Propionibacterium acnes infection induces upregulation of inflammatory genes and cytokine secretion in prostate epithelial cells

Background The immune stimulating bacterium Propionibacterium acnes is a frequent colonizer of benign and malignant prostate tissue. To understand the pathogenesis of the earliest phase of this infection, we examined the P. acnes triggered immune response in cultivated prostate epithelial cells. Results Prostate epithelial cells are triggered to secrete IL-6, IL-8 and GM-CSF when infected with P. acnes. The secretion of cytokines is accompanied by NFκB related upregulation of the secreted cytokines as well as several components of the TLR2-NFκB signaling pathway. Conclusions P. acnes has potential to trigger a strong immune reaction in the prostate glandular epithelium. Upon infection of prostate via the retrograde urethral route, the induced inflammatory reaction might facilitate bacterial colonization deeper in the prostate tissue where persistent inflammation may impact the development of prostate diseases as hyperplasia and/or malignancy.


Background
Asymptomatic histological inflammation is a common feature when prostate tissue is subjected to morphological examination. Varying degree of inflammation is present at both benign (prostatic hyperplasia) and malignant (neoplasia) conditions. A growing amount of research supports the idea that chronic prostatic inflammation contributes to gradual transition of normal epithelial cells to malignant cells [1]. For example, many of the genevariants linked to familiar prostate cancer code for proinflammatory cytokines and chemokines [2]. A plethora of microorganisms have been evaluated for their possible involvement in the etiology of prostate inflammation. Many studies purported E. coli and sexually transmitted agents as likely candidates capable of inducing chronic prostatic inflammation [3][4][5]. A Gram-positive bacterium; Propionibacterium acnes (P. acnes) has been reported to be frequently present in various prostatic diseases (as reviewed in [6]) and its presence has been correlated to inflammation in prostate cancer specimens [7][8][9].
P. acnes, a well studied pathogenetic factor in cutaneous disorders like acne vulgaris, has been demonstrated to stimulate monocytes and endothelial cells to secrete proinflammatory cytokines via activation of Toll-like receptor (TLR) 2 [10,11]. In this study we present an in vitro model to study the inflammatory response of prostate derived epithelial cells to P. acnes infection. We report that P. acnes induces upregulation of numerous proinflammatory substances at the mRNA level accompanied by secretion of respective soluble substances such as interleukins 6, 8 and GM-CSF. Components of the TLR2-NFκB signaling pathway were upregulated, suggesting an involvement of this particular pathway for the response. Blocking of the TLR2 with monoclonal antibodies partly reduced the effects. tion with P. acnes. To set experimental conditions as multiplicity of infection (MOI) and useful infection time, we defined the desired criteria as maximal cytokine secretion after 48 h and no visual cellular detachment or celldeath. A MOI of 16-40:1 fulfilled these criteria (data not shown). We therefore decided to use a MOI of 16:1 for the following experiments.

P. acnes induced secretion of IL-8 is partially blocked by α-TLR-2 antibodies
To determine whether the secretion of IL-6, IL-8, and GM-CSF was TLR2-mediated, TLR2 on RWPE-1 cells were blocked with monoclonal anti-TLR2 antibodies at a concentration of 100 ng/ml prior to infection. This particular mab clone has previously been demonstrated to block TLR2 activation in human cells [13]. Secretion of IL-8 was significantly (p = 0.05) reduced when measured 24 h after infection ( Figure 2). No such blocking effect was recognizable 48 h after infection. Levels of IL-6 and GM-CSF were not significantly affected ( Figure 2). Figure  2 shows means for triplicates with the error bars representing the standard deviation.

P. acnes infection induces up-regulation of several cytokines and components of the TLR-2 signaling pathway
The potent P. acnes stimulated effect on secretion of IL-6, IL-8 and GM-CSF prompted us to investigate an array of genes involved in inflammatory signaling pathways. As our main focus is the early responses, we wanted to collect mRNA as early as possible, yet late enough to allow observation of significant regulatory events. We used the cDNA prepared from cells infected for 24 h for comparison with cDNA from uninfected cells. Of the 84 genes analyzed, 20 were more than two-fold upregulated (p = 0.05): CCL2, CSF2 (GM-CSF), CSF3, CXCL10, IFNB1, IL1A, IL6, IL8, IRAK2, IRF1, JUN, LTA, NFKB2, NFK-BIA, REL, RELA, RIPK2, TLR2, TNF, and TICAM1 (Table 1). Only four genes were downregulated (p = 0.05): FOS, HMGB1, TLR4 and UBE2V1 (Table 2).

