Immunomodulatory Effects of Canine Adipose Tissue Mesenchymal Stem Cell-Derived Extracellular Vesicles on Stimulated CD4+ T Cells Isolated from Peripheral Blood Mononuclear Cells

Adipose tissue-derived mesenchymal stem cells (ADSCs) have anti-inflammatory and immunomodulatory characteristics. Many studies have suggested that the immunomodulation of ADSCs is largely mediated by secreted paracrine factors. Various factors are secreted from ADSCs, among which extracellular vesicles are considered to play a major role in the communication between ADSCs and target cells. Several studies have reported the function of canine ADSC-derived extracellular vesicles (cADSC-EVs), but few studies have reported the immunomodulatory effects of cADSC-EVs on immune cells. The purpose of this study was to investigate the effects of cADSC-EVs on in vitro-stimulated CD4+ T cells isolated from peripheral blood mononuclear cells (PBMCs). cADSC-EVs were isolated from cADSCs under naive conditions or primed conditions by tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). The expression levels of several microRNAs in cADSC-EVs were altered by priming with TNFα and IFNγ. Culturing PBMCs stimulated with concanavalin A in the presence of naive or primed cADSC-EVs inhibited the differentiation of PBMCs and CD4+ T cells and promoted apoptosis of PBMCs. CD4+, CD8+, and CD4+CD8+ T cells were decreased, while CD3+CD4−CD8− T cells were increased. T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cells were analyzed by flow cytometry. cADSC-EVs inhibited the proliferation of Th1 and Th17 cells and enhanced Th2 and Treg cell proliferation. However, CD4+ T cells that had incorporated labeled cADSC-EVs comprised only a few percent of all cells. Therefore, these responses of stimulated CD4+ T cells may be due to not only direct effects of cADSC-EVs but also to indirect effects through interactions between cADSC-EVs and other immune cells. In conclusion, cADSC-EVs exert immunosuppressive effects on stimulated CD4+ T cells in vitro. These findings may be useful for further studies of immune diseases.


Introduction
Mesenchymal stem cells have various biological characteristics that include an immunomodulatory capacity [1][2][3]. Many studies have demonstrated that MSCs suppress the differentiation, proliferation, secretions, and migration of immune cells [4]. It has been documented that MSCs improve abnormal immune responses in autoimmune diseases in vivo and it has been thought that these benefits are partly due to secreted factors from MSCs [5][6][7]. Moreover, the MSC immunomodulatory ability is altered by inflammatory cytokine, such as those present in the inflammatory microenvironment. Stimulation with interferon-γ (IFNγ) enhances the immunosuppressive effects of MSCs. Priming MSCs with IFNγ upregulates indoleamine 2,3-dioxygenase (IDO), secretes important immunomodulatory molecules, such as prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), transforming growth factor-β (TGFβ), and chemokine ligand 2, and increases the expression of human leukocyte antigen class I and II molecules and costimulatory molecules [8]. Priming MSCs with tumor necrosis factor-α (TNFα) also promotes upregulation of immunoregulatory factors, such as PGE2, IDO, and HGF, but is much less pronounced compared with IFNγ priming [9]. However, the combination of inflammatory cytokines to stimulate MSCs may lead to additional effects. Priming MSCs with TNFα and IFNγ increase factor H production [10], which potently inhibits complement activation. Factor H secreted by MSCs is significantly suppressed by the inhibition of PGE2 and IDO. Therefore, priming MSCs with inflammatory cytokines is more useful for the treatment of immune-mediated diseases [11][12][13].
MSCs secrete large numbers of molecules, which include cytokines and growth factors, as well as extracellular vesicles (EVs), and these secretomes contribute to MSC abilities [14,15]. Studies have focused on the immunomodulatory capacity of MSC secretomes for various types of immune cells and MSC-derived EVs (MSC-EVs) may have similar functions to parent cells in terms of immunomodulatory effects [4]. EVs transfer signaling molecules from one cell to another cell through a paracrine mechanism, because EVs enclose various active molecules, such as bioactive proteins, as well as lipids, mRNAs, and microRNAs [16], which contribute to regulating the gene expression and phenotypic transformation of target cells. MSC-EVs are incorporated by target cells through direct membrane fusion, receptor-mediated phagocytosis, and several other internalization mechanisms, which leads to subsequent activation of signal transduction pathways and involvement in various physiological and pathological processes that include immune responses [17][18][19]. Therefore, MSC-EVs are attracting attention as immunomodulators.
Similar to humans, immune-mediated diseases also exist in veterinary medicine, such as atopic dermatitis, inflammatory bowel disease, and immune-mediated arthritis. Almost all of these diseases have unclear mechanisms and some cases become intractable with current treatments. Therefore, MSC-based therapy is also expected to be an alternative treatment method for immune-mediated diseases [11][12][13]. However, few studies have documented the functions of canine ADSC-(cADSC-) EVs [20][21][22]. Furthermore, studies that focus on the effects of cADSV-EVs on T cells have not been reported. In this study, we investigated the immunomodulatory properties of cADSC-EVs on in vitro-stimulated CD4 + T cells isolated from peripheral blood mononuclear cells (PBMCs). Moreover, we evaluated whether immunomodulatory effects of cADSC-EVs on stimulated CD4 + T cells were enhanced by parent cells primed with inflammatory cytokines such as TNFα and IFNγ.

