Neutrophil-Derived Exosomes Induce M1 Macrophage Polarization and Prime Macrophage Pyroptosis via miR-30d-5p in Sepsis


 Background: Polymorphonuclear neutrophils (PMNs) have been demonstrated to play a role in proinflammatory M1 activation and macrophage (Mϕ) pyroptosis in sepsis. Accumulating evidence suggests PMN-derived exosomes as a new subcellular entity acting as a fundamental link between PMN-driven inflammation and tissue damage. However, the role of PMN-derived exosomes in sepsis remains unclear. This study aimed to determine whether PMN-derived exosomes play a role in proinflammatory M1 activation and Mϕ pyroptosis in sepsis and explore the potential mechanisms involved. Methods: Exosomes were isolated from the supernatant of PMNs activated with phosphate buffered saline (PBS) or tumor necrosis factor (TNF)-α, cocultured with Raw264.7 macrophages or BMDMs, and then assayed for macrophage polarization and pyroptosis. To examine the role of exosomes in vivo, PMN-derived exosomes were administered to mice, and then examined for lung inflammation. Results: After activated by TNF-α, PMNs released exosomes (TNF-Exo) to promote M1 macrophage activation both in vivo and in vitro. In addition, TNF-Exo primed macrophages for pyroptosis by upregulating NLRP3 inflammasome expression through NF-κB signaling pathway. Mechanistic studies demonstrated that miR-30d-5p mediated the function of TNF-Exo by targeting SOCS-1 and SIRT1 in macrophages. Furthermore, treatment of miR-30d-5p inhibitors in vivo significantly decreased cecal ligation and puncture (CLP) or TNF-Exo-induced M1 macrophage activation and macrophage death in the lung. Lung injury was also alleviated by miR-30d-5p inhibitors.Conclusions: In this study, we identified a novel mechanism of PMN-Mϕ interaction in sepsis, demonstrating that exosomal miR-30d-5p from PMNs induced M1 macrophage polarization and primed Mϕ for pyroptosis by activating NF-κB signaling. These findings suggest a previously unidentified role of neutrophil-derived exosomes in sepsis and may lead to new therapeutic approaches.


Introduction
Sepsis is de ned as a global health priority by the World Health Organization, with a reported death rate of 30-45% in hospitalized patients (1,2). Mortality in sepsis is primarily due to multiple organ dysfunction syndrome including acute lung injury (ALI) as a common and important component (3).
Sepsis elicits dysregulated immune responses manifested by a cytokine/chemokine elevation (also known as 'cytokine storm'), which correlates well with increased disease severity and poor prognosis.
However, the molecular mechanism remains to be fully elucidated (4,5).
Polymorphonuclear neutrophils (PMNs) are the most abundant leukocytes in mammals, playing a double-edged sword role in sepsis. A number of studies have demonstrated that regulation of neutrophils plays a crucial role in the treatment of sepsis(6). In addition to PMNs, macrophages (Mϕ) are predominant immune cells that mediate the in ammatory process (7). Recent studies have proven that the crosstalk between PMNs and Mϕ plays an important role in regulating in ammation following trauma, hemorrhagic shock, endotoxemia and other pathologies(8-11).
Macrophages are roughly classi ed as M1 macrophage with proin ammatory effects, or M2 macrophage with anti-in ammatory effects. M1 macrophage encourages in ammation by secreting proin ammatory cytokines, such as interleukin 1β (IL-1β), IL-12, IL-6, tumor necrosis factor-α (TNF-α), all of which contribute to the development of sepsis-related lung injury (12,13). Besides, pyroptosis is a programmed cell death process associated with in ammation (6,14). In sepsis, pyroptosis promotes pore formation in the plasma membrane, leading to cell swelling and membrane rupture, nally resulting in the leakage of abundant in ammatory factors out of cells (15,16). Accumulating evidence indicates that PMNs play a role in proin ammatory M1 activation and pyroptosis of Mϕ (4,11,17). However, the exact role of PMNs and the underlying mechanism need to be further addressed.
A recent study (18) reported that circulating plasma exosomes induced macrophage M1 activation during sepsis-related ALI. Exosomes are small extracellular vesicles secreted by various cell types, with size ranging from 30 to 150 nm(18). 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 (19).
Neutrophil-derived exosomes participate in the pathogenesis of chronic lung injury, such as chronic obstructive pulmonary disease, bronchopulmonary dysplasia and asthma (20,21). However, whether neutrophil-derived exosomes play a role in sepsis-related ALI remains unclear.
MicroRNAs (miRNAs) are a group of small single-stranded noncoding RNAs that regulate gene expression by binding to the 3' untranslated region of target mRNAs, resulting in the degradation of mRNA as well as inhibition of translation (22). Exosome-contained miRNAs may serve as an important mediator of intercellular communication in sepsis (23). However, whether neutrophil-derived exosomal miRNAs could modulate Mϕ activation or death during sepsis needs to be further addressed.
In this study, we identi ed a novel mechanism of PMN-Mϕ interaction in sepsis, and demonstrated that exosomal miR-30d-5p from neutrophil induced macrophage M1 polarization and prime Mϕ for pyroptosis by activating NF-κB signaling in sepsis. We further demonstrated that exosomal miR-30d-5p activated NF-κB signaling by targeting SOCS-1 and SIRT1. These ndings suggest a previously unidenti ed pathway of PMN-Mϕ cross-talk, which could enhance Mϕ activation and death, and subsequent exaggerated post-sepsis in ammation.

