Mycoplasma genitalium Lipoproteins Induce Human Monocytic Cell Expression of Proinflammatory Cytokines and Apoptosis by Activating Nuclear Factor κB

This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-κB (NF-κB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-κB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-κB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-κB.


INTRODUCTION
Mycoplasma genitalium is a cell wall-less bacterial pathogen with the distinction of possessing the smallest known genome of any self-replicating cells, it was discovered in 1981 when isolated from the urethra of two men with nongonococcal urethritis [1]. The exact mode of infection and pattern of diseases caused by M. genitalium still remains to be solved, but the pathogen is presumed to be sexually transmitted, and the infections often appear to be chronic and asymptomatic [2,3]. Most investigations have concerned male urethritis patients but M. genitalium has also been implicated in pelvic inflammatory disease, pneumonia, arthritides, and AIDS [4][5][6].
It is known that M. genitalium adheres to host cells by the terminal tip organelles, which is the first step for the pathogenicity. However, the exact molecular pathogenesis of M. genitalium is vague [7]. It has been suggested that during mycoplasmal infection, the damage to host cells is not caused by direct lesion but by immunopathogenesis [8-accelerating cell death, which depends on different cell lines and different stimuli.
No research has been reported on the pathogenicity of M. genitalium to human monocytes, however; and we have studied whether M. genitalium LP could induce human monocytes cell line THP-1 to express proinflammatory cytokines and apoptosis. Therefore, the present study is aimed to investigate whether M. genitalium LP-induced apoptosis in monocytes is associated with the activation of NF-κB, and evaluate the effect of the specific inhibitor pyrrolidine ditheiocarbamate (PDTC) on the production of proinflammatory cytokines and apoptosis.

Cell culture and stimulation
The human monocytic cell line THP-1 (China Center for Type Culture Collection, Wuhan University, Wuhan, China) was cultured in RPMI 1640 medium (Hyclone, Utah, USA) supplemented with 2 mmol/L L-glutamine and 10% heatinactivated fetal bovine serum (FBS, Hyclone, USA) at 37 • C with 5% CO 2 . Cells were cultivated in 1% FBS overnight in 24-microwell plates (Costor, NY, USA) at a concentration of 10 5 cells/mL before stimulation. 0.5 to 5 μg/mL of LP or 0.1 μg/mL of lipopolysaccharide (LPS, Sigma-Aldrich, Mo, USA) was added into the medium for 24 hours. To evaluate the effect of PDTC, cells were preincubated with 25 μM PDTC (Sigma-Aldrich) for 30 minutes before stimulation.

Mycoplasma culture and LP preparation
M. genitalium (G-37 strain, ATCC) was cultivated in modified SP-4 medium until the beginning of the stationary phase and then pelleted by centrifugation. The preparation of LP and the aqueous phase (used as a control) followed the methods described previously [16]. Lipoproteins in the Triton X-114 phase were precipitated by methanol and used for stimulation after being suspended in sterile PBS. The protein concentration was determined by using a bicinchoninic acid kit (Pierce, Oud Beijerlands, the Netherlands). The preparations were preincubated with 100 μg/mL polymyxin B for 2 hours prior to stimulation in order to eliminate the possibility of endotoxin contamination during the process of its preparation.

Assay for cytokine detection
THP-1 cells were cultivated and stimulated in 24-well tissue culture plates as described above. Being stimulated after 24 hours, the cells were lysed by two consecutive cycles of freezing/thawing, thus the samples represented the total amount of cytokines produced (both intracellular and those that have released into the supernatant). The cytokines concentration was measured by using human TNF-α, IL-1β, IL-6 ELISA kits (Jingmei Biotech, Shenzhen, China). Supernatants were used for cytokine determination by the quantitative "sandwich" ELISA technique, using monoclonal antibodies specific for the tested cytokines. The detection limit of the ELISA kits for TNF-α, IL-1β, and IL-6 were 7 pg/mL, 2 pg/mL, and 2 pg/mL, respectively. The assays were performed in accordance with the manufacturer's instructions.

Nuclear protein preparation and electrophoretic mobility gel shift assay (EMSA)
THP-1 cells were cultured in a dish in 1% FBS for 24 hours and then stimulated with LP (3 μg/mL) at indicated time intervals. The cells were collected on ice before isolation of nuclear extracts by the protocol reported by Muller and Homaidan [17,18]. Briefly, the cells (10 6 /mL) were washed with ice-cold PBS, suspended in 200 μL of lysis buffer (10 mM HEPES [pH7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT) and allowed to swell on ice for 15 minutes, and then 12.5 μL of 10% Nonidet P-40 was added. The tube was then mixed thoroughly with a Vortex mixer for 10 seconds prior to centrifugation (20,000 g) at 4 • C for 8 minutes. The nuclear pellets thus obtained were resuspended in 25 μL of ice-cold nuclear extraction buffer (20 mM HEPES [pH7.9], 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT) and kept on ice for 15 minutes with intermittent agitation. The samples were subjected to centrifugation for 5 minutes at 4 • C, and the supernatant was stored at −70 • C after measurement of its protein content with the Bio-Rad protein assay kit (Bio-Rad, Munich, Germany). The detection of the activated NF-κB in the nuclei of unstimulated and stimulated cells was completed by using a biotin-labeled EMSA kit (Viagene, Ningbo , China), according to the manufacture's instruction. The consensus NF-κB oligonucleotides included in the kit was 5 -AGT TGA GGG GAC TTT CCC AGG C-3 .
Yimou Wu et al.

