Tp47-Induced Monocyte-Derived Microvesicles Promote the Adherence of THP-1 Cells to Human Umbilical Vein Endothelial Cells via an ERK1/2–NF-κB Signaling Cascade

ABSTRACT The Treponema pallidum membrane protein Tp47 induces immunocyte adherence to vascular cells and contributes to vascular inflammation. However, it is unclear whether microvesicles are functional inflammatory mediators between vascular cells and immunocytes. Microvesicles that were isolated from Tp47-treated THP-1 cells using differential centrifugation were subjected to adherence assays to determine the adhesion-promoting effect on human umbilical vein endothelial cells (HUVECs). Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in Tp47-induced microvesicle (Tp47-microvesicle)-treated HUVECs were measured, and the related intracellular signaling pathways of Tp47-microvesicle-induced monocyte adhesion were investigated. Tp47-microvesicles promoted THP-1 cell adhesion to HUVECs (P < 0.01) and upregulated ICAM-1 and VCAM-1 expression in HUVECs (P < 0.001). The adhesion of THP-1 cells to HUVECs was inhibited by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies. Tp47-microvesicle treatment of HUVECs activated the extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-κB signaling pathways, whereas ERK1/2 and NF-κB inhibition suppressed the expression of ICAM-1 and VCAM-1 and significantly decreased the adhesion of THP-1 cells to HUVECs. IMPORTANCE Tp47-microvesicles promote the adhesion of THP-1 cells to HUVECs through the upregulation of ICAM-1 and VCAM-1 expression, which is mediated by the activation of the ERK1/2 and NF-κB pathways. These findings provide insight into the pathophysiology of syphilitic vascular inflammation.

Extracellular vesicles constitute a heterogeneous set of cell-derived membranous structures, including exosomes and microvesicles, that are intercellular biomessengers that enable the cell-cell interchange of proteins, lipids, and genetic material (11). Elucidation of the pathophysiological functions and medical applications involving the use or analysis of the above-mentioned vesicles is essential for an understanding of the cellular mechanisms that govern extracellular vesicle biology (12,13). Extracellular vesicles released from endothelial cells or monocytes generate targeted cross talk between endothelial cells and monocytes (14,15). Our previous investigations have demonstrated that both T. pallidum and Tp47 induce the adherence of a human monocytic cell line (THP-1) to human dermal vascular smooth muscle cells by increasing the levels of adhesion-associated cytokines (2,10). It is intriguing whether microvesicles constitute a functional inflammatory mediator of the vascular cell-immunocyte interaction in vascular inflammation.
Here, a series of experiments was conducted to investigate the effect of monocytederived Tp47-induced microvesicles (Tp47-microvesicles) on THP-1 cell adherence to HUVECs. Furthermore, the levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression and the signaling pathways involved in cell adhesion were analyzed.

RESULTS
Identification of microvesicles derived from THP-1 cells. Microvesicles were isolated from the cell culture medium of THP-1 cells that were stimulated with phosphate-buffered saline (PBS) or 10 mg/mL Tp47 for 24 h. Transmission electron microscopy revealed cup-shaped vesicles, which corroborated the microvesicular morphology ( Fig. 1A and 1B). Compared with PBS-stimulated microvesicles (PBS-microvesicles), flow cytometric analysis revealed a significant increase in annexin V positivity in Tp47-microvesicles (t = 17.38; P , 0.001) (Fig. 1C), and Western blotting showed that the protein level of matrix metalloproteinase 2 (MMP2) was high in the vesicles (Fig. 1D).
Tp47-microvesicles promoted the adhesion of THP-1 cells to HUVECs. To investigate the influence of Tp47-microvesicles on THP-1 cell adherence to HUVECs, HUVECs were pretreated for 24 h with Tp47-microvesicles and PBS-microvesicles at different concentrations and then incubated with THP-1 cells for 1 h. As shown in Fig. 2, Tp47-microvesicles dosedependently enhanced THP-1 cell adhesion to HUVECs: the number of adherent THP-1 cells was significantly increased in the presence of 10 mg/mL Tp47-microvesicles (P , 0.05) or 25 mg/mL Tp47-microvesicles (P , 0.01), which indicated the promotion of the adhesion of THP-1 cells to HUVECs by Tp47-microvesicles.
Tp47-microvesicles promoted THP-1 cell adhesion to HUVECs via ICAM-1 and VCAM-1. To analyze the effects of Tp47-microvesicles on the expression of two cell adhesion-associated genes, ICAM-1 and VCAM-1, HUVECs were pretreated with 10 and 25 mg/mL Tp47-microvesicles or PBS-microvesicles for 24 h. Treatment with Tp47-microvesicles markedly increased the expression of ICAM-1 and VCAM-1 mRNAs in HUVECs (P , 0.001) ( Fig. 3A and B), whereas the protein levels of ICAM-1 and VCAM-1 were dosedependently increased when HUVECs were stimulated with increasing concentrations of Tp47microvesicles (Fig. 3C). In contrast, PBS-microvesicles induced no significant change in the mRNA and protein levels of ICAM-1 and VCAM-1 in HUVECs regardless of the dose changes.

