Immature dendritic cells derived exosomes promotes immune tolerance by regulating T cell differentiation in renal transplantation

Objective: To investigate the mechanism of immature dendritic cells-derived exosomes (imDECs) in the regulation of T cell differentiation and immune tolerance in renal allograft model mice. Results: imDECs significantly improved the percent of survival, relieved inflammatory response, and reduced CD4+T cell infiltration. In addition, imDECs reduced the rejection associated cytokines in allograft mice, and increased the percentage of Foxp3+CD4+T cells in spleen and kidney tissues. imDECs suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells under Th17 polarization condition. Moreover, miR-682 was found to be highly expressed in imDECs which suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells. Luciferase reporter assay showed ROCK2 was a target of miR-682, and ROCK mRNA level was negative correlated with miR-682 mRNA level. Conclusion: miR-682 was highly expressed in imDECs, and imDECs-secreted miR-682 promoted Treg cell differentiation by negatively regulating ROCK2 to promote immune tolerance in renal allograft model mice. Methods: Renal allograft model mice were established, and imDECs or mature dendritic cells-derived exosomes (mDECs) were injected into model mice. Rejection associated cytokines IFN-γ, IL-2, IL-17 levels in plasma were detected by ELISA. IL-17A, Foxp3, miR-682, ROCK2, p-STAT3, p-STAT5 expressions were measured by qRT-PCR or western blot.


INTRODUCTION
Kidney transplantation is the most effective treatment for patients with end-stage renal disease (ESRD) [1]. However, it still has challenges such as long-term survival of the recipient and adverse effects of immunosuppressive agents, which induced overall suppression of the immune system and increased infection rate and the incidence of tumor [2,3]. Regulatory T cells (Tregs) inhibit effector responses via multiple mechanisms, including inhibition of proliferation and activation of CD4+ and CD8+ T cells, suppression B cell responses, and regulation of macrophage and natural killer cell functions [4]. It has been demonstrated that induced Tregs could prevent antibody-mediated renal injury and rejection in a mouse model [5]. Indoleamine 2, 3-dioxgenase (IDO) of mesenchymal stem cells (MSCs) enhanced the function and expression of Treg cells (CD4+Foxp3+T cells) and induced allograft tolerance [6]. Hence, Tregs play a vital role in immune tolerance of kidney transplantation.
Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in triggering T cellmediated immunity and tolerance [7]. The potential of DCs to trigger immunity or tolerance depends on the maturation status of DC, in which immature DCs (imDCs) induces T-cell tolerance, whereas mature DCs (mDCs) induces T-cell immunity [8]. It has been found that imDC triggered antigen-specific immunotolerance, prolonged renal allograft survival, and increased the content of Tregs in a rat kidney graft model [9]. Moreover, researchers found that exosomes derived from imDCs combined with Tregs induced immune tolerance in a rat liver allograft model and achieved long-term survival [10]. Exosomes derived from imDCs could suppress collagen-induced arthritis and reduce the severity of arthritis, which suggested that imDCs-derived exosomes (imDECs) suppressed inflammatory and autoimmune responses [11]. imDECs could also increase Tregs percentage and Foxp3 mRNA expression in spleen, and prolong the intestinal allograft survival and induce transplant tolerance [12]. Therefore, imDECs could play critical roles in Treg cell differentiation and induction of immune tolerance. miRNAs exert a post-transcriptional regulatory action on through binding to the 3'-untranslated region (UTR) of the target genes in various biological processes. Studies point out that miRNAs play vital roles in immune responses and Treg cell differentiation [13,14]. However, the role of exosomes-derived miRNAs in the regulation of immune tolerance in renal allograft survival is still not known. According to the online information of GSE33179, there are remarkable differences in miRNA expressions between imDECs and mDECs, and miR-682 is one of them [15]. Besides, researchers have shown that miR-682 is involved in the NF-κB activation induced inflammatory reaction in intestinal epithelial cells (IECs), and miR-682 overexpression-treated mice has lower TNF-α protein expression in IECs from ischemia-reperfusion mice than sham mice [16], indicating miR-682 is an immunerelated miRNA.
Rho-associated kinase 2 (ROCK2) is a member of Rho kinase family that regulates the production of IL-17 and IL-21 and plays an important role in autoimmunity [17]. Recent studies have found that ROCK2 plays an important role in T cell differentiation [18,19]. ROCK2 promotes Th17 cell differentiation and suppresses Treg differentiation, and abnormal activation of ROCK2 may be involved in the development of autoimmune diseases [17]. KD025 (ROCK2 selective inhibitor) promotes Treg function, shifts Th17/Treg balance, and reduces proinflammatory cytokine secretion (IL-17, IL-21, RORγt) in T Cells [18]. Inhibition of ROCK2 remarkably decreases the percentage of IFN-γ+CD4+T cells and IL-17A+CD4+T cells, and increases the percentage of Foxp3+CD4+T cells in inflammatory bowel disease [19]. It has been reported that STAT3 activation promotes Th17 cell differentiation, and STAT5 activation promotes Treg differentiation. ROCK2 inhibitor significantly reduces STAT3 activation and increases STAT5 activation in peripheral blood mononuclear cells [20]. So, ROCK2 may regulate Th17/Treg balance through regulating STAT3 and STAT5 activation. Moreover, bioinformatics database predicted that ROCK2 was a candidate target of miR-682. Therefore, we speculated that imDECs-secreted miR-685 decreased ROCK2 expression in CD4+T cells, thereby inducing Treg cell differentiation and immune tolerance in kidney transplantation.

