Fig 1.
Physical characterization of TV-EVs purified from different isolates.
(A) Coomassie blue staining was used to analyze the protein compositions of TV cell lysate and TV-EVs isolated from two cell lines (ATCC 50143 and ATCC 30236) and the clinical strain (NDMC5). (B) Western blot analysis was utilized to detect the expression of the mammalian exosomal marker CD63 in fractions of TV-EVs isolated from the culture medium in the presence or absence of serum. GAPDH was served as the cytosolic marker. (C) TEM was utilized to visualize the ultrastructure of TV-EVs secreted from different isolates. (D) NTA was used to determine the concentrations and sizes of EVs derived from different TV isolates.
Fig 2.
Internalization of TV-EVs by THP-1 macrophages and Ect.
(A) THP-1 macrophages (4 × 104 cells) and (B) Ect (4 × 104 cells) were co-cultured with PKH67-labeled TV-EVs (30 µg/ml) for different time intervals (30 minutes, 1 hour, 2 hours, 3 hours, and 6 hours). CellMask plasma membrane stain was used to label the plasma membrane, and 4,6-diamidino-2-phenylindole (DAPI) was employed for nuclear staining. Cellular uptake was visualized using a confocal microscope. Scale bar:10 µm. (C) 4 × 104 cells of THP-1 macrophages and (D) Ect were co-cultured with TV-EVs (30 µg/ml) or PBS (Control; Ctrl) for 6 hours, and the ultrastructure was determined using TEM. a. PBS-treated control cells (Ctrl), b. and c. THP-1 macrophages or Ect co-cultured with TV-EVs (THP-1 + EVs or Ect + EVs, respectively), d. Enlarged images of boxed areas are shown. Arrows indicate the presence of numerous vesicle-like structures resembling TV-EVs.
Fig 3.
TV-EVs induce the production of various pro-inflammatory cytokines in the host.
(A) THP-1 macrophages (2 × 105 cells/well) were co-cultured with TV-EVs (30 µg/ml) isolated from different strains (ATCC 30236, ATCC 50143, and NDMC5) for 6 hours. The culture supernatants were collected for screening 48 common human cytokines using a multiplex immunoassay. The culture supernatants were assayed for (B) IL-8, (C) MIP-1β, and (D) CXCL1 using ELISA. (E) Ect (2 × 105 cells/well) were co-cultured with TV-EVs (30 µg/ml) isolated from different strains for 8 hours and the culture supernatants were collected for screening 48 common human cytokines as mentioned above. The culture supernatants were assayed for (F) CXCL1, (G) IL-8, and (H) IL-6 using ELISA. (I) The in vivo model shows the pretreatment strategy for female BALB/c mice before TV-EVs or PBS inoculation. (J) Mice vaginas (n = 6 mice per group) were inoculated with TV-EVs isolated from different strains (50143 and NDMC5) for 3, 6, 9, and 12 days and the vaginal lavages were collected and assayed for IL-8 and CXCL1 using ELISA. The quantitative data are expressed as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 4.
TV-EVs induce NLRP3 inflammasome activation in THP-1 macrophages.
(A) THP-1 macrophages (5 × 106 cells) were treated with the PBS control (Ctrl) or TV-EVs (30 µg/ml) isolated from different strains (ATCC 30236, ATCC 50143, and NDMC5) for 6 hours. Subsequently, the cells were collected (Lysate) and subjected to western blot analysis using anti-NLRP3, anti-caspase-1, anti-ASC, anti-IL-1β, and anti-β-actin antibodies. The supernatant (Sup) was collected for detecting IL-1β secretion. LPS (1 µg/ml) in combination with ATP (30 nM) was used as the positive control for NLRP3 inflammasome activation. The protein expression of β-actin was used as the internal control for western blot analysis. (B) Quantification of protein bands from (A) was performed using Image J. (C) THP-1 macrophages were treated with TV-EVs isolated from different strains or PBS for 6 hours and the supernatants were collected for IL-1β detection using ELISA. (D) Immunofluorescence analysis was performed to detect the distribution of NLRP3 and ASC in THP-1 macrophages (4 × 104 cells/ml) treated with TV-EVs or PBS (Ctrl) using antibodies against NLRP3 (red) and ASC (green), followed by visualization under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar: 10 µm. The data are expressed as the means ± SEM from three independent experiments.* p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 5.
TV-EVs activate NF-κB signaling to regulate NLRP3 inflammasome activation and pro-inflammatory cytokine secretion in THP-1 macrophages.
