Introduction
Sepsis is defined as a global health priority by the World Health Organization (WHO) and characterized by excessive inflammation in response to infection, with a reported death rate of 30–45% in hospitalized patients [
1,
2]. Acute respiratory distress syndrome (ARDS) is the most common severe manifestation of multiple organ dysfunction syndrome and a significant factor contributing to the morbidity and mortality of sepsis [
3].
Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes in mammals, which play a crucial role in the pathogenesis of sepsis-related acute lung injury (ALI) or ARDS [
4]. Exosomes are small extracellular vesicles secreted by various cell types, with size ranging from 30 to 150 nm [
5]. They can transfer a multitude of proteins and genetic material (including DNA, mRNA and microRNA [miRNA]) to target cells, playing a key role in cell-to-cell communications [
6]. Animal studies have implicated the roles of PMN-derived exosomes in many chronic lung injuries, including chronic obstructive pulmonary disease (COPD), bronchopulmonary dysplasia and asthma [
7,
8], but their role in sepsis-related ALI remains unclear.
In addition to PMNs, M1 macrophages, as a proinflammatory phenotype, also promoted the occurrence of sepsis-related ALI. M1 macrophages could be activated by circulating plasma exosomes, which further promote the secretion of proinflammatory cytokines, such as interleukin 1β (IL-1β), IL-12, IL-6 and tumor necrosis factor-α (TNF-α) [
9,
10]. Recently, the pyroptosis of macrophage (Mϕ) has also been highlighted in sepsis-related ALI. Pyroptosis is a caspase-1-dependent proinflammatory cell death type [
4,
11]. Caspase-1 activated by inflammasomes including NOD-like receptor 3 (NLRP3) inflammasomes initiates pyroptosis by cleaving gasdermin D (GSDMD, the core event in pyroptosis) to form pores in the plasma membrane, leading to cell swelling and membrane rupture, finally resulting in the leakage of mature forms of IL-1β and IL-18 out of cells [
12,
13]. Pyroptotic macrophage-released danger signals or danger-associated molecular pattern molecules enhance inflammatory responses in sepsis-related ALI [
14].
The crosstalk between PMNs and macrophages in regulating inflammation has been documented [
15‐
18]. We previously reported that exosomes secreted from macrophages promoted neutrophil necroptosis following hemorrhagic shock [
17]. Macrophage pyroptosis in sepsis could also be primed by neutrophil extracellular traps (NETs) [
19]. However, the effects of PMN-derived exosomes on the behavior of macrophage and the underlying mechanisms in sepsis-related ALI are unknown.
Here, we firstly identified the role of PMN-derived exosomes in sepsis-related ALI by inducing macrophage M1 polarization and priming macrophage for pyroptosis. Bioinformatics analysis and further mechanistic studies revealed that PMN-derived exosomes transferred miR-30d-5p into macrophages and then activated NF-κB signaling pathway by inhibiting SOCS-1 (suppressor of cytokine signaling) and sirtuin 1 (SIRT1), which were both recognized as negative regulators of NF-κB signaling pathway previously [
20,
21]. These findings suggest a previously unidentified pathway of PMN-Mϕ crosstalk, which could enhance macrophage activation and death, and subsequently exaggerate post-sepsis inflammation and induce lung injury.
Materials and methods
Animals
Wild type (WT) male C57BL/6J mice aged 6–8 weeks (Shanghai Sippr-BK Laboratory Animal Co., Ltd., Shanghai, China) were fed under a specific pathogen-free environment in Xinhua Hospital Animal Laboratory (Shanghai, China). All animal experiments were conducted under the rules approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Approval No.: XHEC-F-2020-019).
PMNs isolation and activation
PMNs were induced in the peritoneal cavity of the mice as previously described [
22]. Briefly, mice were injected intraperitoneally (i.p.) with 1 ml 9% casein solution twice overnight and killed 3 h after the second injection to harvest the peritoneal lavage fluid (PLF), which was subsequently centrifuged, and the cell pellets were washed. PMNs were isolated by discontinuous density gradient centrifugation with two commercially available solutions (Histopaque-1077 and Histopaque-1119) of differential density (Sigma (St. Louis, MO; #11191 and #10771) according to the manufacturer’s instructions. The resulting cells consisted of 90% PMNs, and viability of the isolated PMNs was 95% as assessed by flow cytometry and Trypan blue staining, respectively.
After isolation, PMNs were suspended in complete culture medium (RPMI 1640 containing 10% exosome-free FBS, supplemented with 50 mg/ml penicillin/streptomycin) at a concentration of 106 cells/ml. PMNs activation was induced upon 12-h incubation with 20 ng/mL TNF-α at 37 °C. An equal volume of phosphate buffered saline (PBS) to TNF-α was used as negative control.
