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Crucial contributions of protein cargos to the capabilities of macrophage-derived extracellular vesicles | Journal of Nanobiotechnology


Characterization of macrophage-derived EVs

Naïve M0 mouse bone marrow-derived macrophages (BMDMs) have been handled with LPS or IL-4 to induce M1 or M2 macrophages, respectively. Their activating states have been confirmed with move cytometry evaluation utilizing antibodies towards iNOS and Arg1 (Further file 1: Fig. S1A), the prototypical markers for M1 and M2 macrophages, respectively [2]. The tradition media of M0, M1, and M2 BMDMs have been collected to extract EVs (M0-, M1-, and M2-EVs) utilizing the ultracentrifugation protocol [12]. When examined underneath transmission electron microscopy, all three varieties of macrophage-derived EVs demonstrated the standard cup-shaped morphology, and their sizes ranged from 50 to 150 nm in diameter (Fig. 1A). Following the rules of the Worldwide Society for Extracellular Vesicles [7], the macrophage-derived EVs have been validated utilizing antibodies towards two cytosolic proteins with plasma membrane-binding capability (ALIX and TSG101) and one tetraspanin protein (CD81). All three varieties of macrophage-derived EVs contained ALIX, TSG101, and CD81, however not glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Fig. 1B), confirming their EV identities on the molecular stage. Nanoparticle monitoring evaluation confirmed that these three varieties of macrophage-derived EVs had comparable common diameters of roughly 120 nm (Fig. 1C). Apparently, the yield of M1-EVs was considerably greater than that of M0-EVs, indicating that EVs have been extra actively launched by M1 macrophages in comparison with naïve macrophages (Further file 1: Fig. S1B). The purity of those macrophage-derived EVs was decided by evaluating the ratio of EV counts to protein focus [13]. The ratios of M0-, M1-, and M2-EVs have been roughly 0.13–0.3 × 1010/μg (Further file 1: Fig. S1C), corresponding to these of EVs from the tradition media of most cancers cell strains grown in commonplace cell tradition flasks [13].

Fig. 1
figure 1

Characterization of macrophage-derived EVs. EVs have been remoted from the tradition media of M0, M1, and M2 BMDMs and subjected to totally different analyses. A Consultant TEM photographs of M0-, M1-, and M2-EVs. B EV validation utilizing immunoblot evaluation with antibodies towards EV markers. 5 μg proteins from mobile or EV lysates have been loaded in every lane. C Yield and dimension distribution of EVs utilizing NanoSight NS300

Comparative analyses of protein profiles of M0-, M1-, and M2-EVs

As a result of we centered on the protein cargos of macrophage-derived EVs on this research, we extracted proteins from three organic replicates of M0- and M1-EV pairs, in addition to three organic replicates of M0- and M2-EV pairs. The proteins from every EV pattern have been subjected to quantitative proteomics evaluation to acquire their protein profiles (Further file 2: Desk S1, Further file 3: Desk S2). Comparative evaluation of protein profiles of M1- and M0-EV pairs revealed that 79 proteins have been considerably upregulated and 38 downregulated in M1-EVs, in comparison with M0-EVs (Further file 1: Fig. S2A). Principal element evaluation (PCA) of the protein profiles of M0- and M1-EV pairs demonstrated an excellent separation of M0- and M1-EV proteins (Further file 1: Fig. S2B). Comparability of protein profiles of M2- and M0-EV pairs confirmed that 53 proteins have been considerably enriched and 64 decreased in M2-EVs, in comparison with M0-EVs (Further file 1: Fig. S2C). PCA confirmed that M2-EV proteins have been clustered collectively and separated from M0-EV proteins (Further file 1: Fig. S2D). The proteins in M1- or M2-EVs that have been at the least 1.5-fold extra considerable than in M0-EVs with p < 0.05 have been chosen as signature proteins of M1-EVs (73 proteins) or M2-EVs (53 proteins, Further file 4: Desk S3). Volcano plots confirmed that proteins have been considerably modified in M1- or M2-EVs, in comparison with M0-EVs (Further file 1: Fig. S2E, F). Apparently, Cxcl10 and CD40, each extremely induced in M1 macrophages [14, 15], have been among the many most upregulated proteins in M1-EVs (Further file 1: Fig. S2E). Typical M2 markers Arg1 and macrophage galactose N-acetyl-galactosamine-specific lectin 2 (Mgl2) [5, 16] have been prominently enriched in M2-EVs (Further file 1: Fig. S2F).