DISCUSSION
The present consensus among syphilologists is that the clinical manifestations of syphilitic infection emerge from treponeme-driven inflammatory processes (16). Furthermore, the pathology of syphilis is characterized by vascular manifestations such as periarteritis and endarteritis. As the T. pallidum genome does not encode orthologs of either wellcharacterized bacterial toxins or components of secretory systems (17), membrane proteins are considered the virulence factors of T. pallidum. Tp47 induces the adherence of THP-1 cells to human dermal vascular smooth muscle cells by increasing adhesion-associated cytokine production (10). Microvesicles belong to a group of heterogeneous membrane-coated vesicles that can function as signaling components in inflammatory processes (18). However, it is unclear whether microvesicles are involved and mediate the interaction of immunocytes with vascular cells to contribute to vascular inflammation. This study showed that Tp47-microvesicles promoted THP-1 cell adherence to HUVECs and increased the mRNA and protein expression levels of ICAM-1 and VCAM-1. Moreover, the adherence of THP-1 cells to HUVECs was inhibited considerably by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies. Furthermore, Tp47-microvesicle treatment of HUVECs activated the ERK1/2 and NF-k B signaling pathways, which induced ICAM-1 and VCAM-1 expression. These findings confirmed that Tp47-microvesicles increase the adhesion of THP-1 cells to HUVECs through the induction of ICAM-1 and VCAM-1 expression via the activation of the ERK1/2 and NF-k B pathways. Endothelial dysfunction, an early phase of vascular inflammation in humans, commences with ICAM-1 and VCAM-1 expression and immune cell adhesion (5,19,20). In this study, we found that Tp47-microvesicles enhanced THP-1 cell adherence to HUVECs and upregulated ICAM-1 and VCAM-1 mRNA and protein expression, which may contribute to syphilitic vascular inflammation. The identification of the mechanisms that underlie the interaction of immune cells with HUVECs that may downregulate abnormal leukocytic adhesion in the vascular endothelium may serve to prevent and treat T. pallidum-induced vascular inflammation.
Recently, evidence of the importance of extracellular vesicles for intercellular communication processes, with key implications for endothelial homeostasis, cell survival, inflammation, and thrombosis, has been reported (21). The cell adhesion molecules ICAM-1 and VCAM-1 are expressed in HUVECs by microvesicles in lipopolysaccharide-treated THP-1 cells (22). In this study, we found that the coincubation of Tp47-microvesicles with HUVECs increased THP-1 cell adhesion to HUVECs and upregulated ICAM-1 and VCAM-1 expression. Moreover, the increased adhesion of THP-1 cells to HUVECs was inhibited by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies, suggesting that the Cell adhesion is regulated by a network that comprises cell signaling molecules, epigenetic mechanisms, transcription factors, and posttranscriptional regulators. ICAM-1 expression is elicited by the activation of a redox-sensitive intracellular signaling cascade that involves ERK1/2 and p38 mitogen-activated protein kinase (MAPK) and results in NF-k B activation (23). In this study, the ERK1/2 inhibitor PD98059 inhibited the protein expression of adhesion molecules and impaired THP-1 cell adhesion to HUVECs. The pretreatment of HUVECs with the ERK1/2 inhibitor PD98059 inhibited the Tp47microvesicle-induced phosphorylation of ERK1/2 and the nuclear translocation of NF-k B. Although the ERK1/2 inhibitor PD98059 failed to inhibit ICAM-1 mRNA expression, possibly through translational rather than transcriptional effects, these findings indicated that Tp47-microvesicles mediate THP-1 cell adhesion to HUVECs in an ERK1/2-dependent manner.
The activation of the transcription factor NF-k B is primarily responsible for the inflammatory response that occurs after exposure to stimuli. Ik Ba sequesters NF-k B in the cytoplasm, and Ik Ba phosphorylation induces the proteasome-mediated degradation of Ik Ba and the

