Mesenchymal Stromal Cell-Derived Microvesicles Regulate an Internal Pro-Inflammatory Program in Activated Macrophages

Mesenchymal stromal cells (MSCs) are multipotent cells with abilities to exert immunosuppressive response promoting tissue repair. Studies have shown that MSCs can secrete extracellular vesicles (MVs-MSCs) with similar regulatory functions to the parental cells. Furthermore, strong evidence suggesting that MVs-MSCs can modulate several immune cells (i.e., Th1, Th17, and Foxp3+ T cells). However, their precise effect on macrophages (Mϕs) remains unexplored. We investigated the immunoregulatory effect of MVs-MSCs on activated M1-Mϕs in vitro and in vivo using differentiated bone marrow Mϕs and an acute experimental model of thioglycollate-induced peritonitis, respectively. We observed that MVs-MSCs shared surface molecules with MSCs (CD44, CD105, CD90, CD73) and expressed classical microvesicle markers (Annexin V and CD9). The in vitro treatment with MVs-MSCs exerted a regulatory-like phenotype in M1-Mϕs, which showed higher CD206 level and reduced CCR7 expression. This was associated with decreased levels of inflammatory molecules (IL-1β, IL-6, nitric oxide) and increased immunoregulatory markers (IL-10 and Arginase) in M1-Mϕs. In addition, we detected that MVs-MSCs promoted the downregulation of inflammatory miRNAs (miR-155 and miR-21), as well as, upregulated its predicted target gene SOCS3 in activated M1-Mϕs. In vivo MVs-MSCs treatment reduced the Mϕs infiltrate in the peritoneal cavity inducing a M2-like regulatory phenotype in peritoneal Mϕs (higher arginase activity and reduced expression of CD86, iNOS, IFN-γ, IL-1β, TNF-α, IL-1α, and IL-6 molecules). This in vivo immunomodulatory effect of MVs-MSCs on M1-Mϕs was partially associated with the upregulation of CX3CR1 in F4/80+/Ly6C+/CCR2+ Mϕs subsets. In summary, our findings indicate that MVs-MSCs can modulate an internal program in activated Mϕs establishing an alternative regulatory-like phenotype.

inTrODUcTiOn Mesenchymal stromal cells (MSCs) are multipotent cells with mesodermal differentiation capacity into adipocytes, osteoblasts, and chondroblasts (1). MSCs were originally described in bone marrow, but currently these cells can be isolated from various vascularized tissues including the adipose tissue (2). Therapeutically, MSCs are able to migrate toward injured tissues exerting anti-inflammatory and anti-apoptotic functions that consequently promote tissue repair (3). In addition, MSCs also have low immunogenicity when transplanted into patients, indicating their use as promising candidate for cellbased therapies (4). With regard to its regenerative properties, there are more than 600 protocols using MSCs in several diseases which include mainly orthopedic, cardiovascular, and autoimmune disorders (see in https://www.clinicaltrials. gov).
Additionally, other studies have shown that MSCs can secrete extracellular vesicles (EVs) with similar immunoregulatory and regenerative functions found in parental cells (13,14). EVs can be defined as a heterogeneous population of vesicles derived from cell membranes that can be classified into: apoptotic bodies (1-5 µm), microvesicles (MVs) (100-1,000 nm), and exosomes (EXOs) (40-100 nm) according to their size, biogenesis, and density (15,16). Interestingly, pre-clinical therapies based on EXOs or MVs derived from MSCs have been effective in models of cardiovascular, neurologic, and kidney diseases (13,17,18). Furthermore, in the context of inflammation, there is strong evidence that MSCs-derived EVs (EVs-MSCs) can inhibit Th17 cells, induce the conversion of Th1 to Th2 response and promote the proliferation of regulatory Foxp3 + T cells (14,19). Altogether, these findings suggest that EVs-MSCs possess a potent immunoregulatory capacity on lymphocytes and probably in other immune cell subsets involved in the inflammatory response. However, the precise effect of MSCsderived microvesicles (MVs-MSCs) on the immunobiology of Mϕs remains unexplored. Thus, considering the important role of Mϕs in tissue regeneration, the aim of this study was to evaluate the immunoregulatory effect of MVs-MSCs on classically activated M1-Mϕs.