With a purpose to get hold of a systemic view of the protein capabilities of macrophage-derived EVs, we used the web platform Metascape [17] to determine considerably enriched phrases of EV signature proteins in gene ontology (GO) mobile processes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Reactome gene units, and WikiPathways. This pathway enrichment evaluation confirmed that the signature proteins of M1-EVs have been predominantly concerned in proinflammatory phrases, resembling inflammatory response and sample recognition receptor (PRR) signaling (Fig. 2A). The proteins considerably enriched in M2-EVs have been primarily implicated in immunomodulation and tissue transforming phrases, resembling extracellular matrix (ECM) group and the peroxisome proliferator-activated receptor (PPAR) signaling pathway (Fig. 2B). We performed additional comparative analyses of enriched pathways in M1- and M2-EVs to know the similarities and divergences between them (Fig. 2C). A number of the signature proteins in M1- and M2-EVs have been concerned in comparable pathways, resembling phagocytosis, a well-recognized mobile operate of macrophages [18]. Nonetheless, many M1- and M2-EV signature proteins have been concerned in separate and distinct pathways. M1-EVs have been uniquely enriched in proteins associated to antigen processing and presentation, PRR signaling, and response to IFN-γ (Fig. 2C), all of which resembled the first capabilities of M1 macrophages [3, 4]. In regard to M2-EVs, they solely contained proteins collaborating within the detrimental regulation of cell activation and ECM group (Fig. 2C), simulating the principle actions of M2 macrophages [3, 4].

Fig. 2
figure 2

Comparative analyses of protein profiles of M1- and M2-EVs. Complete proteins of M0-, M1-, and M2-EVs (3 organic replicates/pattern) have been subjected to quantitative proteomics evaluation. The proteins whose abundances in M1- or M2-EVs have been at the least 1.5-fold greater than these in M0-EVs with P < 0.05 have been chosen as signature proteins of M1- or M2-EVs. These proteins have been used for the pathway enrichment analyses (A, B) and pathway comparative evaluation (C). FC: fold change

To additional perceive the useful connections of signature proteins in M1- and M2-EVs, we subjected their signature protein profiles to protein interplay evaluation with Search Instrument for the Retrieval of Interacting Genes/Proteins (STRING) [19] to disclose protein–protein interplay (PPI) networks. Primarily based on the counts and energy values, the foremost protein clusters within the M1-EV protein community have been discovered to be extremely related to pathways involving antigen processing and presentation, inflammatory response, T cell activation, and endocytosis (Fig. 3A), reflecting the pro-inflammatory nature of M1-EV proteins. In the meantime, the foremost protein clusters within the M2-EV protein community have been intimately related to pathways concerned in detrimental regulation of immune response, ECM group, response to wounding, and collagen-containing ECM (Fig. 3B), indicating their involvement in irritation decision and tissue transforming.

Fig. 3
figure 3

PPI networks of M1-EV and M2-EV signature proteins. PPI evaluation was performed with the signature proteins of M1-EVs (A) and M2-EVs (B). The main protein clusters inside every community have been highlighted with totally different colours. The GO pathways that every protein cluster was enriched have been indicated under the PPI networks. The clusters of enriched proteins in M1- or M2-EVs with (a) rely in community > 5, (b) energy > 0.9, and (c) false discovery price < 0.05 in GO phrases have been highlighted within the community. Depend in community: the primary quantity signifies variety of proteins within the community that have been annotated with a selected time period, and the second quantity signifies variety of proteins in complete (within the community and within the background) that had this time period assigned. Energy: describes the dimensions of the enrichment impact; False discovery price: describes the importance of the enrichment. FC: fold change

Gene set enrichment evaluation (GSEA) is a strong analytical software initially developed to interpret gene expression profiles [20]. In comparison with different analytical approaches that focus solely on considerably modified genes, GSEA emphasizes gene units (teams of genes that share frequent organic capabilities) and ranks all of the genes in a given knowledge set with out setting any arbitrary threshold. GSEA additionally has been utilized in analyzing proteomics knowledge in a cardiomyopathy research [21]. We in contrast all of the recognized proteins in M1- or M2-EVs with the present ontology gene units (mouse collections) from the Molecular Signature Database (MsigDB) in GSEA. The evaluation (utilizing all EV proteins) confirmed remarkably comparable outcomes (Further file 1: Fig. S3A, B) to these of pathway enrichment and PPI community analyses (utilizing solely considerably upregulated proteins). M1-EV proteins have been primarily concerned in proinflammatory pathways (Further file 1: Fig. S3A) and M2-EV proteins have been primarily associated to tissue transforming (Further file 1: Fig. S3B). We used the lately developed on-line software PSEA-Quant (which makes use of protein units from GO and MsigDB to particularly analyze proteomics knowledge [22]) to investigate the overall proteins from M1- and M2-EVs. The obtained outcomes (Further file 1: Fig. S4A, B) have been extremely in line with these of our GSEA, pathway enrichment, and PPI community analyses.

Collectively, the outcomes of pathway enrichment, PPI community, GSEA, and PSEA-Quant analyses have been extremely constant and supportive of the notion that protein cargos loaded in macrophage-derived EVs appeared to confer on them capabilities much like these of their parental macrophages.