Discussion
Prostate specimens commonly display signs of chronic histological inflammation, along with occasional acute inflammation. Numerous studies have explored a possible link between prostate inflammation and cancer development and recent reviews of epidemiologic, genetic, and molecular studies have collectively suggested that the two cellular processes may indeed interact [2,[14][15][16]. Exposure to environmental factors such as infectious agents can lead to injury of the prostate and to the development of chronic inflammation [17]. The intrinsic interplay between microbes and urogenital cells is a key feature in the understanding of the microbial involvement in prostate disease. Both Chlamydia and Mycoplasma have been demonstrated to induce IL-6 and IL-8 production in immortalized normal prostate epithelial cells (PNT2) [18,19]. Given the emergence of P. acnes as an infecting agent in prostate tissue [7][8][9] we investigated the effect of the bacterium on prostate epithelial cells of non-malig-nant origin (RWPE-1). In vitro, P. acnes induced considerable secretion of IL-6 and IL-8 and, to a lesser extent, GM-CSF. Secretion of IL8 was shown to be mediated via TLR2, as the receptor blockage with anti-TLR2 monoclonal antibodies reduced its secretion. In contrast, we did not observe any significant reduction in secretion of IL-6 and GM-CSF by blockage of TLR2. Earlier reports present evidence that P. acnes is able to stimulate monocytes and endothelial cells to secrete pro-inflammatory cytokines via activation of TLR2 [10,11]. Our results partly confirm this. Even toll-like receptors 4 and 9 have been implicated in P. acnes mediated immune modulatory effects [20]. Both human and rat prostate epithelial cell lines are known to express TLR2, TLR4, and TLR9 [21,22] and since blockage of TLR2 in our experiment has not totally inhibited cytokine secretion, the involvement of other TLR may also be hypothesized. However, possible TLR4 involvement is compromised by the observed downregulation of the gene expression. Another mechanism may involve auto inducing capability of the released cytokines that generates a self-perpetuating inflammatory process.
The increased secretion of such cytokines was accompanied by concordant mRNA up-regulation. Moreover, the broader analysis of inflammation associated genes revealed that chemokine ligands and pro-inflammatory substances CCL2, CXCL10, TNF-α, TNF-β (lymphotoxin-α), CSF3, IL1-α, and IFN-β were also significantly upregulated. Further studies are required to determine if upregulation of aforementioned genes is accompanied by enhanced cytokine production by prostate epithelial cells. The upregulation of the transcriptional regulators JUN, REL, RIPK2, NFKB2, NFKBIA, IRF1, IRAK2 and the TLR/IL1-receptor co-factor TICAM1 is coherent with earlier studies of TLR2 signaling cascade leading to Fib activation [23,24].
Secretion of IL-6, IL-8 and GM-CSF are central for recruitment and differentiation of macrophages and neutrophils in inflamed tissue [25][26][27]. A prolonged time of increased cytokine levels might have adverse effects on the tissue. P. acnes induced elevation of IL-8 expression in hair-follicle endothelial cells is associated with epidermal hyperplasia and follicular hyperkeratosis in acne vulgaris and psoriasis [28,29]. There is also a correlation between the more pronounced IL-8 expression and dermal angiogenesis [29]. Interestingly, both IL-6 and IL-8 have been suggested as contributors to prostate cancer development. The expression of IL-6 and its receptor has been demonstrated in clinical specimens of both prostate cancer and benign prostate hyperplasia [30], and levels of IL-6 increase in organ-confined tumors [31]. In vitro experiments have shown that IL-6 may play a role in prostate cancer cell growth and differentiation and that it stimulates cell growth of malignant cells [32]. Following pro- longed treatment with IL-6, prostate cancer cells can alter the responsiveness to the cytokine and acquire the ability to proliferate at a higher rate and become more tumorigenic [33,34]. IL-8 has been shown to increase the transcriptional activity of the androgen receptor in prostate cancer cell lines, suggesting a potential role of this chemokine in modulating the transition of prostate cancer to an androgen-independent state [35]. Other studies report that IL-8 contribution to prostate cell proliferation is independent of the androgen receptor [36]. Our data indicate that the prostate epithelium significantly contributes to locally increased levels of both IL-6 and IL-8 when infected with P. acnes, thus potentially promoting adverse effects as increased proliferation and angiogenic activities by autocrine and/or endocrine mechanisms. The pathogenesis of P. acnes in locations other than the hair-follicle is still poorly understood. We currently address questions about its involvement in prostate disease such as prevalence, genetic variability and impact on histological inflammation and neoplasia (Elgh et al., manuscripts in preparation).

