Fig 1.
Production of cytokines by human prostate epithelial cells (RWPE-1) stimulated with T. vaginalis.
(A) RWPE-1 cells were incubated with T. vaginalis (R:T = 1:10) for 3 hr. Production of cytokines was determined by ELISA assays. (B) Cytokine mRNAs were determined by real-time PCR. Data are means ± SD of four independent experiments. *p<0.05 versus untreated RWPE-1 (C).
Fig 2.
Migration of monocytes (THP-1) induced by conditioned medium of human prostate epithelial cells (RWPE-1) stimulated with T. vaginalis.
THP-1 cells (1 × 105) were added to the upper compartment of Boyden chambers, and the following chemicals or conditioned media were added to lower chambers; recombinant human (rh) CCL2 (100 ng/ml), rhCXCL8 (100 ng/ml), rhIL-6 (100 ng/ml), conditioned medium of RWPE-1 cells alone (CM), conditioned medium of RWPE-1 cells stimulated with T. vaginalis (TCM), or TCM preincubated with 100 ng/ml of neutralizing antibody to IL-6. After 6 hr, cells that had migrated to the lower chambers were counted. Data are means ± SD of four independent experiments. *p<0.05 versus untreated THP-1 cells (C). NS = not statistically significant.
Fig 3.
Polarization of PMA-THP-1 macrophages to the M2-like macrophage phenotype by conditioned medium of human prostate epithelial cells (RWPE-1) stimulated with T. vaginalis.
THP-1 monocytes were treated with 100 nM PMA for 24 hr and then cultured in absence of PMA for 72 hr (M0). The cells were then treated with 100 ng/ml LPS and 20 ng/ml rhIFN-γ for M1 macrophages (M1), 20 ng/ml rhIL-4 and 20 ng/ml rhIL-13 for M2 macrophages (M2), 5 ng/ml rhIL-6, CM, or TCM for 72 hr. IL-10, TGF-β, CD36 and arginase-1 was determined as M2 macrophage markers. (A) Production of IL-10 and TGF-β measured by ELISA assays. (B) Expression of CD36 and arginase-1 was determined by western blot. Graph represent densitometric analysis (means of three independent western blot experiments). (C) IL-12, CXCL-10, CCL2 and NOS2 were measured as M1 macrophage markers. Production of IL-12, CXCL-10 and CCL2 measured by ELISA assays. Expression of NOS2 was analyzed by real-time PCR. Data are means ± SD of three independent experiments. *p<0.05 versus THP-1-derived macrophage (M0). CM: conditioned medium of RWPE-1 alone, TCM: conditioned medium of RWPE-1 stimulated with T. vaginalis.
Fig 4.
Polarization of human monocyte-derived macrophages (HMDM) to the M2-like macrophage phenotype is induced by conditioned medium of human prostate epithelial cells (RWPE-1) stimulated with T. vaginalis.
Human peripheral blood mononuclear cells (PBMCs) incubated with 50 ng/ml M-CSF for 2 hr to allow monocyte adhesion. Non-adherent cells were removed and adherent monocytes were treated with 50 ng/ml M-CSF for 6 days for macrophage differentiation, and then cultured with 100 ng/ml LPS and 20 ng/ml rhIFN-γ for M1 macrophages (M1), 20 ng/ml rhIL-4 and 20 ng/ml rhIL-13 for M2 macrophages (M2), CM, or TCM for 48 hr. Macrophages were identified with M1 markers such as IL-12, CCL2, TNF-α and NOS2, and M2 markers such as IL-10, TGF-β, CD36 and arginase-1. (A) Production of IL-10 and TGF-β was measured by ELISA assay. Expression of CD36 was evaluated by real-time PCR. (B) Production of IL-12, CCL2 and TNF-α was measured by ELISA assay. (C) mRNA expression of arginase-1 and NOS2 were detected by RT-PCR. Densitometry of mRNA bands were quantified by three independent experiments. Data are means ± SD of three independent experiments. *p<0.05 versus HMDM (M0). CM: conditioned medium of RWPE-1 cells alone, TCM: conditioned medium of RWPE-1 cells stimulated with T. vaginalis.