MaTerials anD MeThODs animals
Around 8-to 10-week-old C57BL/6 male mice (20-25 g) were obtained from the Center for Experimental Models Development for Medicine and Biology (CEDEME) of the Federal University of São Paulo (UNIFESP). Additionally, B6:129P CX3CR1 +/GFP and B6:129 (Cg) CCR2 +/RFP mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA), and these inbred strains were backcrossed to generate CCR2 +/RFP CX3CR1 +/GFP mice. All mice were housed in a clean room with constant temperature (22°C) and a light/dark cycle of 12/12 h. The mice received a solid diet and filtered water ad libitum. All animal protocols and experiments were approved by the Ethics Committee on Animal Experimentation of UNIFESP with accession number CEP 685598.

isolation and characterization of adipose-Derived Mscs
Adipose-derived MSCs were isolated from epididymal fat of C57BL/6 mice (n = 10) as previously published (20). Briefly, adipose tissue was washed with PBS-containing penicillin/ streptomycin 50 U/mL (Gibco, Thermo, USA) and minced with surgical scissors. Subsequently, the tissue was incubated with collagenase type IA solution 0.075% (Sigma-Aldrich, USA) at 37°C for 30 min. After digestion, the enzyme activity was neutralized by adding DMEM Low Glucose culture medium (Gibco) with 10% fetal bovine serum (FBS, Hyclone, Thermo Fisher Scientific). The digested material was centrifuged at 300 g for 10 min, and the pellet was subjected to red blood cells lyse solution treatment with NH4Cl 0.83%. Then, the cell pellet was twice washed with PBS and centrifuged at 300 g for 5 min. The cells were incubated at 37°C in humid atmosphere with 5% CO2 with DMEM Low Glucose with antibiotics (penicillin/streptomycin 100 U/mL) and 10% FBS. At confluence of 80-90% of the cell monolayer, MSCs were trypsinized (0.05%, Trypsin-EDTA, Gibco) and further expanded to 10-12 passages. The immunophenotypic characterization and of multilineage differentiation of MSCs was performed according to previous works from our group (20,21).

isolation of MVs-Mscs
Mesenchymal stromal cells were expanded until 80% of confluence with DMEM low glucose (10% FBS). At high density confluence, culture growth medium was changed to serum-free DMEM low glucose. After 48 h, the conditioned medium was collected and centrifuged at 2,000 g for 30 min at 4°C to remove cells and cellular debris. Then, the conditioned medium was subjected to ultracentrifugation at 100,000 g for 2 h at 4°C, using a SW28 rotor (Optima L-90K, Beckman Coulter, USA). Finally, the pellet containing MVs-MSCs was re-suspended in PBS and stored at −80°C. MVs-MSCs protein content was quantified by Pierce method (Thermo Fisher Scientific) and characterized using flow cytometry (BD LSRFortessa, BD biosciences) and nanoparticle tracking analysis (NTA)-based methods (NanoSight, Malvern, UK). Transmission electron MSC-Derived MVs Regulates Activated Macrophages Frontiers in Immunology | www.frontiersin.org July 2017 | Volume 8 | Article 881 microscopy was performed using protocols established in our center of facilities for electron microscopy at Federal University of São Paulo (CEME).

MVs-Mscs nTa
Microvesicles-MSCs were analyzed by NTA using NanoSight LM10 (NanoSight, UK). The laser was set at 405 nm and the speed of the high-sensitivity camera shutter was set at 30.01 ms (OrcaFlash2.8). The videos were analyzed using NTA software (version 2.3 NanoSight), and recordings were made for 30 s per sample. All samples were diluted in ultrapure PBS (Gibco, Thermo, USA) to achieve a concentration of 2 × 10 8 and 9 × 10 10 particles/mL. Samples were administered and registered under controlled flow, using NanoSight syringe pump system. Finally, histograms were generated as results of NTA analyzes to evaluate particle concentration and average size of MVs-MSCs.
Then, drops of 20 μL were placed on parafilm in a flat surface. Formvar-carbon-coated electron microscopy grids were placed on the drops for 10 min for MVs adsorption. Grids were washed with 0.1M sodium cacodylate buffer, post fixed in 1% buffered glutaraldehyde, and washed with deionized water. Negativepositive staining was performed according to Théry et al. (22). Briefly, grids were contrasted in oxalic uranyl, then embedded in a mixture of saturated uranyl and methylcellulose 2% (1:9) on ice. Fluid excess was gently blotted on filter paper and air dried. Images acquisition and observation was run under a JEOL 1200 EX II (Japan) transmission electron microscope at 80 kV.

isolation and Polarization of Bone Marrow-Derived Macrophages (BMDM)
Femurs and tibia of C57BL/6 male mice were dissected, and the epiphyses were surgically removed with scissors to perform internal flushing with sterile PBS. The cells thus obtained from bone marrow were filtered using Cell Steiner 70 µm filter (Corning, USA), and the flow-through cells were twice centrifuged and washed at 300 g for 5 min. Subsequently, cells were lysed with 0.83% NH4Cl (3 min/4°C) and cultured in DMEM high glucose medium (Gibco) containing 10% FBS (Gibco) and 30% of supernatant medium from L929 cells (ATCC, Cell Lines) (23  In Vivo Model of Peritonitis induced by Thioglycollate