(A) THP-1 macrophages (5 × 106 cells) were treated with the PBS control (Ctrl) or TV-EVs (ATCC 50143) for 15, 60, 120, and 240 minutes. Whole cell lysates were collected and subjected to western blot analysis. The protein expression of NF-κB (p65) and phosphorylated NF-κB (p-p65) was detected using specific antibodies. (B) Quantification of protein bands from (A) using Image J. (C) Immunofluorescence analysis was performed in THP-1 macrophages treated with TV-EVs for 4 hours using an antibody against NF-κB (green), followed by visualization under a confocal microscope. Nuclei were stained with DAPI (blue). (D) THP-1 macrophages were pretreated with the NF-κB inhibitor BAY 11–7082, and then co-cultured with TV-EVs isolated from different strains (ATCC 30236, ATCC 50143, and NDMC5) for 6 hours. Whole cell lysates were analyzed by western blot analysis using anti-NLRP3 and anti-IL-1β antibodies. Protein bands were quantified as mentioned above. (E) The supernatants collected from THP-1 macrophages (2 × 105 cells/well) treated with different conditions as mentioned in (D) were analyzed for IL-1β, IL-8, MIP-1β, and CXCL-1 secretion using ELISA. The protein expression of β-actin was used as the internal control for western blot analysis. The quantitative data are expressed as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 6.
TV-EVs activate the AKT, NF-κB, p38 MAPK, and ERK pathways through PI3K signaling to mediate inflammation in Ect.
(A) Ect (2 × 106 cells) were treated with the PBS control (Ctrl) or TV-EVs (ATCC 50143) for 2, 4, 6, 8, and 10 hours. Subsequently, whole cell lysates were collected and subjected to western blot analysis. The phosphorylation of PI3K, AKT, NF-κB, p38 MAPK, and ERK was detected using specific antibodies. (B) Quantification of protein bands from (A) using Image J. (C) Immunofluorescence analysis was performed to detect the localization of NF-κB in Ect treated with TV-EVs compared with the PBS-treated control (Ctrl) using an antibody against NF-κB (green), followed by visualization under a confocal microscope. Nuclei were stained with DAPI (blue). Scale bar: 10 µm. (D) Ect were pretreated with the PI3K inhibitor, wortmannin, and then co-cultured with TV-EVs isolated from different strains for 4 hours. The cells were collected and analyzed by western blot analysis using anti-p-PI3K, anti-p-AKT, anti-p-p38 MAPK, and anti-p-ERK antibodies. The protein bands were quantified as mentioned above. (E) Ect (4 × 104 cells) were pretreated with the PI3K inhibitor wortmannin and then co-cultured with TV-EVs for 4 hours. Immunofluorescence analysis was performed using an anti-NF-κB antibody (green). Nuclei were stained with DAPI (blue). Scale bar:10 µm. (F) Ect (2 × 105 cells/well) were pretreated with pathway inhibitors, including NF-κB (BAY 11-7082), p38 MAPK (SB203580), or ERK (PD98059), followed by treatment with TV-EVs isolated from different strains (ATCC 30236, ATCC 50143, and NDMC5) for 8 hours. The culture supernatants were collected and assayed for IL-6, IL-8, and CXCL1 by ELISA. Ect treated with the PBS control (Ctrl) and TV-EVs isolated from different strains (50143-EV and NDMC5-EV) were served as a negative and positive control, respectively. The protein expression of β-actin was used as the internal control for western blot analysis. The quantitative data are expressed as the means ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 7.
TV-EVs activate the NF-κB/NLRP3 signaling pathway by TLR3 in THP-1 macrophages.
(A) THP-1 macrophages (5 × 106 cells) were treated with the PBS control (Ctrl) or TV-EVs (ATCC 50143) for 15, 60, 120, and 240 minutes. Whole cell lysates were collected and analyzed by western blot analysis using an anti-TLR3 antibody. Quantification of protein bands using Image J. (B) THP-1 macrophages were pretreated with the TLR3 inhibitor (614310) or siRNA targeting TLR3 (si-TLR3), and then co-cultured with TV-EVs for 15 minutes. The protein expression levels of TLR3, p-NF-κB, and NLRP3 in whole cell lysates of THP-1 macrophages were analyzed by western blot analysis using specific antibodies. Quantification of protein bands as mentioned above. (C) THP-1 macrophages (2 × 105 cells/well) were pretreated the TLR3 inhibitor or si-TLR3, and then co-cultured with TV-EVs from different isolates for 6 hours. The culture supernatants were collected and analyzed for CXCL-1, MIP-1β and IL-8 by ELISA. Ect treated with the PBS control (Ctrl) and TV-EVs isolated from different strains (50143-EV and NDMC5-EV) were served as a negative and positive control, respectively. The protein expression of β-actin was used as the internal control for western blot analysis. The quantitative data are expressed as the means ± SEM from three independent experiments.*p < 0.05, ** p < 0.01, *** p < 0.001. ns: not significant.