Materials And Methods
More detailed information on methods is provided in Additional le 1: supplementary 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 speci c pathogen-free environment in Xinhua Hospital Animal Laboratory (Shanghai, China). All animal experiments were conducted under the rules approved by Ethics Committee of Xinhua Hospital A liated to Shanghai Jiao Tong University School of Medicine, and the approval number is XHEC-F-2020-019.

PMNs isolation and activation
PMNs were induced in the peritoneal cavity of the mice as previously described (24). 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 10 6 cells/ml. PMNs activation was induced upon incubation with phosphate buffered saline (PBS) or 20 ng/mL TNF-α for 12 h at 37°C. Raw264.7 macrophages were then transfected with microRNA control (50nM) and miR-30d-5p inhibitor (Guangzhou Ribobio Corporation, Guangzhou, China) for 24 h prior to be cocultured with exosomes as per the manufacturer's instructions.

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 calculating the least signi cant difference to compare means. A value of P < 0.05 was considered statistically signi cant.

Results
Characterization of exosomes isolated from the supernatant of PMNs stimulated ex vivo. TNF-α plays an important role as a potent inducer of in ammatory response and a key regulator of innate immunity in sepsis-related ALI, and is often used to activate neutrophils (25)(26)(27). We thus used TNF-αactivated PMNs to mimic sepsis in vitro. Exosomes isolated from the supernatant of PMNs stimulated with PBS (PBS-Exo) or TNF-α (TNF-Exo) were rst characterized morphologically by transmission electron microscopy (TEM). The isolated microvesicles displayed a round, cup-shaped morphology with a diameter about 100 nm ( Fig. 1A-1B). Western blot showed high expression of exosome-speci c markers CD9, CD63 and TSG101 (Fig. 1C). The protein concentration in TNF-Exo was higher than that in PBS-Exo (Fig. 1D). After 3-h incubation of BMDMs in vitro with Dil-labeled exosomes, Mϕ internalization of exosomes was observed (Fig. 1E). To determine the signaling pathway changes after PMN-Exo treatment, BMDMs were collected for TMT-based proteomics. The results showed that TNF-Exo upregulated the expressions of in ammatory cytokines (IL-1α and IL-1β), M1 macrophage marker (inducible nitric oxide synthase, iNOS) and NLRP3 complexes (NLRP3, Caspase-1) (Fig. 1F, Additional le 2: Table 1). Enrichment pathway analysis showed that NF-κB signaling pathway was within the 20 most enriched pathways (Fig. 1G). In addition, the protein levels of NLRP3, pro-IL-1β and NF-κB p-p65 were increased in TNF-Exo exposed BMDMs as demonstrated by Western blot (Fig. 1H). All these results indicate that PMNs released exosomes to upregulate NF-κB signaling activity, promote M1 activation and increase NLRP3 in ammasome expression in macrophages during sepsis.
All these data indicate that TNF-Exo promoted M1 macrophage activation and then induced pulmonary in ammation in vivo.
As indicated by TMT-based proteomics, TNF-Exo increased the expression of NLRP3 in ammasomes and gasdermin D (GSDMD, the core event in pyroptosis) in macrophages. We thus determined the role of PMN-Exo in macrophage pyroptosis, and found that TNF-Exo increased PMϕ death after injection intraperitoneally, as shown by double staining of Annexin V and PI (Fig. 3A). To further determine the type of PMϕ death, PMϕ were detected for nuclear fragmentation, caspase-1 activation (the characteristics of pyroptosis) by staining the cells with TMR-Cell Death Reagent and Alexa Flour 488-labeled caspase-1 FLICA. Flow cytometry showed ~ 8% pyroptotic PMϕ at 24 h after TNF-Exo injection (Fig. 3B).
