Medicine, Baltimore, USA.4Core Facility Microarray, Max Planck Institute forInfection Biology, Berlin, Germany.5Department of Biomedicine, Aarhus University, Aarhus,Denmark.

Summary

The contribution of the human microbiota tohealth and disease is poorly understood. Propioni-bacterium acnes is a prominent member of theskin microbiota, but is also associated withacne vulgaris. This bacterium has gained recentattention as a potential opportunistic pathogen atnon-skin infection sites due to its associationwith chronic pathologies and its isolation fromdiseased prostates. We performed comparativeglobal-transcriptional analyses for P. acnes infec-tion of keratinocytes and prostate cells. P. acnesinduced an acute, transient transcriptional inflam-matory response in keratinocytes, whereas thisresponse was delayed and sustained in prostatecells. We found that P. acnes invaded prostate epi-thelial cells, but not keratinocytes, and was detect-able intracellularly 7 days post infection. Furthercharacterization of the host cell response to infec-tion revealed that vimentin was a key determinantfor P. acnes invasion in prostate cells. siRNA-mediated knock-down of vimentin in prostate cells

attenuated bacterial invasion and the inflammatoryresponse to infection. We conclude that host celltropism, which may depend on the host proteinvimentin, is relevant for P. acnes invasion and inpart determines its sustained inflammatory capac-ity and persistence of infection.

Introduction

The Gram-positive bacterium Propionibacterium acnes isa ubiquitous member of the skin microbiota and is found insebaceous follicles located on the face and back of themajority of the human population. In addition to its preva-lence on human skin, P. acnes has also been detected atother body sites such as the stomach, large intestine, oralcavity, conjunctiva and prostate (Hentges, 1993; Cohenet al., 2005; Hori et al., 2008; Delgado et al., 2011; Perryand Lambert, 2011).

Propionibacterium acnes is generally regarded as acommensal of the skin. Certain properties suggest amutualistic role of the bacterium (Cogen et al., 2008);however, the bacterium is known for its association withacne vulgaris, a highly prevalent skin condition with aninflammatory component that affects up to 80% of ado-lescents (Kurokawa et al., 2009). Despite this association,the exact role and significance of P. acnes in acne vulgarisremains undetermined. Cell culture experiments usingskin-derived keratinocytes and sebocytes showed thatP. acnes can trigger an inflammatory response, includingthe production of a range of pro-inflammatory chemokinesand cytokines (Graham et al., 2004; Nagy et al., 2006;Lee et al., 2010). Pattern recognition receptors of thetoll-like receptor (TLR) protein family have been identifiedas P. acnes responsive receptors, and the expression ofTLR2 and TLR4 is elevated in P. acnes infected keratino-cytes (Jugeau et al., 2005). Furthermore, P. acnes infec-tion triggers the production of reactive oxygen species inkeratinocytes, which is associated with interleukin (IL)-8production and host cell apoptosis (Grange et al., 2009a).

Besides its role in inflammatory acne initiation and/orprogression, emerging evidence suggests that P. acnescan also act as an opportunistic pathogen in other dis-eases. It has been reported to be causatively involved incases of endocarditis, intravascular and central nervous

Received 13 March, 2012; revised 2 June, 2012; accepted 27 June,2012. *For correspondence. E-mail brueggemann@microbiology.au.dk; Tel. (+45) 87168067; Fax (+45) 87168067.†Present address: Unité de Pathogenie Microbienne Moleculaire,Institut Pasteur, Paris, France.‡Present address: Novartis Vaccines & Diagnostics, Siena, Italy.

Cellular Microbiology (2012) 14(11), 1720–1733 doi:10.1111/j.1462-5822.2012.01833.xFirst published online 22 July 2012

© 2012 Blackwell Publishing Ltd

cellular microbiology

system infections, endophthalmitis, and has been particu-larly associated with infections following surgical interven-tion and the implantation of prosthetic devices (Perry andLambert, 2011). Recently, independent studies reported alink between P. acnes and prostate pathologies. P. acneswas found to be associated with histological inflammationin the prostate, and we, along with others, detected it incancerous prostates (Cohen et al., 2005; Alexeyev et al.,2007; Fassi Fehri et al., 2011). Investigations with pros-tate epithelial cells showed that P. acnes induced thesecretion of cytokines and chemokines such as IL-6, IL-8and GM-CSF (Drott et al., 2010; Fassi Fehri et al., 2011).Moreover, infection with P. acnes modulated host celladhesion and proliferation properties, which resulted inthe initiation of cellular transformation (Fassi Fehri et al.,2011). These studies indicate that the pathological out-comes of P. acnes infection might be highly dependent onthe induction of tissue-specific host responses.

The consequences of P. acnes’ omnipresence forthe human host are poorly understood; circumstantialevidence suggests that the host response to P. acnesdepends on host predisposition (e.g. immune status),P. acnes strain-specific properties, and the anatomicalsite of infection (Dessinioti and Katsambas, 2010;Lomholt and Kilian, 2010; Szabó and Kemény, 2011).Here, we have compared the global transcriptional pro-files of skin and prostate epithelial cells during infectionwith P. acnes strain P6, a clinical prostate isolate. Micro-array analyses revealed that the host cell responsesto infection fundamentally differed in keratinocyte andprostate cell lines, particularly with regard to the temporalregulation of inflammatory response genes. Subsequentanalyses showed host cell-specific modifications ofthe canonical MAP kinase and NF-kB pathways byP. acnes. Immunofluorescence and electron microscopydemonstrated host cell-specific invasion and intracellularpersistence of P. acnes. Bacterial invasion was partiallydependent on the prostate expressed host cell proteinvimentin. This study reveals, for the first time, thatP. acnes host cell tropism has biological significance forthe pathogenic properties of this ubiquitous bacterium.