Comparative analyses of protein signatures of M1- and M2-EVs with their respective parental macrophages

Subsequent, we sought to know the extent of the protein signature resemblance between EVs and their parental macrophages. Complete proteins of M0, M1, and M2 BMDMs have been subjected to quantitative proteomics evaluation. Proteins that have been at the least 1.5-fold extra considerable in M1 or M2 BMDMs with p < 0.05 than in M0 BMDMs have been thought of to be signature proteins of M1 BMDMs (387 proteins) or M2 BMDMs (351 proteins); we used these to conduct the comparative analyses with signature proteins of M1- or M2-EVs, respectively. Thirty-one out of 73 signature proteins in M1-EVs overlapped with proteins that have been considerably enriched in M1 BMDMs (Fig. 4A). These embody CD40, Cxcl10, and lots of different proteins concerned in inflammatory responses (together with serum amyloid A 3 [Saa3], tumor necrosis issue alpha-induced protein 2 [Tnfaip2], and toll-like receptor 2 [Tlr2]). The Circos plot (Fig. 4B) depicted the shared signature proteins and pathways between M1-EVs and M1 BMDMs. CD40 and Cxcl10 are extremely induced in M1 macrophages and generally thought of to be M1 markers [14, 15]. Immunoblot evaluation confirmed that Cxcl10 was particularly enriched in M1-EVs, in comparison with M0- or M2-EVs (Fig. 4C). Thus, Cxcl10 might doubtlessly function a brand new particular protein marker for M1-EVs. Amongst 53 signature proteins in M2-EVs, 11 have been discovered within the protein signature of M2 BMDMs (Fig. 4D). They embody M2 markers (Arg1 and Mrc1/CD206) [5] and proteins associated to ECM group and cell adhesion (syndecan-4 [Sdc4], matrix metallopeptidase 12 [Mmp12], polypeptide N-acetylgalactosaminyltransferase 6 [Galnt6]). The Circos plot (Fig. 4E) confirmed the signature proteins and pathways that overlapped between M2-EVs and M2 BMDMs. Arg1 was verified to be extremely current in M2-EVs, in comparison with M0- or M1-EVs (Fig. 4C), a discovering in line with the volcano plot evaluation of M2-EV signature proteins (Further file 1: Fig. S2F). Due to this fact, our knowledge recommended that Arg1 was a brand new particular protein marker for M2-EVs.

Fig. 4
figure 4

Comparative analyses of protein signatures of M1- and M2-EVs with protein signatures of their respective parental macrophages. A Venn diagram exhibiting the overlap of protein signatures of M1-EVs with these of M1 BMDMs. B Circos plot depicting the signature proteins and pathways shared between M1-EVs and M1 BMDMs. C Immunoblot evaluation of Cxcl10 and Arg1. In every lane, 5 μg proteins from EV lysates have been loaded. CD81 served as a loading management. D Overlap of enriched proteins in M2-EVs with these of M2 BMDMs. E Circos plot demonstrating the mutual signature proteins and pathways of M2-EVs and M2 BMDMs. F Heatmap exhibiting that M1-EV signature proteins shared 16 of the highest 20 pathways of M1 BMDMs. G Heatmap indicating that M2-EV signature proteins shared 5 of the highest 12 pathways of M2 BMDMs

The enriched phrases of each parental macrophages and their EVs have been mixed to run a comparative evaluation utilizing the Metascape platform to elucidate the useful similarities and variations between macrophages and their EVs. M1-EV signature proteins shared 16 (or 80%) of the highest 20 pathways of the protein signature of M1 BMDMs, most of which have been concerned in inflammatory responses (Fig. 4F). Of the highest 100 enriched phrases of M1-EVs and M1 BMDMs, 33% have been shared by M1-EVs and their parental cells, 53% have been uniquely enriched in M1 BMDMs, and 14% have been discovered solely in M1-EVs (Further file 1: Fig. S5). The overlap of pathways between M2-EV proteins and M2 BMDM proteins was not as hanging because the pathway overlap between M1-EV and M1 BMDM proteins. Among the many high 20 phrases of M2-EVs and M2 BMDMs, M2-EVs shared 5 (or 42%) of 12 pathways with the protein signature of M2 BMDMs (Fig. 4G). As a result of there have been fewer enriched phrases in M2 BMDMs in comparison with M1 BMDMs, solely the highest 70 enriched phrases from M2-EVs and M2 BMDMs have been mixed within the comparative evaluation. Amongst them, 10% of the pathways have been shared by M2-EVs and their donor cells; 66% belonged solely to M2 BMDMs; and 24% have been solely current in M2-EVs (Further file 1: Fig. S6). Of observe, M2-EV signature proteins, in comparison with M2 BMDM proteins, have been extremely enriched within the ECM group pathway, detrimental regulation of cell activation, and the PPAR signaling pathway (Fig. 4G and Further file 1: Fig. S6), suggesting their potential distinctive roles in these processes.