Next, we assessed the effect of PMN-Exo on macrophage pyroptosis in vitro. Surprisingly, treatment with TNF-Exo did not promote macrophage death or pyroptosis, while ATP/nigericin signi cantly upregulated pyroptotic cell death in TNF-Exo-primed macrophages (Fig. 3C-3D). The cleaved GSDMD N-terminus and IL-1β secretion were also increased in TNF-Exo plus ATP or nigericin group (Fig. 3E-3F). In addition, TNF-Exo increased NLRP3 and caspase-1 mRNA expression (Fig. 3G-3H). NLRP3 in ammasome expression levels are known to be regulated by the proin ammatory transcription factor NF-κB(28), and the previous result of the present study showed that TNF-Exo upregulated NF-κB signaling activity (Fig. 1H).
Collectively, these results indicate that TNF-Exo primed macrophages for pyroptosis by upregulating NLRP3 in ammasome expression through NF-κB signaling pathway. miRNA analysis of PMN-derived exosomes.
We screened PMN-derived exosomes for miRNAs and detected 26 miRNAs that were increased ≥ 2-fold in TNF-Exo compared with PBS-Exo (Fig. 4A, Additional le 2: Table 2). Enrichment pathway analysis was also performed to identify the most enriched pathways related to signaling transduction for these 26 miRNAs, and the data showed that NF-κB signaling pathway was within the 20 most enriched pathways (Fig. 4B).
Next, we sought to understand the mechanism through which miR-30d-5p activated NF-κB signaling pathway. Bioinformatics analysis showed that SOCS-1 (suppressor of cytokine signaling) and sirtuin 1 (SIRT1) were putative target genes for miR-30d-5p, and also negative regulators of NF-κB signaling pathway. As predicted by Targetscan in our study, miR-30d-5p may conserve the binding sites in the 3' UTR of SOCS-1 and SIRT1 (Fig. 6A). To validate this bioinformatic prediction, we conducted a dual luciferase reporter assay and found that luciferase activity was markedly reduced by miR-30d-5p overexpression in SOCS-1/SIRT1 3′-UTR WT group, but not in 3′-UTR Mut group (Fig. 6B). In Raw264.7 macrophages, overexpression of miR-30d-5p suppressed both the mRNA and protein levels of SOCS-1 and SIRT1 (Fig. 6C-6D). All these results demonstrate that SOCS-1 and SIRT1 were the direct target genes of miR-30d-5p.
All the above results demonstrate that exosomal miR-30d-5p targeted SOCS-1 and SIRT1 in macrophage, and subsequently activated NF-κB partly by increasing acetylation of Lysine 310 of p65.
We next investigated the functional role of miR-30d-5p in PMN-derived exosomes during sepsis by using the cecal ligation and puncture (CLP) mouse model to mimic sepsis in vivo. It was found that miR-30d-5p expression was signi cantly increased in the lung tissue of CLP group (Fig. 7A). Injection of TNF-Exo also upregulated miR-30d-5p level in the lung tissue (Fig. 7B). Next, miR-30d-5p inhibitor or scrambled negative control was administered via the tail vein of mice before conducting the CLP model or injecting TNF-Exo, and the expression levels of proin ammatory cytokines and NLRP3 in ammasome in the lung tissues were examined. The results showed that miR-30d-5p inhibitor signi cantly reversed the upregulation of IL-6, iNOS, NLRP3 mRNA levels induced by CLP, and repressed IL-1β, iNOS, NLRP3, caspase-1 mRNA expression following TNF-Exo injection (Fig. 7C-7F). In addition, inhibition of miR-30d-5p decreased CLP or TNF-Exo-induced M1 macrophage activation and macrophage death in the lung ( Fig. 7G-7J). All these data demonstrate that exosomal miR-30d-5p enhanced M1 macrophage activation and macrophage death, and then promoted lung in ammation during sepsis.

