In vitro naive CD4 + T cells differentiation upon treatment with miR-29b-loaded exosomes from Mesenchymal Stem Cells

Background: Gene regulation by microRNA is central in T cell differentiation processes. Here, we investigate miR-29b roles in the reprogramming of T cell differentiation, which can be a promising therapeutic avenue for various types of in�ammatory disorders such as rheumatoid arthritis and multiple sclerosis. Methods and Results: Adipose Mesenchymal Stem Cell derived exosomes (AMSC-Exo) enriched with miR-29b were delivered into naive CD4 + T cells (nCD4 + ) cells. The expression level of some important transcription factors including ROR γ t, GATA3, T-bet, and Foxp3 was determined by quantitative Real-Time PCR (qPCR). Moreover, �ow cytometry and Enzyme-linked Immunosorbent Assay (ELISA) was respectively used to measure the frequency of T regulatory cells and the levels of cytokines production (IL-17, IL-4, IFN-γ , and TGF-β ). This study indicates that the transfection of miR-29b mimics into T lymphocytes through AMSC-Exo can alter the CD4 + T cells differentiation into other types of T cells. Conclusions: In conclusion, AMSC-Exo-based delivery of miR-29b can be considered as a new fascinating avenue for T cell differentiation inhibition and the future treatment of several in�ammatory disorders.


Introduction
CD4 + lymphocytes are key players in the adaptive immunity [1].Based on cytokines and cellular environment, the naive CD4 + T cells (nCD4 + ) activation leads to differentiation into effector and regulatory subsets of T helper (Th) cells, which play central roles in ghting against tumor cells and infectious diseases [2].Therefore, the appropriate regulation of Th cell differentiation and programming is an integral part of the immune system response against pathogens.Disruption of the regulation of Th cell differentiation can cause numerous autoimmune diseases [3,4].
It has been demonstrated that cytokines, costimulatory molecules, and antigens are important contributors in determining the differentiation fate of nCD4 + cells [5].Although using cytokines is a common strategy for in vitro differentiation of nCD4 + cells as they regulate the expression of transcription factors, several recent studies elucidated that microRNA-based systems can be e ciently used as a robust strategy to control CD4 + T cells differentiation because they control the expression of not only lineage-speci c transcription factors but also cytokines [6][7][8].
miR29b is a vital contributor in nCD4 + cell fate decision.It has been shown that miR-29 family can inhibit nCD4 + cells differentiation into Th1 cells by suppressing of IFNγ, T-bet, and Eomesodermin expression [9].Further studies suggest that the suppression of miR-29a/b expression upon intracellular bacterial infections increases the production of IFNγ by initiating a positive feedback loop, which increases cell resistance to infections [10].
Several delivery approaches have been developed for safe and e cient delivery of miRNAs [11][12][13].
Here, we have investigated the roles of miR-29b-enriched Adipose Mesenchymal Stem Cell derived exosomes (AMSC-Exo) in nCD4 + cell fate decision.Hence, the miR-29b-enriched exosomes were delivered into nCD4 + cells, and then their differentiation was studied by measuring the pro le of gene expression, cytokine production, and regulatory T cells frequency.

Mice
Female mice (6-8 weeks) of the C57BL/6 strain were purchased from the Royan Institute (Tehran, Iran) and were housed and manipulated in accordance with the ethical committee's guidelines of Shahid Beheshti University of Medical Sciences (66001030).Three to 5 mice were used in each experimental group.

AMSCs isolation and culture from adipose tissue
Liposuction aspirates were obtained from abdomen of ve different donors (healthy adult aged 25-40 years) with written informed consent.All procedures were performed in accordance with the ethical committee guidelines of Shahid Beheshti University of Medical Sciences (66001030).A previously described collagenase digestion method was used to AMSCs isolation [18].In brief, 0.075% type I collagenase (Gibco, Grand Island, NY) was used for adipose tissue dissociation at 37°C for 20 min.After collagenase neutralization by FBS, the samples were centrifuged and the pellet was cultured at 37°C in DMEM/F12 (Dulbecco modi ed Eagle medium) plus 10% Fetal Bovine Serum (FBS) (Gibco, Rockville, MD) and 100 U/ml streptomycin/penicillin in a 5% CO2 humidi ed incubator.Next day nonadherent cells were removed and medium was changed.The half of media was changed every 3 days and adherent cells were grown to 80% con uency.The cells were used at passage 3 for the following experiments.
Besides, the AMSCs were also identi ed by the induction of mesenchymal lineages differentiation via osteogenic and adipogenic differentiation media as described previously [18].