RESULTS imDEC relieved acute rejection after kidney transplantation
As shown in Figure 1A, the percent of survival in isograft mice was significantly higher than the allograft mice. The injection of imDECs significantly improved the percent of survival, whereas the injection of mDEC didn't significantly change the percent of survival ( Figure 1A). The median survival time of allograft group, allograft+mDEC group, and allograft+imDEC group was 10 d, 12 d, and 16 d ( Figure 1A). H&E staining of kidney tissue showed that the inflammatory response was relieved in allograft+imDEC group compared with allograft group ( Figure 1B). In addition, CD4 staining of kidney tissue showed that CD4+T cell infiltration was significantly reduced in allograft+imDEC group ( Figure  1C). The percentage of CD4+ area was significantly decreased in allograft+imDEC group than allograft or allograft+mDEC group ( Figure 1C). These findings indicated that the injection of imDEC relieved acute rejection after kidney transplantation.

imDEC reduced the production of cytokines
Cytokines are important mediators in the induction of immune response in kidney transplantation. Herein, the levels of rejection associated cytokines in the grafts of recipients were measured. We found renal function indicator serum Scr, and rejection associated cytokines IFN-γ, IL-2, IL-17 levels in plasma were significantly upregulated in allograft mice than that of isograft mice ( Figure 2A). And the injection of imDEC significantly reduced the rejection associated cytokines in allograft mice ( Figure 2A). Compared with allograft group, the percentage of Foxp3+CD4+T cells in spleen and kidney tissues was significantly higher in allograft+imDEC group, whereas there was no significant change in the percentage of Foxp3+CD4+T cells between allograft+mDEC group and allograft group ( Figure 2B). According to the immunofluorescence images, the expression of ROCK2 in allograft group was higher than isograft group, and the expression of ROCK2 in allograft+imDEC group was reduced than allograft group ( Figure 2C). Next, we detected the ROCK2, STAT3, STAT5 expressions in the spleen. The expression of ROCK2 and p-STAT3 were significantly up-regulated in CD4+T cells from spleen of allograft mice, whereas p-STAT5 expression was significantly down-regulated in CD4+T cells from spleen of allograft mice ( Figure 2D). The injection of imDEC could decrease the expression of ROCK2 and p-STAT3, and increase the expression of p-STAT5 ( Figure 2D).

imDEC suppressed the IL17+CD4+T cells and promoted the Foxp3+CD4+T cells
As shown in Figure 3A and 3B, imDECs and mDECs from imDCs and mDCs were determined by transmission electron microscope or western blot. Calnexin was detected in cell lysis of imDCs and mDCs, whereas it was not detected in exo lysis of imDCs and mDCs ( Figure 3B). In addition, exosome markers CD63, Alix and TSG101 were found in in exo lysis of imDCs and mDCs, whereas they were not found in cell lysis of imDCs and mDCs ( Figure 3B). Next, we determined the effect of imDEC on the differentiation of IL17+CD4+T cells and Foxp3+CD4+T cells, and primary CD4+T cells were collected from the spleen. Under Th17 polarization condition, imDEC suppressed the percentage of IL17+CD4+T cells and IFN-γ+CD4+T cells, and promoted the percentage of Foxp3+CD4+T cells ( Figure 3C). Besides, imDEC promoted the mRNA transcription of Foxp3 and inhibited the mRNA transcription of IL-17A under Th17 polarization condition for 72 h ( Figure 3D). imDEC also promoted the expression of p-STAT5, and inhibited the expression of p-STAT3 and ROCK2 ( Figure 3E and 3F).