Discussion
In the current study, we demonstrated a previously unidenti ed role of PMN-Mϕ interaction in promoting in ammation in sepsis. In addition, exosomal miR-30d-5p from neutrophils induced macrophage M1 polarization and primed Mϕ for pyroptosis in sepsis by activating NF-kB signaling, which in turn enhanced in ammation. We further demonstrated that exosomal miR-30d-5p activated NF-kB signaling by targeting SOCS-1 and SIRT1.
In the early stage of sepsis, neutrophils are thought to be the primary innate immune cells that cause 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 in ammation and tissue damage (21). 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, and found an increased number of PMN-derived exosomes after TNF-α stimulation.
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 in ammation during sepsis-related ALI, but the study did not address the cellular origin of 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 signi cant lung in ammatory response, suggesting that exosomes released from TNF-α-activated PMNs are a kind of pro-in ammatory exosome and play an important role in sepsis-related ALI.
In addition, we observed that ATP/nigericin signi cantly upregulated pyroptotic cell death in TNF-Exoprimed macrophages. Induction of pyroptotic cell death in vitro usually needs two signals: the priming signal and the secondary signal. The priming signal upregulates NLRP3 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, ASC and procaspase-1, by regulating the formation of the ASC pyroptosome and splicing of caspase-1 into its active form (37). Based on our results, TNF-Exo serves as a priming signal to increase NLRP3 in ammasome expression through activating NF-κB signaling pathway, which needs the secondary signal to nally induce pyroptotic cell death. High concentrations of extracellular ATP have been implicated in multiple in vivo in ammatory responses, including lung in ammation and brosis, systemic in ammation and tissue damage during endotoxemia(38, 39). Almost all mammalian cells, including myeloid cells, platelets, leukocytes, epithelial and endothelial cells, can release ATP (40). That may explain why TNF-Exo could promote macrophage pyroptosis in vivo, as TNF-Exo or cytokines 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 in sepsis. The exosome-mediated in ammatory pathway may be a new mechanism responsible for the development of lung in ammation by promoting PMN-Mϕ communication.
Finally, we demonstrated that miR-30d-5p played a functional role in PMN-derived exosomes during sepsis-related ALI. The expression level of miR-30d-5p was signi cantly increased in the lung tissue after TNF-Exo injection, 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 in ammation, indicating that miR-30d-5p contributed to sepsis-related ALI.

