Impaired pathogen-induced autophagy and increased IL-1β and TNFα release in response to pathogenic triggers in secretory phase endometrial stromal cells of endometriosis patients

Highlights

• Tightly controlled inflammatory responses in endometrium of healthy fertile women.

• Impaired pathogen-induced autophagy in endometrium of patients with endometriosis.

• Increased inflammatory responses in secretory endometrial stromal cells of endometriosis patients.

Abstract

Research question

It is not clear whether innate immunity along with autophagy is altered in endometrial cells of patients with endometriosis.

Design

This study evaluated the effects of lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) stimulation on autophagy induction, pro-IL-1β expression, and secretion of interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα) in endometrial epithelial and/or stromal cells of patients with endometriosis (EE-endo, ES-endo, respectively), those of patients with hydrosalpinx (EE-hydro, ES-hydro, respectively) and those of healthy fertile women (EE-healthy, ES-healthy, respectively), with and without inhibition of autophagy by autophagy-related (ATG)13 gene small interfering RNA (siRNA).

Results

Stimulation with either LPS or poly I:C triggered autophagy in EE/ES-healthy, whereas no significant induction was observed in either EE/ES-endo or EE/ES-hydro. In EE- and/or ES-healthy, IL-1β and/or TNFα secretion after stimulation with LPS or poly I:C was significantly higher in cells with ATG13 knockdown compared with those with siRNA control (P < 0.03), whereas no significant difference was observed in either EE/ES-endo or EE/ES-hydro. In the secretory phase ES-endo without autophagy inhibition, IL-1β and TNFα secretion were significantly higher compared with those of ES-healthy after stimulation with either LPS or poly I:C for 4 h (P < 0.001) and for 24 h (P < 0.01).

Conclusion

Pathogen-induced autophagy was impaired in EE/ES-endo. Increased IL-1β and TNFα release in response to pathogenic triggers in the secretory phase ES-endo may result in the development of an inflammatory uterine microenvironment detrimental to successful embryo implantation.

Introduction

Numerous studies have shown that immunological dysfunction and chronic inflammation are involved in the pathophysiology of endometriosis (reviewed in Ahn et al., 2015; Symons et al., 2018). However, it is not clear whether innate immune function is altered in endometrial cells of patients with endometriosis. The immune system plays an essential role in protecting the host against infections (Albiger et al., 2007). The innate immune system comprises the first line of defence against invading microbial pathogens and largely depends on a large family of pattern recognition receptors (PRR) such as Toll-like receptors (TLR) and nucleotide-binding domain (NOD)-like receptors (NLR), which detect distinct evolutionarily conserved structures on pathogens, termed pathogen-associated molecular patterns (PAMP) (Albiger et al., 2007). Most TLR and NLR are detected in human endometrial epithelial and stromal cells (Sheldon and Bromfield, 2011).

A growing body of evidence suggests that the uterus is a non-sterile compartment (Benner et al., 2018; Moreno et al., 2018). Moreno et al. (2018) showed that pathological modification of a stable profile of endometrial microbiota during the acquisition of endometrial receptivity is associated with poor reproductive outcomes for patients undergoing IVF. The human endometrium is an important site of innate immune defence (Quayle, 2002; Sheldon and Bromfield, 2011). The innate immune response appears to be carried out by epithelial and stromal cells of the endometrium, which then recruit more professional immune cells to the site of infection (Quayle, 2002; Sheldon and Bromfield, 2011). Previous studies demonstrated the involvement of TLR-4 and LPS in the pathophysiology of endometriosis (Khan et al., 2013; 2014; Kobayashi et al., 2014). Intrauterine bacterial contamination may be a cause of endometriosis (Khan et al., 2013, 2014; Kobayashi et al., 2014; Koninckx et al., 2019). However, not only bacterial, but also viral pathogens are involved in female genital tract infection (Amjadi et al., 2014; Sheldon et al., 2011). TLR-3, TLR-7/8 and TLR-9 are involved in antiviral responses; among these, TLR-3 recognizes dsRNA viruses (Lin and Staudt, 2013). TLR-3 activation in vitro by poly I:C reduces the attachment of trophoblast cells to endometrial cells (Montazeri et al., 2015).

Interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα) are central mediators of innate immunity (Newton et al., 2012). Numerous studies have demonstrated the involvement of these two proinflammatory cytokines in the pathophysiology of endometriosis (reviewed in Ahn et al., 2015; Symons et al., 2018). The IL-1β and TNFα genes are tightly regulated at the transcriptional, post-transcriptional and post-translational levels (Fan et al., 2005; Falvo et al., 2011; Newton et al., 2012; Palanisamy et al., 2012; Quayle, 2011; Sheldon and Bromfield, 2011). The mechanism of IL-1β release does not follow the conventional endoplasmic reticulum (ER)–Golgi route of secretion (Lopez-Castejon and Brough, 2011). IL-1β gene transcription is induced upon nuclear factor (NF)-κB activation caused by PAMP or damage-associated molecular patterns (DAMP) binding to TLR, resulting in production of pro-IL-1β, which is not biologically active (Lopez-Castejon et al., 2011). The second step is inflammasome-dependent; NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation is negatively regulated by autophagy, which controls excess release of the mature, active form of IL-1β (Harris et al., 2011; Lopez-Castejon et al., 2011). Autophagy balances inflammation during the innate immune response and prevents excessive inflammation (Netea-Maier et al., 2016). As evolution progressed, nearly all innate immune system components, such as conventional PRR and inflammasomes, became integrated with autophagy. Impaired autophagy can lead to inflammatory, autoimmune or general immunity disorders. Recent studies have shown that impaired autophagy may be involved in the pathophysiology of endometriosis (Mei et al., 2015; Zhan et al., 2018).

Based on these findings, it was postulated that innate immunity along with autophagy is altered in endometrial cells of patients with endometriosis, causing chronic inflammation and/or endometrial receptivity defects.

The present study evaluated the effects of lipopolysaccharide (LPS; a TLR-4 ligand) and poly I:C (a TLR-3 ligand) stimulation for 4 h (acute) (Dinarello, 2018; Gurung et al., 2015; Swangchan-Uthai et al., 2012; Zhu and Kanneganti, 2017) and 24 h (chronic) (Dinarello, 2018; Gurung et al., 2015; Swangchan-Uthai et al., 2012; Zhu and Kanneganti, 2017) on secretion of mature IL-1β and TNFα in endometrial epithelial and stromal cells of infertile patients with endometriosis, with and without autophagy inhibition by the autophagy-related (ATG)13 gene small interfering RNA (siRNA) (Matsuzaki et al., 2018). The autophagic machinery is encoded by ATG genes (Mizushima, 2007). In the present study, among the different ATG genes, the ATG13 gene was selected, which is a part of the UNC51-like kinase (ULK) complex (Mizushima, 2007; Yang and Klionsky, 2009). ATG genes coordinate the six steps of autophagy: initiation, nucleation, elongation, vesicle (autophagosome) formation, autophagosome–lysosome fusion, and autophagosome degradation and recycling (Mizushima, 2007; Yang and Klionsky, 2009). The ATG13 is essential for autophagy initiation (Mizushima, 2007; Yang and Klionsky, 2009).