Measurement of cell apoptosis
Two different techniques were used as follows. (1) AO-EB staining, an exclusion dye method which enables to differentiate between live, early-apoptotic, late-apoptotic, and necrotic cells [19]. At 12 hours after induction of apoptosis, cells were collected, centrifuged at 500 g for 10 minutes, and resuspended in 100 μL PBS. Samples of 25 μL from each culture were stained with AO-EB (final concentration 1 μg/mL for each AO and EB), and observed under a fluorescence microscope. At least 200 cells were randomly counted in each sample (in duplicates), and the percentage of apoptotic cells was calculated; (2) Annexin-V-FITC-PI staining, which detects the exposure of phosphatidylserine (PS) to the external leaflet of the plasma membrane in early apoptosis, and enables to differentiate between live, early apoptotic, and dead cells (it does not differentiate between late apoptotic and necrotic cells) [20]. At 12 hours after induction of apoptosis, cells were washed with PBS, centrifuged and resuspended in 400 μL of a binding bufter (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl 2 , PH7.4). Annexin-V-FITC (5 μL; 10 μg/mL) was added to a sample of 195 μL of cell suspension, mixed, incubated for 10 minutes at room temperature in the dark, washed with PBS, and resuspended in 190 μL of binding buffer containing 10 μL of PI (1 μg/mL). The double-stained cells were analyzed by the FACS within 10 minutes (10 4 cells/sample).
In all measurements of apoptosis, the percentage of cells that underwent apoptosis was determined by subtracting the percentage of spontaneous apoptosis (unstimulated cells) from the total apoptosis (stimulated cells).

Statistical analysis
Data obtained from the three independent experiments were expressed as mean±SE. The data were analyzed by a one-way ANOVA test followed by an independent-samples t test using SPSS software. A P-value of less than 0.05 was considered significant.

Marked production of cytokines in THP-1 by M. genitalium LP
LP prepared from M. genitalium stimulated THP-1 cells to produce TNF-α, IL-1β, and IL-6 in dose-dependent manner ( Figure 1) Interestingly, when the concentration of LP was increased from 3 μg/mL to 5 μg/mL, the level of cytokines decreased. The contamination of LP by LPS was not responsible for this since polymyxin B pretreated LP had no effect on TNF-α production ( Figure 2). After being incubated for 24 hours, cells were harvested, and the concentration of TNF-α in the medium was determined by ELISA as described in Section 2.

Induction of TNF-α, IL-1β, and IL-6 mRNA expression after treatment with LP
Because the cytokine mRNA are regulated mainly at the transcriptional level, we examined the expression levels of TNF-α, IL-1β, and IL-6 mRNA using RT-PCR. As shown in Figure 3, the addition of 3 μg/mL LP after 18-hour stimulation increased these three genes' mRNA expression and β-actin as an internal control was similar in all samples.

PDTC-induced downregulation of TNF-α, IL-1β, and IL-6 production and its mRNA expression
We found that the NF-κB inhibitor PDTC significantly inhibited the production of TNF-α, IL-1β, and IL-6 in THP-1 cells stimulated by M. genitalium LP. As shown in Figure 1, the levels of TNF-α, IL-1β, and IL-6 in the conditioned media from cells treated with 3 μg/mL of LP in combination with 25 μM PDTC were significantly lower than in media from cells treated 3 μg/mL of LP alone (P < .05). The effects of PDTC on the expression of TNF-α, IL-1β, and IL-6 mRNA showed similar patterns in LP-stimulated cells, as determined by RT-PCR (Figure 3).

M. genitalium LP induced NF-κB activation by EMSA
In this study, we investigated whether M. genitalium LP was capable of triggering NF-κB activation. For this purpose, the THP-1 cells were stimulated with LP at different time intervals, NF-κB DNA binding activities in nuclear extracts were assessed by nonradioactive EMSA as described in Section 2. As depicted in Figure  were maximal by 2 hours of stimulation and then declined. The specificity of NF-κB DNA binding was verified by competition analysis with an excess of unlabeled specific or unspecific oligonucleotides.