Conclusions
We demonstrated that exosomal miR-30d-5p from neutrophil induced macrophage M1 polarization and primed Mϕ for pyroptosis through activating NF-kB signaling. Exosomal miR-30d-5p mediated the crosstalk between PMNs and macrophages by targeting SOCS-1 and SIRT1. miR-30d-5p played a critical role in the development of lung in ammation and injury during sepsis. These ndings suggest that exosomal miR-30d-5p derived from PMNs may represent a new therapeutic target for the progression of sepsis-

Availability of data and materials
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions YJ, TZ and CMZ contributed equally to this work. YJ, ZLM and XYS contributed to the conception and design. YJ, TZ, CMZ and HYJ performed the experiments, analyzed data, and wrote the manuscript. XYT and RX participated in the animal experiments. WW helped to revise the manuscript. All authors read and approved the nal manuscript.

Ethics approval
All animal experiments were conducted under the rules approved by Ethics Committee of Xinhua Hospital A liated to Shanghai Jiao Tong University School of Medicine, and the approval number is XHEC-F-2020-019.

Consent for publication
All listed authors consent to the submission and all data are used with the consent of the person generating the data. Protein quanti cation of exosomes by BCA. E Immuno uorescence images showing bone marrowderived macrophages (BMDMs) incubated with Dil-labeled exosomes (green) for 3 h. Cell membranes and nuclei were counterstained with DiR (red) and Hoechst (blue) respectively. Scale bar, 10 μm. F-H Treatment of BMDMs with PBS-Exo/TNF-Exo for 24 h. F BMDMs for tandem mass tags (TMT)-based proteomic quanti cation. G Enrichment pathway analysis showed that NF-κB signaling pathway was within the 20 most enriched pathways. H Western blot of NLRP3, pro-IL-1β, NF-κB p-p65 and p65 in BMDMs after coculture with exosomes. Student's t test (D) or one-way analysis of variance with Tukey's multiple comparisons test (H) was used for the analysis. Graphs represent means ± SEM, n≥3; *P < 0.05, **P < 0.01 compared within two groups.  and caspase-1 mRNA expression by real-time PCR. One-way analysis of variance with Tukey's multiple comparisons test was used for the analysis. Graphs represent means ± SEM, n ≥ 3; *P < 0.05, **P < 0.01 compared within two groups. indicates the number of genes in the miRNA target list over the total genes in the respective canonical pathway. C-E Expression of miR-30d-5p in PMN-derived exosomes, PMNs stimulated with PBS/TNF-α and recipient Raw264.7 macrophages treated with PBS/PBS-Exo/TNF-Exo. Student's t test (C-D) or one-way analysis of variance with Tukey's multiple comparisons test (E) was used for the analysis. Graphs represent means ± SEM, n≥3; *P < 0.05, **P < 0.01 compared within two groups.  Exosomal miR-30d-5p activates NF-κB in macrophage via targeting SOCS-1 and SIRT1. A Sequence alignment between miR-30d-5p and its putative binding sites (in red letters) in the SOCS-1/SIRT1 3′-UTR. Mutation of the miR-30d-5p target sites (in blue letters) is also shown. B Detection of the relative luciferase activities of WT and Mut SOCS-1/SIRT1 reporters by luciferase reporter assay, using Renilla luciferase vector as the internal control. qRT-PCR analysis of relative SOCS-1/SIRT1 mRNA levels (C) and Exosomal miR-30d-5p promotes lung injury during sepsis in vivo. Mice were subjected to sham or CLP for 24 h, or treated with exosomes (300 μg/mouse) isolated from the supernatant of PMNs stimulated ex vivo through i.p. injection. The miR-30d-5p inhibitor or negative control was injected into each mouse 1 day before CLP surgery or TNF-Exo injection. Relative expression levels of miR-30d-5p (A-B), in ammatory cytokine mRNA (IL-6, IL-1β, TNF-α) and iNOS mRNA (C-D), NLRP3 and caspase-1 mRNA