Results

Transcriptional profiles of P. acnes infected HaCaT andRWPE1 cells

In order to compare the transcriptional responses of skinand prostate epithelia to P. acnes infection we infected thekeratinocyte-derived cell line, HaCaT (Boukamp et al.,1988) and the prostate-derived cell line, RWPE1 (Belloet al., 1997). First, we performed a comparative analysisof the genome-wide transcriptional profiles of these twocell lines in response to infection with P. acnes strain P6.

The transcriptional responses at 24 h post infection (h p.i.)(short-term) and 7 days post infection (d p.i.) (long-term)were determined by microarray (Fig. 1). It was ascer-tained that cell viability (HaCaT and RWPE1 cell lines)was not adversely affected by P. acnes infection (Fig. S1).Our analysis focused on genes that were not differentiallyexpressed at a basal level (i.e. in the absence of infection)between the two cell lines. A total of 1212 and 1308 geneswere differentially expressed between the two cell lines at24 h p.i and 7 d p.i., respectively, including 307 genes thatwere differentially expressed at both time points. The datashowed an upregulation of inflammation-associatedgenes such as IL-8, IL-6, LTB and IL-1b at 24 h p.i. in bothHaCaT and RWPE1 cells (Fig. S2). However, P. acnesupregulated inflammatory targets more strongly in HaCaTcells compared with RWPE1 at 24 h p.i.; for example, theupregulation of IL-8 was 22-fold stronger in HaCaT cells at24 h p.i. (Fig. 1A). This was also true for several otherchemokines, including CCL1, CCL3, CCL8 and CXCL1.At 7 d p.i. the situation was reversed and P. acnes infec-tion upregulated inflammation-associated genes inRWPE1 cells more strongly compared with HaCaT cells.For example, IL-6 and TLR2 were 9.5- and 6-fold, respec-tively, more strongly upregulated in RWPE1 cells. Func-tional network analysis was performed with de-regulatedgenes in the two cell lines at 24 h p.i and 7 d p.i. (Fig. S3).This again illustrated the upregulation of concerted func-tions of the inflammatory response in HaCaT cells at 24 hp.i and in RWPE at 7 d p.i. All significantly de-regulatedgenes were categorized according to their assigned bio-logical functions/diseases (Fig. 1B). Besides the terms‘inflammatory responses’ and ‘inflammatory disease’ themost enriched functions/diseases were ‘dermatologicaldiseases and conditions’, ‘cancer’ and ‘cellular growthand proliferation’, all of which assigned to de-regulatedgenes in RWPE1 cells at 7 d p.i.

Thus, HaCaT cells strongly upregulated inflammatoryresponse genes during acute infection with P. acnes;however, this response did not persist considerablybeyond 24 h p.i. In contrast, the transcriptional responseof RWPE1 cells to acute infection was limited, but becamemore pronounced up to 7 d p.i.

P. acnes-dependent activation of the MAPK pathwaydiffers in RWPE1 and HaCaT cells

The observed differences in the acute (24 h p.i.) transcrip-tional response to P. acnes infection between the two celllines prompted us to investigate the initial cellular signal-ling events during infection. MAP kinase (MAPK) cas-cades play crucial roles in the response to externalstimuli, and mediate a first line reaction towards patho-gens (Pearson et al., 2001). There are three major MAPKsignalling pathways that employ the ERK, p38 and JNK

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kinases; the activation of each of these kinases, andhence pathway specificity, can be determined by proteinphosphorylation at specific sites. P. acnes infection ofHaCaT cells led to phosphorylation of ERK1/2, p38 andJNK (Fig. 2A and B) in a time-dependent manner asdetermined by immunoblot. Increased phosphorylationwas observed at 30 min p.i. (p38) and 4 h p.i. (ERK1/2and JNK). Phosphorylation of these three MAP kinasesdecreased at 8 h p.i., but was strongest at 24 h p.i., indi-cating an oscillatory activation pattern of MAPK cascadesin HaCaT cells in response to P. acnes infection. In con-trast, basal expression levels of phospho-ERK1/2 andphospho-JNK were higher in RWPE1 cells in the absenceof infection and only phospho-p38 increased in RWPE1cells 24 h p.i. (Fig. 2C and D). Moreover, ERK was slightlydeactivated from 8 h p.i., as judged by decreasing phos-phorylation levels of ERK1/2. Similar to observations in

HaCaT cells, p38 activation oscillated: the first peak ofp38 phosphorylation occurred approximately 2 h p.i., andstrongest phosphorylation was observed at 24 h p.i.

P. acnes activates the NF-kB pathway in RWPE1, butnot in HaCaT or HEKa cells

The NF-kB signalling pathway is another importantmediator of the inflammatory response and is oftenactivated during innate immune recognition of bacterialpathogens. NF-kB activation requires rapid phosphoryla-tion, ubiquitination and subsequent degradation of IkBa,the inhibitor protein of NF-kB (Natoli and Chiocca, 2008).To monitor NF-kB signalling in infected host cells, IkBadegradation was determined by immunoblot analysis. Nonotable IkBa degradation was observed in P. acnesinfected HaCaT or RWPE1 cells during the first 24 h of

Fig. 1. Gene expression differences in P. acnes-infected HaCaT and RWPE1 cells.A. Direct comparison of microarray analyses of transcript levels between P. acnes-infected HaCaT and RWPE1 cells at 24 h p.i and 7 d p.i.revealed cell type-specific upregulation of inflammation-associated genes in a time-dependent manner. Green and red indicate strongerexpression in HaCaT and RWPE1 cells respectively. Numbers are fold changes. Only genes that were not differentially expressed at a basallevel (i.e. in the absence of infection) between the two cell types were considered in this analysis. Grey indicates non-significant (n.s.)expression differences.B. Functional analysis of host cell transcriptome responses to P. acnes infection. Biological functions and diseases were assigned tode-regulated genes upon infection of HaCaT and RWPE1 cells. P-values indicate significance of enrichment of biological functions/diseases inthe respective transcriptomes. At 24 h p.i. the transcriptional response in HaCaT cells dominates over that of RWPE1 cells in most pathways.However, at 7 d p.i. the transcriptional response in RWPE1 cells is largely dominant. The microarray data are based on eight and fourexperiments for the 24 h and 7 days time points respectively. In order to compensate specific effects of the dyes and to ensure statisticallyrelevant data analysis, a colour-swap dye reversal was performed.