Materials and Methods
2.1. Stimulation of cADSCs with TNFα and IFNγ. cADSCs were isolated and used as described previously [23]. In brief, Adipose tissue was aseptically collected from falciform ligament fat of three anaesthetized dogs. The tissue was washed extensively in phosphate buffer solution (PBS), minced, and digested with collagenase type I (Sigma-Aldrich, Tokyo, Japan) at 37°C for 45 min with intermittent shaking. After washing with PBS and centrifuging, the pellets containing the stromal vascular fraction were resuspended, filtered through a 100-μm nylon mesh, and incubated overnight in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and a 1% antibiotic-antimycotic solution (Thermo Fisher Scientific, Tokyo, Japan) in a humidified atmosphere with 5% CO 2 at 37°C. Unattached cells were removed by changing the medium, and the attached cells were washed twice with PBS. Thereafter, the medium was replaced every 3-4 days. At 80-90% confluence, the cells were detached with trypsin-EDTA solution (Sigma-Aldrich) and passaged repeatedly. The expression of several markers, such as CD14-FITC, CD29-PE, CD34-PE, CD44-PE, CD45-FITC, and CD90-PE, on these cells was determined by flow cytometry using a CytoFLEX (BECKMAN COULTER, Tokyo, Japan) [24]. cADSCs at passage 3 were seeded in 150-mm dishes (4 × 10 6 cells/dish) and cultured in high glucose DMEM with 10% exosome-free FBS (Thermo Fisher Scientific) and a 1% antibiotic-antimycotic solution in a humidified atmosphere with 5% CO 2 at 37°C. After 24 h, unattached cells were removed by changing the medium, and the attached cells were stimulated for 24 h with tumor necrosis factor-α (TNFα, 20 ng/ml) and interferon-γ (IFNγ, 20 ng/ml). Naive and primed cADSCs were cultured for 72 h.