ROCK2 was a target of miR-682
The targeted mRNAs of miR-682 were analyzed by three online bioinformatics database (Targetscan, miRDB, and microT-CDS), and there were 37 common candidates in the three database using Venn diagram analysis for evaluation based on R language ( Figure 5A). ROCK was one of these mRNAs, and the binding sites between miR-682 and ROCK2 3′UTR were shown in Figure 5B. Luciferase reporter assay showed that miR-682 mimic significantly suppressed luciferase activity of ROCK2 WT, indicating that miR-682 targetedly regulated ROCK2 ( Figure 5B). T cells were collected from the spleen in allograft group, allograft+imDEC group and allograft+mDEC group to detect the correlation of miR-682 and ROCK2 mRNA. We found ROCK was negative correlated with miR-682, which further proved the negative role of miR-682 on ROCK2 ( Figure 5C).

miR-682 regulated T cell differentiation via ROCK2 under Th17 polarization condition
Under Th17 polarization stimulation, si-ROCK significantly suppressed the expression of ROCK2.
KD025 (ROCK2 selective inhibitor) and small interference RNA targeting ROCK2 (si-ROCK2) significantly decreased the expression of p-STAT3 and increased the expression of p-STAT5 ( Figure 6A). Moreover, KD025 and si-ROCK2 significantly decreased IL-17 secretion and mRNA level, and increased Foxp3 mRNA level ( Figure 6B and 6C). miR-682 mimic downregulated the expression of ROCK2 and p-STAT3, inhibited IL-17 secretion, up-regulated the expression of p-STAT5, and promoted the percentage of Foxp3+CD4+T cells ( Figure 6D-6G). And overexpression of ROCK2 could reverse the effect of miR-682 on the regulation of T cell differentiation ( Figure 6D-6G).

imDEC regulated acute rejection after kidney transplantation through miR-682
To demonstrate the effect of imDEC-derived miR-682 on survival rate, 10 μg control-sponge-imDEC, miR-682-sponge-imDEC or miR-682 mimic was injected into the allograft model mice via the tail vein 24 hours before and after transplantation (ten mice in each group). We found that the survival rate in miR-682sponge-imDEC group was lower than control-sponge-imDEC group, and the survival rate in miR-682 mimic group and control-sponge-imDEC group was higher than NC group ( Figure 7A). H&E and CD4 staining showed that the remission effect of miR-682sponge-imDEC on renal inflammation indicating that imDEC could regulate acute rejection after kidney transplantation through miR-682 ( Figure 7B). In addition, serum IL-2, IL-17 and IFN-γ levels were significantly reduced in miR-682 mimic group and control-sponge-imDEC group, whereas miR-682sponge-imDEC increased serum IL-2, IL-17 and IFN-γ levels compared with control-sponge-imDEC ( Figure  7C). We further extracted the proteins of T cells from the spleen and allograft kidney tissues, and detected the ROCK2, IL-17 and Foxp3 expressions. The effect of miR-682-sponge-imDEC on inhibiting IL-17 and promoting Foxp3 was reduced ( Figure 7D-7G).