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infection (Fig. 3A and B). However, degradation of IkBacould be detected in RWPE1, but not in HaCaT cells, atlater infection time points (48 h to 7 d p.i.) (Fig. 3C and D),which was indicative of cell type-dependent activation ofNF-kB. Since HaCaT cells are reported to have an aber-rant NF-kB signalling pathway (Lewis et al., 2006), werepeated the experiments with primary human epidermalkeratinocytes (HEKa cells). As in HaCaT, no IkBa degra-dation could be observed in HEKa cells upon P. acnesinfection (data not shown).

IkBa forms a dimer with the transcriptional regulatorp65; once IkBa is degraded upon stimuli, the dimer dis-aggregates and p65 translocates into the nucleus tocontrol DNA transcription of NF-kB target genes (Hoff-mann and Baltimore, 2006). Thus, p65 nuclear transloca-tion is a sign of NF-kB activation. By immunofluorescenceanalysis we observed p65 nuclear translocation inP. acnes-infected RWPE1 cells at 48 h p.i., but not inHaCaT cells (Fig. 3E and 3F). These data indicated thatNF-kB was exclusively activated in RWPE1 cells followinglong-term infection with P. acnes.

Intracellular localization and persistence of P. acnes inRWPE1 cells, but not in HaCaT or HEKa cells

Based on the results obtained, we suspected that theinfection process was fundamentally different in the twocell lines, which could reflect cell type-specific invasionand/or adhesion properties of P. acnes. P. acnes has pre-viously been shown to invade RWPE1 cells (Fassi Fehriet al., 2011), but investigations were not extended tokeratinocytes. Thus, we compared the invasiveness ofP. acnes in both cell lines, using a streptomycin/penicillinprotection assay. Invasion was 6- and 10-fold higher inRWPE1 compared with HEKa and HaCaT cells, respec-tively, as determined from cfu counts at 24h p.i. (Fig. 4A).To confirm the invasion phenotypes, we performed extra-/intracellular double staining of P. acnes. Immunofluores-cence microscopy (after stringent washing to removeunattached bacteria) revealed higher numbers of intrac-ellular, as well as extracellularly attached, P. acnesbacteria in RWPE1 cells at 24 h p.i. compared withHaCaT cells (Fig. 4B–E). This indicated cell type-specific

Fig. 2. Host cell-dependent activation of MAPK pathways by P. acnes.A and B. In P. acnes infected HaCaT cells, three MAPK pathways are activated in a time-dependent manner, determined by phosphorylationof ERK1/2, JNK and p38.C and D. In P. acnes infected RWPE1 cells, only p38 is activated at 24 h p.i.Quantification of Western blots was based on the band intensity normalized to b-actin (B, D). Representative blots of three independentexperiments are shown. NI, non-infected.

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adhesion and invasion properties of P. acnes. Further-more, P. acnes bacteria could be detected intracellularlyup to 3 weeks p.i. in RWPE1 cells (Fig. 5A). Heat-inactivated bacteria were not detected 3 weeks p.i., whichdemonstrated that bacterial viability was required forintracellular persistence. Notably, P. acnes could not bedetected in HaCaT cells 3 weeks p.i. (data not shown).Intracellular P. acnes could also be detected by electronmicroscopy at 24 h p.i. in RWPE1 cells (Fig. 5B–E). Thus,P. acnes intracellular invasion and persistence is cell type-specific; this host cell tropism could explain the observed

differences in sustained NF-kB activation and transcrip-tional de-regulation.

Functional analysis of P. acnes host cell tropism

To investigate the mechanistic basis for the observedhost cell tropism of P. acnes we identified endogenousdifferences in the transcriptional profiles of RWPE1 andHaCaT cells. Employing a stringent fold change cut-off,we identified a number of genes that were highly differen-tially expressed between the two cell lines. We focused on

Fig. 3. P. acnes triggers NF-kB activation in RWPE1 but not in HaCaT cells.A–D. Western blot analysis reveals no degradation of IkBa in HaCaT of RWPE1 cells during acute infection (� 24 h). During long-terminfection (48 h to 7 days) significant degradation could be observed in RWPE1, but not in HaCaT cells. b-Actin was used as loading control.E and F. Immunofluorescence analysis of p65 translocation in infected RWPE1 and HaCaT cells at 48 h p.i. Nuclear translocation of p65could only be detected in infected RWPE1 cells. p65, green; P. acnes, red; cellular nuclei, blue. NI, non-infected; inf, infected. Scale bar,20 mm. Representative blots and images of three independent experiments are shown.

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genes that were upregulated at least 25-fold in RWPE1cells, i.e. RPS4Y1, VIM, IGFBP2, LOC150763, IL1B andFN1 (Table S1). Since previous studies had demonstrateda role for vimentin (VIM) in host–pathogen recognitionand/or interactions (Kumar and Valdivia, 2008; Miller andHertel, 2009; Das et al., 2011; Ghosh et al., 2011), wefocused further functional analyses on this protein.

Vimentin facilitates P. acnes host cell invasion

Vimentin is a type III intermediate filament protein andhas various functional roles; for example, it is involved inregulating cell shape and integrity, adhesion, migrationand signalling events (Satelli and Li, 2011). We initiallyconfirmed the microarray data with the observationthat vimentin was expressed in RWPE1 cells, but wasnot detectable in HaCaT cells by immunoblot (Fig. 6A).