To additional verify the resemblance between parental macrophages and their EVs, the gene expression profiles of murine M1 and M2 BMDMs obtained in an impartial research [23] have been used to conduct a comparative evaluation with the signature proteins of M1- and M2-EVs. On this new evaluation, twenty out of 73 signature proteins in M1-EVs overlapped with genes that have been considerably elevated in M1 BMDMs (Further file 1: Fig. S7A); their shared signature proteins and pathways have been proven by the Circos plot (Further file 1: Fig. S7B). Amongst 53 signature proteins in M2-EVs, 12 have been discovered within the gene signatures of M2 BMDMs (Further file 1: Fig. S7C); their overlapped signature proteins and pathways have been demonstrated within the Circos plot (Further file 1: Fig. S7D). Pathway evaluation confirmed that M1-EV signature proteins shared 15 of the highest 20 pathways (75%) of the M1 BMDM gene signature (Further file 1: Fig. S7E); M2-EVs shared 9 of 18 pathways (50%) of the M2 BMDM gene signature (Further file 1: Fig. S7F). Due to this fact, evaluating protein profiles of M1-/M2-EVs to gene expression profiles of M1/M2 macrophages led to strikingly comparable outcomes to these discovered within the protein profile comparability between EVs and their parental macrophages. Of observe, our macrophage tradition circumstances have been barely totally different from these within the gene expression profile research. In that research [23], naïve BMDMs have been handled with 100 ng/mL of LPS and 20 ng/mL of INF-γ for twenty-four h to induce M1 BMDMs, or they have been handled with 20 ng/mL of IL4 for twenty-four h to induce M2 BMDMs. The identical situation was utilized in our research to induce M2 BMDMs; nevertheless, to induce M1 BMDMs, we incubated naïve BMDMs with 10 ng/mL LPS for 8 h.

Collectively, comparability of protein profiles of EVs with the protein/gene signatures of their parental macrophages demonstrated that EVs shared solely a restricted variety of proteins with their respective parental macrophages, but the capabilities of their proteins manifested excessive similarity with these of their parental cells. M1-EV signature proteins have been predominantly enriched within the inflammatory course of, reflecting an M1-like profile, and M2-EV signature proteins shared many options of M2 macrophages and thus confirmed an M2-like phenotype, with additional enrichments of proteins associated to particular pathways.

Protein cargos of M1-EVs induced M1 polarization and activated T lymphocytes

As a result of GSEA permits us to create new reference units, we used the signature proteins of M1 BMDMs to create a protein set known as “M1 protein signature,” and we used the considerably upregulated genes in M1 BMDMs [23] to create a gene set “M1 gene signature” in GSEA. When complete proteins of M1-EVs have been in contrast with these new reference units, they have been discovered to be extremely associated to the M1 protein signature (Fig. 5A) or M1 gene signature (Further file 1: Fig. S7G). As well as, the outcomes from pathway enrichment, PPI community, GSEA, and PSEA-Quant analyses (Figs. 2, 3, Further file 1: Fig. S3, S4) all indicated that M1-EV proteins have been extremely associated to lymphocyte activation. The enrichment of the “lymphocyte activation” gene set within the ranked M1-EV proteins in GSEA was additional visualized in Fig. 5B.

Fig. 5
figure 5

Protein cargos in M1-EVs induced M1 polarization and stimulated T lymphocytes. A GSEA confirmed that M1-EVs have been considerably enriched with proteins associated to M1 protein signature. B GSEA recommended that M1-EV proteins positively correlated with lymphocyte activation. C Expression of M1 marker genes in naïve macrophages handled with 1.5 × 109/mL of M0- or M1-EVs for 8 h. D Expression of M1 marker genes in naïve macrophages handled with 1.5 × 109/mL of standard or heated M1-EVs for 8 h. EVs have been heated at 95 °C for 10 min to denature the proteins. E, F Move cytometry evaluation exhibiting the protein ranges of iNOS and Arg1 in BMDMs handled with 6 × 109/mL of standard (E) or heated (F) M0- and M1-EVs for 8 h. GJ Murine splenocytes have been primed with anti-CD3 antibody (1 µg/mL) within the absence or presence of 1 × 1010/mL M1-EVs for 72 h, adopted by move cytometry evaluation and cytokine measurement. GH Zombie Violet staining to evaluate viability of complete splenocytes (G), T cells (CD4+ and/or CD8+) and non-T cells (CD4 and CD8 detrimental, H). I Proliferation of CD4+ and CD8+ lymphocytes measured utilizing CellTrace CFSE dye. J IFN-γ launch from anti-CD3-primed splenocytes handled with common or heated M1-EVs. Knowledge introduced as imply ± STD (N = 3). **P < 0.01

