Exosomes derived from LPS-induced MHs cells prompted an inflammatory response in sepsis-induced acute lung injury

https://doi.org/10.1016/j.resp.2021.103711Get rights and content

Highlights

  • GW4869 inhibited exosome generation and pro-inflammatory cytokine production.

  • The exosomes derived from MHs cells maintained the pro-inflammatory effect.

  • Treatment with LPS prompted the production of pro-inflammatory cytokines and exosomes.

Abstract

Exosome is a novel tool with an essential role in cell communication. However, its role in the pathogenesis of sepsis-induced acute lung injury is currently unknown. Here, we first found that lipopolysaccharide (LPS) could up-regulate the expression of pro-inflammatory cytokines and promote exosomes release in the murine alveolar macrophage cell line (MHs cells). Moreover, we found MHs cells derived exosomes also maintain the pro-inflammatory effect after LPS stimulation. Treating with hydrochloride hydrate (GW4869) could dose-dependently downregulated the release of exosomes and inhibited the upregulation of inflammatory cytokines in MHs cells with LPS treatment. Also, we further identified GW4869 administration induced the remission of histopathologic changes, the reduction of pro-inflammatory cytokines in lung tissue, and inhibit serum exosomes release. These results indicate that the downregulation of exosome release by GW4869 might protect lung tissue from LPS induced injury through the suppression of excessive inflammatory responses, suggesting its potential therapeutic effects on sepsis-induced acute lung injury.

Introduction

Sepsis is a common complication in the clinic with a high mortality rate at about 50 % over the past two decades (Lelubre and Vincent, 2018; Vincent et al., 2014). It has been proved that sepsis develops as the response to infection, including initiation of generalized inflammatory and immune suppression (Lelubre and Vincent, 2018). Unfortunately, several inflammatory cells, such as macrophages and neutrophils, were aberrantly recruited and continue activated during this progression as compared to the normal or optimal response (Stearns-Kurosawa et al., 2011). With the robust accumulation of these cells and related pro-inflammatory mediators, including nuclear factor-kappa B (NF-κB), tumor necrosis factor (TNF)-α and interleukin (IL-6, IL-8, and IL-1β), many acute life-threatening organ dysfunctions in the patients would be initiated (Agrawal et al., 2013; Wu et al., 2007; Zhang et al., 2013). Numerous studies have demonstrated that the lung tissue was first to be damaged during this progression, which is termed sepsis-induced acute lung injury (SALI) (Martin et al., 2003). During the development of ALI, IL-1β, IL-6, and TNF-α are considered as important pro-inflammatory mediators (Li et al., 2019; Meng et al., 2018). They had reported that when the SALI occurred, the pulmonary neutrophil sequestration and capillary integrity had been disrupted, and thus resulting in pulmonary capillary leak and edema, as well as impairment of lung function (Chatterjee et al., 2007). As the essential defender, alveolar macrophage plays a key role in the pathogenesis of SALI. The crosstalk between macrophages and epithelial cells has been reported to influence balance of lung inflammation, epithelial injury, and alveolar fluid clearance, thereby controlling the severity of SALI (Lee et al., 2018). Recently, previous studies demonstrated that suppression the functions of macrophages and neutrophils might reduce acute lung injury (Herold et al., 2011). For example, Kojima et al. reported that galectin-9 has conserved carbohydrate recognition domains and affinity for β-galactoside, which plays a crucial role in inflammatory and autoimmune diseases. Administration of galectin-9 would interfere with the release of pro-inflammatory cytokines derived from macrophages (Kojima et al., 2011). Another group suggested that melatonin could significantly inhibit the infiltration macrophages into the lung via blocking the activation of nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome and related downstream targets like caspase 1 and IL-1β (Zhang et al., 2016). However, there are no effective therapies aimed to improve the ALI directly. Hence, there is a compelling need to explore novel target facilitating the development of therapeutic interventions for this disease. Also, further understanding of the underlying mechanisms of macrophages to protect lung tissue during SALI is of the utmost clinical importance.

