Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2 - PubMed (original) (raw)

Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2

Ling Li et al. Front Microbiol. 2018.

Abstract

Trichomoniasis, caused by Trichomonas vaginalis infection, is the most prevalent sexually transmitted disease in female and male globally. However, the mechanisms by innate immunity against T. vaginalis infection have not been fully elucidated. Toll-like receptor2 (TLR2) has been shown to be involved in pathogen recognition, innate immunity activation, and inflammatory response to the pathogens. Nonetheless, the function of TLR2 against T. vaginalis remains unclear. In the present study, we investigated the role of TLR2 in mouse macrophages against T. vaginalis. RT-qPCR analysis revealed that T. vaginalis stimulation increased the gene expression of TLR2 in wild-type (WT) mouse macrophages. T. vaginalis also induced the secretion of IL-6, TNF-α, and IFN-γ in WT mouse macrophages, and the expression of these cytokines significantly decreased in TLR2-/- mouse macrophages and in WT mouse macrophages pretreated with MAPK inhibitors SB203580 (p38) and PD98059 (ERK). Western blot analysis demonstrated that T. vaginalis stimulation induced the activation of p38, ERK, and p65 NF-κB signal pathways in WT mouse macrophages, and the phosphorylation of p38, ERK, and p65 NF-κB significantly decreased in TLR2-/- mouse macrophages. Taken together, our data suggested that T. vaginalis may regulates proinflammatory cytokines production by activation of p38, ERK, and NF-κB p65 signal pathways via TLR2 in mouse macrophages. TLR2 might be involved in the defense and elimination of T. vaginalis infection.

Keywords: MAPK; NF-κB; TLR2; TLR2-/-; Trichomonas vaginalis; cytokines.

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Figures

FIGURE 1

FIGURE 1

Trichomonas vaginalis induces cytokines secretion in a TLR2-dependent way in mouse peritoneal macrophages. RT-qPCR analysis for TLR2 in total RNA isolated from mouse peritoneal macrophages incubated in medium alone, with T. vaginalis or Pam3CSK4 (10 μg/ml), respectively. RT-qPCR demonstrated that T. vaginalis stimulation significantly enhances TLR2 gene expression in WT macrophages (A). WT macrophages were co-incubated with T. vaginalis or Pam3CSK4 for 2 h (B). Relative Gray analysis of western blot (C). WT and TLR2-/- mouse peritoneal macrophages were co-incubated with T. vaginalis. The levels of IL-6, TNF-α, and IFN-γ in cell culture supernatant were detected by ELISA. Compared with TLR2-/- mouse peritoneal macrophages, the production of IL-6, TNF-α, and IFN-γ in WT macrophages were significantly increased (D–F). Data are expressed as the mean ± SD from three separate experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

FIGURE 2

FIGURE 2

Trichomonas vaginalis activates p38 and ERK signal pathways in WT mouse peritoneal macrophages. WT macrophages were co-incubated with T. vaginalis for different times (0, 0.5, 1, 2, and 4 h), phosphorylation of p38 and ERK were detected by western blot (A). Relative Gray analysis of western blot (B). The phosphorylated of p38 (at 0.5 h) and ERK (at 2 h) were observed obviously. Data are expressed as the mean ± SD from three separate experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

FIGURE 3

FIGURE 3

Trichomonas vaginalis induces cytokines production regulated by p38 and ERK via TLR2. Phosphorylationof p38 and ERK in TLR2-/- mouse peritoneal macrophages were significantly reduced after co-incubated with T. vaginalis for 0.5 h (p38) or 2 h (ERK) compared to in WT mouse peritoneal macrophages (A). Inhibitors of p38 (SB203580; 30 μM) or ERK (PD98059; 40 μM) were used to pretreated WT mouse peritoneal macrophages for 1 h before co-incubated by T. vaginalis. Phosphorylation of p38 and ERK in SB203580 and PD98059 pre-treated WT macrophages (C). Relative Gray analysis of western blot (B,D). The production of IL-6, TNF-α, and IFN-γ induced by T. vaginalis were significantly inhibited by the inhibitors compared to in WT mouse peritoneal macrophages (E–G). Data are expressed as the mean ± SD from three separate experiments (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

FIGURE 4

FIGURE 4

Trichomonas vaginalis induces changes of subcellular localization of NF-κB p65 via TLR2. Confocal microscopy analysis revealed the effects of T. vaginalis or Pam3CSK4 on the translocation of NF-κB p65 from the cytoplasm to the nucleus in WT macrophages after the incubation with T. vaginalis for 1 h (A). The percentage of colocalization of NF-κB with the nuclear signal under the different treatments (B). WT mouse macrophages were incubated with T. vaginalis for different times (0–4 h), cell lysates were used for western blot analysis (C). WT and TLR2-/- mouse macrophages were incubated with T. vaginalis for 1 h, phosphorylation of NF-κB p65 and IκBα was detected by western blot (E). Phosphorylation of NF-κB p65 in Pam3CSK4 treated macrophages (G). Relative Gray analysis of western blot (D,F,H).

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