It has been reported that M1-EVs from LPS-treated RAW264.7 cells have been able to repolarizing IL-4-induced M2 BMDMs into M1 macrophages [24] and that M1 BMDM-derived EVs promoted expression of IFN-γ in T lymphocytes [25]. The capabilities of M1-EVs recognized in these earlier research supported the outcomes of our bioinformatic analyses, though the accountable lively cargos in M1-EVs weren’t decided. Due to this fact, we first sought to validate the function of M1-EVs in macrophage polarization. M1- or M0-EVs have been incubated with naïve BMDMs, adopted by gene expression evaluation. M1-EVs dramatically enhanced expression in BMDMs of M1 marker genes, together with the Nos2 (encoding iNOS protein) and Il1b genes [3], in comparison with phosphate-buffered saline (PBS) or M0-EV-treated cells (Fig. 5C). The transcription of the M2 marker Arg1 gene was not affected by M1-EVs (Further file 1: Fig. S8A), indicating the specificity of M1-EVs in inducing M1 macrophages. Move cytometry evaluation additional confirmed that M1-EVs induced the protein stage of iNOS, however not that of Arg1 (Fig. 5E). Our outcomes have been in line with conclusions of the earlier research [24] and validated the outcomes of our bioinformatic analyses. Contemplating that our bioinformatic analyses have been primarily based on M1-EV proteins, we assessed the function of M1-EV proteins in inducing M1 polarization. M1-EVs have been heated at 95 °C for 10 min to denature the proteins. Despite the fact that most proteins within the heated M1-EVs misplaced operate, their uptake by naïve BMDMs was corresponding to the uptake of untreated M1-EVs (Further file 1: Fig. S8B). Nonetheless, heated M1-EVs largely did not induce iNOS protein ranges (Fig. 5F) or to reinforce the mRNA ranges of the Nos2 and Il1b genes (Fig. 5D). Due to this fact, the protein cargos in M1-EVs performed a key function in inducing M1 polarization.

Subsequent, we assessed the consequences of M1-EVs on lymphocyte activation. Incubation of M1-EVs with splenocytes from C57BL/6J mice led to elevated launch of proinflammatory cytokines IFN-γ and TNF-α in a dose-dependent method (Further file 1: Fig. S9A, B), suggesting that M1-EVs activated splenocytes. Warmth therapy of M1-EVs abolished their stimulatory results on splenocytes (Further file 1: Fig. S9A, B), which pointed to the important function of M1-EV proteins in such capabilities. To additional examine the consequences of M1-EVs particularly on T cells, splenocytes have been stimulated with soluble anti-CD3 antibody [26] and concomitantly handled with M1-EVs. Staining of a fixable viability marker Zombie Violet dye indicated that M1-EVs dramatically promoted splenocyte viability in tradition (Fig. 5G), in addition to the viability of complete T cells (Fig. 5H). Proliferation of T lymphocytes was monitored utilizing 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling. Elevated cell counts and variety of divisions have been noticed for each CD4+ and CD8+ T cells after M1-EV therapy in comparison with management cells (Fig. 5I), illustrating promotion of CD3-primed T cell proliferation by M1-EVs. Lastly, M1-EVs dose-dependently enhanced the discharge of IFN-γ from anti-CD3-stimulated splenocytes and this impact was considerably curbed by warmth therapy of M1-EVs (Fig. 5J). Collectively, our knowledge not solely confirmed outcomes of the earlier research [25] exhibiting that M1-EVs promoted IFN-γ manufacturing in T cells, but in addition revealed that M1-EVs fostered the viability and proliferation of T lymphocytes. All these capabilities of M1-EVs have been primarily mediated by their protein cargos.

Protein cargos of M2-EVs induced M2 polarization in naïve BMDMs and guarded tight junction from disruption in Caco-2 cells

The same strategy was utilized in GSEA to evaluate the potential correlation of M2-EV proteins with their parental cell proteins. The signature proteins and genes [23] of M2 BMDMs have been used to create “M2 protein signature” and “M2 gene signature” units, respectively. M2-EV proteins have been discovered to be extremely associated to the M2 protein signature (Fig. 6A) and the M2 gene signature (Further file 1: Fig. S7H). M2 BMDM-derived EVs have been proven to induce M2 polarization in M1 BMDMs presumably by synergistic results of their cytokine cargos [9], supporting the outcomes of our bioinformatic analyses. For validation functions, naïve BMDMs have been incubated with M0- or M2-EVs, adopted by gene expression evaluation. The Arg1, Cd36, and Ccl17 genes are extremely induced in M2 macrophages and sometimes thought of to be M2 markers [3, 27]. M2-EVs have been able to growing mRNA ranges of those genes (Further file 1: Fig. S10A) however did not induce expression of the Nos2 gene (Further file 1: Fig. S10B), suggesting that M2-EVs particularly induced M2 polarization in naïve BMDMs. Move cytometry evaluation confirmed that M2-EVs enhanced the protein stage of M2 marker Arg1 however not that of M1 marker iNOS (Further file 1: Fig. S10C). Though the uptake of heated M2-EVs by BMDMs was corresponding to that of untreated M2-EVs (Further file 1: Fig. S10D), heat-treated M2-EVs did not induce both the protein stage of Arg1 (Further file 1: Fig. S10E) or expression of the Arg1 and Cd36 genes (Further file 1: Fig. S10F). Collectively, our cell tradition checks confirmed that M2-EVs particularly induced M2 polarization in naïve BMDMs by their protein cargos.