To investigate its function in the context of P. acnes infec-tion we ectopically overexpressed vimentin in HaCaTcells (Fig. 6B). In parallel, vimentin knock-down (siVIM)RWPE1 cells were generated using siRNA (Fig. 6C).Employing the streptomycin/penicillin protection assay,we then compared numbers of intracellular P. acnes invimentin-overexpressing HaCaT cells and siVIM RWPE1cells with those in control cells (HaCaT carrying a GFP-containing vector and RWPE1 transfected with AllStarssiRNA respectively) at 24 h p.i. Overexpression of vimen-tin in HaCaT cells led to a 1.7-fold increase in intracellularbacteria (Fig. 6D). Conversely, silencing of vimentinexpression in RWPE1 cells led to a 2.3-fold decreasein intracellular P. acnes (Fig. 6E). Both experimentsshowed that manipulation of vimentin expression alteredthe number of intracellular bacteria at 24 h p.i. Thedata revealed that P. acnes invasion depends on the

A

Fig. 4. Host cell type-specific attachment and invasion of P. acnes.A. A streptomycin/penicillin protection assay revealed a 5- to 10-fold difference of viable intracellular P. acnes in RWPE1 and HaCaT/HEKacells at 24 h p.i. **P � 0.01.B–E. Confocal immunofluorescence microscopy revealed cell type-specific differences in attachment and invasion of P. acnes.B and D. No extracellular and very few intracellular P. acnes (red) could be detected in infected HaCaT cells after stringent washing.C and E. More extra- (green) as well as intracellular (red) bacteria are found in infected RWPE1 cells. Scale bar, 20 mm.D and E. Higher magnification of P. acnes infected HaCaT and RWPE1 cells. Scale bar, 8 mm. Actin stained in blue (blue).

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Fig. 5. Persistence of intracellular P. acnes inRWPE1 cells.A. Viable, intracellular P. acnes (green), butnot heat-inactivated bacteria, were detected inRWPE1 cells after infection for 21 days andstringent washing. Nuclei, blue; actin, red.Scale bar, 20 mm.B–E. Electron microscopy images of infectedRWPE1 show P. acnes in intracellularvacuoles (double arrowhead) as singlebacterium or in small clusters. P. acneslocalizes close to the nuclear (n) envelope(single arrowhead). Scale bars, 2 mm and8 mm. Representative images from threeindependent experiments are shown.

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expression of vimentin. To further address the role ofvimentin in the uptake of P. acnes, we used an anti-VIMantibody to block membrane-exposed vimentin. Invasionof P. acnes into anti-VIM-treated cells was approximatelytwofold decreased at 24 h p.i. compared with anti-IgG-treated cells (Fig. S4A), indicating that the invasionprocess of P. acnes is partially vimentin-dependent. Sincevimentin is an intermediate filament protein with supposedstructural roles, for example in supporting the cellularmembrane and the cytoskeleton integrity (Eriksson et al.,2009), we investigated if disturbance of the cytoskeletonnetwork would influence P. acnes invasion. Indeed,P. acnes invasion was significantly reduced at 24 h p.i. inRWPE1 cells treated with chemical inhibitors of actin andmicrotubule polymerization, e.g. up to 10-fold in the caseof cytochalasin B treatment (Fig. S4B). This implicatedvimentin and the cytoskeleton network in the adhesionand cellular invasion of P. acnes.

Vimentin-dependent bacterial host cell entry and its rolein the inflammatory response of RWPE1 cells toP. acnes infection

Given the finding that vimentin is involved in P. acnesinvasion, further investigations into the effects of vimentinexpression manipulation were performed to elucidatewhether this would influence the inflammatory responseof RWPE1 cells to P. acnes. Therefore, we compared

gene expression profiles of P. acnes-infected RWPE1cells transfected with either vimentin-specific siRNA orAllStars control siRNA at 24 h p.i. We focused on genesthat were not de-regulated in non-infected siVIM RWPE1(compared with control cells). A total of 1329 genes weredifferentially expressed between infected siVIM RWPE1and infected AllStars transfected control cells, indicating astrong influence of vimentin on the host cell response toP. acnes. Inflammation-associated genes were a promi-nent group of downregulated genes (Fig. S5A and B); forexample, expression of IL-8 was reduced more than40-fold in infected siVIM RWPE1 cells compared withinfected control cells. These data show that silencing ofvimentin expression in RWPE1 cells – thus partially pre-venting P. acnes internalization – reduced the inflamma-tory response. Taken together, we have shown thatvimentin plays an important role in establishing intracel-lular P. acnes infection, by facilitating or supporting bac-terial invasion in a host cell-dependent manner. The datafurther indicate that the capability of the microorganism toinvade intracellularly has direct consequences on hostcell-derived inflammation.

Vimentin is expressed in prostate tissue

Vimentin facilitated the invasion of P. acnes into prostatecells in vitro. It has been previously reported that vimentinis focally expressed in prostate gland epithelium (Wernert

Fig. 6. Role of vimentin in host cell invasionof P. acnes.A. Western blot analysis revealed thatvimentin (VIM) is expressed in RWPE1 butnot in HaCaT cells.B. HaCaT cells transformed with aVIM-containing expression vector expressvimentin. Cells transformed with aGFP-containing plasmid were used as acontrol.C. Knock-down of VIM in RWPE1 by RNAisuccessfully reduced vimentin protein levels.AllStars siRNA- transfected cells were usedas a control.D. Overexpression of VIM in HaCaT cellsincreased P. acnes invasion.E. Knock-down of VIM in RWPE1 cellsreduced P. acnes invasion.b-Actin was used as loading control.Representative blots and results of at leastthree independent experiments are shown.**P � 0.01.