Flow cytometry analyses
Thioglycollate medium 3% (Sigma-Aldrich) was prepared in sterile conditions and protected from light at least 6 months before experiments. Mice were injected intraperitoneally (IP) with 2 mL of thioglycollate solution and euthanized on day 4. Mice treated with MVs-MSCs were injected IP with four doses of MVs-MSCs (8.38 × 10 9 particles) at 0, 24, 48, and 72 h. In contrast, control mice were treated at the same time-points but using vehicle (PBS). Finally, on the 4th day after thioglycollate infusion, an peritoneal lavage was performed with 5 mL of cold sterile PBS. The peritoneal exudate was centrifuged at 300 g for 5 min and cells were processed by FACS and the supernatant was analyzed by MILLIPLEX (Merck Millipore, USA). During the standardization of the model of peritonitis, washes at 24, 48, 72, and 96 h were performed to identify the cell cluster with GR1, CD19, and CD11b phenotype by FACS ( Figures S4A,B in Supplementary Material). Furthermore, the predominant presence of Mϕs in the last day of the induction of peritonitis was confirmed by morphological analysis (HE) and immunohistochemistry for CD11b ( Figure S4C in Supplementary Material).

cytokine assessment in intraperitoneal lavage
The concentrations of IFN-γ, IL-1α, IL-1β, TNF-α, IL-6, and IL-10 cytokines in the mouse peritoneal lavage were measured using MILLIPLEX MCYTOMAG-70K kit (Millipore Corporation, USA). Controls and samples were processed according to manufacturer's instructions. The sample detection was performed by Luminex SD device (Luminex, USA) using the software MILLIPLEX Analyst (Millipore Corporation).

enzymatic activity of arginase
The enzymatic activity of arginase was measured using an in house method. Total protein lysate from Mϕs was diluted 1:10 in 50 mM Tris-HCl buffer pH 7.4 containing 0.5 M PMSF and protease inhibitors. Then, 120 µL of each sample was transferred to 1.5 mL tubes and 120 µL of MnCl2 10 mM were added to each tube, which were further heated to 55°C for 10 min. After incubation, the tubes were placed on ice and 240 µL of L-Arg (0.5 M) were added. Then each sample was divided into additional four 0.6 mL tubes with 100 µL per tube. Two tubes were kept at 37°C for 1 h and the others left in ice for 1 h (control reaction). After this second incubation period (1 h), all tubes were taken to a fumes hood and 500 µL of the mix of acid solution (H2SO4:H3PO4:H2O) (1:3:7) were added, as well as 25 µL of ISPF 9%, always protected from light. Subsequently, tubes were sealed with parafilm. Samples were heated for 45 min at 100°C in a water bath, with 200 µL of each reaction product then pipetted on an ELISA plate. Samples were read in a spectrophotometer at 540 nm. The final results were expressed in nmol of protein (urea/min/mg).

Dosage of nitric Oxide (nO)
Nitric oxide estimation was performed by indirect colorimetric Griess method. First, a standard curve was constructed with NaNO2 using concentrations of 5, 10, 30, and 60 µM (Sigma-Aldrich, USA). Then, 50 µL of Mϕs cell lysate was incubated with 50 µL of Griess reagent for 1 h. After incubation, the plates were analyzed spectrophotometrically at 550 nm. The results were expressed in micromolar and normalized by the total protein concentration (μM/μg).

statistical analysis
For statistical analyses to compare two samples we used Student's t-test, and in analyses that comprised more than two independent samples one-way ANOVA was performed followed by post hoc Tukey test. The results are presented as mean and SD for parametric variables. Differences were considered significant if P < 0.05. Statistical analyses and graphs were performed with GraphPad Prism version 5.0 (GraphPad Software, USA).

