Characterization of exosomes isolated from the supernatant of PMNs stimulated ex vivo.
TNF-α plays an important role as a potent inducer of inflammatory response and a key regulator of innate immunity in sepsis-related ALI, and is often used to activate neutrophils(25–27). We thus used TNF-α-activated PMNs to mimic sepsis in vitro. Exosomes isolated from the supernatant of PMNs stimulated with PBS (PBS-Exo) or TNF-α (TNF-Exo) were first 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-specific 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 inflammatory cytokines (IL-1α and IL-1β), M1 macrophage marker (inducible nitric oxide synthase, iNOS) and NLRP3 complexes (NLRP3, Caspase-1) (Fig. 1F, Additional file 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 inflammasome expression in macrophages during sepsis.
PMN-derived exosomes promote M1 macrophage activation both in vivo and in vitro.
To verify whether PMN-derived exosomes promoted M1 macrophage activation in vivo, C57bl/6 WT mice were injected with PBS-Exo or TNF-Exo (300 µg/mouse, i.p.) for 24 h. It was found that TNF-Exo significantly increased the proportion of M1 macrophage (CD11c + CD206-) in peritoneal macrophage (PMϕ) (Fig. 2A). To explore whether PMN-Exo mediated sepsis-related ALI, Dil-labeled exosomes were injected intraperitoneally into naive mice. Fluorescence imaging showed that PMN-Exo accumulated in the lung 12 h after injection (Fig. 2B). 24 h after exosome injection, the lung morphological changes were observed by H&E staining, demonstrating that TNF-Exo promoted lung injury as compared with PBS-Exo (Fig. 2C). Furthermore, qRT-PCR showed the same result that the expressions of pro-inflammatory mediators (iNOS, IL-1β and TNF-α) in the lung were increased by TNF-Exo (Fig. 2D). The immunofluorescence results showed that the numbers of macrophages (F4/80 + cells) and M1 macrophages (iNOS + F4/80 + cells) were markedly increased in the lung after TNF-Exo injection (Fig. 2E). All these data indicate that TNF-Exo promoted M1 macrophage activation and then induced pulmonary inflammation in vivo.
To further confirm that TNF-Exo promoted M1 macrophage activation in vitro, PMN-Exo and Raw264.7 macrophages/BMDMs were cocultured. The results showed that TNF-Exo promoted macrophage M1 polarization as assessed by flow cytometry, qRT-PCR and ELISA (Fig. 2F-2K).
PMN-derived exosomes prime macrophage for pyroptosis.
As indicated by TMT-based proteomics, TNF-Exo increased the expression of NLRP3 inflammasomes 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 significantly 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 inflammasome expression levels are known to be regulated by the proinflammatory 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 inflammasome 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 file 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 searched the literature and found that miR-30d-5p was reported to positively regulate NF-κB signaling pathway(29). We thus used qRT-PCR to verify the expression of miR-30d-5p in TNF-Exo was significantly higher than that in PBS-Exo (Fig. 4C). However, in PMNs exposed to TNF-α, miR-30d-5p was decreased, indicating that miR-30d-5p relocated from the cellular compartment to exosomes (Fig. 4D). Interestingly, macrophages treated with TNF-Exo exhibited higher levels of miR-30d-5p than PBS-Exo (Fig. 4E). These results indicate that TNF-α could enhance miR-30d-5p loading into exosomes and transfer to recipient macrophages. Thus, we hypothesized that PMN-derived exosomes may transfer miR-30d-5p into macrophages and then activate NF-κB signaling pathway during sepsis.
PMN-derived exosomes promote M1 macrophage activation and prime macrophage for pyroptosis through miR-30d-5p.
To test the above hypothesis, we transfected Raw264.7 macrophages with miR-30d-5p inhibitors prior to coculture with PMN-derived exosomes. It was found that transfection of miR-30d-5p inhibitors reversed the upregulation of M1 macrophage markers and pro-inflammatory cytokines induced by TNF-Exo (Fig. 5A-5C). In addition, inhibition of miR-30d-5p significantly decreased NF-κB p-p65 protein expression in recipient macrophages treated with TNF-Exo (Fig. 5D).
Moreover, treatment of Raw264.7 macrophages with an miR-30d-5p inhibitor prior to coculture with TNF-Exo decreased mRNA levels of NLRP3 and caspase-1 (Fig. 5E). Intracellular caspase-1 activation was also measured by flow cytometry and Western blot. Transfection of miR-30d-5p inhibitors exhibited a significant suppressive effect on caspase-1 activation in response to TNF-Exo and ATP (Fig. 5F-5G). The cleaved GSDMD N-terminus upregulated by TNF-Exo plus ATP stimulation was also inhibited by miR-30d-5p inhibitors (Fig. 5H).
Altogether, these data show that exosomal miR-30d-5p promoted M1 macrophage activation and primed macrophage for pyroptosis via an miR-30d-5p-NF-κB signaling-dependent pathway.
Exosomal miR-30d-5p activates NF-κB in macrophage via targeting SOCS-1 and SIRT1.
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.
To determine whether exosomal miR-30d-5p targeted SOCS-1/SIRT1 in recipient macrophages, we investigated SOCS-1 and SIRT1 levels in PMN-Exo-treated PMNs, and found that both mRNA and protein expressions of SOCS-1 and SIRT1 were concomitantly inhibited in recipient macrophages (Fig. 6E-6F), while miR-30d-5p inhibitor significantly upregulated the expression of SOCS-1 and SIRT1 (Fig. 6G). In addition, previous studies demonstrated that SIRT1 reduced NF-κB activity by decreasing the acetylation levels of lysine 310 of the NF-κB p65 subunit(30). We thus examined lysine 310 acetylation of p65 subunit and found that TNF-Exo enhanced p65 lysine 310 acetylation (Fig. 6F), while inhibition of miR-30d-5p decreased lysine 310 acetylation of p65 (Fig. 6G).
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.
Exosomal miR-30d-5p promotes lung injury during sepsis in vivo.
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 significantly 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 proinflammatory cytokines and NLRP3 inflammasome in the lung tissues were examined. The results showed that miR-30d-5p inhibitor significantly 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 inflammation during sepsis.