cADSC-EV Isolation and Characterization.
After 72 h of culture, the medium was harvested, and then, EVs were isolated by a MagCapture Exosome Isolation Kit PS (FUJIFILM Wako Pure Chemical, Osaka, Japan). In brief, the medium was centrifuged at 300 g for 5 min and then 1,200 g for 20 min at 4°C to remove cells and debris. The supernatant was added to 1/100 volumes of EV-Save Extracellular Vesicles Blocking Reagent (FUJIFILM Wako Pure Chemical, Osaka, Japan) and concentrated using an ultrafiltration unit (Vivaspin; SARTORIUS, Tokyo, Japan). After concentrating the supernatant, extracellular vesicles including exosomes were isolated in accordance with the manufacturer's instructions. Finally, the EVs were suspended in PBS.
The concentration and size of EVs were determined by nanoparticle tracking analysis (NTA) using a NanoSight LM10 (Malvern Panalytical, Tokyo, Japan) with the following parameters: camera level 12, threshold 8, 21.4°C, and five videos per analyzed sample. Visualization of EVs was assessed by transmission electron microscopy (H-7600; Hitachi High-Tech, Tokyo, Japan).
The total protein concentration of naive and primed EVs was measured by a BCA assay kit. Each 0.5 μg protein sample in 20 μl with 2.5 μl NuPAGE LDS sample buffer was electrophoresed in 4%-12% Bis-Tris Gels and then transferred to a 0.45-μm PVDF membrane. The membranes were blocked for nonspecific binding with Tris-buffered saline with 0.1% Tween-20 and 5% dry nonfat milk overnight at 4°C. After blocking, the membranes were incubated with a primary ice for 20 min. PBMCs were stained with anti-CD3-FITC (Clone: CA17.2A12, Bio-Rad), anti-CD4-APC (Clone: YKIX302.9, eBioscience), and anti-CD25-RPE (clone: P4A10, Thermo Fisher Scientific) or their respective isotype controls. Then, PBMCs were fixed and permeabilized using True-Nuclear Transcription Factor Buffer Set (BioLegend). Finally, PBMCs were stained with anti-Foxp3-eFluor450 (clone: FJK-16 s, Thermo Fisher Scientific) or that of isotype controls. Analysis by flow cytometry was performed by measuring the frequency of Foxp3 expression on gated CD3 + CD4 + CD25 + cells.
2.11. Uptake of EVs by CD4 + T Cells. cADSC-derived naive and primed EVs were stained with Vybrant DiI Celllabeling Solution (Thermo Fisher Scientific), and then, unincorporated dye was removed from labeled EVs using an Exosome Spin Column (Thermo Fisher Scientific) in accordance with the manufacturers' instructions. PBMCs were stimulated with ConA (5 μg/ml) for 3 days and then collected, washed with PBS, and resuspended in RPMI-1640 medium with 10% FBS, 1% antibiotic-antimycotic solution, 1% nonessential amino acid, 1% GlutaMAX, and 50 μM 2mercaptoethanol. One million stimulated PBMCs were cultured in a 12-well plate with 1 μg/ml naive or primed labeled-EVs for 3 or 12 hours. Cells were fixed with 2% paraformaldehyde and then covered with a mounting medium with DAPI (VECTASHIELD: H-1200: VECTOR LABORA-TORIES, CA, USA). Immunofluorescence images were captured under a BZ-X700 multi-purpose microscope (KEY-ENCE, Osaka, Japan). The same samples without fixation were incubated with a canine Fc receptor binding inhibitor on ice for 20 min. Then, the cells were stained with anti-CD3-FITC (Clone: CA17.2A12, Bio-Rad) and anti-CD4-APC (Clone: YKIX302.9, eBioscience) or their respective isotype controls. Fluorescence was evaluated by flow cytometry.
2.12. Statistical Analysis. All data are presented as the mean ± standard deviation. Differences among multiple groups were assessed by one-or two-way analysis of variance and differences were compared using the Tukey-Kramer post hoc test. P < 0:05 was considered statistically significant. Statistical analyses were performed using Excel 2019 with add-in software Statcel 3.

Characterization of cADSC-Derived
EVs. cADSCs were pretreated with or without TNFα and IFNγ, and then, an enriched fraction of EVs was collected from the supernatant using the PS affinity method. The size of isolated naive EVs was 166 ± 7:7 nm and that of primed EVs was 145 ± 1:5 nm. The concentrations of EVs were 34:4 ± 3:1 and 42:8 ± 1:9 × 10 9 particles/ml for naive and primed EVs, respectively ( Figure 1(a)). Both naive and primed EVs expressed specific exosomal markers CD9, CD63, and TSG101 [25]. The size of collected vesicles and marker expression indicated that 4   Journal of Immunology Research cADSC-derived EVs isolated from the supernatant were an enriched fraction of exosomes (Figure 1(b)). Expression of exosome-associated microRNAs was investigated by a miRNA PCR array. Seventy microRNAs were detected in both naive and primed EVs, and expression levels of 14 microRNAs in primed EVs were significantly different compared with that in naive EVs ( Figure 2). All data of fold regulation and P values of each microRNA are shown in Supplemental Material 1.
3.7. cADSC-Derived EV Uptake by CD4 + T Cells. T cellenriched PBMCs were coculture with cADSC-derived EVs for 3 or 12 hours. Both naive and primed EVs were incorporated into T cells and CD4 + T cells treated with EVs were observed by flow cytometry (Figure 11). The ratio of CD4 + T cells with cADSC-derived EVs at 12 h was significantly elevated in both naive and primed EVs compared to those at 3 h.