M. genitalium LP induced cell apoptosis by Annexin-V-FITC-PI and AO-EB staining
The percentage of apoptotic cells was examined by two techniques as follows. (1) a typical experiment of Annexin-V-FITC-PI staining was shown in Figure 5. By this technique,   Table 1).

DISCUSSION
Although the molecular basis of mycoplasma pathogenicity remains unclear, modulatory effect on the immune system induced by LP appears to play an important role in the development of mycoplasma-associated diseases [21]. One of the best documented effects of mycoplasma is the induction of numerous cytokines by monocytic cells [9]. For example, M. fermentans-derived LP has been demonstrated to induce inflammatory mediators such as TNF-α, IL-1β, and IL-6 released from mouse macrophages [4,22]. In the present study, we have demonstrated that lipoproteins derived from the human M. genitalium are capable of inducing THP-1 cells to produce proinflammatory cytokines and to induce apoptosis by activating NF-κB.
In an attempt to clarify the potential pathogenicity of M. genitalium, we have demonstrated in this study that LP from M. genitalium could trigger THP-1 cells to produce TNF-α, IL-1β, and IL-6 in a dose-dependent manner. However, when the concentration of LP increased from 3 to 5 μg/mL, the level of inflammatory cytokines decreased. This may be explained by assuming that excessively high concentrations of LP may be toxic to THP-1 cells, and thus, decreased inflammatory cytokine production [8]. We have also found that PDTC, an inhibitor of NF-κB [23], could significantly inhibit THP-1 cells treated with LP from producing inflammatory cytokines, and inhibit the expression of their mRNA. TNF-α, IL-6, and IL-1β are important inflammatory mediators: they can stimulate rapid neutrophil influx, and these cells are effective mediators of host defense until antigen-specific mechanisms are induced to eliminate the pathogen. However, the stimulation of cytokine production could also influence the development of inflammatory reactions inevitably, which may directly or indirectly contribute to disease pathogenesis and tissue damage [24].
Recent studies have demonstrated that there are NF-κB binding sites in the 5 transcriptional regulation regions of the cytokine gene. NF-κB is known as a widespread rapid-response transcription factor that is normally expressed in the cytoplasm of a variety of cells [25,26]. Since the induction of gene-specific recognition elements located in the upstream promoter region, we investigated whether the production of TNF-α, IL-1β, and IL-6 and the expression of their mRNA in THP-1 cells treated with M. genitalium LP were associated with the activation of NF-κB. By using electrophoretic mobility shift and transactivation assays, we have clearly demonstrated that LP can induce the transcriptional activation of NF-κB; the activation peaked 2 hours after stimulation. The above results indicate that LP from M. genitalium are potent activators of NF-κB, and NF-κB activation may be of great importance for inducing the production of TNF-α, IL-1β, and IL-6 and the expression of their mRNA. Apoptosis is a major form of cell death, characterized initially by a series of stereotypical morphological changes. These changes reflect complex biochemical events carried out by a family of cysteine proteases called caspases [27][28][29]; but a question remains to be answered whether the M. genitalium LP could induce apoptosis by macrophages in addition to inducing inflammatory cytokine production. In this study, cell apoptosis was detected in THP-1 cells treated with the M. genitalium LP by Annexin V-FITC-PI and AO-EB staining, and the apoptosis-inducing activity was inhibited by PDTC, which demonstrated that M. genitalium LP-induced THP-1 cell death was partially associated with the activation of NF-κB [4]. In terms of infectious diseases, apoptosis appears to be one of the defense mechanisms against microbes hiding in cells which do not necessarily possess any mechanism against them. However, excessive immune cells apoptosis may affect the immune response in the primary infection site, and thus make it easy for mycoplasma to diffuse [30,31]. It is very likely that apoptosis-related cytokines, such as TNF-α produced by macrophages and lymphocytes in response to mycoplasmal lipoproteins, play an important role in the expression of the cytotoxicity. This speculation is supported by findings that M. fermentans enhances concanavalin A-induced apoptosis of mouse splenic T-cells and that TNF-α plays a key role in the activity [32]. However, whether these cytokines exert some effects on the cytotoxicity of M. genitalium LP to THP-1 cells is still unknown. Further studies are in progress to clarify the molecular mechanism.
The above results indicate that LP from M. genitalium are potent activators of NF-κB, and that NF-κB activation may be of great importance for inducing the production 7 of proinflammatory cytokines and its mRNA and apoptosis following stimulation with LP from M. genitalium. Since proinflammatory cytokines play important roles in the pathogenesis of infectious disease sequelae, the success of anticytokine therapy in the inflammatory reaction may relieve the abnormal immune response brought about by mycoplasma infection. It is now clear that NF-κB were involved in this modulation, and these findings contribute to unraveling the complex mechanisms of immune reactivity to mycoplasma infection and may ultimately prove useful in the development of new therapeutic strategies to prevent tissue damage in mycoplasma-associated diseases [33].