MiR-21^flox/flox mice were on the C57BL/6 background and purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China). Foxp3^Cre-YFP transgenic mice were kindly provided by Dr. Bin Li from Shanghai Jiao Tong University, P. R. China. Foxp3^Cre-YFP mice were crossed with miR-21^flox/flox mice to produce mice with Treg-specific deletion of miR-21 (Foxp3^Cre-YFPmiR-21^f/f). 6 to 8-week old mice in the C57BL/6 background were purchased from Beijing Vital River Laboratory Animal Technology Company Limited. Mice were kept under pathogen-free conditions at the animal core facility of Shandong Eye Institute, Shandong First Medical University &Shandong Academy of Medical Sciences. All experiments were carried out in accordance with the Committee guidelines of Shandong Eye Institute and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

For the induction of EAU in mouse, mice were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg) and then immunized with 400 ug inter-photoreceptor retinoid-binding protein (IRBP)^1-20 (5 mg/ml, GPTHLFQPSLVLDMAKVLLD, purchased from China Peptides) emulsified 1:1 in complete Freund’s adjuvant (Chondrex, USA) with an additional 100 µl mycobacterium tuberculosis H37R (5 mg/ml, BD biosciences, USA). An additional 200 ng bordetella pertussis toxin (Millipore, USA) was intravenously injected immediately after peptide injection. Fundoscopic exam was used to assess the severity of EAU on a scale of 0–4 using previously published criteria (29).

For the induction of EAE in mouse, 8 to 12-week old female mice were injected subcutaneously into both flanks with 100 μg myelin oligodendrocyte glycoprotein (MOG)^35–55 peptide (3 mg/ml, MEVGWYRSPFSRVVHLYRNGK, purchased from China Peptides) dissolved in PBS and emulsified in an equal volume of complete Freund’s adjuvant (Chondrex, USA) supplemented with 5 mg/ml Mycobacterium tuberculosis H37R. Mice were also injected intraperitoneally (i.p.) with 200 ng pertussis toxin (Millipore, USA) on the day of immunization and 48 h later. Clinical assessment of EAE was performed daily after disease induction according to the following criteria: 0, no disease; 1, tail paralysis; 2, hind limb weakness or partial paralysis; 3, complete hind limb paralysis; 4, forelimb and hind limb paralysis; 5, moribund state. Mean clinical scores were calculated by adding the scores of individual mice and dividing by the total number of mice in each group.

For the induction of RA in mouse, 6 to 8-week old male mice were intradermally immunized with 100 μg chicken type II collagen (4 mg/ml, purchased from China Peptides) emulsified in complete Freund’s adjuvant at the base of the tail, followed by a booster immunization with 100 μg chicken type II collagen emulsified in incomplete Freund’s adjuvant. The cumulative score of all four paw scores were calculated on a scale of 0–16, where each paw was scored as follows: 0, normal paw; 1, one toe inflamed and swollen; 2, more than one toe, but not entire paw inflamed and swollen, or mild swelling of entire paw; 3, entire paw inflamed and swollen; 4, very inflamed and swollen or ankylosed paw.

Flow cytometry analyses were performed on freshly isolated cells from mouse thymus, spleen, and mesenteric lymph node (MLN) or cultured cells. Cells were labeled with a combination of the following fluorescence-conjugated mouse mAbs: PE or PerCP-Cy5.5-anti-CD4, APC or PerCP-Cy5.5-anti-CD8, PerCP-Cy5.5-anti-CD25, PE-cy7-anti-GITR, PerCP-Cy5.5-anti-Nrp1, PerCP-Cy5.5-anti-CD44, APC-anti-CD62L, PerCP-Cy5.5-anti-CD11c. For the intracellular staining of Foxp3, RORγT and CTLA4, cells were fixed, permeabilized, and stained with PE or APC-anti-Foxp3, PE-anti-RORγT and APC-anti-CTLA4 per manufacturer’s instructions (Invitrogen, USA). For the intracellular staining of IL-17A, IL-17F and IL-10, cells were stimulated with cell stimulation cocktail (PMA+Ionomycin) plus protein transport inhibitors (eBioscience, USA) for 4 h and then stained with APC-anti-IL-17A, PE or APC-anti-IL-17F, or APC-anti-IL-10 per manufacturer’s instructions (Invitrogen, USA). For the intracellular staining of phospho-Stat3, cells were fixed and permeabilized by transcription factor phospho buffer set, and then stained with Alexa Fluor 647-anti-Stat3 (PY705) antibody (BD Bioscience, USA) per manufacturer’s instructions (BD Bioscience, USA). Stained cells were examined on CytoFLEX flow cytometry system (Beckman Coulter Inc., USA). All antibodies were purchased from BioLegend.

