Inhibition of TLR2/TLR4 alleviates the Neisseria gonorrhoeae infection damage in human endometrial epithelial cells via Nrf2 and NF-Kβsignaling

https://doi.org/10.1016/j.jri.2020.103192Get rights and content

Highlights

  • N.g suppressed cell viabilities and induced cell apoptosis and inflammation.

  • TLR2/TLR4 downregulation inhibited the infection damage.

  • Nrf2 was activated while NF-κB/p65 was depleted as TLR2/ TLR4 was knocked down.

  • Activation of Nrf2 and inhibition of NF-κB resulted in decrease of TLR2/TLR4.

Abstract

Background

Neisseria gonorrhoeae (N.g) is Gram-negative bacteria and can lead to endometritis in female. Toll-like receptors regulate immune response in various diseases. However, the roles of TLR2 and TLR4 in. Neisseria gonorrhoeae-induced infection damage in human endometrial epithelia were investigated.

Methods

hEECs were infected with N.g (MOI 10 and 100) and cell viability and apoptosis were measured by CCK8 and flow cytometry assays in both infected groups with the uninfected normal hEECs as negative control. TLR2/TLR4 proteins were measured by ELISA method. Pro-inflammatory markers NLRP3, PGES (PGE2) and TNF-α were assessed by RT-qPCR (mRNA expression) and Elisa (protein concentrations). Transfection assays were performed to up- or down- regulate expression of TLR2 and TLR4 so as to study the functions of TLR2/TLR4 in. N.g-infected hEECs, followed by apoptosis and inflammation assessment. Similarly, we explored the interactions between TLR2/TLR4 and Nrf2/NF-κB/p65 by knocking down TLR2/TLR4 to detect the signaling and further regulating the signaling to evaluate TLR2/ TLR4, apoptosis and inflammation in cells.

Results

N.g suppressed cell viabilities and induced cell apoptosis and inflammation. TLR2/TLR4 downregulation inhibited the infection damage. Nrf2 was activated while NF-κB/p65 was depleted as TLR2/ TLR4 was knocked down. Activation of Nrf2 and inhibition of NF-κB resulted in decrease of TLR2/TLR4, which could retard apoptosis and inflammation induced by N.g infection.

Conclusion

TLR2/TLR4 depletion could alleviate the N.g-infected hEECs via Nrf2/NF-kB signaling, suggesting that TLR2/TLR4 inhibitors might serve as a treatment to reduce N.g infection in human endometrial epithelia.

Introduction

Pelvic inflammation disease (PID) usually takes place after sexually transmitted pathogens rise from the cervix to the uterus and the oviducts, leading to endometritis and salpingitis (Zheng et al., 2018).

Endometritis is a disease featuring inflammation in the endometrial mucosa, which is normally caused by bacteria like Chlamydia trachomatis, Neisseria gonorrhoeae, etc.(Moreno et al., 2018). Endometrial epithelial cells are important part of the endometrial microenvironment (Sekulovski et al., 2019). Neisseria gonorrhoeae is a gram-negative diplococcus that can infect mucosal tissues (endocervix, pharynx, urethra, rectum and conjunctiva, etc.) (Britigan et al., 1985). Moreover, Neisseria gonorrhoeae can enter bloodstream, giving rise to disseminated gonococcal infection (DGI) (Lee et al., 2015). As is extensively reported previously, Neisseria gonorrhoeae is one of the pathogens which contribute to endometritis in human (Ferris, 2016). In addition, there are more than 86 million cases per year infected with gonorrhoeae globally, estimated by World Health Organization (Mendes et al., 2020, Rowley et al., 2019). Previous review has introduced that Neisseria gonorrhoeae adheres to a surface protein or receptor and then invades epithelial cells through evading cellular autophagy (Mendes et al., 2020). Later, the large scale proliferation of Neisseria gonorrhoeae causes lysis of host cells and results in inflammation response after invading submucosa (Ortiz et al., 2015). Furthermore, resistance to antibiotics like penicillin, fluoroquinolones and cephalosporins has made Neisseria gonorrhoeae no direct treatment, which attracts much academic attention (Ortiz et al., 2015). Earlier there was a study revealing that Neisseria gonorrhoeae induced secretion of chemokines (IL-8 and TNF-α) and suppressed the secretion of IL-6 in human endometrial epithelial cells (Christodoulides et al., 2000). Other scientists have discovered recently that Neisseria gonorrhoeae could activate the pro-inflammatory cytokines and induce changes in epithelial cells in 3D endometrial epithelium of endometritis (Łaniewski and Herbst-Kralovetz, 2019). However, the potential mechanism remains to be explored. Hence, it is urgent to dig out more about the interactions between Neisseria gonorrhoeae and epithelial cells in endometrium as well as potential mechanisms beneath so as to further discover the possible treatments against Neisseria gonorrhoeae infection in endometritis.