Exosomes puri cation from AMSCs conditioned media
To exosomes isolation, rst, AMSCs were adapted to serum-free medium [19].AMSCs were cultured in descending concentration of FBS in presences of 1% insulin/transferrin/selenium (Invitrogen, Carlsbad, CA, USA).After reaching 0% FBS, conditioned media was collected every 3 days, and exosomes were isolated using an isolation kit from Cibbiotech (Tehran, Iran) according to the instructions provided with the kit.In short, supernatants were ltered by a 0.22 µm lter and centrifuged for 20 min at 3000g to remove cell debris.Supernatants were then incubated with exosome precipitation buffer (5:1 ratio) overnight at 4°C to precipitate exosomes.The samples were centrifuged at 4°C for 40 min at 3000g and pellets were resuspended in 200µL PBS and stored at -70°C.The protein content of isolated exosomes was calculated using a bicinchoninic acid protein assay kit (Parstous, Mashad, Iran).

Scanning electron microscopy examination
The shape and size of the puri ed exosomes were examined with Scanning Electron Microscopy (SEM).To do this, Specimens were prepared by xing 10µL exosomes with 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO).After dehydration in increasing concentrations of ethanol, they were left to dry at room temperature.Finally, the samples were evaluated under SEM (MIRA3 TESCAN, Brno, Czech Republic) at 30 kV following gold-palladium sputtering.

Transmission electron microscopy examination
For morphology analysis of puri ed exosomes from AMSCs, after xing with 2.5% glutaraldehyde and dehydration in ascending sequence of ethanol, the samples were further proceed for epoxy resin (PolySciences, Warrington, PA, USA) embedding in accordance with the manufacturer's instruction.
Ultrathin sections (a thickness of maximum 70 nm) were loaded onto formvar coated copper grids, contrasted with uranyl acetate, and visualized by Zeiss-EM10C (Germany) transmission electron microscope (TEM).

Exosomes size distribution analysis
Dynamic light scattering (DLS) measured the size distribution and average size of small particles.Exosomes obtained from the supernatants of AMSCs were diluted 1:10 in PBS and examined by Malvern Zetasizer (Worcestershire, UK).

Exosomes Immunophenotyping
To further characterization, exosomes immunophenotyping was performed using antibodies against CD63 and CD81 (important markers of exosomes).The data were collected via ow cytometry.The relative uorescence was calculated by Flowjo (Tree Star, Inc., Ashland, OR).

Splenocytes isolation and nCD4 cell puri cation
Spleens were removed from mice and subjected to mechanically disruption and homogenization.The homogenates were centrifuged for 5 min in 400g at 4°C.Then, erythrocytes were lysed at room temperature in ACK lysis buffer for 4 min, followed by additional centrifugation.The cell pellets were resuspended in RPMI-1640 medium (Life Technologies, Carlsbad, CA) plus 5% FBS and the count of splenocytes was measured by a Neubauer counting chamber.Splenocytes were subjected to magneticactivated cell sorting for nCD4 + cells isolation.nCD4 + cells were puri ed from splenocytes by Miltenyi Biotech naive CD4 + T cell isolation kit II (Bergisch Gladbach, Germany) according to the instructions provided with the kit.First, non-CD4 + T cells were depleted from splenocytes via a cocktail of biotinconjugated antibodies.Then, positive selection was used to isolate CD62L + cells.The CD4 + CD62L + T cells was analyzed by ow cytometry and the purity was 86% (Fig. 3a).

PKH67 labeling of exosomes
For exosome uptake examination, puri ed exosomes (200 µg protein) were uorescently labeled by PKH67 (Sigma-Aldrich, St. Louis, MO).Exosomes were resuspended in 250 µL Diluent C and 2 µL of PKH67 dye was also diluted in the same amount of Diluent C and then they were mixed gently.After 5 min incubation at room temperature, the staining reaction was stopped by 250 µL of FBS and exosomes re-isolated via isolation kit to get rid of the excess dyes.Labeled exosomes were co-incubated with nCD4 + cells for 24 hours, and then the cells were washed with PBS, xed with 2.5% glutaraldehyde, and observed using a Leica TCS SPE (Leica, Mannheim, Germany) confocal microscope.For the negative control, above labeling procedure was performed on a mixture without exosomes.

ELISA of cytokines
The ELISA assay was used to determine IL-17, IL-4, IFN-γ, and TGF-β concentrations.After 5 days of cell culture, cell-free supernatants were collected and the cytokine were quanti ed via R&D Systems ELISA kit (Minneapolis, MN, USA) following the manufacturer's protocol.Cells were harvested, washed, and used for qPCR and ow cytometry analysis (see below).

Real-time PCR assay
To miR-29b and transcription factors expression analysis 5 days following nCD4 + cells co-culturing with exosomes, TRIzol Reagent (Life Technologies, Grand Island, NY) was used for total RNA isolation.The RNA concentration was determined via a NanoDrop spectrophotometer and then reverse transcribed to cDNA.RNA quanti cation was performed using a standard SYBR Green I reaction mix (Ampliqon, Odense, Denmark) on an ABI StepOne™ (Applied Biosciences, Foster City, CA).U47 and β-2M were used as background control [13].