Exosome isolation and characterization
Exosomes were isolated from the supernatant of PMNs treated with PBS (PBS-Exo) or TNF-α (TNF-Exo) ex vivo using Total Exosome Isolation Reagent (#4484450; Thermo Fisher Scientific, Waltham, MA, USA). The detailed isolation procedure and the methods used to determine exosomal morphology, size distribution, and surface marker expression are described in Additional file
1.
In vivo exosome administration to WT C57BL/6 mice
To explore exosome function in vivo, five WT C57BL/6 mice in each group were injected with PBS-Exo or TNF-Exo (300 μg/mouse) intraperitoneally using a 31-gauge insulin syringe, respectively. An equal volume of PBS was used as negative control. After 24 h, the obtained peritoneal lavage fluid was centrifuged, and peritoneal macrophages were detected by flow cytometry after gating with F4/80. To visualize changes in morphology and macrophage polarization, lung tissues were harvested and fixed in 4% paraformaldehyde for H&E and immunofluorescence staining. H&E staining was evaluated by a pathologist who was blinded to the experimental groups. To evaluate the lung injury, five independent random lung fields were evaluated per mouse for neutrophils in alveolar spaces, neutrophils in interstitial spaces, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening, and weighed according to the official American Thoracic Society workshop report on features and measurements of experimental ALI in animals [
23]. The resulting injury score is a continuous value between 0 and 1. For immunofluorescence staining, paraffin-embedded lung tissues were sectioned, blocked with PBS containing 1% goat serum and 3% BSA, permeabilized with PBS/Triton 0.01%, and incubated with F4/80 and iNOS antibodies, and then with species-specific secondary antibodies coupled with Alexa Fluor Dyes. DNA was stained using DAPI. The sections were treated with autofluorescent quenching solution (#G1221; Servicebio, Wuhan, China) and mounted in Vectashield Mounting Media.
The in vivo miR-30d-5p inhibitors were transfected into the mouse lung through tail vein injection using the in vivo-jetPEI (Polyplus-transfection SA, New York, NY). Briefly, the miR-30d-5p inhibitor or negative control (50 μg, N/P ratio = 6, i.e., 0.12 μl of in vivo-jetPEI per μg nucleic acid) dissolved in 200 μl 5% glucose solution was injected into each mouse 1 day before exosome injection, according to the manufacturer’s protocol.
In vitro co-culture experiments
Raw264.7 macrophages or BMDMs were treated with PMN-derived exosomes (100 μg/ml) at 37 °C for 24 h. To induce pyroptosis, macrophages were primed with exosomes for 24 h before stimulation with 5 mM ATP (#HY-B2176; MedChemExpress, Monmouth Junction, NJ, USA) or 20 μM nigericin (#HY-100381; MedChemExpress) for 2 h.
For miR-30d-5p inhibition, Raw264.7 macrophages were transfected with microRNA control or miR-30d-5p inhibitor (Guangzhou Ribobio Corporation, Guangzhou, China) at a concentration of 50 nM using Lipofectamine 3000 for 24 h prior to be co-cultured with exosomes as per the manufacturer’s instructions. For miR-30d-5p overexpression, Raw264.7 macrophages were transfected with microRNA control or miR-30d-5p mimic at a concentration of 50 nM using Lipofectamine 3000 for 48 h.
Flow cytometry analysis of M1 polarization and pyroptosis
Macrophages were centrifuged and resuspended in PBS for FACS analysis. According to the manufacturer’s instructions, anti-CD11c (#117307; BioLegend, San Diego, CA, USA), anti-CD86 (#105106; BioLegend), anti-CD206 (#141706; BioLegend) and anti-F4/80 (#123116; BioLegend) antibodies were used for fluorescent staining. Isotype antibody controls were used to exclude nonspecific staining. Data were obtained using a CytoFLEX flow cytometer (Beckman, USA). Programmed cell death was analyzed with apoptosis detection kit (#559763; BD Biosciences, Franklin Lakes, NJ). Macrophages were incubated with Annexin-V and PI for 15 min at room temperature in the dark and then analyzed by flow cytometry. Cells double-stained positive for Annexin V and PI were considered as undergoing programmed death. Cell pyroptosis was detected by two-color flow cytometry. Macrophages were incubated with Alexa Fluor 488-labeled caspase-1 FLICA (#ICT098; ImmunoChemistry, Bloomington, MN, USA) at 37 °C for 1 h. After being fixed with 4% paraformaldehyde, cells were stained with TMR red-labeled In-Situ Cell Death Detection reagent (#12156792910; Roche Applied Science, Indianapolis, IN, USA), following the manufacturer’s instructions. Double-stained cells were identified as pyroptotic cells. Background and autofluorescence were determined by a control antibody with the same isotype staining.