Fig. 6
figure 6

Proteins in M2-EVs have been the important thing parts in defending tight junction construction and barrier integrity from disruption in Caco-2 cells. A GSEA confirmed that M2-EV proteins have been extremely associated to M2 protein signature. B GSEA recommended excessive enrichment of M2-EV proteins in ECM structural constituent. C Heatmap exhibiting the relative abundance of consultant ECM constituents and enzymes in signature proteins of M2-EVs, in comparison with these of M1-EVs. D Consultant photographs of occludin IF of Caco-2 cells and quantification of occludin sign depth. The differentiated Caco-2 monolayers have been handled with PBS or common or heated M2-EVs in PBS (6 × 109/mL) for 48 h within the presence of 1% DSS. E M2-EVs prevented DSS-induced TEER discount, however heated M2-EVs misplaced such protecting results. The differentiated Caco-2 monolayers have been pretreated with PBS or common or heated M2-EVs in PBS (6 × 109/mL) for 36 h, adopted by therapy with 1.5% DSS for 48 h. F Consultant photographs of occludin IF of Caco-2 cells and quantification of occludin sign depth. The differentiated Caco-2 cells have been handled with PBS, M2-EVs in PBS (6 × 109/mL), FMOD (3 µg/mL), MFGE8 (3 µg/mL), or FMOD (1.5 µg/mL) and MFGE8 (1.5 µg/mL) collectively for 48 h within the presence of 1% DSS. G FMOD and MFGE8 proteins protected cells from DSS-induced TEER discount. The differentiated Caco-2 monolayers have been pretreated with PBS, M2-EVs in PBS (6 × 109/mL), FMOD (3 µg/mL), MFGE8 (3 µg/mL), or FMOD (1.5 µg/mL) and MFGE8 (1.5 µg/mL) collectively for 36 h, adopted by therapy with 1.5% DSS for 48 h. *P < 0.05 and **P < 0.01 relative to cells handled with DSS alone (black bar)

Our pathway enrichment, PPI community, GSEA, and PSEA-Quant analyses (Fig. 2, 3, Further file 1: Fig. S3, 4) all pointed to the essential involvement of M2-EV proteins in ECM regulation. In GSEA, the ECM structural constituent was the highest ontology time period recognized with M2-EV proteins (Fig. 6B). These bioinformatic findings led us to experimentally study the function of M2-EVs in ECM regulation. ECM, primarily comprised of fibrous proteins (resembling collagens and elastin) and glycoproteins (resembling fibronectin and laminin), not solely supplies structural scaffolds for cells but in addition participates in regulation of tissue operate and homeostasis [28]. The comparative proteomics evaluation confirmed that signature proteins of M2-EVs have been enriched with a wide range of ECM constituents and enzymes, in contrast with these of M1-EVs (Fig. 6C). A number of the ECM enzymes resembling Mmp12 and trypsin 1 (Prss1) have been reported to contain collagen degradation [29, 30]. Thus, we first examined whether or not ECM enzymes in M2-EVs have been concerned in ECM degradation. M2-EVs have been sonicated or lysed to launch their protein cargos, adopted by incubation with a collagen combination. Though the optimistic management collagenase successfully degraded collagens, proteins in M2-EVs had a marginal impact on collagen degradation (Further file 1: Fig. S11A, B).

ECM regulates cell–cell junction positioning and promotes tight junction formation [31, 32]. Contemplating the enrichment of ECM constituents in M2-EVs, we hypothesized that M2-EVs presumably engaged in barrier capabilities. Colorectal adenocarcinoma Caco-2 cells have been chosen because the mobile mannequin as a result of they spontaneously differentiate right into a polarized epithelial monolayer with tight junction construction when reaching confluence [33]. As a result of dextran sulfate sodium (DSS) was proven to disrupt the tight junction construction of the Caco-2 monolayer [34], we handled the differentiated Caco-2 monolayer with DSS with or with out M2-EVs. According to outcomes of the earlier research [34], DSS disrupted the tight junction mesh construction of Caco-2 cells, as demonstrated by immunofluorescence (IF) staining utilizing an antibody towards the tight junction protein occludin (Fig. 6D). Remarkably, M2-EVs largely protected the tight junction construction from disruption by DSS, which was abolished by warmth therapy of M2-EVs (Fig. 6D). IF staining utilizing an antibody towards claudin1, one other key protein element of tight junction [35], confirmed an identical sample (Further file 1: Fig. S12A), additional confirming that M2-EVs, by their protein cargos, have been critically engaged in upkeep of tight junction construction.