Analysis of regulatory T cells frequency
The frequency of regulatory T cells was determined by staining with Miltenyi Biotech CD4/CD25/Foxp3 Treg detection kit (Germany) in accordance with the manufacturer's instructions.Single-cell suspensions were incubated rst with FITC-conjugated-CD4 Abs and APC-conjugated-CD25 Abs at 4°C in dark for 10 min.These cells were subsequently xed and permeabilized to intracellular transcription factor staining by PE-conjugated-Foxp3 Abs.Data was acquired using FACSCalibur and analysis was performed by Flowjo.

Statistical data analysis
All data are the mean of 3 independent experiments with standard deviation (SD) in triplicate.One-way analysis of variance in GraphPad Prism 7.0 (GraphPad, San Diego, CA) was used to measure p-values and a p-value less than 0.05 was considered statistically signi cant.

AMSCs characterization
Fibroblast-like stem cells were obtained from adipose tissue of healthy individuals and light microscopy analysis (Fig. 1a) showed plastic adherence cells with spindle-shaped morphology.The differentiation potential of these cells was evaluated by culturing in the appropriate induction media.Three weeks following culture in adipogenic or osteogenic differentiation medium, accumulated triacylglycerol was identi ed by Oil red O staining (Fig. 1b) and calcium depositions were assessed using Alizarin red S (Fig. 1c).Finally, immunophenotyping of isolated cells was displayed as histograms in Figs.1d and showed that 73.5%, 93.3%, and 76.4% of the cells were respectively expressed CD105, CD90, and CD73, but they had a little expression of CD45, CD34, and CD14.These data con rmed the purity and identity of AMSCs.

Identi cation of exosomes
The particles secreted from AMSCs were characterized by DLS, TEM, and SEM.The morphology examination by SEM showed nano-sized cup-shaped vesicles (Fig. 2a).Furthermore, the assessment of puri ed particles with TEM showed typical vesicular structures (Fig. 2b).As shown in Fig. 2c, DLS analysis also revealed that these particles ranged almost from 30 to 90 nm in diameters (average size 48 nm).Finally, immunophenotyping showed isolated vesicles were positive for CD63 and CD81 exosome markers (Fig. 2d).All these data indicated that the vesicles obtained from the conditional media have the characteristics of exosomes.

Exosomes were internalized by nCD4 cells and miRNA-29b were delivered into the cells
To evaluate the internalization capacity of exosomes by nCD4 + cells, exosomes were stained by PKH67 dye and then co-cultured with nCD4 + cells.24 hours later, the uptake of the uorescent exosomes by nCD4 + cells was visualized under uorescence and also confocal microscopy.As shown in Fig. 3c, uorescence microscopy images indicated that almost all nCD4 + cells revealed a green signal, suggesting exosomes have been e ciently taken up by nCD4 + cells.These ndings were validated by data from confocal microscopy that showed exosomes internalization and accumulation in the cytoplasm, especially in the perinuclear region (Fig. 3d).PKH26 micelles were not detected in control negative group, which showed they could not develop artefacts.
In addition, following the exosomes uptake, miR-29b levels increased in nCD4 + cells and con rmed miR-29b delivery to nCD4 + cells.qPCR analysis showed that 24 hours and 5 days upon co-culturing a signi cant increase (p ≤ 0.0001) in the expression level of miR-29b was observed The cytokine production change upon miR-29b loaded exosomes treatment Next, we investigate the underlying mechanism of nCD4 + cells differentiation.After ve days of treatment with miR-29b-enriched exosomes, the secretion of IL-17, IL-4, IFN-γ, and TGF-β from T cells during activation and differentiation was assessed.miR-29b-enriched exosomes can signi cantly suppress the production of IL-17 and IL-4 in comparison with other groups (p ≤ 0.05).In addition, no change was seen in the IFN-γ expression in miR-29b-enriched exosomes compared to TGF-β treated and control cells.Moreover, no induction was detected in the expression of TGF-β in miR-29b-enriched exosomes groups (Fig. 4).