resUlTs isolation and characterization of MVs-Mscs
Previous studies have clearly demonstrated the effectiveness of MSCs to educate and reprogram M1-Mϕs and M2-Mϕs (11,25).
Considering this perspective, we tested the ability of adiposederived MSCs to regulate M1-Mϕs ( Figure S1 in Supplementary Material). Using the gate exclusion strategy with Mϕs specific marker (F4/80), it was possible to discriminate the Mϕs and MSCs independent sub-populations ( Figure S1A in Supplementary Material). Further coculture analysis confirmed that MSCs decreased the expression of costimulatory molecules (CD86/B7-2) and chemokine receptor (CCR7) in M1-Mϕs previously stimulated with LPS/IFN-γ (Figures S1B,C in Supplementary Material). Additionally, the MSCs also increased the mannose receptor (CD206) frequency, a molecule associated with M2 profile, in M1-Mϕs cocultures ( Figure S1D). Interestingly, unstimulated Mϕs presented similar behavior to MSCs-modulated M1-Mϕs ( Figures  S1B,D). These findings indicate that MSCs in direct contact with M1-Mϕs can promote a prominent immunomodulatory effect. According to these results, we isolated MVs from adipose-derived MSCs supernatants to investigate if MVs-MSCs could assist a similar regulatory function as that observed for its parental cells. Initially, we characterized the MVs-MSCs population and defined its size, morphology, and immunophenotypic characteristics. Transmission electron microscopy analysis revealed that MVs-MSCs exhibit double membrane structures and present spheroid shape with a diameter inferior to 200 nm ( Figure 1A). Additionally, to confirm size and concentration of the MVs-MSCs, we performed a NTA using the NanoSight equipment. This assay showed that purified MVs-MSCs samples had a size around 147.1 ± 13.26 nm and an approximate concentration of 1.0 × 10 11 particles/mL (Figures 1B,C). Finally, we investigated if markers associated with MSCs also could be found in MVs-MSCs. We used predetermined particle size (beads with 3.8 and 7 µm) to discriminate the MVs-MSCs population location in FACS workflow ( Figure 1D). MVs-MSCs constitutively expressed the classical MSCs antigens such as CD44, CD105, CD90, CD73, but not endothelial (CD31) and hematopoietic (CD45) molecules ( Figure 1E). Additionally, as an internal control, we found that MVs derived from macrophages (MVs-Mϕs) do not express classical MSCs antigens (i.e., CD73, CD90, and CD105) on its surface, suggesting the high specificity of our MVs-MSCs immunophenotypic profile ( Figure S2 in Supplementary Material). Moreover, MVs-MSCs presented annexin V (microvesicle marker) and tetraspanin (CD9, an EXOs marker) surface expression (Figure 1F). In summary, our structural and immunophenotypic analysis suggest that our EVs-MSCs population, defined here as MVs-MSCs, can be comprised as a heterogeneous subpopulation predominantly enriched with MVs (particles with 100-1,000 nm).

MVs-Mscs interacts with M1-Mϕs to regulate activation, Oxidative stress, and secretory Profile
To investigate the capacity of MVs-MSCs to interact and regulate the pro-inflammatory profile of M1-Mϕs, we initially isolated bone marrow-derived Mϕs and extensively differentiated and characterized these cells to M1 and M2 phenotypes ( Figure S3 in Supplementary Material). We observed by fluorescent confocal microscopy the ability of MVs-MSCs to associate/incorporate into M1-Mϕs. Live cell imaging showed that MVs-MSCs pre-labeled with PKH26 (red fluorescent dye) had the ability to attach and partially incorporate into the M1-Mϕs cytoplasm right after their administration to M1-Mϕs (i.e., in less than 30 min) (Figure 2A). Visible MVs-MSCs aggregates were detected on M1-Mϕs surface or in intracytoplasmic space during the timecourse (Figure 2A) (Video S1 in Supplementary Material). Moreover, we also confirmed that a single dose of MVs-MSCs suspension can be rapidly incorporated into a confluent macrophage culture (Figure 2A) (Video S3 in Supplementary Material). As a control, the PKH26 red dye alone did not incorporate into M1-Mϕs (Figure 2A) (Video S2 in Supplementary Material).
To confirm the transfer of vesicles into the cytoplasm, we evaluated granularity in M1-Mϕs treated with serial doses of MVs-MSCs. Pre-treated M1-Mϕs had their side scatter (SSC, granularity) parameter increased when compared with untreated and activated M1-Mϕs ( Figure 2B). This suggests that M1-Mϕs may incorporate MVs-MSCs and subsequently enhance its internal granularity, probably by the transfer or indirect production of bioactive molecules such as mRNA, miRNAs, proteins, and lipids (26).
We then investigated the ability of MVs-MSCs to modulate pro-inflammatory markers in M1-Mϕs. The treatment with MVs-MSCs decreased M1-related molecules such as CCR7, but not CD86, a classical marker of activation ( Figure 2C). On the other hand, MVs-MSCs administration increased the expression of M2-associated markers in M1-Mϕs such as mannose receptor (CD206) (Figure 2C). Furthermore, MVs-MSCs potentially reduced the mRNA expression of inflammatory cytokines such IL-1β and IL-6 in M1-Mϕs, while increasing anti-inflammatory molecules such as IL-10 (Figures 3A-C). Interestingly, the expression of CXCL9, a strong chemoattractant protein, remained unaltered in M1-Mϕs treated or not with MVs-MSCs ( Figure 3D). Finally, we attempted to show the MVs-MSCs ability to reduce oxidative stress in M1-Mϕs. We identified that MVs-MSCs treatment increased arginase activity (M2 marker) in M1-Mϕs and reduced endogenous NO production (M1 marker) (Figures 3E,F). In most experiments, unstimulated Mϕs presented a pattern similar to the observed in MVs-MSC-treated M1-Mϕs. Thus, our results suggest that MVs-MSC were effective to reduce activation, oxidative stress, chemokine receptor expression, and pro-inflammatory cytokines on M1-Mϕs, thereby inducing a switch from M1 to a M2-like phenotype.