Sequencing of exosomal miRNA and data analysis
Total RNA was extracted from PBS-Exo/TNF-Exo using the miRNeasy Serum/Plasma Kit (Qiagen, Valencia, CA, USA). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI Group, Shenzhen, China). After acquiring the raw data, the differentially expressed miRNAs were calculated using the t test. Those with ≥ twofold upregulation and a P value < 0.05 were regarded as significantly different. A heat map was generated using the R 3.5.3 software. Pathway analysis was conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. The 20 most enriched pathways related to signaling transduction are listed and were used to reveal the most associated pathways.
Quantitative real-time PCR (RT-qPCR)
Total RNA isolation was performed using TRIzol following the manufacturer’s instruction (TAKARA, Japan). mRNA was reverse transcribed using PrimeScript RT reagent Kit (#RR036; TAKARA, Tokyo, Japan), and PCR was conducted using TB Green™ Premix Ex Taq™ (Tli RNaseH Plus) (#RR420A; TAKARA) and QuantStudio™ 3 System (Applied Biosystems). Data were normalized to the expression of GAPDH. Primer sequences are shown in Additional file
1: Table S1.
For exosomal miRNA quantification, total RNA was extracted from 200 μl PMN-derived exosomes using miRNeasy Serum/Plasma kit (#217184; Qiagen, Valencia, CA, USA). RNA pellets were resuspended in 14 μl RNase-free water. Twelve microliters of RNA solution were used for reverse transcription, according to the protocol of miScript RT Kit (#218161; Qiagen). miRNA expression was quantified using a miScript SYBR Green PCR Kit (#218075; Qiagen). qPCR analysis was also performed for miR-30d-5p expression in cells or the lung tissue. Briefly, RNA was extracted by TRIzol reagent (TAKARA, Japan) and RT-qPCR was conducted using the Mir-X miRNA qRT-PCR SYBR Kit (#638316; TAKARA). Relative expression was calculated using the comparative cycle threshold (Ct) method (2−ΔΔCT) normalized to U6. The miRNA qPCR primers were purchased from Guangzhou Ribobio Corporation.
Luciferase assay
The 3′-UTR of the SOCS-1/SIRT1 sequence containing the predicted miR-30d-5p binding sites and its mutant was cloned into the plasmid vector and transfected into HEK293 cells. A renilla luciferase vector was co-transfected in all transfections described to monitor transfection efficiency. All luciferase results were reported as relative light units: the average of the photinus pyralis firefly activity observed was divided by the average of the activity recorded from the renilla luciferase vector.
Mouse model of cecal ligation and puncture (CLP)
The CLP mouse model was prepared as previously described [
22]. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (5 mg/kg) via i.p. injection. After disinfection, a 1 cm midline laparotomy was made in the abdomen. The cecum was then exteriorized, and ligated below the cecal valve, and punctured with an 18-gauge needle to induce sepsis. A small drop of cecal content was extruded. The cecum was then returned to the peritoneal cavity and the abdominal incision closed with sutures. Mice were resuscitated with (5 ml/100 g) saline. Sham animals underwent the same surgical procedures without cecum ligation and puncture. The in vivo miR-30d-5p inhibitors were transfected into the mouse lung through tail vein injection using the in vivo
-jetPEI one day before CLP surgery, according to the manufacturer’s protocol. At 24 h after surgery, the animals were euthanized with phenobarbital overdose (100 mg/kg body weight), followed by collection of the lung tissues as described previously.
Statistics
The normal distribution of the data was tested using the Shapiro–Wilk test. Normally distributed data are presented as means ± SEM. Comparisons between 2 groups were performed by the 2-tailed Student’s t test. Multiple group comparisons were performed by one-way ANOVA followed by Tukey's multiple comparisons test with GraphPad Prism 8 software. Comparison of survival rates between groups was performed using Log-rank test. A value of P < 0.05 was considered statistically significant.
Discussion
It was found in our study that exosomal miR-30d-5p from PMNs induced macrophage M1 polarization and primed macrophages pyroptosis by activating NF-kB signaling via targeting SOCS-1 and SIRT1. In addition, we discovered a previously unidentified role of PMN-Mϕ interaction in promoting inflammation in sepsis-related ALI.