Transepithelial electrical resistance (TEER) is a measurement {of electrical} resistance throughout a mobile monolayer. Due to its excessive sensitivity and non-invasiveness, it’s a extensively used quantitative methodology to guage the integrity of tight junction in cultured epithelial monolayers [36]. According to the IF staining outcomes of tight junction proteins, DSS therapy considerably decreased TEER of Caco-2 monolayers, in comparison with untreated cells (Fig. 6E). Remarkably, M2-EVs, however not heated M2-EVs, blocked such discount (Fig. 6E), additional supporting the notion that proteins in M2-EVs have been the important thing parts contributing to the upkeep of barrier integrity.

In M2-EVs, fibromodulin (FMOD), a small leucine-rich glycoprotein concerned in ECM composition, has been proven to modulate expression of tight junction proteins and its depletion aggravated DSS-induced destruction of the epithelial barrier in mice [37]. Milk fats globule epidermal progress issue (EGF) issue 8 protein (MFGE8), a cysteine-rich secretory glycoprotein, maintained the integrity of the epididymal epithelium [38]. Administration of recombinant MFGE8 preserved colon integrity in DSS-induced colitis mice [39]. The presence of each FMOD and MFGE8 in M2-EVs was verified by immunoblot utilizing their particular antibodies (Further file 1: Fig. S12B). Remarkably, recombinant FMOD and MFGE8 proteins partially rescued the tight junction construction of Caco-2 cells from disruption by DSS, as proven by IF staining of occludin (Fig. 6F) and claudin1 (Further file 1: Fig. S12C). Cotreatment of Caco-2 cells with FMOD and MFGE8 proteins synergistically reserved their tight junction construction within the presence of DSS (Fig. 6F, Further file 1: Fig. S12C). Apparently, both FMOD or MFGE8 alone was capable of largely curb the decreased TEER brought on by DSS therapy, and these two proteins collectively reversed DSS-triggered TEER lower (Fig. 6G). Collectively, it appeared that ECM parts like FMOD and MFGE8 in M2-EVs labored collectively to take care of the tight junction construction and barrier integrity of epithelial cells.

M2-EVs protected mice from DSS-induced colitis

Inflammatory bowel illness (IBD), consisting of Crohn’s illness and ulcerative colitis, is characterised by power and recurrent irritation of the digestive tract [40]. Compromised integrity of the epithelial barrier has typically been noticed in IBD sufferers [41]. As a result of our cell tradition knowledge recommended that proteins in M2-EVs induced polarization of anti-inflammatory M2 macrophages in naïve macrophages and guarded Caco-2 cells from disruption of the tight junction construction, we hypothesized that M2-EV proteins might have useful capabilities within the administration of IBD.

Acute colitis was induced in C57BL/6J mice by administering 1.5% (w/v) DSS in consuming water for 7 days; PBS, M2-EVs in PBS, or heated M2-EVs in PBS have been intravenously injected into the mice on the indicated time factors (Fig. 7A). M2-EVs, however not heated M2-EVs, considerably prevented the shortened colon size in mice induced by DSS therapy (Fig. 7B, C). Measurement of inflammatory cytokine ranges within the media of ex vivo cultured colon tissues confirmed that M2-EV administration considerably decreased launch of cytokines IL-1β and IL-18, however warmth therapy of M2-EVs abolished such results (Fig. 7D). On the transcription stage, M2-EVs inhibited expression of the Il1b and Il6 genes, whereas heated M2-EVs had marginal results (Fig. 7E). On the pathological stage, hematoxylin and eosin (H&E)-stained colon sections demonstrated that M2-EVs, however not heated M2-EVs, inhibited immune cell infiltration and reserved the intactness of the colon’s mucosa layer, in comparison with DSS-treated mice that acquired PBS solely (Fig. 7F). Unbiased animal research utilizing extra mice additional confirmed that M2-EV administration strongly protected the colon from colitis improvement (Further file 1: Fig. S13A–C). Due to this fact, it appears that evidently M2-EVs had potent protecting capabilities within the colon with colitis, primarily mediated by their protein cargos.