MVs-Mscs exerts a Down-regulation of Pro-inflammatory mirnas in M1-Mϕs
After we had identified that MVs-MSCs exerted a regulatory effect on M1-Mϕs through modulation of mRNAs and cell signaling proteins, we studied the ability of MVs-MSCs to modulate some important miRNAs related to M1/M2 phenotype (Figure 4). Analysis of these miRNAs showed that miR-155 and miR-21 were significantly more expressed in M1-Mϕs than in unstimulated Mϕs, considering that miR-146 expression, a regulatory miRNA, was not different between both Mϕs groups (Figures 4A-C). Surprisingly, we detected that MVs-MSCs-treated M1-Mϕs reduced miR-21 and miR-155 expression when compared with  Figures 4B,C). Thus, these data show that MVs-MSCs may modulate M1-Mϕs via downregulation of miR-155 and miR-21 but not by upregulation of miR146a.
In the next step, we set to investigate the predicted target genes of MVs-MSCs-modulated miRNAs in M1-Mϕs. In this sense, we evaluated the expression of SOCS1 and SOCS3  molecules, which possess immunomodulation potential and have been reported as potential targets of these altered miRNAs (Figures 4D,E) (27,28). Indeed, M1-Mϕs exposed to MVs-MSCs increased the expression of SOCS3 transcript, suggesting that MVs-MSCs upregulate SOCS3 expression by downregulating its associated miR-155, which is also related to M1 activation ( Figure 4E). In contrast, the expression of SOCS1 was slightly increased in M1-Mϕs when pulsed with MVs-MSCs, but this was not statistically significant ( Figure 4A).