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et al., 1987; Heatley et al., 1995). In addition, vimentinwas found to be abundantly expressed in poorly differen-tiated prostate cancer samples (Wei et al., 2008; Zhaoet al., 2008); the latter studies suggested that vimentinaffects prostate cancer cell motility and invasiveness. Weimmunostained clinical skin and prostate tissue samplesto analyse tissue-specific vimentin expression patterns.Vimentin was absent from skin epithelium (Fig. 7A), butwas focally expressed in prostate epithelium (Fig. 7B andC). Interestingly, a distinct subset of cells that lined glan-dular structures were vimentin-positive (Fig. 7C).

Discussion

We have shown that a P. acnes clinical prostate isolateexhibits profound host cell tropism. Alongside the role ofP. acnes strain determinants in predisposition to, andpathogenesis of, P. acnes infection (Holland et al., 2010;Lomholt and Kilian, 2010; Brzuszkiewicz et al., 2011;McDowell et al., 2011), host factors are also importantfor the pathological outcome of infection (Szabó andKemény, 2011). Moreover, the likelihood exists that thepathogenic potential of P. acnes depends on the anatomi-cal site of infection. Experimental comparison of hostcell-specific responses to P. acnes infection had not pre-viously been explored; thus, we compared infections ofkeratinocyte (HaCaT) and prostate (RWPE1) derived celllines using a P. acnes clinical isolate. We observed cleardifferences in both the magnitude of the transcriptionalresponses and their temporal regulation between the twocell lines. In essence, P. acnes-induced inflammation inkeratinocytes is acute but transient, whereas the bacte-rium triggers delayed but sustained inflammation in pros-

tate cells, which is associated with prolonged NF-kBactivation. Other inflammatory stimuli such as lipopolysac-charide (LPS) do not elicit such differential effects;LPS triggers an acute NF-kB-dependent response inboth HaCaT and RWPE1 cells (Seo et al., 2001; Grangeet al., 2009b; Kim et al., 2011), indicating that thehere described differential inflammatory responses areP. acnes-specific.

The capacity of P. acnes to induce a sustainedde-regulation of the transcriptional response in RWPE1-infected cells was associated with invasion. To date, theinvasion capacity of P. acnes has not been well studied.We previously showed that P. acnes isolates couldbe visualized intracellularly at 24 h p.i. in prostateepithelial cells (Fassi Fehri et al., 2011). In a studyattempting to find an aetiological link between sarcoido-sis and P. acnes infection, invasion of HEK293T cells byP. acnes was correlated with bacterial serotype andgenotype, although no association with disease could bemade (Furukawa et al., 2009). In another sarcoidosisstudy, Tanabe and co-workers observed the invasion ofA549 cells by clinical P. acnes isolates (Tanabe et al.,2006). Here, we present evidence that host cell entrymarks the beginning of a persistent stage of infection,characterized by continuous NF-kB activation that impli-cates sustained upregulation of inflammatory markers.The data add to our previous findings that long-terminfection of RWPE1 cells by P. acnes altered host cellfate; in particular, increasing cell proliferation and reduc-ing E-cadherin expression and anchorage-independentgrowth (Fassi Fehri et al., 2011), which are markers ofthe epithelial-to-mesenchymal transition (EMT) (Kalluriand Weinberg, 2009).

Fig. 7. Vimentin expression in skin andprostate tissues.A. Skin tissue sample showing somescattered VIM-positive stromal cells;epidermal cells (outer layer) are VIM-negative.B. Prostate tissue sample with VIM-positivecells, representing blood vessel-liningendothelial cells and stromal cells.C. Benign prostatic hyperplasia tissue samplewith strong VIM expression in cells in theglandular epithelium.Green, VIM; blue, nuclei.

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Here, vimentin was expressed in RWPE1 but not inHaCaT cells. Moreover, we confirmed that expression ofvimentin was absent from human keratinocyte tissue, butpresent in the prostate. Our data suggest that vimentinexpression is partially responsible for the observed differ-ences in P. acnes invasion between the two cell lines.Vimentin has been described as a cytoskeletal compo-nent responsible for maintaining cell integrity in mesen-chymal cells (Eriksson et al., 2009). It is expressed in awide range of cell types including pancreatic precursorcells, neuronal precursor cells, fibroblasts, endothelialcells, renal tubular cells, macrophages, neutrophils, leu-cocytes and renal stromal cells (Satelli and Li, 2011). Inaddition, vimentin has been recognized as an EMTmarker (Thiery, 2002) and is overexpressed in variousepithelial cancers, including those of the prostate andgastrointestinal tract (Wei et al., 2008; Zhao et al., 2008).Our observation that vimentin was not produced in kerati-nocytes, which are of ectodermal origin, supports thegeneral consensus (Katagata et al., 1999). RWPE1 cellsare secretory epithelial cells and are derived from theendoderm; although lineage dependence has not beenunequivocally determined, an endothelial origin has beenexcluded (Bello et al., 1997). RWPE1 cells are frequentlyused as a prostate epithelial cell model since they organ-ize into acini in 3D matrigel and secrete PSA into thelumen when exposed to androgen (Bello-Deocampoet al., 2001). A recent study revealed that mesenchymaland epithelial cadherins were coexpressed in RWPE1cells (Härmä et al., 2010); the authors speculated thatthese cells (or subpopulations within RWPE1) might haveundergone a (partial) EMT. Interestingly, all cell lines,which were reported to be invaded by P. acnes, includingHEK293T and A549 cells, are vimentin-positive (Lahatet al., 2010; Bhattacharyya and Hope, 2011), supportingour findings that vimentin is required for efficient bacterialinvasion.