Discussion
Mesenchymal stem cells (MSCs) have immunomodulatory and anti-inflammatory abilities by secreting numerous factors such as extracellular vesicles, cytokines, chemokines, and growth factors. As research on MSC-based therapies has proceeded, exosomes have been focused on as important factors that exert immunomodulatory effects. Exosomes are typically small membrane vesicles (30-150 nm) that include extracellular vesicles (30-1000 nm) secreted by various cell types. The classically used protocol to isolate exosomes is ultracentrifugation, but there are currently several exosome isolation methods based on different principles [26,27]. The ultracentrifugation method is based on the principles of precipitation and sedimentation. Therefore, ultracentrifugation   Journal of Immunology Research  also concentrates nonspecifically sedimentable particles, which include non-EV proteins, and is associated with low reproducibility because of the loss of unstable and invisible pellets after centrifugation. Conversely, the PS affinity method is based on the high affinity of T cell immunoglobulin domain and mucin domain-containing protein 4 that strongly binds to phosphatidylserine expressed on the surface of EVs. The PS affinity method only requires general laboratory equipment. Moreover, this method isolates EVs with higher quality and reproducibility compared with ultracentrifugation [28]. In this study, we collected cADSC-EVs using the PS affinity method. The results of NTA and analyses of exosome markers (CD9, CD63, and TSG101) suggested that the cADSC-EVs isolated from culture supernatants were an enriched exosome fraction.
In this study, we examined the microRNA expression profile of naive and TNFα/IFNγ-primed cADSC-EVs. micro-RNAs, which are a class of noncoding RNAs of 20-22 base pairs, regulate the expression of multiple RNAs and play an important role in various biological processes that include the immune response. Several microRNAs affect the immune response in T cell development, proliferation, differentiation, and function [29,30]. The microRNA profiles showed that several microRNAs in cADSC-EVs varied after stimulation with TNFα and IFNγ. For example, miR-146a, which was overexpressed in primed cADSC-EVs, acts as a negative regulator of T cells and promotes Treg cell functions [30]. MiR-34a, which was downregulated in primed cADSC-EVs, plays an important role in T cell activation by targeting >30 genes across different cellular pathways that control the immune response [31].
Several studies have reported that primed EVs increase immunomodulatory functions compared with naive EVs [32,33], whereas other reports have indicated that primed EVs do not enhance the immunomodulatory effects of MSC-EVs [34,35]. In our study, primed cADSC-EVs did not enhance any functions of PBMCs or stimulated CD4 + T cells. The reason that the immunomodulatory functions of primed cADSC-EVs were not enhanced remains unclear, but several hypotheses were proposed. First, packaged soluble factors inside EVs may be related [34]. The properties of factors secreted from MSCs change depending on MSC culture conditions [5]. There is evidence suggesting that immunomodulatory effects of MSCs are enhanced in response to inflammatory stimulation [5]. Inflammatory stimuli by TNFα and IFNγ have been commonly used to research the immune and regenerative functions of MSCs and are known to enhance the immunosuppressive properties of MSCs [6]. For example, immunomodulatory effects of T cells are attributed to the upregulation of IDO. MSC-EVs also packaged various active proteins, which included IDO, but previous studies have demonstrated no significant change of IDO in PBMCs cocultured with or without MSC-EVs and thus suggested that the mechanism that underlies the immunomodulatory capacities of MSC-EVs is different from that of MSC soluble factors [36]. Second, stimulation methods of T cells may be related [35]. In this study, PBMCs were stimulated with ConA. ConA is an antigen-independent mitogen and is widely used as a T cell stimulus. However, T cell activation by an anti-CD3/28 antibody can be used in humans and mice as an antigen-dependent method. Antigen-presenting cells partially mediate T cell suppression induced by MSCs [37]. Therefore, another explanation might be a lack or small number of antigen-presenting cells in our experimental setting. In our study, we analyzed characteristic features by only comparing the expression of microRNAs between naive and primed cADSC-EVs, but there are many other components such as proteins and mRNAs. Furthermore, several cytokines and growth factors have been reported in MSCs, such as TGFβ, TNFα, IFNγ, IL-4, and IL-10 [6]. Further studies are required to clarify the alteration of components in cADSC-EVs with or without priming and using other stimulation methods.
MSCs suppress T cell proliferation [38], B cell activity [39], and NK cell proliferation [40] and interfere with the differentiation, maturation, and function of dendritic cells [41]. The purpose of this study was to investigate the immunomodulatory capacity of cADSC-EVs for T cells. In this study, PBMCs were stimulated with ConA. The immunomodulatory effects exerted by MSC-EVs on activated T cells remain a widely discussed topic. Primed and naive human MSC-EVs cocultured with PBMCs suppress the proliferation of T cells but do not affect that of B and NK cells [42]. Other studies have shown that MSC-EVs inhibit the proliferation of NK and B cells, but their effects on the proliferation of T cells remain unclear [32,43]. Our results demonstrated that cADSC-EVs suppressed the proliferation of PBMCs and CD4 + T cells and enhanced apoptosis of PBMCs. It has been demonstrated that MSC-EVs carry various active molecules that may contribute to the MSC-EV capacity to inhibit T cell proliferation and activation and induce T cell apoptosis [4]. However, the mechanisms of suppressive proliferation and induced apoptosis in T cells by MSC-EVs remain unclear. Table 1: Comparison of CD4 + , CD8 + , CD4 + CD8 + , and CD4 -CD8 -T cell populations after coculture of PBMCs with cADSC-EVs.

