The meibomian gland (MG) is a modified functional sebaceous gland (SG) anatomically arranged within the tarsal plates of the upper and lower eyelids in the human body (Obata, 2002). Matured meibocytes disintegrate via holocrine secretion and release a lipids-enriched mixture (Meibum) into the MG duct, which is further transported to the ocular surface, constructing the tear film lipid layer (TFLL) (Butovich, 2017; Knop et al., 2011). Any pathological factors that can result in MG duct obstruction and qualitative and quantitative abnormalities of the meibum would induce meibomian gland dysfunction (MGD) (Knop et al., 2011). MGD has detrimental effects on the health of the ocular surface microenvironment, leading to tear film instability, accelerated tear evaporation rate, and increased tear osmolarity, which is the prime pathogeny of evaporative dry eye (Bai et al., 2021; Barna et al., 2019; Eom et al., 2013). According to a recent meta-analysis on the global prevalence of MGD, the incidence of this condition in certain regions has reached as high as 51.2%–71.0% (Hassanzadeh et al., 2021). Despite its prevalence, the etiology of MGD remains unclear, and there is currently a lack of radical therapy, which seriously affects the quality of life of patients (Asiedu et al., 2022).

Meibum comprises non-polar lipids such as wax esters, sterol esters, cholesterol, and triglycerides, as well as polar lipids including phospholipids, sphingolipids, (O-acyl)-omega-hydroxy fatty acids, and free fatty acids (FFAs) (Butovich, 2017; Chen et al., 2010; McCulley and Shine, 2003). Nonpolar lipids make up the majority of meibum (∼90%), which are located in the outermost layer of the TFLL and serve as lubricants and water barrier (Brown et al., 2013). Although polar lipids account for a lower percentage of meibum (∼10%), they act as surfactant molecules to facilitate the diffusion of TFLL over the ocular surface and provide the crucial anti-evaporation effect (Paananen et al., 2020). Notably, FFAs in meibum are considered products of bacterial degradation (Chhadva et al., 2017; McCulley and Shine, 2003). Commensal bacteria in meibum, such as Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes (Dougherty and McCulley, 1984; Knop et al., 2011), possess lipolytic enzymes that break down neutral fats and esters, releasing trace amounts of FFAs under physiological conditions (Chhadva et al., 2017). However, the meibum of patients with MGD demonstrated significantly higher culture positivity and more complex bacterial composition compared to healthy individuals (Jiang et al., 2018; Zhang et al., 2017). This phenomenon resulted in the increased production of bacteria-derived lipolytic enzymes that broke down nonpolar esters thus elevating the release of toxic FFAs within MG (Baudouin et al., 2016; Dougherty and McCulley, 1986b). Clinical studies have reported that the level of FFAs in the meibum was positively correlated with MG obstruction and worse meibum quality (Arita et al., 2016; Suzuki et al., 2022), indicating the accumulated FFAs may contribute to the pathology of MGD.

Oleic acid (OA), a monounsaturated omega-9 fatty acid, is the most abundant degraded FFAs in human meibum due to its wide presence in the wax esters, sterol esters, and triglycerides (Butovich et al., 2012; Chen et al., 2010; Ziemanski et al., 2021a, 2021b). Previous clinical research revealed that the relative content of OA in FFAs was significantly increased in the meibum from patients with MGD and chronic blepharitis (Dougherty and McCulley, 1986a; Joffre et al., 2008). Furthermore, the higher amount of meibum OA was associated with MG seborrheic changes (Shine and McCulley, 2000). In the SG, OA has been identified as a robust stimulator of seborrhea in human sebocytes (Brami-Cherrier et al., 2022). OA could induce lipogenic abnormalities and proinflammatory response in the pilosebaceous unit, resulting in inflammatory acne and skin keratinization (Katsuta et al., 2005; Li et al., 2017; Zhu et al., 2021). OA could also activate the NLRP3 inflammasome signal in human liver cells, leading to the production of proinflammatory cytokines and the development of hepatic steatosis (Wang et al., 2023b). However, whether abnormal OA elevation would cause MGD-related meibum lipids disorder and proinflammatory response remains unclear.

Therefore, in the present study, we aimed to investigate the potential role of OA in MGD using an in vitro model of organotypic cultured mouse MG, which recapitulated the pathological features of MGD characterized by lipid deposition, ductal abnormalities, and inflammasome activation, and further verified the role in human meibomian gland epithelial cells (HMGECs).