Investigations into the role of vimentin in microbialinfections have shown that infectious agents can modu-late vimentin to establish an intracellular niche and foroptimal positioning of bacteria-containing vacuoles. Sucha mechanism has been studied for African swine fevervirus infection, which initiated vimentin rearrangement toform a cage surrounding viral factories (Stefanovic et al.,2005). Similar observations have been made withChlamydia trachomatis, which remodelled and recruitedcytoskeletal components of the host cell, includingvimentin, to form a dynamic scaffold that provided struc-tural stability to the inclusion (Kumar and Valdivia, 2008)and Salmonella typhimurium, which induced the forma-tion of aggresome-like structures and dramaticallyremodelled vimentin and cytokeratin networks in epithe-lial cells and macrophages (Guignot and Servin, 2008).Interestingly, that study indicated that vimentin remodel-

ling was required to maintain Salmonella microcoloniesin the juxtanuclear area, a prerequisite for the initiation ofSalmonella replication. We also observed that P. acnesbacteria preferentially localized close to the nucleus, butthe significance of this localization for the intracellularfate of P. acnes requires further exploration. Studieshave also shown that pathogens can exploit vimentin forthe purpose of invasion; it has been shown that vimentinis found on the extracellular surface of various cells,and some bacteria interact specifically with surface-exposed vimentin. For example, meningitic Escherichiacoli K1 strains bound to surface-exposed vimentin asprimary receptor to gain entry into human brain microv-ascular endothelial cells (Chi et al., 2010). Our data, inparticular the effect of antibody-mediated neutralizationof surface-bound vimentin on P. acnes’ invasion, alsopoint towards a role for vimentin as a cell surface-associated mediator of bacterial invasion. The mechanis-tic basis of this interaction is an interesting question forfuture investigation.

Despite its ubiquitous presence on the human skin,our understanding of the role of P. acnes in health anddisease, at both skin and non-skin sites, remains frag-mentary. Although an aetiological role for P. acnes inbenign prostatic hyperplasia, prostate cancer or otherinflammatory conditions is not established, mounting evi-dence suggests that this bacterium has strong inflamma-tory capacity, which could initiate or contribute to disease.The pronounced pro-inflammatory potential of P. acnes isalso the key argument for its driving role in inflammatoryacne. Our data contribute to the ongoing debate concern-ing the putative role of P. acnes in prostate cancer. Wehave demonstrated that P. acnes can induce a sustainedinflammatory response in prostate cells permissive tobacterial invasion in a host cell-specific manner. Giventhat vimentin is expressed in the human prostate, ourdata support the hypothesis that P. acnes acts as anopportunistic invader of prostate cells, and could contrib-ute to prostate cancer aetiology owing to its inflammatoryprofile. In summary, we propose that host cell tropism isan important facet of P. acnes-associated pathologies. Itis very likely that host cell tropism is affected by bacterialheterogeneity, i.e. phylogenetically distinct P. acnes iso-lates might differ substantially in eliciting host cell-specificresponses. The exact interdependence of bacteriaI andhost cell entities and its relevance for health and diseasehas to be investigated in the future.

Experimental procedures

Bacteria, cell cultures and reagents

Propionibacterium acnes strain P6 (type I-2, ST33, according tothe MLST scheme of Lomholt and Kilian, 2010), an isolate from acancerous prostate (Fassi Fehri et al., 2011), was cultured on

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Brucella agar plates for 3 days at 37°C under anaerobic condi-tions. For heat inactivation P. acnes was incubated at 60°C for30 min.

The human keratinocyte cell line HaCaT (CLS) was cultured inDMEM medium (Gibco) supplemented with 10% heat-inactivatedFCS. HEKa (Invitrogen) cells are primary human epidermalkeratinocytes isolated from adult skin; they were cultured inEpiLife® Medium supplemented with human keratinocyte growthsupplement (Gibco). The human prostate epithelial cell lineRWPE1 (ATCC CRL-11609) was cultured in keratinocyte mediumsupplemented with 50 mg ml-1 bovine pituitary extract and5 ng ml-1 epidermal growth factor (Gibco).

Antibodies and inhibitors

Polyclonal anti-P. acnes antibody (Fassi Fehri et al., 2011)was diluted 1:1000 for immunofluorescence analysis. Polyclonalrabbit antibodies against phospho-JNK, phospho-p38, IkBa (CellSignaling), against vimentin (H84 and V9) (Santa Cruz), and apolyclonal mouse antibody against phospho-ERK1/2 (Sigma-Aldrich) were diluted 1:1000 for Western blotting. The monoclonalanti-b-actin antibody (Sigma-Aldrich) was used at a 1:5000 dilu-tion. Polyclonal rabbit antibody against p65 (Santa Cruz) andmonoclonal mouse antibody against vimentin (Zymed) werediluted 1:50 and 1:400, respectively, for immunofluorescence. Forneutralization of vimentin, host cells were treated with anti-vimentin H84 antibody (Santa Cruz) at a 1:10 dilution immediatelyprior to P. acnes infection. The following chemical inhibitors (dis-solved in DMSO) were used: cytochalasin B (Serva) and cytocha-lasin D (Fluka) at 1 mg ml-1; colcemide (Roche) at 10 ng ml-1 andnocodazole (Sigma) at 100 ng/ml. All inhibitors were added toRWPE1 cells immediately prior to bacterial challenge.

Infection of epithelial cells

HaCaT, HEKa and RWPE1 cells were seeded into 12- or 24-wellplates. P. acnes, cultured for 3 days on Brucella plates, washarvested, washed in the respective cell culture medium anddiluted. Viability of bacteria was assessed by colony-forming unit(cfu) counts; it was ascertained by cfu counts that P. acnes wasviable in all used cell culture media over the indicated infectiontime periods. Cells were infected at a multiplicity of infection (moi)of 50 in a humidified 5% CO2 atmosphere at 37°C. Infectionswere stopped 30 min, 1 h, 2 h, 4 h, 8 h, 24 h or 48 h after initialinfection. For long-term infections (1–3 weeks), media werechanged every third day and cells were split once a week.