Fig. 7
figure 7

M2-EVs protected mice from DSS-induced colitis by their protein cargos. A Schematic diagram exhibiting the experimental process. 2-month-old male C57BL/6J mice have been intravenously injected with PBS or common or heated M2-EVs in PBS (3 × 108/g) on day 1, 4, and 6. All mice got 1.5% (w/v) DSS in consuming water from day 4–11 and sacrificed on day 11. N = 4–5/group. B Consultant colon photographs of colitis mice that acquired PBS or common or heated M2-EVs in PBS. C M2-EVs, however not heated M2-EVs, prevented the shortened colon size in mice induced by DSS therapy. D The degrees of cytokines within the media of ex vivo cultured colonic tissues. Launched cytokine was normalized to the protein focus of tissue lysates. E Expression of pro-inflammatory cytokine genes in colon tissues. F Consultant photographs of H&E-stained sections of colon tissues. Within the bar graphs, every dot represents one mouse. Knowledge introduced as imply ± SEM. * P < 0.05 and ** P < 0.01 relative to the management colitis mice acquired PBS (bar with black dots)

Contemplating these outstanding protecting properties of M2-EVs in colitis, we assessed whether or not intravenously injected M2-EVs might bodily accumulate within the colon. The membrane proteins of M2-EVs have been covalently labeled with a fluorescent dye in close to infrared ranges from an ExoGlow-Vivo EV labeling package. The close to infrared fluorescence spectrum of this dye allows deep tissue illumination and eliminates background from auto-fluorescence. The labeled M2-EVs have been intravenously given to C57BL/6J mice with DSS-induced colitis, their vital accumulation have been discovered within the liver, spleen, and lung (Further file 1: Fig. S14A, B), in line with many research reporting that EVs typically accumulate in these tissues after intravenous injection [42,43,44]. Apparently, M2-EVs additionally markedly collected within the colon, in addition to within the cecum and higher gastrointestinal tract (Further file 1: Fig. S14A, B). Such colonic localization probably helped M2-EVs exert their protecting capabilities within the colon.

Lastly, we investigated the consequences of M2-EVs on macrophage polarization and tight junction construction in colitis. Immunohistochemistry (IHC) staining utilizing an antibody towards F4/80 (macrophage marker [45]) confirmed that M2-EV therapy suppressed infiltration of macrophages (Fig. 8A). Concerning the macrophage polarization standing, M2-EVs decreased the extent of the M1 marker, iNOS protein (Fig. 8B), whereas enhancing the extent of the M2 marker, Mrc1/CD206 protein (Fig. 8C), suggesting that M2-EV administration drastically promoted polarization of intestinal macrophages into the M2 state. Immunohistofluorescence (IHF) of colon sections utilizing anti-occludin antibody demonstrated that M2-EVs not solely elevated the degrees of tight junction proteins, but in addition reserved the tight junction construction (Fig. 8D). Claudin1 IHF of colon sections confirmed comparable outcomes (Fig. 8E). Persistently, immunoblot evaluation confirmed that ranges of each occludin and claudin1, in addition to CD206, in colon tissues have been enhanced by M2-EVs (Fig. 8F). Collectively, M2-EVs induced M2 macrophage polarization and guarded tight junctions within the colons of colitis mice, in help of the in vitro capabilities we recognized or validated in cell tradition.

Fig. 8
figure 8

M2-EVs promoted polarization of M2 macrophages and improved barrier capabilities in DSS-induced colitis. The mice from Further file 1: Fig. S13 have been subjected to additional evaluation. N = 3–4/group. AC Antibodies towards macrophage marker F4/80 (A), M1 marker iNOS (B), and M2 marker CD206 (C) have been used to conduct IHC of colon sections. Their consultant photographs and quantifications have been proven. D, E Antibodies towards tight junction protein occludin (D) and claudin1 (E) have been used to conduct IHF of colon tissues. Their consultant photographs and quantifications have been proven. F Immunoblot evaluation of colon tissues. GAPDH served as a loading management. Within the bar graphs, every dot represents one mouse. Knowledge introduced as imply ± SEM. *P < 0.05 and **P < 0.01 relative to the management colitis mice acquired PBS (bar with black dots)

In a latest research [46], Wang et al. reported that M2-EVs from mouse peritoneal macrophages decreased immune cell infiltration and reserved colon size in DSS-induced colitis mice, which allied to our findings. Nonetheless, the research’s authors proposed a distinct mechanism: M2-EVs elevated the extent of an extended non-coding RNA (lncRNA), maternally expressed 3 (MEG3), in colon tissues, which in flip competitively sure to miR-20b-5p to advertise expression of the Creb1 gene. In our colitis mouse mannequin, we didn’t observe any affect of M2-EV therapy on ranges of the Meg3 and Creb1 gene in colon tissues (Further file 1: Fig. S15). This mechanistic discrepancy could also be because of the totally different experimental settings. Whereas we gave 1.5% DSS in consuming water for 7 days and intravenously injected M2-EVs (from BMDMs, at 3 × 108/g) 3 days earlier than, on the beginning date, and a pair of days after the administration of DSS, Wang et al. [46] administered 5% DSS in consuming water for 7 days, adopted by distilled water for 3 days and intravenously injected M2-EVs (from mouse peritoneal macrophages, at 50 μg/mouse) each 2 days.

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