MVs-Mscs inhibit Macrophage recruitment and regulate Proinflammatory Microenvironment in experimental Peritonitis
In order to elucidate the immunoregulatory effect of MVs-MSCs on activated Mϕs in vivo, we examined the influence of MVs-MSCs treatment in an experimental model of thioglycollateinduced peritonitis. This classic acute model is characterized by intense Mϕs recruitment to the peritoneal cavity and release of several inflammatory mediators such as IFN-γ, IL-6, MCP-1, IL-17A, and IL-1β (29). We verified 96 h after thioglycollate injection that there was a massive recruitment of CD11b + cells (macrophages) in the peritoneal space ( Figure S4A in Supplementary Material). Complementary, we observed a considerable increase in the total number of inflammatory cells in the peritoneal cavity of animals exposed to thioglycollate over the timecourse ( Figure S4B in Supplementary Material).
To validate the predominant presence of Mϕs at day 4, we performed H.E. and IHC staining for CD11b, which confirmed the consistent presence of macrophages in the peritoneal cavity ( Figure S4C in Supplementary Material). We also observed that around 55% of neutrophils (GR1 + cells) were recruited within 24 h of thioglycollate infusion, but these numbers abruptly decreased over time (20% at 48 h and 5% at 72 h) ( Figure S4A in Supplementary Material). In contrast, we detected that the number of peritoneal Mϕs increased considerably after thioglycollate infusion, whose highest concentration in the peritoneal cavity (>80% of CD11b + cells) was observed at 96 h. Together, these results show that at day 4 after peritonitis induction there Subsequently, we assessed the role of MVs-MSCs on peritoneal Mϕs in two experimental groups: the control group, which was submitted to thioglycollate and treated with vehicle (named: THIO), and the treated group, which was submitted to thioglycollate and exposed to serial doses of MVs-MSCs (named: THIO+MVs-MSCs). Initially, we compared the total number of peritoneal cells present in both groups. Mice administered MVs-MSCs surprisingly showed a reduction in the absolute number of total cells present in the peritoneal exudate (Figures 5A,B). We observed that MVs-MSCs inhibited the recruitment of approximately 20 × 10 6 inflammatory cells in peritoneal space (Figures 5A,B). Consistently, we found that mice treated with MVs-MSCs also had reduction in the relative number of peritoneal Mϕs (F4/80 + CD11b + ) when compared to the control animals (Figures 5C,E). Next, we built a global profile of the inflammatory cells in the peritoneum, considering the absolute and relative number of cells, and we found that mice treated with MVs-MSCs had 7-fold less peritoneal Mϕs than untreated mice ( Figure 5F). These data show that MVs-MSCs were effective o inhibit macrophage recruitment after thioglycollate stimulus ( Figure 5F). Information on the absolute and relative numbers of peritoneal Mϕs, as well as of eosinophils in mice not treated with thioglycollate (NON-THIO) can be consulted in the Figures S5A-C in Supplementary Material.
To demonstrated the ability of MVs-MSCs to modulate the function of infiltrating Mϕs in the peritoneal cavity, we evaluated the expression of M1 and M2 markers on these specific Mϕs sub-populations (F4/80 + CD11b + ). We found that MVs-MSCs decreased the expression of CD86 in peritoneal Mϕs (Figure 5G). Similarly, Mϕs exposed to MVs-MSCs also reduced the expression of iNOS ( Figure 5H). On the other hand, the CD206 and CCR7 levels had a slight reduction after MVs-MSCs treatment but did not reach statical significance (Figures 5I,J). Importantly, when we analyzed the arginase activity in the peritoneal exudate of mice submitted to MVs-MSCs treatment, we found a significant increase when compared to the control group ( Figure 5K). Furthermore, we investigated the release of pro-inflammatory cytokines in the peritoneal lavage. Multiplex analysis revealed that, in fact, mice treated with MVs-MSCs have reduced concentrations of most inflammatory mediators in the peritoneal lavage, such as IFN-γ, IL-1β, and TNF-α, while that of IL-1α did not change (Figure 5L). Interestingly, IL-6 was not detected in the washout of the peritoneal cavity from mice treated with MVs-MSCs ( Figure 5L). These results suggest that MVs-MSCs were highly effective in regulating classically functional M1-Mϕs during peritonitis in vivo, promoting an anti-inflammatory microenvironment.
In addition, we also identified the participation of an eosinophil population that has been reported as interfering cells in this model (30). Here, these cells were detected in the peritoneal exudate as a F4/80 low CD11b -SSC-A high gate population in Figures 5D,E, and as F4/80 low SSC-A high SIGLEC F + gate population in Figures 6A,B,D. Although we have observed that MVs-MSCs provided slight increase on relative number of eosinophils in the peritoneum, this difference was not statistically significant ( Figure 5D) and, more importantly, these eosinophils were positive for Ly6C but did not exhibit chemokine-associated receptors (CX3CR1 and CCR2) as monocytes, suggesting a nonpivotal participation in inflammatory peritonitis ( Figure 6C).

MVs-Mscs induce higher expression of cX3cr1 in ly6c + ccr2 + Macrophages subsets
Currently, it is known that two different monocyte subtypes have opposite roles in murine inflammation: Ly6C + CCR2 + CX3CR1 low and LY6C − CCR2 − CX3CR1 hi monocytes (31). Previous findings suggest that thioglycollate-induced peritonitis promote infiltration of inflammatory Ly6C + monocytes, which subsequently differentiate into peritoneal activated Mϕs (32). In this context, we decided to investigate the influence of MVs-MSCs in the expression of CX3CR1 and CCR2 receptors on activated peritoneal Mϕs. Thus, we performed the thioglycollate-induced peritonitis in heterozygous mice with a florescent gene report for chemokine receptors CX3CR1 +/GFP (green) and CCR2 +/RFP (red) in inbred mice strains CCR2 +/RFP CX3CR1 +/GFP . Flow cyto metry analysis showed that MVs-MSCs were again effective in reducing the relative frequency of peritoneal Mϕs in these fluorescent report mice (Figures 6A,B,E). Next, we found that MVs-MSCs decreased the absolute number of infiltrated cells in the peritoneum cavity ( Figure S5F in Supplementary Material), whereas the overall profile of inflammatory cells was similar to the previous experiments depicted in Figure 6F. In addition, we similarly documented some baseline information on macrophage and eosinophil frequencies in NON-THIO mice CX3CR1 + / GFP CCR2 + / RFP (Figures S5D-I