Fig 8.
TV-EVs upregulate TLR3 expression to modulate the PI3K, NF-κB, p38 MAPK, and ERK-mediated inflammation in Ect.
(A) Ect (2 × 106 cells) were treated with the PBS control (Ctrl) or TV-EVs (ATCC 50143) for 2, 4, 6, 8, and 10 hours. Whole cell lysates were collected and analyzed by western blot using an anti-TLR3 antibody. Quantification of protein bands using Image J. (B) Ect were pretreated with the TLR3 inhibitor (614310) or siRNA targeting TLR3 (si-TLR3) and then co-cultured with TV-EVs for 4 hours, and the protein expression levels of TLR3, p-PI3K, p-NF-κB, p-ERK, and p-p38 MAPK were analyzed using specific antibodies. (C) Quantification of protein bands from (B) using Image J. (D) Ect (2 × 105 cells/well) were pretreated with the TLR3 inhibitor or si-TLR3, then co-cultured with TV-EVs from different isolates for 8 hours. The culture supernatants were collected and analyzed for IL-6, IL-8, and CXCL1 by ELISA. Ect treated with the PBS control (Ctrl) and TV-EVs isolated from different strains (50143-EV and NDMC5-EV) were served as a negative and positive control, respectively. The data are expressed as the means ± SEM from three independent experiments.*p < 0.05, **p < 0.01, ***p < 0.001.
Fig 9.
Proteomic analysis identifies novel proteins involved in the TV-EV-induced immune response in Ect.
Enrichment analysis of differentially expressed proteins revealed enriched gene sets in the Ect proteome treated with TV-EVs purified from different strains for different time intervals (2, 4, and 8 hours). The enrichment plots showed the most (A) up-regulated or (B) down-regulated pathways in Ect treated with TV-EVs using the KEGG database. The enrichment score (ES) represents the degree to which the genes in the set are over-represented at either the top or bottom of the ranked list. The positive ES and negative ES indicate upregulation and downregulation of a specific gene set, respectively. (C) Validation of proteomic results by western blot analysis. Ect (2 × 106 cells) were treated with the PBS control (Ctrl) or TV-EVs for 2, 4, and 8 hours, and whole cell lysates were collected and analyzed by western blot using antibodies against MICB, MBNL2, TRAF3IP2, and KLC4. (D) The role of TLR3 in regulating the expression levels of MICB and TRAF3IP2. Ect pretreated with the siRNA targeting TLR3 (si-TLR3) were co-cultured with TV-EVs for 4 hours, and the whole cell lysates were analyzed by western blot using specific antibodies. (E) The role of MICB and TRAF3IP2 in regulating the inflammatory pathways induced by TV-EVs. Ect pretreated with si-MICB and si-TRAF3IP2 were co-cultured with TV-EVs for 4 hours, and the whole cell lysates were analyzed using specific antibodies. Quantification of protein bands was performed using Image J. The protein expression of β-actin was used as the internal control for western blot analysis. The data are expressed as the means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns: not significant.
Fig 10.
Proposed model for TV-EV-induced inflammatory cascade in host cells.
In THP-1 macrophages, TV-EVs activate the TLR3-mediated NF-κB/NLRP3 pathway, inducing the secretion of IL-8, MIP-1β, CXCL1, and IL-1β. In Ect, TV-EVs activate PI3K signaling to positively regulate the NF-κB, p38 MAPK, and ERK pathways, inducing the secretion of IL-6, IL-8, and CXCL1. Specifically, TV-EV-induced TLR3 overexpression in Ect upregulates PI3K and NF-κB pathways while downregulating the p38 MAPK and ERK pathways. Furthermore, proteomic analysis identifies that TV-EVs induce overexpression of MICB and TRAF3IP2 in Ect, which are positively regulated by TLR3. MICB enhances PI3K-mediated activation of the NF-κB and ERK pathways, while TRAF3IP2 activates PI3K signaling but suppresses the ERK pathway.