Progesterone Suppresses Neisseria gonorrhoeae-Induced Inflammation Through Inhibition of NLRP3 Inflammasome Pathway in THP-1 Cells and Murine Models

Abstract

Asymptomatic/subclinical gonococcal infections in females continue to be prevalent within the general population, thus emerging as a global health problem. However, the reasons for these clinical manifestations are unknown. Our group had previously found out that in females, asymptomatic gonococcal infections correlate with higher serum progesterone (P4) levels and lower IL-1β levels in cervical secretions. We used murine infection model and THP-1 cells to determine whether P4 exerts anti-inflammatory effects on gonococcal infections. In the murine infection model, P4 (1 mg/day) inhibited the inflammatory effects induced by gonococcal infections which led to decreased neutrophil infiltration, reduced polymorphonuclear neutrophils (PMNs) numbers, IL-1β, TNF-α, and IL-6 levels in vaginal secretions. In addition, P4 down-regulated the mRNA and protein levels of NLRP3, associated with lower mRNA levels of pro-IL-1β, repressed caspase-1 activity in genital tissues and THP-1 cells. Moreover, P4 suppressed the phosphorylation levels of NF-κB and attenuated Neisseria gonorrhoeae (N. gonorrhoeae, gonococci or GC)-induced ROS generation. This is consistent with the two signals required for activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome. In conclusion, our result shows that P4 suppresses the gonococci induced-inflammation, especially through the NLRP3 inflammasome pathway, and partially explains the pathogenesis of asymptomatic GC infection in women.

Keywords: NF-κB; NLRP3 inflammasome; Neisseria gonorrhoeae; inflammation; reactive oxygen species.

FIGURE 1

Progesterone suppresses N. gonorrhoeae-induced neutrophils infiltration in murine models. (A) Time line for the N. gonorrhoeae infection model. (B) The average duration of gonococci recovery among the three groups. (C) The average number of gonococci recovered from vaginal swabs of BALB/c mice, the single vaginal swab was suspended in 100 μl of PBS. (D) The percent of PMNs in stained vaginal smears from experimental mice were obtained on fifth day after inoculation. (E,F) Paraffin embedded sections (vaginal and cervical tissues of infected mice and uninfected controls) were stained with hematoxylin and eosin, and tissue sections were examined under an OLYMPUS light microscope. All data are representative of at least three independent experiments. Data are presented as the mean ±SD, n = 5. OVX^–, ovariectomy; GC, Neisseria gonorrhoeae; P4, progesterone; CFU, Colony-Forming Units.

FIGURE 2

Progesterone suppresses N. gonorrhoeae-induced cytokines secretion in murine models. 50 μl of vaginal mucus was continuously collected from test and control mice for 10–12 days, and detection of IL-1β (A), TNF-α (B), and IL-6 (C) levels were carried out using commercially available LEGEND plex^TM Multi-Analyte Flow Assay Kits. The IL-1β (D) and IL-18 (E) levels from the vaginal secretions of N. gonorrhoeae infected mice on fifth day after inoculation. All data are representative of at least three independent experiments. *p < 0.05, **p < 0.01. Data are presented as the mean ± SD, n = 5. Ovx^–, ovariectomy; P4, progesterone.

FIGURE 3

Progesterone suppresses N. gonorrhoeae-induced NLRP3 inflammasome activation in murine models. The mRNA levels of NLRP3 were assayed in the vaginal (A) and cervical (B) tissues on day 5 after inoculation. The mRNA levels of pro-IL-1β were assayed in the vaginal (C) and cervical (D) tissues on day 5 after inoculation. All data are representative of at least three independent experiments. **p < 0.01. Data are presented as the mean ± SD, n = 5. Ovx^–, ovariectomy; P4, progesterone.

FIGURE 4

Immunofluorescence of NLRP3 and Caspase-1 p20 in vaginal and cervical tissues. (A) Immunofluorescence and fluorescence microscope were used to estimate the number of NLRP3-positive cells (red fluorescence) and caspase-1p20-positive cells (green fluorescence) on day5 after inoculation. (B) Semi-quantification analysis of relative fluorescence intensity of NLRP3 using ImageJ software. (C) Semi-quantification analysis of relative fluorescence intensity of caspase-1p20 using ImageJ software. (D) Immunofluorescence of anti-CD68 (red fluorescence) and anti-IL-1β (green fluorescence) of vaginal and cervical tissues. **p < 0.01. Data are presented as the mean ± SD, n = 5. Ovx^–, ovariectomy; P4, progesterone.

FIGURE 5

Progesterone inhibits N. gonorrhoeae -induced NLRP3 inflammasome activation and IL-1β secretion in THP-1 macrophages. (A) PMA-differentiated THP-1 cells were pre-treated with N. gonorrhoeae overnight and then stimulated with P4. Four hours later supernatants were analyzed by ELISA for IL-1β release. (B) Cells were selected to detect the protein levels of NLRP3 and Caspase-1p20. (C,D) Semi quantification analysis of NLRP3 and caspase-1p20 western blot films using ImageJ software. (E,F) The mRNA levels of pro-IL-1β and NLRP3 on N. gonorrhoeae infected human differentiated THP-1 macrophages. (G,H) Cell supernatants from activated THP-1 cells were evaluated for active IL-1β by Western blot, semi quantification analysis of active IL-1β western blot films using ImageJ software. *p < 0.05, **p < 0.01. Data are presented as the mean ± SD, n = 5. GC, Neisseria gonorrhoeae; P4, progesterone.

FIGURE 6

Progesterone inhibits the activation of NF-κB signal pathway. (A) PMA-differentiated THP-1 cells were pre-treated with N. gonorrhoeae overnight and then stimulated with P4. Four hours later, cells were selected to detect the mRNA levels of NF-κB p65. (B) Semi-quantification analysis of NF-κB western blot films using ImageJ software. (C) Phosphorylation of NF-κB and total NF-κB expression were detected by immunoblotting. Supernatants were analyzed by ELISA for IL-6 (D) and TNF-α release (E). Data are representative or means ± SD of three independent experiments. *p < 0.05, **p < 0.01. GC, Neisseria gonorrhoeae; P4, progesterone.

FIGURE 7

Progesteroneattenuates N. gonorrhoeae-induced ROS generation. (A) PMA-differentiated THP-1 cells were pre-treated with N. gonorrhoeae overnight and then stimulated with P4. Four hours later, oxidized DCF fluorescence was detected by fluorescence microscope, and semi-quantification analysis of the oxidized DCF fluorescence using ImageJ software. (B) P4 pre-treated differentiated THP-1 cells were stimulated with 5 mM H₂O₂ and N. gonorrhoeae, supernatants were analyzed by ELISA for IL-1β release. (C) Fluorescent images were visualized using an OLYMPUS Fluorescence microscope. Data are representative or means ± SD of at least three independent experiments. *p < 0.05, **p < 0.01. NG, Neisseria Gonorrhoeae; P4, progesterone.

FIGURE 8

Overview of mechanisms by which progesterone inhibited NF-κB and NLRP3 inflammasome activated by N. gonorrhoeae. Progesterone inhibits N. gonorrhoeae induced-inflammation in murine models and macrophages through (1) inhibiting the activation of NF-κB signal pathway, thus decreasing NLRP3 and pro-IL-1β expression; (2) suppressing ROS production, thus inhibiting caspase-1 activation and IL-1β/IL-18 secretion.