Journal of Immunology Research
To clarify the alteration of the immune response of Th cells induced by cADSC-EVs, intracellular cytokines were detected by flow cytometry. Both naive and primed cADSC-EVs increased the ratio of Th2 cells but decreased Th1 cells. Some studies of autoimmune diseases have reported that MSC-EVs drive a shift from Th1 toward Th2 cells and rebalances Th1/Th2 cells by downregulating proinflammatory cytokines TNFα and IFNγ and upregulating anti-inflammatory cytokines IL-10 or IL-4 [36,50]. Moreover, cADSC-EVs inhibited activated T cell differentiation into Th17 cells and promoted differentiation into Treg cells. Such regulation has also been observed in human MSC-EVs [4], but the mechanism is unclear. On the basis of the immune balance effect exerted by cADSC-EVs on Th and Treg cells, our results indicate that cADSC-EVs may act as an ameliorating agent for autoimmune diseases.
In this study, we examined the effects of cADSC-EVs on T cells and focused on CD4 + T cells isolated from PBMCs. cADSC-EVs affected CD4 + T cell proliferation, apoptosis, and differentiation, but CD4 + T cells that took up cADSC-EVs were only a few percent of all cells. This result was similar to previous studies of unfractionated PBMCs or purified T cells, and MSC-EVs were almost entirely incorporated by monocytes [32,51]. MSC-EVs also act on monocytes/macrophages [4]. Thus, the influence of T cells was not only a direct effect of cADSC-EVs but also included indirect effects of monocytes affected by cADSC-EVs. Further studies of the interactions between cADSC-EVs, T cells, and monocytes are needed to clarify the effects of cADSC-EVs on the immune response.

Conclusions
Our study shows that cADSC-EVs have an immunoregulatory function by inducing PBMC apoptosis, suppressing the proliferation of PBMCs and stimulated CD4 + T cells as well as the differentiation of CD4 + and CD8 + T cells, and changing Th1/Th2/Treg cell populations in vitro. To evaluate whether cADSC-EVs have beneficial effects on immune diseases and are practical for use in treatments of immune diseases, further study is needed to analyze which components of cADSC-EVs exert the individual immunosuppressive effect and the pathways by which these immunosuppressive effects occur.

Data Availability
The data used to support the findings of this study are available from the corresponding authors upon reasonable request.  Journal of Immunology Research