in Supplementary Material).
We have also observed that peritoneal Mϕs (F4/80 + gate), both treated and untreated with MVs-MSCs, had high expression of the monocyte marker Ly6C + (Figure 6F,G). These data indicate that MVs-MSCs did not alter the subset of monocytes that infiltrated the peritoneum (Ly6C + or Ly6C − ) during inflammatory stimulus. Furthermore, we observed the expression of chemokine receptors CCR2 and CX3CR1 in this Mϕs population gated to F4/80 + Ly6C + (Figure 6H). We detected that Ly6C + Mϕs were positive for CCR2 independently of treatment (Figure 6H, Histograms). This information is consistent with the innate ability of Ly6C + monocytes to exhibit CCR2 in order to migrate from bone marrow to inflammatory tissues. In this context, the MVs-MSCs treatment did not alter the expression of CCR2 in Ly6C + Mϕs ( Figure 6H). In addition, peritoneal Mϕs from NON-THIO mice showed low expression of CCR2 and CX3CR1 ( Figures S5G,H in Supplementary Material).
Surprisingly, when we evaluated the expression of fractalkine receptor (CX3CR1) on Ly6C + Mϕs population, we could identify three major additional sub-populations based on the fluorescence intensity expression of this molecule: (1) population G1 (Ly6C + CCR2 + CX3CR1 − Mϕs), population G2 (Ly6C + CCR2 + CX3CR1 low Mϕs), and population G3 (Ly6C + CCR2 + CX3CR1 high Mϕs) (Figures 6H-M). Importantly, the frequency of the G3 population was significantly increased in mice treated with MVs-MSCs (Figure 6M), compared to the G1 population, that was slightly elevated, and the G2 population, that did not change. In this context, we may partially conclude that MVs-MSCs regulate peritoneal Mϕs to both M1 and M2 phenotype, as well as upregulate fractalkine receptor (CX3CR1) in specific Mϕs subsets.