Toll-like receptors (TLRs) are membrane receptors first discovered in macrophages and dendritic cells and can be activated by pathogens (Underhill et al., 1999, Krutzik et al., 2005). TLR2/4 was discovered to mediate LPS-induced endometritis in animal models (Zhang et al., 2019, Wu et al., 2018, Jiang et al., 2019). Specifically, TLRs were triggered in Neisseria gonorrhoeae-induced endometrial epithelial cells yet the underlying modulatory mechanisms behind TLRs in endometritis need to be unveiled (Łaniewski et al., 2017). Despite of the above mentioned, due to the feature that human is the only nature host of Neisseria gonorrhoeae, interaction between TLRs and Neisseria gonorrhoeae-decoyed endometritis was seldom explored (McGowin and Totten, 2017). In this study, interactions between Neisseria gonorrhoeae and TLR2/4 in endometrial epithelial cells were analyzed and potential regulatory mechanism was explored.

Section snippets

Cell culture

The primary human endometrial epithelial cells (hEECs) were bought from ATCC (USA) and cultured in DMEM medium (Gibco™, USA) with 10 % fetal bovine serum (FBS, Gibco™, USA), 100 ng/ml penicillin and 100U/ml streptomycin at 37 °C, 5 %CO2. After 24 h of culture, cells were seeded into 24-well plates with the density of 105 per well and cultured for 12 h for further use.

Cell transfection and treatment

Cell transfection was performed to regulate the TLR2 and TLR4 expression in hEECs. The si-TLR2, si-TLR4, oe-TLR2 and oe-TLR4 were

Neisseria gonorrhoeae induced apoptosis and inflammation of human endometrial epithelial cells

In order to study the effects of Neisseria gonorrhoeae, cell viabilities were examined in the groups infected by differential concentrations of Neisseria gonorrhoeae. Results indicated that cell viabilities were significantly decreased in infected groups and the group with higher MOI(100) displayed lower viability than the one with lower MOI(10) (Fig. 1A). Moreover, apoptosis assays revealed that infected groups presented higher cell apoptosis rates compared to the normal hEEC group and the

Discussion

N.g sticks to and invades the mucosal epithelia, which forms the initial barrier against bacteria or virus (Naumann et al., 1997). Therefore, we established the N.g-induced endometritis model in vitro with hEECs. TLRs are innate immune receptors, recognizing pathogen-related molecular patterns (Kannaki et al., 2011). TLR2 and TLR4 are both surface-expressed receptors and TLR4 recognizes the LPS toxicity of Gram-negative bacteria, inducing the secretion of IL-6, IL-8 and PGE2 (Sheldon et al.,

Conclusion

TLR2 and TLR4 were both activated by Neisseria gonorrhoeae in human endometrial epithelial cells and downregulation of TLR2 and TLR4 deterred cell apoptosis and inflammation damage induced by N.g infection via activating Nrf2 and suppressing NF-κB signaling pathway, suggesting that inhibitors of TLR2 or TLR4 might be helpful to alleviate N.g-induced endometritis. However, experiments in vivo and clinical validations remain to be performed in the near future.

Declaration of Competing Interest

The authors report no declarations of interest.

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    Authors contributed equally to this study.

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