Fig 5.
Proliferation of macrophages in response to conditioned medium of human prostate epithelial cells (RWPE-1) stimulated with T. vaginalis.
THP-1 monocytes were treated with 100 nM PMA for 24 hr to obtain M0 macrophages and then cultured in complete medium for 72 hr. The cells were then treated with 100 ng/ml LPS and 20 ng/ml IFN-γ for M1 macrophages, 20 ng/ml IL-4 and 20 ng/ml IL-13 for M2 macrophages, 5 ng/ml IL-6, CM, or TCM for 72 hr. Human peripheral blood mononuclear cells (PBMCs) incubated with 50 ng/ml M-CSF for 2 hr to allow monocyte adhesion. Non-adherent cells were removed and adherent monocytes were treated with 50 ng/ml M-CSF for 6 days for macrophage differentiation, and then cultured with CM, or TCM for 48 hr. (A) Proliferation of THP-1-derived macrophages was measured by CCK-8 assay. (B) mRNA expression of cyclin D1 and c-Myc was determined by RT-PCR. Densitometry of mRNA bands were quantified by three independent experiments. (C) Expression of PCNA, cyclin D1 and Bcl-2 was determined by western blot. Graph represent densitometric analysis (means of three independent western blot experiments). (D) Immunofluorescence assays were performed using an anti-c-Myc primary antibody and an anti-rabbit Alexa Fluor-594-labeled secondary antibody (DNA stained with DAPI/blue) and images were selected from five random fields. (E) Proliferation of human PBMCs-derived macrophages was measured by CCK-8 assay. (F) Expression of Bcl-2 and c-Myc was determined by RT-PCR. Densitometry of mRNA bands were quantified by three independent experiments. Data are means ± SD of three independent experiments. *p<0.05 versus THP-1-derived macrophages or HMDM (M0). CM: conditioned medium of RWPE-1 alone, TCM: conditioned medium of RWPE-1 stimulated with T. vaginalis.
Fig 6.
Activation of the IL-6R/JAK2/STAT3 signal pathway in THP-1-derived macrophages is induced by conditioned medium of human prostate epithelial cells (RWPE-1) stimulated with T. vaginalis.
THP-1 cells were treated with 100 nM PMA for 24 hr, and cultured with rhIL-6 (5 ng/ml), CM, or TCM for 24 hr. (A) Expression of IL-6 receptor was examined by immunofluorescence using an anti-IL-6R primary antibody and an anti-rabbit Alexa Fluor-594-labled secondary antibody and detected three randomly chosen fields. (B) Gp130, p-JAK2, p-STAT3 and iNOS were determined by western blot. Graph represent densitometric analysis (means of three independent western blot experiments). (C) Expression of IL-6R, STAT3 and CD206 mRNA was evaluated by real-time PCR. Data are means ± SD of three independent experiments. *p<0.05 versus THP-1-derived macrophages (M0). CM: conditioned medium of RWPE-1 alone, TCM: conditioned medium of RWPE-1 stimulated with T. vaginalis.
Fig 7.
Involvement of the IL-6 signaling pathway in M2-type macrophage polarization of THP-1 cells.
THP-1 cells were treated with 100 nM PMA for 24 hr, and cultured with CM, or TCM for 72 hr. To examine involvement of IL-6 signaling in M2 macrophage polarization, M0 macrophages were pretreated with anti-gp-130 antibody (100 ng/ml) or ruxolitinib (JAK inhibitor, 10 μM) before addition of TCM. (A) Production of IL-10 and TGF-β as M2 macrophage markers was measured by ELISA assay. (B) Production of IL-12 and CXCL-10 as M1 macrophage markers was measured by ELISA assays. (C) Proliferation of macrophages was evaluated by CCK-8 assays. (D) Expression of p-JAK2, p-STAT3, cyclin D1 and PCNA protein was determined by western blot. Graph represent densitometric analysis (means of three independent western blot experiments). (E) Immunofluorescence assays were performed using an anti-c-Myc primary antibody and an anti-rabbit Alexa Fluor-594-labled secondary antibody and images selected from five random fields. Data are means ± SD of three independent experiments. *p<0.05 versus THP-1-derived macrophage (M0). #p<0.05 versus conditioned medium of RWPE-1 stimulated with T. vaginalis (TCM). NS = not statistically significant. CM: conditioned medium of RWPE-1 alone, TCM: conditioned medium of RWPE-1 stimulated with T. vaginalis, Rux: Ruxolitinib.