Host cell viability

A WST-1 assay (Rapid Cell Proliferation Kit, Merck) was used todetermine host cell viability in the absence and presence ofP. acnes. The assay measured the increased activity of cellularmitochondrial dehydrogenases that can cleave the tetrazoliumdye WST-1 to formazan. Formazan formation was quantified bymeasuring the change in absorbance at 450 nm in a microplatereader (procedure as described by the manufacturer).

Microarray and transcriptome analysis

Microarray experiments were performed as dual-colour hybridi-zations. To compensate for dye-specific effects, a dye-reversal

colour-swap was applied (Churchill, 2002). Samples were iso-lated with TRIzol (Invitrogen) and total RNA prepared accordingto manufacturer’s instructions using glycogen as carrier. TotalRNA yield and purity was assessed using an Agilent 2100 bio-analyser (Agilent Technologies) and a NanoDrop 1000 spectro-photometer (Kisker). RNA labelling was performed with the QuickAmp Labelling Kit (Agilent Technologies). In brief, mRNA wasreverse transcribed and amplified using an oligo-dT-T7-promotorprimer and resulting cRNA was labelled with either Cyanine3-CTP or Cyanine 5-CTP. After precipitation, purification andquantification, 1.25 mg of each labelled cRNA was fragmentedand subsequently hybridized to whole human genome 44k micro-arrays (AMADID-014850), according to the supplier’s protocol(Agilent Technologies). Hybridized microarrays were washedusing the SSC washing protocol (Agilent Technologies). Scan-ning of microarrays was performed with 5 mm resolution usinga DNA microarray laser scanner (Agilent Technologies). Rawmicroarray image data were analysed with the Image Analysis/Feature Extraction software G2567AA (Version A.9.5.1, Agilent).The extracted MAGE-ML files were further analysed with theRosetta Resolver Biosoftware, Build 7.2 (Rosetta Biosoftware).Ratio profiles comprising single hybridizations were combinedin an error-weighted fashion to create ratio experiments. Atwofold change expression cut-off for ratio experiments wasapplied together with anti-correlation of ratio profiles renderingthe microarray analysis highly significant (P-value < 0.01).The data presented in this publication have been depositedin NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Acces-sion No. GSE33731. Ingenuity Pathway Analysis (IPA; http://www.ingenuity.com) software was used for functional analyses oftranscriptome data. The tool identified the biological functionsand/or diseases that were most significant to the expressiondata. Genes which were de-regulated at least twofold and withP-values < 0.01 were included in the analysis and associatedwith biological functions and/or diseases in the Ingenuity Knowl-edge Base. Right-tailed Fisher’s exact test was used to calculatea P-value determining the probability that each biological functionand/or disease assigned to data sets was due to chance alone.

Western immunoblotting

Cells were lysed in Laemmli buffer. Resulting protein extractswere loaded on SDS-PAGE gels; separated proteins weretransferred to PVDF membranes. Membranes were probed withprimary antibodies and HRP-conjugated secondary antibodies(Amersham), and detected with ECL reagent (ICN). The mono-clonal anti-b-actin antibody (Sigma) was used as a loadingcontrol. All blots shown are representative of at least three inde-pendent experiments.

Immunofluorescence

RWPE1 and HaCaT cells were grown on 12 mm coverslips. AfterP. acnes infection, cells were fixed with 4% paraformaldehyde inPBS for 15 min at room temperature. Cells were then stained bypermeabilization with 0.2% Triton X-100 for 15 min and blockingwith 0.2% BSA (Biomol) in PBS for 10 min, all at room tempera-ture. Primary antibodies (anti-P. acnes and anti-p65) were incu-bated for 1 h, followed by a detection step using Cy2- or Cy3-

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conjugated anti-mouse/anti-rabbit IgG secondary antibodies(1:150, 1 h; Jackson Immunoresearch). Actin was stained withPhallodin (Invitrogen). Nuclei were stained with Draq5 (Cell Sig-naling). Coverslips were mounted in Mowiol and analysed byconfocal laser scanning microscopy using a Leica TCS SP andepifluorescence microscopy. Images were taken using appropri-ate excitation and emission filters for the fluorescent dyes used.Overlay images of the single channels were obtained usingAdobe Photoshop. Extra-/intracellular staining was achieved asfollows: after fixation of infected cells with 4% paraformaldehydeand blocking with 0.2% BSA for 10 min, extracellular bacteriawere stained with primary anti-P. acnes antibody for 1 h andsubsequently detected with secondary Cy2-conjugated anti-mouse IgG antibody (1:150, 1 h; Jackson Immunoresearch).Cells were then permeabilized with 0.2% Triton X-100 for 15 minand intracellular bacteria were stained with primary anti-P. acnesantibody for 1 h, and detected with secondary Cy3-conjugatedanti-mouse IgG antibody (1:150, 1 h; Jackson Immunoresearch).Coverslips were mounted in Mowiol and analysed.

For immunofluorescence-staining of human skin and prostatetissues, formalin-fixed, paraffin-embedded tissue samples weredeparaffinized and hydrated through a graded ethanol seriesbefore undergoing heat-induced antigen retrieval for 45 min intarget retrieval solution (Dako). Tissues were then incubated withmouse anti-vimentin antibody at a dilution of 1:400. Slides werethen incubated with Alexa-488-conjugated secondary antibodyand counterstained with DAPI. All tissue samples were obtainedunder a Johns Hopkins Institutional Review Board (IRB)approved protocol.