DiscUssiOn
Currently, the cell-free therapies with focus on regeneration have been considered to be very attractive and promising approaches (33). Clinically, the use of cell-derived inert particles such as MVs-MSCs to modify target cells by transferring bioactive molecules has strong advantages to traditional interventions, considering they are easily stored, handled, and are safer than parental MSCs (34). With this perspective, in 2011 an intriguing pilot study was conducted with steroid-refractory graft-versus-host diseases patients. Interestingly, that study reported that intravenous administration of MVs-MSCs promoted an improvement in clinical parameters such as diarrhea episodes, skin rash, and mucosal lesions without additional intercurrences (35). Later, the authors showed, using an in vitro approach, that the reduction in TNF-α and IFN-γ indexes in NK cells could be partially considered as modulatory mechanism in these MVs-MSCs-treated patients. Considering this evidence, it is plausible that MVs-MSCs could also modulate other immune cells involved in autoimmune processes, such as Th1-and Th17-responses (14,19). In this sense, we have extensively investigated and demonstrated herein that MVs-MSCs possess in vitro and in vivo specific abilities to immunomodulate activated Mϕs promoting a pro-resolution microenvironment.
In order to achieve our objective, we first characterized and confirmed the ability of MSCs to regulate M1-Mϕs. This strategy was employed as a quality control step to verify prior of MVs-MSCs isolation its usability. Thus, we can avoid some misconception that cell-derived particles represent only a marginal reference of the biological function status of their parental cells (36). Indeed, our MSCs treatment modulated two pivotal surface antigens required for migration and amplification of the inflammatory response in M1-Mϕs (i.e., CCR7 and CD86) (37,38). In addition, we also found that CD206 was more expressed in MSCs-treated   (33). However, it is possible to define and identified these sub-populations based on size, phenotypic and structural characteristics. MVs are double membrane particles with sizes between 100 and 1,000 nm and contain high amounts of phosphatidylserine, ceramide, and sphingomyelin. In contrast, EXOs are smaller than MVs (40-100 nm) and contain tetraspanins (e.g., CD9 and CD63), as well as some heat shock proteins. We determined that the population of EVs present in our MVs-MSCs suspension was compatible with an heterogeneous subpopulation enriched with MVs (Figure 1). Thus, our MVs-MSCs expressed surface antigens associated with parental cells and annexin V, which classically define our MSCsderived particles as predominantly composed by MVs.
One report has demonstrated that MVs-MSCs have elevated distribution of CD44, a receptor related to the ability to incorporate into cell surfaces (39). In this sense, our MVs-MSCs population also exhibited a higher index of CD44, suggesting a promising potential to incorporate and regulate M1-Mϕs. In fact, our findings show that MVs-MSCs were quickly incorporated into the intracytoplasmic region of M1-Mϕs during cocultured experiments, without nuclear co-localization. These data suggest that the preferred site of MVs-MSCs incorporation in Mϕs, in our experimental conditions, would be the cytoplasm. In line with this, previous studies have shown that the therapeutic effect of MVs-MSCs is mainly mediated through transfer and modification of miRNAs that occur primarily in the cytoplasm compartment (40). On the other hand, MVs-MSCs not only can transfer genetic material, but also may transport growth factors and receptors with bioactive functions into target cells. These data support the idea that the elevated M1-Mϕs cellular granularity observed after incubation with MVs-MSCs could be attributed to the transfer of bioactive biomolecules.
In order to investigate the functional immunoregulatory potential of MVs-MSCs, we reported that MVs-MSCs were effective in downregulating M1 markers (i.e., CCR7, IL-1b IL-6, and NO) and subsequently upregulating M2 molecules (IL-10 and CD206) in M1-Mϕs. Surprisingly, M1-Mϕs when exposed to MVs-MSCs exhibited higher arginase activity, which has been recently shown to be a key enzyme associated with M2 regulatory phenotype. This enzyme catalyzes the conversion of l-arginine to l-ornithine, thus suppressing NO production, a pro-inflammatory mediator (41), what was indeed detected in our coculture experiments with MVs-MSCs-modulated M1-Mϕs. Furthermore, arginase also has additional pleiotropic functions such as being a main mediator of cell proliferation, of tissue healing and of inhibition of T lymphocytes (41,42).
After verifying that MVs-MSCs promoted a M2-like phenotype in activated M1-Mϕs, we attempted to elucidate the mechanism involved in this process. We set out to study the main miRNAs that regulate the Mϕs polarization. Our results show that MVs-MSCs promoted the reduction of miRNAs associated with M1 profile (40) such as miR-21 and miR-155. Importantly, the main function of miR-155 is to suppress IL-13 receptor expression, which regulates the activation of inflammatory Mϕs (40). Moreover, we found that SOCS3 protein, a specific target of miR-155 and miR-21, had its expression reduced in MVs-MSCs-treated M1-Mϕs. In line with this, we further detected the lack of IL-6 expression, a strong regulator of SOCS3, on M1-Mϕs exposed to MVs-MSCs both in vivo and in vitro experiments. The possible explanation for this molecular interplay would be the fact that SOCS3 is a strong inhibitor of the STAT6 signaling pathway, thus interfering with IL-6 expression, which seems to be regulated downstream by miR-155 (43). Thus, this information suggests that the reduction of miR-21 and miR-155 could be the mechanism by which M1-Mϕs exhibit M2-like phenotype.
To investigate the immunomodulatory role of MVs-MSCs in vivo, we evaluated the impact of MVs-MSCs treatment in an experimental model of acute peritonitis. We detected that MVs-MSCs injection promoted a significant reduction in the global number of infiltrating Mϕs into the peritoneal cavity with downregulation of inflammatory markers (CD86 and iNOS) and upregulation of arginase activity. These results complement our in vitro previous investigation, demonstrating the functional ability of MVs-MSCs to also regulate M1 peritoneal Mϕs. Supporting this evidence, we detected that pro-inflammatory cytokines such as IL-1β, IFN-γ, IL-6, and TNF-α were broadly reduced in peritoneal Mϕs from mice treated with MVs-MSCs. Additionally, we observed that MVs-MSCs also expressively increased the expression of CX3CR1 in a subpopulation of Mϕs defined here as Ly6C + CCR2 + . Interestingly, Ly6C + CCR2 + Mϕs are commonly CX3CR1 low or negative and are widely associated with pro-inflammatory functions (31). However, in some special situations, it has been documented that Ly6C + CCR2 + Mϕs may also express CX3CR1 high , and, in this case, perform anti-inflammatory functions (44). Hence, we can speculate that MVs-MSCs can make use of an alternative mechanism of modulation, that yet needs to be extensively described, involving the upregulation of CX3CR1 in classically inflammatory Mϕs.
In summary, our results indicate that MVs-MSCs have immunomodulatory effects on M1-Mϕs inducing a switch from M1 to M2-like pattern. Thus, our innovative findings suggest that MVs-MSCs can modulate activated Mϕs to an alternative regulatory-like phenotype, thus relevantly contributing to further investigations in the field of inflammatory diseases and tissue regeneration.

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All animals' protocols and experiments were approved by the Ethics Committee on Animal Experimentation of UNIFESP with accession number CEP 685598.

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The authors would like to thank Prof. Dr. Reinaldo Salomão and Laboratory of Immunology of the Federal University of São Paulo for the availability and assistance with the flow cytometer. We also express our recognition for Research Facility Center (CEFAP) of the University of Sao Paulo for all service with confocal microscopy experiments. Finally, we thank The Electronic Microscopy Center (CEME) of the Federal University of São Paulo for support with electron microscopy experiments.