Tp47-Induced Microvesicles Promote Monocyte Adhesion
Microbiology Spectrum subsequent activation and nuclear translocation of NF-k B; this mechanism is a key activator of the genes that encode cytokines and adhesion molecules (23). In this study, Tp47-induced ICAM-1 and VCAM-1 expression and THP-1 cell adhesion were completely abolished by the NF-k B pathway inhibitor BAY11-7085. This indicates that Tp47-microvesicle-stimulated Ik Ba phosphorylation and NF-k B nuclear translocation are crucial factors for ICAM-1 and VCAM-1 upregulation and mediate the adherence of THP-1 cells to HUVECs. In addition, the ERK1/2 inhibitor PD98059 hindered Ik Ba phosphorylation, whereas BAY11-7085 did not block ERK1/2 phosphorylation (data not shown). These findings suggest that the activation of an intracellular signaling cascade involving ERK1/2, culminating in the activation of NF-k B signals, is the cause of TP47-microvesicle-induced ICAM-1 and VCAM-1 expression. Several potential limitations of this study should be acknowledged. First, based on centrifugation protocols, the separation of exosomes usually requires a centrifugal force of more than 100,000 Â g (24); however, the microvesicles were collected at only 20,000 Â g, and thus, it is inevitable that microvesicles with exosomes were isolated. Second, extracellular vesicles represent an important mode of intercellular communication as vehicles for the cell-cell transfer of membrane and cytosolic proteins, lipids, and RNA (25). Proteomic analysis FIG 5 Tp47-microvesicles (Tp47-MVs) promoted the adhesion of THP-1 cells to HUVECs via ICAM-1 and VCAM-1. Optical/fluorescence microscopy revealed the adhesion of THP-1 cells to HUVECs when HUVECs were incubated with 10 mg/mL of an anti-ICAM-1 antibody or an anti-VCAM-1 neutralizing antibody. ImageJ was used to calculate the number of THP-1 cells that adhered to HUVECs. Multigroup comparisons were undertaken using one-way ANOVA. Data were presented as the mean 6 SD of three biological replicates. **, P , 0.01; ***, P , 0.001.

Tp47-Induced Microvesicles Promote Monocyte Adhesion
Microbiology Spectrum of microvesicles is needed to determine their biophysical and biochemical properties. Finally, there is the limitation that microvesicles secreted by individual immune cells cannot mimic the infection process in a syphilitic host. Therefore, animal experiments are preferable for comprehensively ascertaining the role of microvesicles in vascular inflammation and the pathogenesis of syphilis.
Conclusion. In summary, this study showed that Tp47-microvesicles promote the adherence of THP-1 cells to HUVECs by inducing ICAM-1 and VCAM-1 expression. This suggests that microvesicles constitute a functional inflammatory mediator between vascular cells and immunocytes and thereby contribute to vascular inflammation in syphilis. Furthermore, the above-mentioned process is mediated by the ERK1/2 and NF-k B signaling pathways (Fig. 9). Recognition of the relevant mechanisms of ICAM-1 and VCAM-1 expression and monocyte adhesion may enable a better understanding of the pathophysiology of syphilitic vascular inflammation.

