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  and malignant  conditions.
a growing amount of research supports the idea that chronic prostatic inflammation contributes to gradual transition of normal epithelial cells to malignant cells  <cit> .
for example, many of the gene-variants linked to familiar prostate cancer code for proinflammatory cytokines and chemokines  <cit> .
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  <cit> .
a gram-positive bacterium; propionibacterium acnes  has been reported to be frequently present in various prostatic diseases  and its presence has been correlated to inflammation in prostate cancer specimens  <cit> .
p. acnes, a well studied pathogenetic factor in cutaneous disorders like acne vulgaris, has been demonstrated to stimulate monocytes and endothelial cells to secrete pro-inflammatory cytokines via activation of toll-like receptor   <dig>  <cit> .
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 pro-inflammatory substances at the mrna level accompanied by secretion of respective soluble substances such as interleukins  <dig>   <dig> 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 tlr <dig> with monoclonal antibodies partly reduced the effects.
RESULTS
pilot studies to define experimental conditions for p. acnes infection of epithelial cells
secretion of cytokines is one of the end results of innate immune response at a cellular level.
we therefore assessed the secretion of three key cytokines, il- <dig>  il- <dig> and gm-csf  from the prostate-derived epithelial cell-line rwpe- <dig> in response to infection with p. acnes.
to set experimental conditions as multiplicity of infection  and useful infection time, we defined the desired criteria as maximal cytokine secretion after  <dig> h and no visual cellular detachment or cell-death.
a moi of 16-40: <dig> fulfilled these criteria .
we therefore decided to use a moi of 16: <dig> for the following experiments.
prostate epithelial cells secrete il- <dig>  il- <dig>  and gm-csf in response to propionibacterium acnes infection
secretion of cytokines il- <dig>  il- <dig> and gm-csf by rwpe- <dig> was measured  <dig> h and  <dig> h after infection with p. acnes.
<dig> h after infection, the levels of secreted il- <dig>  il- <dig> and gm-csf were:  <dig>  ±  <dig> ,  <dig>  ±  <dig> , and  <dig>  ±  <dig>  , respectively.
the corresponding values from the uninfected control cells were:  <dig>  ±  <dig>  , not detectable, not detectable .
<dig> h after infection, the concentrations increased to:  <dig>  ±  <dig> ,  <dig>  ±  <dig> , and  <dig>  ±  <dig>  .
uninfected:  <dig>  ±  <dig> ,  <dig>  ±  <dig> , and  <dig>  ±  <dig>  .
the diagram shows means for triplicates with the error bars representing the standard deviation  <cit>  .
p. acnes induced secretion of il- <dig> is partially blocked by α-tlr- <dig> antibodies
to determine whether the secretion of il- <dig>  il- <dig>  and gm-csf was tlr2-mediated, tlr <dig> on rwpe- <dig> cells were blocked with monoclonal anti-tlr <dig> antibodies at a concentration of  <dig> ng/ml prior to infection.
this particular mab clone has previously been demonstrated to block tlr <dig> activation in human cells  <cit> .
secretion of il- <dig> was significantly  reduced when measured  <dig> h after infection .
no such blocking effect was recognizable  <dig> h after infection.
levels of il- <dig> and gm-csf were not significantly affected .
figure  <dig> 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- <dig> signaling pathway
the potent p. acnes stimulated effect on secretion of il- <dig>  il- <dig> 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  <dig> h for comparison with cdna from uninfected cells.
of the  <dig> genes analyzed,  <dig> were more than two-fold upregulated : ccl <dig>  csf <dig> , csf <dig>  cxcl <dig>  ifnb <dig>  il1a, il <dig>  il <dig>  irak <dig>  irf <dig>  jun, lta, nfkb <dig>  nfkbia, rel, rela, ripk <dig>  tlr <dig>  tnf, and ticam <dig> .
only four genes were downregulated : fos, hmgb <dig>  tlr <dig> and ube2v <dig> .
semiconfluent rwpe- <dig> monocell-layers were infected with p. acnes at a moi of 16: <dig>  after  <dig> h infection, the cells were harvested, mrna was collected and cdna was prepared.
the cdna corresponding to  <dig> inflammation-associated genes was quantified with qrt-pcr and compared with cdna prepared from non-infected cells.
inclusion criteria: > 2-fold up-regulation, .
semiconfluent rwpe- <dig> monocell-layers were infected with p. acnes at a moi of 16: <dig>  after  <dig> h infection, the cells were harvested, mrna was collected and cdna was prepared.
the cdna corresponding to  <dig> inflammation-associated genes was quantified with qrt-pcr and compared with cdna prepared from non-infected cells.
inclusion criteria: > 2-fold down-regulation, .
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  <cit> .
exposure to environmental factors such as infectious agents can lead to injury of the prostate and to the development of chronic inflammation  <cit> .
