Interleukin-18 levels and mouse Leydig cell apoptosis during lipopolysaccharide-induced acute inflammatory conditions
Introduction
Post-intensive-care syndrome is defined as new or worsening impairments in physical, cognitive, or mental health status arising after a critical illness and persisting beyond acute care hospitalization (Elliott et al., 2014). Several physical manifestations of post-intensive-care syndrome, such as respiratory and neuromuscular problems, have been reported, but few reports have described reproductive dysfunction and gonad physiology after a severe illness (Herridge et al., 2003; Stevens et al., 2007). Every year 20–30 million people worldwide suffer from sepsis, a life-threatening complication of severe infections, and ∼20 % of adult male patients suffering sepsis are between the peak reproductive ages of 18–45 years old (Beale et al., 2009; Reinhart et al., 2013). Recently, long-term mortality and quality of life after sepsis have been examined (Winters et al., 2010). Understanding the effects of severe illnesses on the gonads and reproductive function is crucial to understanding how quality of life may be affected.
Interleukin (IL)-18 is an important cytokine to maintain the homeostasis of testicular cells. IL-18 is produced by germ cells, Leydig cells, and resident macrophages and may regulate testicular function via autocrine and paracrine signaling under physiologic conditions (Abu Elhija et al., 2008a,d; Komsky et al., 2012; Strand et al., 2005). IL-18 is also a known inflammasome-mediated proinflammatory cytokine, and IL-18 levels are increased in mouse testes by inflammatory stimulation, such as lipopolysaccharide (LPS) administration (Abu Elhija et al., 2008b,c). We previously showed using a knockout mouse model that endogenous IL-18 has both pro-apoptotic and anti-apoptotic effects on mouse testicular germ cells during endotoxemia depending on the inflammatory stage. Endogenous IL-18 induced germ cell apoptosis during the acute phase of inflammation and suppressed germ cell apoptosis during the recovery phase (Inoue et al., 2015). We could not elucidate the effect of IL-18 on Leydig cells using this mouse model.
The primary function of Leydig cells is to synthesize and secrete androgens (Shalet, 2009). Several in vivo studies have demonstrated that LPS causes dysfunctional testicular steroidogenesis, reduces the levels of steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase in the testes and decreases serum testosterone (Allen et al., 2004; O’Bryan et al., 2000; Reddy et al., 2006; Wang et al., 2020). Shortages of androgen lead to male infertility (Jungwirth et al., 2012). Thus, we hypothesized that high-dose IL-18, which is induced by inflammatory stimuli or conditions such as sepsis, would induce Leydig cell apoptosis, affecting steroidogenesis and male fertility. In this study, we investigated the impact of IL-18 on Leydig cell apoptosis under inflammatory conditions in vitro.
Section snippets
Cell culture, LPS and rIL-18 stimulation, and cell viability
A Leydig cell line (TM3) and a macrophage cell line (RAW264.7) were purchased from the American Type Culture Collection (Manassas, VA, USA). TM3 cells and RAW264.7 cells that had undergone 4 passages were used for the experiments. The TM3 cells were cultured in Dulbecco's modified eagle medium (DMEM)/F12 medium (American Type Culture Collection) supplemented with penicillin (50 U/mL), streptomycin (50 μg/mL; MP Biomedicals, Illkirch, France), 5 % horse serum (American Type Culture Collection),
LPS induced Leydig cell apoptosis via a death- receptor-mediated pathway
To establish inflammatory conditions, we treated a Leydig cell line, TM3 cells, with LPS and examined apoptosis as indicated by cleaved caspase-3 protein expression. The amount of cleaved caspase-3 in TM3 cells increased after 12 h of LPS treatment and remained elevated after 48 h of treatment with either 200 ng/mL LPS or 1000 ng/mL LPS (Fig. 1A, B). These results suggest that endotoxins induce Leydig cell apoptosis via caspase-3 dependent pathways. We also examined cleaved caspase-8 levels, a
Discussion
This study demonstrated that both LPS and high-dose rIL-18 induce apoptosis in Leydig cells via the Fas/FasL/caspase-8-dependent apoptotic pathway. Additionally, they demonstrated that LPS can induce Leydig cell apoptosis via a TNF/TNFR/caspase-8-dependent pathway. Zhou et al. (2016) showed that Leydig cell apoptosis was induced by LPS via the mitochondrial pathway. To our knowledge, our study is the first to demonstrate that Leydig cell apoptosis is also induced by LPS via
Funding
This work was supported by JSPS KAKENHI Grant Number 16K20391.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.
Acknowledgements
We thank Shannon Wyszomierski, PhD for editing the manuscript.
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