Tbx21 and RORγt expression inhibition in nCD4 cells following miR-29b enriched exosomes treatment
Next, we explored the regulatory mechanism of miR-29b-enriched exosomes on nCD4 + cells differentiation.nCD4 + cells were treated with miR-29b-enriched exosomes.After 5 days of incubation, total RNA was prepared and the expression of some important transcription factors in the differentiation of T cells such as Foxp3 (Treg), Tbx21 (Th1), GATA3 (Th2), and RORγt (Th17) was assessed by qPCR assay.Figure 5 shows the expression of Tbx21 was considerably decreased in exosomes and miR-29benriched exosomes compared to the control group (p ≤ 0.05 and p ≤ 0.001, respectively).Similarly, the Tbx21 expression level was also reduced in TGF-β1 treated cells (p ≤ 0.001).Both miR-29b and TGF-β1 inhibit the differentiation of nCD4 + cells to Th1 cells.Our results also showed a decrease in the expression of GATA3 in miR-29b-loaded exosomes compared to that of exosomes treated cells (p ≤ 0.01).Interestingly, the RORγt expression, a critical transcription factor in Th17 cell differentiation, was signi cantly decreased in miR-29-enriched exosomes (p ≤ 0.01).Furthermore, an increase in the Treg transcription factor, Foxp3, expression was detected in the exosomes group (p ≤ 0.05) in comparison with the control group.TGF-β1 increased both RORγt and Foxp3 expression compared to other groups (p ≤ 0.0001).

Increased CD4CD25Foxp3 Cells frequency upon miR-29b loaded exosomes treatment
Flow cytometry was used to delineate the role of miR-29b on the frequency of CD4 + CD25 + Foxp3 + T Cells.

Discussion
According to the previous studies, miR-29b has a signi cant role in CD4 + T cell fate regulation and it has immunomodulatory effects [9,10,[20][21][22].Due to poor immunogenicity, low toxicity, and high packaging capacity, herein we used AMSC-Exo as promising delivery systems for miRNAs.This study revealed the ability of AMSC-Exo loaded with miR-29b to regulate the T cells differentiation to different type of helper T cells, via not only suppression of IL-4 and IL-17 but also inhibition of several transcription factors including Tbx21 and RORγt.
Previous studies demonstrated that the regulation of the IFN-γ production has a key role in proper immune responses and has been regarded as a central contributor in setting the balance between regulatory and helper T cells during in ammatory immune responses and cancer development [23].In addition, Steiner and his co-workers established that miR-29a and miR-29b could be vital inhibitors of the Th1 cell fate by using DGCR8-/-miR-de cient CD4 + T cells [9].In agreement with these observations, we con rmed that miR-29b-loaded AMSCs exosomes could decrease the expression of the Tbx21, a transcription factor that plays signi cant role in the process of Th1 cell differentiation, in nCD4 + cells.Some reports revealed that MSCs-derived exosomes are the main contributors in the conversion of Th1 into Th2 cells by changing the expression of several cytokines [24,25].Furthermore, our results concur well with the fact that Tbx21 could suppress T cells differentiation to Th2 [26].As a result, we believe that the suppression of Th1 differentiation is highly related to miR-29b because the expression of GATA3 was suppressed in the presence of the exosomes enriched with miR-29b compared to the exosomes group.We also observed Tbx21 downregulation following the TGF-β addition, which is in complete agreement with a study by Gorelik and et. al., where they demonstrated the inhibitory role of TGF-β on Tbx21 expression [27].
We also detected an increase in the population of CD4 + CD25 + Foxp3 + Treg cells while the expression of Foxp3 was increased in the presence of miR-29b-enriched AMSCs derived exosomes and TGF-β.This lends support to studies showed that Foxp3 induction is essential for e cient Treg differentiation of nCD4 + cells [28].
Simultaneously, we revealed a decrease in the expression of the RORγt following the treatment of nCD4 + cells with miR-29b-enriched AMSCs derived exosomes, which leads to a high miR-29b-mediated Th17 differentiation inhibition.In addition, the production of IL-17, which is one of the most important cytokines in Th17 cells, was reduced, con rming inhibition of nCD4 + cells differentiation into the Th17 cells.Our experiments are in line with previous results by Xuan who showed downregulation of IL-17 and decreased quantity in Th17 cells upon miR-29 mimic delivery into CD4 + cells [29].One mechanism for RORγt inhibition by miR-29 could be the inhibitory effect of miR-29 on L-12p40 and IL-23p19, which leads to decreased expression of IL-23 [30].
Our research showed the impacts of miR-29b carried by AMSC-Exo on CD4 + T cell subsets differentiation.These ndings suggest that miR-29b is a key player in both inhibition of Th1 and Th17 differentiation and induction of Treg differentiation from nCD4 + cells.However, further studies seem to be essential to reveal the molecular pathway for DIFFERENTIATION regulation BY miR-29b.Moreover, clinical studies should be also addressed in future researches.

Conclusion
In this study, we established that miR-29b-enriched AMSCs derived exosomes are important contributors in the inhibition of nCD4 + cells differentiation to Th1, and Th17.These ndings will greatly lead us to develop novel strategies for the treatment of various in ammatory diseases like autoimmune diseases.