DISCUSSION
Studies have reported that DC-derived exosome could play important roles in immune regulation, such as binding Toll-like-receptor ligands to innate-immunity effector cells, or inducing the specific anti-tumor immune response [26,27]. imDC is a status of DC that induces Tcell tolerance, which can prolong the survival of renal allograft and increase Treg numbers in a rat kidney graft model [9]. Immune tolerance and long-term survival were also achieved in a rat liver allograft model after combined treatment of imDECs and Tregs [10]. In addition, imDECs increased the percentage of Tregs and Foxp3 mRNA expression in the spleen and induced transplant tolerance [12]. In this study, we purified exosomes from mDC and imDC and injected them into renal allograft model mice. We determined imDECs remarkably increased survival rate, decreased rejection associated cytokines IFN-γ, IL-2, IL-17 levels, promoted the percentage of Foxp3+CD4+T cells in allograft mice, miRNAs are emerging as vital modulators of gene expression in the immune response which control the differentiation and function of Treg cells [28,29].
Evidence also showed that DECs could secrete miRNAs (miR-155 and miR-146a) to regulate inflammatory response to LPS [30]. So the DECs secreted miRNAs might be involved in the regulation of Tregs differentiation and function. In this study, we detected the expressions of miR-682, miR-649, miR-805 and miR-467f in imDEC and mDEC, and found that miR-682 and miR-805 showed abnormal expressions in imDEC and mDEC. After miR-682 and miR-805 overexpression or blockade of exosomal miR-682 and miR-805, we further found only the abnormal expression of miR-682 changed the IL-17A and Foxp3 mRNA levels, which indicated that only miR-682 might be involved in the regulation of Tregs differentiation. So, we focused on the role of miR-682 in kidney transplantation, and found miR-682 was highly expressed in imDECs. exosomal miR-682 overexpression decreased IL-17A mRNA level and increased Foxp3 mRNA level, and the blockade of exosomal miR-682 achieved the opposite effect. Herein, we first proved that imDECs-secreted miR-682 could promote Tregs differentiation to induce immune tolerance. In addition, we further determined the underlying mechanism of exosomal miR-682 in the regulation of Tregs differentiation after kidney transplantation.
Recently, several studies have proved that ROCK2 inhibition promoted Tregs differentiation [18,29]. In this study, we demonstrated that silencing ROCK2 reduced IL-17 level and increased Foxp3 level, which was consistent with previous reports [18,29]. Luciferase reporter assay showed miR-682 could target to 3' UTR of ROCK2, and ROCK2 mRNA level was negatively correlated with miR-682 mRNA level. Moreover, we found exosomal miR-682 suppressed IL-17 secretion and p-STAT3, and up-regulated p-STAT5 through regulating ROCK2. All these findings indicated imDECs-secreted miR-682 induced STAT3 and STAT5-dependent Treg cell differentiation by inhibiting ROCK2 in CD4+ T cells.
In vitro experiments showed exosomal miR-682 blockade reduced the remission effect of imDEC on renal inflammation and promotion effect of Treg differentiation, indicating that imDEC regulated acute rejection after kidney transplantation through secreting miR-682.
In conclusion, this study demonstrated that miR-682 was highly expressed in imDECs, and imDECs-secreted miR-682 promoted Treg cell differentiation by negatively regulating ROCK2 in CD4+ T cells. In vitro experiments proved that the blockade of exosomal miR-682 reduced the survival rate, increased the secretion of rejection associated cytokines and IL-17 expression, and decreased Foxp3 expression, which indicated that imDECs-secreted miR-682 promoted immune tolerance in renal allograft model mice.

Establishment of renal isograft and allograft model mice
All animal experiments were approved by Ethic Committee of The First Affiliated Hospital of Zhengzhou University. For the establishment of mice allograft kidney transplantation, male C57BL/6 mice were used as kidney donors and female BALB/c mice were used as recipients under isoflurane inhalation anesthesia (Sinopharm Chemical Reagent, China). For isogenic kidney transplantation, BALB/c mice were used as kidney donors and recipients under isoflurane inhalation anesthesia and subcutaneously injected the butorphanol (1mg/kg). In donor mice, the left kidney, the aorta and inferior vena cava and the ureter were removed. Then, in recipient mice, the left kidney was removed, and the allograft was placed into the left lower abdomen. The vessels of the allograft were anastomosed to the aorta and inferior vena cava of recipient, and the ureter was implanted into the bladder. The time for cold ischemia and warm ischemia time was 60 and 20 min, respectively. Mice were divided into isograft group (n=10), allograft group (n=10), allograft+mDEC group (n=10) and allograft+imDEC group (n=10). In isograft group, BALB/c mice were used as kidney donors and recipients. In allograft group, C57BL6 mice were used as kidney donors and BALB/c mice were used as recipients. Contralateral nephrectomy was performed at the time of kidney transplantation, so the mice survived only on transplanted kidneys. In allograft+mDEC group, 10 μg mDECs (exosome from mature DC) were injected into the recipient mice via the tail vein 24 hours before and after transplantation. In allograft+imDEC group, 10 μg imDECs (exosome from immature DC) were injected into the recipient mice via the tail vein 24 hours before and after transplantation. All the recipient mice were single-pass injected with Ampicillin. Mice (seven mice in each group) were sacrificed six days after transplantation, and kidneys were collected for hematoxylin and eosin (H&E) and CD4 staining. Blood was also collected from inferior vena cava on the 6th day after transplantation for the measurement of renal function.
To observe the effect of exosome-derived miR-682 on acute rejection after kidney transplantation, controlsponge-imDEC, miR-682-sponge-imDEC, or miR-682 mimic (10 μg) was injected into the allograft model mice via the tail vein 24 hours before and after transplantation (ten mice in each group). Allograft mice (n=10) were used as negative control (NC). Mice (five mice in each group) were sacrificed six days after transplantation, and kidneys and spleens were collected for H&E and CD4 staining. Blood was also collected from inferior vena cava on the 6th day after transplantation for the measurement of renal function.