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- <dig> and il- <dig> production in immortalized normal prostate epithelial cells   <cit> .
given the emergence of p. acnes as an infecting agent in prostate tissue  <cit>  we investigated the effect of the bacterium on prostate epithelial cells of non-malignant origin .
in vitro, p. acnes induced considerable secretion of il- <dig> and il- <dig> and, to a lesser extent, gm-csf.
secretion of il <dig> was shown to be mediated via tlr <dig>  as the receptor blockage with anti-tlr <dig> monoclonal antibodies reduced its secretion.
in contrast, we did not observe any significant reduction in secretion of il- <dig> and gm-csf by blockage of tlr <dig>  earlier reports present evidence that p. acnes is able to stimulate monocytes and endothelial cells to secrete pro-inflammatory cytokines via activation of tlr <dig>  <cit> .
our results partly confirm this.
even toll-like receptors  <dig> and  <dig> have been implicated in p. acnes mediated immune modulatory effects  <cit> .
both human and rat prostate epithelial cell lines are known to express tlr <dig>  tlr <dig>  and tlr <dig>  <cit>  and since blockage of tlr <dig> in our experiment has not totally inhibited cytokine secretion, the involvement of other tlr may also be hypothesized.
however, possible tlr <dig> 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 ccl <dig>  cxcl <dig>  tnf-α, tnf-β , csf <dig>  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, ripk <dig>  nfkb <dig>  nfkbia, irf <dig>  irak <dig> and the tlr/il1-receptor co-factor ticam <dig> is coherent with earlier studies of tlr <dig> signaling cascade leading to fib activation  <cit> .
secretion of il- <dig>  il- <dig> and gm-csf are central for recruitment and differentiation of macrophages and neutrophils in inflamed tissue  <cit> .
a prolonged time of increased cytokine levels might have adverse effects on the tissue.
p. acnes induced elevation of il- <dig> expression in hair-follicle endothelial cells is associated with epidermal hyperplasia and follicular hyperkeratosis in acne vulgaris and psoriasis  <cit> .
there is also a correlation between the more pronounced il- <dig> expression and dermal angiogenesis  <cit> .
interestingly, both il- <dig> and il- <dig> have been suggested as contributors to prostate cancer development.
the expression of il- <dig> and its receptor has been demonstrated in clinical specimens of both prostate cancer and benign prostate hyperplasia  <cit> , and levels of il- <dig> increase in organ-confined tumors  <cit> .
in vitro experiments have shown that il- <dig> may play a role in prostate cancer cell growth and differentiation and that it stimulates cell growth of malignant cells  <cit> .
following prolonged treatment with il- <dig>  prostate cancer cells can alter the responsiveness to the cytokine and acquire the ability to proliferate at a higher rate and become more tumorigenic  <cit> .
il- <dig> 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  <cit> .
other studies report that il- <dig> contribution to prostate cell proliferation is independent of the androgen receptor  <cit> .
our data indicate that the prostate epithelium significantly contributes to locally increased levels of both il- <dig> and il- <dig> 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 .
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  <cit> .
a possible mechanism may involve intracellular transport in recruited macrophages, as p. acnes has been demonstrated to withstand degradation by phagocytosing mononuclear cells  <cit> .

 asymptomatic histological inflammation is a common feature when prostate tissue is subjected to morphological examination. varying degree of inflammation is present at both benign and malignant conditions. a growing amount of research supports the idea that chronic prostatic inflammation contributes to gradual transition of normal epithelial cells to malignant cells. for example, many of the gene-variants linked to familiar prostate cancer code for proinflammatory cytokines and chemokines. 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. a gram-positive bacterium; propionibacterium acnes has been reported to be frequently present in various prostatic diseases and its presence has been correlated to inflammation in prostate cancer specimens. p. acnes, a well studied pathogenetic factor in cutaneous disorders like acne vulgaris, has been demonstrated to stimulate monocytes and endothelial cells to secrete pro-inflammatory cytokines via activation of toll-like receptor. 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 pro-inflammatory substances at the mrna level accompanied by secretion of respective soluble substances such as interleukins 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 tlr with monoclonal antibodies partly reduced the effects.  pilot studies to define experimental conditions for p. acnes infection of epithelial cells secretion of cytokines is one of the end results of innate immune response at a cellular level. we therefore assessed the secretion of three key cytokines, il- il- and gm-csf from the prostate-derived epithelial cell-line rwpe- in response to infection with p. acnes. to set experimental conditions as multiplicity of infection and useful infection time, we defined the desired criteria as maximal cytokine secretion after h and no visual cellular detachment or cell-death. a moi of 16-40: fulfilled these criteria. we therefore decided to use a moi of 16: for the following experiments. prostate epithelial cells secrete il- il- and gm-csf in response to propionibacterium acnes infection secretion of cytokines il- il- and gm-csf by rwpe- was measured h and h after infection with p. acnes. h after infection, the levels of secreted il- il- and gm-csf were: ±, ±, and ±, respectively. the corresponding values from the uninfected control cells were: ±, not detectable, not detectable. h after infection, the concentrations increased to: ±, ±, and ±. uninfected: ±, ±, and ±. the diagram shows means for triplicates with the error bars representing the standard deviation. p. acnes induced secretion of il- is partially blocked by α-tlr- antibodies to determine whether the secretion of il- il- and gm-csf was tlr2-mediated, tlr on rwpe- cells were blocked with monoclonal anti-tlr antibodies at a concentration of ng/ml prior to infection. this particular mab clone has previously been demonstrated to block tlr activation in human cells. secretion of il- was significantly reduced when measured h after infection. no such blocking effect was recognizable h after infection. levels of il- and gm-csf were not significantly affected. figure 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- signaling pathway the potent p. acnes stimulated effect on secretion of il- il- 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 h for comparison with cdna from uninfected cells. of the genes analyzed, were more than two-fold upregulated: ccl csf, csf cxcl ifnb il1a, il il irak irf jun, lta, nfkb nfkbia, rel, rela, ripk tlr tnf, and ticam. only four genes were downregulated: fos, hmgb tlr and ube2v. semiconfluent rwpe- monocell-layers were infected with p. acnes at a moi of 16: after h infection, the cells were harvested, mrna was collected and cdna was prepared. the cdna corresponding to inflammation-associated genes was quantified with qrt-pcr and compared with cdna prepared from non-infected cells. inclusion criteria: > 2-fold up-regulation,. semiconfluent rwpe- monocell-layers were infected with p. acnes at a moi of 16: after h infection, the cells were harvested, mrna was collected and cdna was prepared. the cdna corresponding to inflammation-associated genes was quantified with qrt-pcr and compared with cdna prepared from non-infected cells. inclusion criteria: > 2-fold down-regulation,. 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. exposure to environmental factors such as infectious agents can lead to injury of the prostate and to the development of chronic inflammation. 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- and il- production in immortalized normal prostate epithelial cells. given the emergence of p. acnes as an infecting agent in prostate tissue we investigated the effect of the bacterium on prostate epithelial cells of non-malignant origin. in vitro, p. acnes induced considerable secretion of il- and il- and, to a lesser extent, gm-csf. secretion of il was shown to be mediated via tlr as the receptor blockage with anti-tlr monoclonal antibodies reduced its secretion. in contrast, we did not observe any significant reduction in secretion of il- and gm-csf by blockage of tlr earlier reports present evidence that p. acnes is able to stimulate monocytes and endothelial cells to secrete pro-inflammatory cytokines via activation of tlr. our results partly confirm this. even toll-like receptors and have been implicated in p. acnes mediated immune modulatory effects. both human and rat prostate epithelial cell lines are known to express tlr tlr and tlr and since blockage of tlr in our experiment has not totally inhibited cytokine secretion, the involvement of other tlr may also be hypothesized. however, possible tlr 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 ccl cxcl tnf-α, tnf-β, csf 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, ripk nfkb nfkbia, irf irak and the tlr/il1-receptor co-factor ticam is coherent with earlier studies of tlr signaling cascade leading to fib activation. secretion of il- il- and gm-csf are central for recruitment and differentiation of macrophages and neutrophils in inflamed tissue. a prolonged time of increased cytokine levels might have adverse effects on the tissue. p. acnes induced elevation of il- expression in hair-follicle endothelial cells is associated with epidermal hyperplasia and follicular hyperkeratosis in acne vulgaris and psoriasis. there is also a correlation between the more pronounced il- expression and dermal angiogenesis. interestingly, both il- and il- have been suggested as contributors to prostate cancer development. the expression of il- and its receptor has been demonstrated in clinical specimens of both prostate cancer and benign prostate hyperplasia, and levels of il- increase in organ-confined tumors. in vitro experiments have shown that il- may play a role in prostate cancer cell growth and differentiation and that it stimulates cell growth of malignant cells. following prolonged treatment with il- prostate cancer cells can alter the responsiveness to the cytokine and acquire the ability to proliferate at a higher rate and become more tumorigenic. il- 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. other studies report that il- contribution to prostate cell proliferation is independent of the androgen receptor. our data indicate that the prostate epithelium significantly contributes to locally increased levels of both il- and il- 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.  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. a possible mechanism may involve intracellular transport in recruited macrophages, as p. acnes has been demonstrated to withstand degradation by phagocytosing mononuclear cells. 
to understand the pathogenesis of the earliest phase of this infection, we examined the p. acnes triggered immune response in cultivated prostate epithelial cells. prostate epithelial cells are triggered to secrete il- il- and gm-csf when infected with p. acnes. p. acnes has potential to trigger a strong immune reaction in the prostate glandular epithelium. the immune stimulating bacterium propionibacterium acnes is a frequent colonizer of benign and malignant prostate tissue. 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.
 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.  prostate epithelial cells are triggered to secrete il- il- 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.  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. 