MATERIALS AND METHODS
Preparation of the recombinant T. pallidum Tp47 protein. The endotoxin in the recombinant T. pallidum Tp47 protein (Boson Biotech Co., Ltd., Xiamen, China) was removed as described previously (8). Endotoxin contamination of the Tp47 preparation was quantified using Tachypleus amebocyte lysate (Chinese Horseshoe Crab Reagent Manufactory, Ltd., Xiamen, China), which detected ,0.05 endotoxin units (EU)/mL. Cell culture. HUVECs (ScienCell Research Laboratories, Carlsbad, CA, USA) were incubated in endothelial cell medium, which contained 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement , which was supplemented with 10% fetal bovine serum (American Type Culture Collection), 1% penicillin, and streptomycin at 37°C with 5% CO 2 . Next, the cell culture dishes were transferred to serum-free medium, and THP-1 cells were stimulated with PBS or 10 mg/mL Tp47 for 24 h; thereafter, culture media were collected for microvesicle isolation. Isolation of microvesicles. THP-1 cells (1 Â 10 6 cells/mL) were processed with PBS or 10 mg/mL Tp47 for 24 h in culture medium as described above. The cells were centrifuged at 500 Â g for 10 min at 4°C. The supernatant was collected and centrifuged at 2,000 Â g for 20 min to ensure the pelleting of the cellular debris. The microvesicle pellets were obtained by centrifuging the microvesicle-containing supernatant at 20,000 Â g for 30 min at 4°C (24). The microvesicle pellets were washed, resuspended in PBS, and stored at 280°C until use. Microvesicles derived from Tp47-stimulated or PBS-stimulated THP-1 cells were denoted Tp47-microvesicles or PBS-microvesicles, respectively. The microvesicle concentration was quantified by measuring the total protein concentration using a bicinchoninic acid (BCA) protein assay kit (TaKaRa, Shanghai, China).
Identification of microvesicles. To observe the morphology of the microvesicles, the samples were examined by transmission electron microscopy using a negative staining method as previously described (26). Microvesicles were stained with annexin V (a marker of microvesicles [27]) and detected by flow cytometry. Briefly, microvesicles were stained with annexin V-fluorescein isothiocyanate (FITC) (Beyotime, Shanghai, China) in annexin V-FITC binding buffer for 30 min in the dark at room temperature. The samples were then centrifuged at 20,000 Â g for 15 min at 4°C. After the removal of the supernatant, the microvesicles were resuspended in 200 mL PBS and immediately subjected to fluorescence-activated cell sorting (FACS), wherein the percentage of annexin-positive microvesicles was analyzed by utilizing a FACSCanto II flow cytometer (BD Biosciences, San Diego, CA, USA). The results were analyzed using FlowJo7.6.4 software (TreeStar, Ashland, OR, USA). Western blot analysis was used to quantify the concentration of matrix metalloproteinase 2 (MMP2) (another marker of microvesicles [28]) in microvesicles as described previously (29).
Analysis of ERK1/2 and NF-jB activation in Tp47-microvesicle-treated HUVECs. HUVECs were stimulated with 25 mg/mL Tp47-microvesicles for various durations, and the cell lysates were collected for Western blotting to detect phosphorylated and total ERK1/2, Ik Ba, and p65 proteins. To confirm the Tp47microvesicle-induced activation of the ERK1/2 and NF-k B signaling pathways, HUVECs were preincubated with the ERK1/2 inhibitor PD98059 and the NF-k B inhibitor BAY11-7085 for 1 h before stimulation with 25 mg/mL Tp47-microvesicles. The expression of phosphorylated and total proteins of the ERK1/2 and NF-k B signaling pathways was detected. For determining the ICAM-1 and VCAM-1 expression levels, the cells were stimulated for 24 h as described above. The anti-phosphorylated ERK1/2 (p-ERK1/2), anti-p-Ik Ba, anti-p-p65, anti-ERK1/2, anti-Ik Ba, and anti-p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).
Analysis of the nuclear translocation of the NF-jB p65 subunit. HUVECs cultured on a Millicell EZ Slide 4-well glass slide box (Millipore, Burlington, MA, USA) were stimulated with 25 mg/mL Tp47-microvesicles for 1 h, and the nuclear translocation of the NF-k B p65 subunit was analyzed as mentioned previously (2). For the inhibition assay, HUVECs pretreated with 3 mM BAY11-7085 and 20 mM PD98059 for 1 h were stimulated with 25 mg/mL Tp47-microvesicles for 1 h, and the nuclear translocation of the NF-k B p65 subunit was analyzed.
Statistical analysis. All data in this research are presented as means 6 standard deviations (SD). Student's t test or one-way analysis of variance (ANOVA) was used to compare two or multiple groups, respectively. All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). P values of ,0.05 were deemed statistically significant.
Data availability. The data that support the findings of this study are available from the corresponding authors upon reasonable request.