Transmission electron microscopy

Propionibacterium acnes infected RWPE1 cells were fixed with2.5% glutaraldehyde, post-fixed with 1% osmium tetroxide, con-trasted with uranylacetate and tannic acid, dehydrated andembedded in Ultra-Low Viscosity Embedding Media (Poly-sciences). After polymerization, specimens were cut at 60 nmand contrasted with lead citrate. Specimens were analysed in aLeo 906E transmission electron microscope (Zeiss SMT) using adigital camera (Morada, SIS).

Antibiotic protection assay

To quantify bacterial entry into host cells an antibiotic protectionassay was performed. Both HaCaT and RWPE1 cells wereseeded to 24-well plates prior to infection. At 24 h p.i., 30 mg ml-1

streptomycin/penicilin (Invitrogen) was added for 2 h to kill extra-cellular bacteria. Cells were washed and 1% saponin (Sigma)was added to permeabilize cells, followed by plating of appropri-ate dilutions of lysate on Brucella agar. Assays were performed induplicate wells. Each experiment was repeated at least threetimes. Data were tested for significance using unpaired t-test(GraphPad Prism).

siRNA and plasmid transfection

For RNA interference experiments RWPE1 cells were seededinto 12-well plates (1 ¥ 105 cells well-1) 1 day prior to transfection.Cells were transfected with 10 nM siRNAs using HiPerFecttransfection reagent (Qiagen), according to the manufacturer’s

instructions. Silencing efficiency on protein level was validatedafter 48 h by Western blot. The following siRNAs were used:siVIM 5′-CAGGTTATCAACGAAACTTCT-3′ and AllStars (QiagenNo. 1027281).

For vimentin overexpression keratinocytes were seeded into12-well plates (5 ¥ 104 cells well-1) 1 day prior to transfection.HaCaT cells were transfected with 1 mg of vimentin cDNA(Origene No. SC111054) or control GFP plasmids using 4 ml ofFuGENE (Roche) according to the manufacturer’s instructions.Overexpression of vimentin and GFP was determined after 48 hby Western blot.

Acknowledgements

The authors thank Meike Sörensen for excellent technical assist-ance and Kate Holden-Dye for critically reading the manuscript.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. P. acnes infection did not reduce host cell viability. Cellswere infected with P. acnes P6 at an moi of 50. After 48 h (A) and7 days (B), cell viability was assessed by a WST-1 assay.Fig. S2. Selection of genes strongly de-regulated in HaCaT andRWPE1 cells upon P. acnes infection. Comparison of geneexpression profiles of P. acnes infected and non-infected cells at24 h p.i. (in contrast to Fig. 1, where genes are listed whoseexpression differs between infected HaCaT and infected RWPE1cells). In HaCaT 1269 genes are significantly de-regulated at24 h p.i., 58% of these are upregulated upon infection. In RWPE1

only 688 genes are significantly de-regulated at 24 h p.i. (73%are upregulated). Two hundred and sixty-four genes are com-monly de-regulated in both cell lines upon infection comparedwith non-infected cells. The data illustrates that P. acnes inducesa much stronger response in HaCaT than in RWPE1 cells at 24 hp.i., in terms of the number of de-regulated genes as well as thefold change differences. Upregulated genes are shown in red,while downregulated ones are depicted in green. Non-significantexpression differences are marked in black. All genes depicted inFig. 1 are included in this figure.Fig. S3. Network analysis of de-regulated genes in P. acnes-infected HaCaT and RWPE1 cells. Interactions between at leastthreefold upregulated genes were depicted with STRING(EMBL), which visualizes known and predicted protein–proteininteractions based on reports within the literature, databaseentries and/or experimental evidence. The blue line thicknessindicates the strength of interaction. Extensive networks largelybased on inflammation-associated genes can be found inP. acnes-infected HaCaT cells at 24 h p.i. (A; key nodes are: IL-8,JUN, ICAM1, CXCL1, CCL4, NFKBIA, ATF3, NOS2) and inRWPE1 cells at 7 d p.i. (D; key nodes are: IL-8, JUN, FOS,PTGS2, ICAM1, MMP9, CSF2, CSF3, STAT1, TLR2). In contrast,only few genes de-regulated in RWPE1 at 24 h p.i. (B) and inHaCaT cells at 7 d p.i. (C) were functionally connected.Fig. S4. Anti-VIM antibody and cytoskeleton-interfering inhibitorspartially block P. acnes invasion into RWPE1 cells. (A) P. acnesless efficiently invaded anti-VIM antibody-treated-RWPE1 cells.Non-treated and anti-IgG antibody-treated cells were used ascontrols. (B) Various chemical inhibitors interfering with actinpolymerization (CB, cytochalasin B; CD, cytochalasin D) andmicrotubule polymerization (Col, colcemide; Noco, nocodazole)block P. acnes invasion. Representative results are at least threeindependent experiments are shown.*P � 0.05, **P � 0.01,***P � 0.005.Fig. S5. Vimentin mediates the inflammatory response toP. acnes in RWPE1. Gene expression profiles of P. acnes-infected siVIM RWPE1 cells and infected AllStars knock-downcontrol cells were compared at 24 h p.i. The inflammatoryresponse to P. acnes is strongly reduced in siVIM RWPE1 cells.(A) Interactions between genes downregulated at least fourfoldwere depicted with STRING (EMBL), which visualizes known andpredicted protein–protein interactions based on reports within theliterature, database entries and/or experimental evidence. Thethickness of the blue line connecting genes correlates withthe strength of interaction. (B) Expression fold changes upon VIMknock-down in infected (INF) RWPE1 cells for some prominentinflammation-associated genes are given. The data wereadjusted to fold change differences between non-infected (NI)siVIM RWPE1 cells and NI AllStars knock-down control cells.Table S1. Strongly differentially expressed genes betweenHaCaT and RWPE1 cells. Comparative microarray analysis onnon-infected HaCaT and RWPE1 cells revealed six genes thatare highly expressed in RWPE1 but not in HaCaT. Only geneswith expression fold changes of � 25 were considered here.

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