In the early stage of sepsis, neutrophils are thought to be the primary innate immune cells that causing damage to host tissues [
31]. In addition to releasing important cytokines, chemokines, ROS and NETs, some studies suggested that PMN-derived exosomes were a new subcellular entity, working as a fundamental link between PMN-driven inflammation and tissue damage [
8]. TNF-α plays a central role in the pathogenesis of sepsis and is an early regulator of the immune response [
32]. Previous studies [
33,
34] showed that TNF-α and IL-1β produced by macrophages activated neutrophils during sepsis, and high concentrations of TNF-α and IL-1β have been reported in BALF from ARDS patients. Therefore, in this study, we used TNF-α to activate PMNs from healthy mice and isolated exosomes from the supernatant.
Macrophages have been shown to be the recipient cells for exogenous exosomes and in direct contact with peripheral serum exosomes [
35]. A recent study showed that peripheral serum exosomes promoted M1 macrophage polarization and inflammation during sepsis-related ALI, but the study did not address the cellular origin of the circulating exosomes [
36]. Our results showed that exosomes isolated from the supernatant of PMNs stimulated with TNF-α promoted M1 macrophage activation both in vivo and in vitro. We also observed that TNF-Exo resulted in a significant lung inflammatory response, suggesting that exosomes released from TNF-α-activated PMNs are a kind of pro-inflammatory exosomes and play an important role in sepsis-related ALI.
In addition, we observed that ATP/nigericin significantly upregulated pyroptotic cell death in TNF-Exo-primed macrophages. Induction of pyroptotic cell death in vitro usually needs two signals: the priming signal and the secondary signal. The priming signal upregulates NLRP3 inflammasome and pro-IL-1β expression levels through the transcription factor NF-κB. After the priming phase, the secondary signal, such as ATP/nigericin, initiates the assembly of several protein complexes, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC) and pro-caspase-1, by regulating the formation of the ASC pyroptosome and splicing of caspase-1 into its active form [
27]. Based on our results, TNF-Exo served as a priming signal to increase NLRP3 inflammasome expression through activating NF-κB signaling pathway, which still required the secondary signal to finally induce pyroptotic cell death. High concentrations of extracellular ATP have been implicated in multiple in vivo inflammatory responses, including lung inflammation and fibrosis, systemic inflammation and tissue damage during endotoxemia [
37,
38]. Almost all mammalian cells, including myeloid cells, platelets, leukocytes, epithelial and endothelial cells, can release ATP in response to stimulation [
39], which may explain why TNF-Exo could promote macrophage pyroptosis in vivo, as TNF-Exo or cytokines upregulated by TNF-Exo may stimulate other cells to release high concentrations of ATP. However, the exact mechanisms need to be further addressed.
Exosomally transferred miRNAs have emerged as novel regulators of cellular function. miRNA sequencing and literature search in our study suggest that miR-30d-5p may be the functional molecule within TNF-Exo. Our study here reported a novel function of miR-30d-5p in exosomes as a regulator of macrophage polarization and pyroptosis. The exosome-mediated inflammatory pathway may be a new mechanism responsible for the development of sepsis-related ALI by promoting PMN-Mϕ communication.
Finally, we further demonstrated the role of miR-30d-5p in TNF-Exo and CLP-induced lung injury. The expression level of miR-30d-5p was significantly increased in the lung after TNF-Exo administration, suggesting that miR-30d-5p could be transferred to the lung tissue via exosomes. miR-30d-5p loss-of-function markedly reduced M1 macrophage activation and death in the lung, and ameliorated lung injury, indicating that miR-30d-5p contributed to TNF-Exo-induced lung injury. Furthermore, inhibition of miR-30d-5p was found to be highly related to the improvement of lung injury and survival rate in the experimental sepsis model, which may provide a novel molecular target for the treatment of sepsis-related ALI.
There were several limitations in our study. Firstly, more studies about the correlations of miR-30d-5p with clinical parameters such as oxygenation index and mortality of sepsis-related ARDS patients are required to make our conclusions more informative and reliable. Secondly, we found that TNF-Exo in vivo could promote macrophage pyroptosis and TNF-Exo in vitro needed a second signal to finally induce macrophage pyroptosis, based on which raised the hypothesis that TNF-Exo or cytokines increased by TNF-Exo may stimulate other cells to release high concentrations of ATP in vivo, which served as a second signal to induce TNF-Exo-primed macrophage pyroptosis. However, the exact mechanisms need to be further addressed. Lastly, although specially inhibition of miR-30d-5p in PMNs in vivo is not easily manipulated, administration of imR-30d-5p inhibitors via the tail vein of mice before CLP in our study did exhibit a protective effect on lung injury and survival, suggesting that miR-30d-5p may represent a new therapeutic target for the progression of sepsis-related ALI.
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