Conclusions
In conclusion, we demonstrate that prostate epithelial cells secrete inflammatory cytokines in response to P. acnes, partly through a TLR2-mediated mechanism. We propose that this strong immune-stimulating effect facilitates the bacterial colonization deeper into the prostate tissue where P. acnes can form long-lasting biofilm-like aggregates [7]. A possible mechanism may involve intracellular transport in recruited macrophages, as P. acnes has been demonstrated to withstand degradation by phagocytosing mononuclear cells [37].

Propionibacterium acnes
P. acnes, serotype 1a, isolated from craniopharyngeom fluid was grown in Brain-Heart Infusion Broth + 5% horse serum at 37C under microaerobic conditions. The  bacteria were grown to a density of 10 9 per ml, pelleted and resuspended into sterile PBS.

Cytokine ELISA
RWPE-1 cells were seeded into 24-well plates at a density of 1 × 10 5 cells per well in one ml normal growth medium.
After 48 h, cells were washed in PBS and the medium was changed to DMEM without FCS and PEST. Cells were infected with P. acnes at a MOI of 16:1 and immediate close contact between bacteria and cells was achieved by centrifugation of the flask for 10 min at 700 g. Noninfected cells were used as controls.
Supernatants were harvested after 24 h and 48 h. Supernatants were cleared from particles by centrifugation 10 min at 12000 g, stored at -20C and later assayed for IL-6, IL-8 and GM-CSF by ELISA (R&D systems, Minneapolis, Minnesota) according to manufacturer's instruction.

RNA preparation and Reverse Transcription PCR
Cells were seeded at a density of 1 × 10 6 in a 25 cm 2 culture flask in normal growth medium. After 48 h, cells were washed in PBS and the medium were changed to DMEM without FCS and PEST. Cells were infected with P. acnes at a MOI of 16:1 and immediate close contact between bacteria and cells were achieved by centrifugation of the flask for 10 min at 700 g. Total RNA was prepared after 0 h and 24 h using RNeasy Mini kit (Qiagen, Hilden, Germany) with the on-column DNase treatment step according to manufacturer's instruction. Cells were trypsinised using 0,05% (w/v) trypsin/EDTA, lysed in 350 μl RTL buffer and homogenized in a TissueLyser with Stainless steel Beads, 5 mm (Qiagen, Hilden, Germany).

Real-time Quantitative PCR
Gene expression analysis measuring transcription of 84 inflammation associated genes was conducted using the RT 2 Profiler PCR Array, Human Toll-Like Receptor Signaling Pathway PAHS-018A (SABiosciences, Frederick, MD, USA) according to manufacturer's instruction. Realtime PCR detection was performed with an IQ™5 instrument (Bio-Rad, Hercules, CA, USA).
Complete list of genes analyzed by the array can be found at: http://www.SABiosciences.com

Data Analysis
Relative gene expression was calculated with the ΔΔC t method in the web-based software package for RT 2 Profiler PCR array systems (SABiosciences, Frederick, MD, USA).

Statistical Methods
Due to the small sample size (n = 3), a permutation test was used to test possible regulation [38]. A null hypothesis corresponding to no regulation was tested for each gene and each protein concentration and rejected for p = 0.05.
Authors' contributions JBD carried out the tissue culture infections, the mRNA assays and the protein quantification. OA participated in the experimental design. PB performed the statistical analysis. FE initiated the study and participated in its design. JO participated in the design of the study, performed pilot studies of experimental conditions and drafted the manuscript. All authors read and approved the final manuscript. Semiconfluent RWPE-1 monocell-layers were infected with P. acnes at a MOI of 16:1. After 24 h infection, the cells were harvested, mRNA was collected and cDNA was prepared. The cDNA corresponding to 84 inflammation-associated genes was quantified with qRT-PCR and compared with cDNA prepared from non-infected cells. Inclusion criteria: > 2-fold down-regulation, (p = 0.05).