Nowadays, the exosomes have emerged as an intercellular mediator participating in cell-cell communication, pathogenesis, and physiology progression by transferring its contents among different cells (Samir et al., 2013; Thery et al., 2002). It is a novel kind of nanoscale membrane-formed compartments with a diameter range from 50 to 150 nm naturally released by almost all types of cells and contents various specific features of their origin (Gao et al., 2017). Moon et al. reported that majority of exosomes in the bronchoalveolar lavage fluid were derived from macrophages at the normal state of mice. Besides, this type of exosome increased robustly at the early stage of LPS stimulation (Moon et al., 2015). It suggested that macrophage-derived exosomes might play an essential role in the formation of SALI. The exosomes from macrophage could be uptake by the target cells such as monocytes, endothelial cells, epithelial cells, and fibroblasts. Based on this observation, they demonstrated that the exosomal microRNAs (miRNAs) or enzymes played an essential role in the regulation of host defense and inflammation (Ismail et al., 2013) (Esser et al., 2010). Besides, Bhatnagar et al. also found when the macrophages infected by the intracellular pathogens, the related exosomes maintained the pathogen-associated molecules, and thus stimulating pro-inflammatory factors production in naive macrophages (Bhatnagar et al., 2007). Given the importance and obscurity of the macrophage-derived exosomes, we sought whether suppression of exosome production would be benefit for SALI therapy.

As an important inhibitor for exosome release, GW4869, has been reported to block the budding of multivesicular bodies, and thus regulated the maturation of exosomes (Kosaka et al., 2010; Li et al., 2013). It has been widely used in several cells, including macrophages (Chen et al., 2019; Wang et al., 2019). Therefore, in this study, we first tried to use the LPS treatment to stimulate the sepsis condition in vivo and in vitro. Using the LPS-induced model, we directly identified the alteration of pro-inflammatory signature in the exosomes and evaluated the effect of them in the formation of SALI. Further, we assessed the protective effect of GW4869 against SALI in vitro and in vivo. These findings reported herein shed light on the molecular mechanism and therapeutic target for the SALI treatment and diagnosis.

Section snippets

Cell culture and treatment

The murine alveolar macrophage cell line (MHs cells) was purchased from American Type Culture Collection (Manassas, VA, USA) and maintained in the Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10 % fetal bovine serum (FBS, Gibco, USA) and 100 U/mL penicillin/streptomycin (Thermo, USA). Cells were cultured in the humidified incubator with 5% CO2 at 37 ℃.

1*106 cells were treated by 1 μg/mL LPS (MedChem Express, China) for 24 h according to the manufacture’s protocol. The

Treatment with LPS prompted the production of pro-inflammatory cytokines and exosomes in MHs cells

First, we treated MHs cells with 1 μg/mL LPS for 24 h to stimulate the sepsis condition in vitro. As shown in Fig. 1A, compared with the control group, levels of TNF-α and IL-1β were increased about 5-folds after LPS treatment (p < 0.05). Besides, the level of IL-6 was significantly up-regulated from 0.05 ± 0.02 ng/mL to 1.44 ± 0.17 ng/mL with the same treatment (p < 0.05). Meanwhile, the exosomes were isolated by ultracentrifugation from the MHs cells with different treatments.

As shown in Fig.

Discussion

Sepsis, a lethal complication of several diseases, often causes injury of multiple organs and even death. As such, understanding the constitutive mechanisms of sepsis and related acute injury is still critical for the improvement in the outcomes of patients with severe sepsis. Previous studies have reported that the occurrence of sepsis was associated with LPS stimulation. Injection of LPS into the animal or treated the cells in vitro activated the inflammatory response, which was similar to

Funding

The study was supported by the National Natural Science Foundation of China (grant no. 81571882; no. 81772053).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

SXT designed the study, performed experiments and wrote the manuscript. SXT, LW and LZ contributed to designing the study and revising the manuscript critically for important intellectual content. The final version of the manuscript has been read and approved by all authors.

Ethics approval and consent to participate

The study was approved by the Committee of International Association for the Protection of Animal and Experimental Medicine and Laboratory Animal Ethics Committee of First Hospital of China Medical University (Shenyang, China).

Patient consent for publication

Not applicable.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

Not applicable.

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