H&E and CD4 staining
Kidney tissues were fixed in 10% neutral buffered formalin for 48 h, dehydrated in ethanol, embedded in paraffin, and cut into 5 μm slices. Slices were prepared and stained with H&E, and observed under a light microscope (Olympus, Japan). CD4 (1:200; Abcam, USA) was stained according to the manufacturer's instructions.

Renal function analysis
Serum creatinine (SCr) level was measured by enzymatic-colorimetric method using an automatic biochemistry analyzer (BS-480; Mindray, China). Plasma IFN-γ, IL-2 and IL-17 levels were detected by ELISA assay (Signalway antibody, USA) according to the manufacturer's instructions.

Generation and culture of immature DCs
Immature DCs were generated from bone marrow flushed from the femurs and tibias of recipient mice as previously reported with 20ng/ml recombinant mouse IL-4 (eBioscience, USA) and 30ng/ml recombinant mouse GM-CSF (eBioscience, USA) [21]. Cells were cultured from day 8 to 10 without cytokines. Then, non-adherent and adherent cells were harvested at day 10. After removal of adherent cells, cells were identified as imDCs, and then cultured from day 10 to 12 with 200 ng/mL LPS (Sigma, USA) to obtain mDCs.

Isolation of exosomes
mDCs and imDCs were cultured for 3 days, and 1×10 6 DCs were collected in the DMEM-Low Glucose medium (Gibco, USA). The medium containing DCs was centrifuged at 300×g, 1200×g and 10,000×g for 10 min, 20 min, and 30 min to remove large fragments. Then, the exosome pellet was washed in saline, passed through a 0.22 μm filter, centrifuged at 100,000×g for 60 min. After determined with CD63 and CD81, the pellet was resuspended in 2 ml PBS. About 15 μg exosomes were harvested from 1×10 7 mDCs or imDCs. The amount of exosomes (imDECs, exosome from immature DC; mDECs, exosome from mature DC) was evaluated based on the amount of protein using Pierce™ BCA Protein Assay Kit (Thermo Scientific, USA).

Electron microscopy
Exosomes were diluted ten times, loaded on a Formvar/carbon coated grid, negatively stained with 10 μl of neutral 1% aqueous phosphotungstic acid, and viewed using an electron microscope (CM120; Philips, Netherlands).

Isolation of CD4+T cells
Spleen was dissociated through a 40 μm nylon mesh and then cultivated in DMEM medium containing 10% fetal bovine serum (FBS; Gibco, USA). MagniSort™ Mouse CD4 T cell Enrichment Kit (Invitrogen, USA) was used to isolate CD4+T cells according to the manufacturer's instructions.

Flow cytometer
CD4+T cells isolated from spleen and kidney were resuspended at 3×10 6 cells/ml. Cells were detected by BD FACSCanto II flow cytometry (BD, USA) and analyzed using CELLQuest software. eBioscience™ Mouse Regulatory T Cell Staining Kit (Invitrogen, USA) was used for the observation of CD4+Foxp3+ T cells. Anti-IL-17A was purchased from eBioscience (ThermoFisher Scientific, USA) for the identification of IL-17A+CD4+T cells.

Western blot
Kidney or spleen tissues and CD4+T cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Thermo Scientific, USA). Protein was separated by SDSpolyacrylamide gel electrophoresis (PAGE), then

Statistical analysis
The data corresponded to mean±standard errors (SE), and were analyzed by SPSS software (version 18.0). Results were analyzed using t test or one-way analysis of variance (ANOVA), with p<0.05 considered statistically significant.