Fig 8.
Increased CD206 expression in prostate tissue is induced by TRAMP-C2 cells co-injected with T. vaginalis.
C57BL/6 mice (n = 5/group) were injected in the prostate with TRAMP-C2 mouse cancer cell line cells (without or with live T. vaginalis). After four weeks, prostate tissues were harvested. (A) Experimental scheme for the animal study. Representative macroscopic images show the resected prostate tissues from mice. (B) Expression of CD206 was observed by immunohistochemistry (IHC). For negative control (NC), only secondary antibody was reacted (insets). Red number in IHC panels was represented as a numerical score from 0 to 10 based on proportion of clusters of immunopositive cells. (C) Data represented as histograms. Bars, means ± SD of five independent experiments (*p = 0,0317 with the one-tailed Mann-Whitney U test). WT: wild-type, NC: negative control.
Fig 9.
Proliferation of prostate cancer cells in response to conditioned medium of M2-like macrophages cultured with TCM.
To prepare the conditioned medium of M2-like macrophages, THP-1-derived macrophages were incubated in RPMI1640 media containing 20% TCM with or without pretreatment of gp130 antibody for 72 hr, and the culture supernatants were collected. The supernatants was named αgp130+M-TCM and M-TCM. Prostate cancer cells (PC3, DU145 and LNCaP) were cultured with CM, TCM, M-CM, M-TCM or αgp130+M-TCM for 24 hr. (A) Proliferation of the prostate cancer cells was measured by CCK-8 assay. (B) Cyclin D1 and PCNA were determined by western blot. Graph represent densitometric analysis (means of three independent western blot experiments). Data are means ± SD of three independent experiments. *p<0.05 versus untreated prostate cancer cells (C). #p<0.05 versus conditioned medium of RWPE-1 stimulated with T. vaginalis (TCM). †p<0.05 versus conditioned medium of RWPE-1 stimulated with T. vaginalis (TCM). CM: conditioned medium of RWPE-1 alone, TCM: conditioned medium of RWPE-1 stimulated with T. vaginalis, M-CM: conditioned medium of THP-1-derived macrophage stimulated with CM, M-TCM: conditioned medium of THP-1-derived macrophage stimulated with TCM, αgp130+M-TCM: conditioned medium of THP-1-derived macrophage stimulated with TCM after pretreatment of gp130 antibody.
Fig 10.
Migration of prostate cancer cells is induced by conditioned medium of M2-like macrophages cultured with TCM.
Prostate cancer cells (PC3, DU145 and LNCaP) were cultured with CM, TCM, M-CM, M-TCM or αgp130+M-TCM for 24 hr. (A) Migration of the prostate cancer cells was assessed by wound healing assays. Representative images photographed by light microscope. (B) Migration was measured as: {(mean wounded breadth–mean remaining breadth)} / mean wounded breadth × 100. Data are means ± SD of three independent experiments. *p<0.05 versus untreated prostate cancer cells (C). #p<0.05 versus conditioned medium of RWPE-1 stimulated with T. vaginalis (TCM). CM: conditioned medium of RWPE-1 alone, TCM: conditioned medium of RWPE-1 stimulated with T. vaginalis, M-CM: conditioned medium of THP-1-derived macrophages stimulated with CM, M-TCM: conditioned medium of THP-1-derived macrophages stimulated with TCM. αgp130+M-TCM: conditioned medium of THP-1-derived macrophage stimulated with TCM after pretreatment of gp130 antibody.