For the isolation of Treg cells (CD4⁺CD8^-YFP⁺), CD4⁺ T cells from spleen were first enriched using EasySep Mouse CD4⁺ T Cell Isolation Kit (STEMCELL Technologies, Canada). Cells were then stained with PE-anti-CD4 and APC-anti-CD8, and CD4⁺CD8^-YFP⁺ Treg cells were sorted by FACSAria III cell sorter (Becton Dickinson, USA). For in vitro Treg suppression of T cell proliferation, naïve CD4⁺ T cells were isolated from the spleen of WT mice using EasySep™ Mouse naïve CD4⁺ T Cell Isolation Kit (STEMCELL Technologies, Canada). Cells were further labeled with 4 μM eFluor-670 (Invitrogen, USA) for 10 min at 37°C followed by washing with ice-cold complete RPMI-1640 media. Sorted Treg cells were mixed with eFluor-670 labeled naïve CD4⁺ T cells (2.5×10⁴/well) at different ratios and seeded in 96-well plate. Mouse T-Activator CD3/CD28 beads (Gibco, USA) were added at a ratio of 1: 1 (beads: naïve CD4⁺ T cells). Dilution of eFluor-670 was analyzed by flow cytometry. For in vitro Treg suppression of dendritic cell activation, bone marrow-derived dendritic cells (BMDCs) were generated from femoral and tibial bones cells as previously described (30). BMDCs (0.5×10⁵/well) were either cultured alone or mixed with sorted Treg cells at a ratio of 1:1 and seeded in 96-well plate. Cells were treated with 2 ug/ml anti-CD3 (ebioscience, USA), 2 ug/ml anti-CD28 (ebioscience, USA), 100 IU/ml IL-2 (PeproTech, USA) and 10 ng/ml LPS (Sigma, USA). After co-culture for 12 h, cells were collected and stained with PerCP-Cy5.5-anti-CD11c, APC-anti-CD86 and APC-anti-CD80 according to manufacturer’s instructions (Invitrogen, USA), and analyzed by flow cytometry.

For cytokine assays, flow sorted Treg cells (CD4⁺CD8^-YFP⁺) from spleen, inguinal lymph node (ILN), or cervical lymph node (CLN) were cultured at a density of 0.2×10⁶/well (96-well plate) in 100 µl of complete RPMI-1640 culture medium (Corning, USA) and stimulated with 2 ug/ml anti-CD3 (ebioscience, USA), 2 ug/ml anti-CD28 (ebioscience, USA) and 100 IU/ml IL-2 (PeproTech, USA) in the presence or absence of 50 ng/ml IL-6 (PeproTech, USA), 20 ng/ml IL-1β (PeproTech, USA), 20 ng/ml IL-23 (PeproTech, USA) or 10 ng/ml IL-12 (PeproTech, USA). After 48 h, the concentrations of IL-17A, IL-17F, IFN-γ, IL-10 and TGF-β in the culture supernatants were determined by quantitative enzyme-linked immunosorbent assay (ELISA) per manufacturer’s instructions (Invitrogen, USA). For the preparation of tissue extract, mouse eye, brain or paw were homogenized in PBS containing complete protease inhibitor mixture (Roche, USA) using TissueLyser II per manufacturer’s instructions (QIAGEN, Germany). The concentrations of IL-17A, IL-17F, IFN-γ, and TNF-α in the tissue extract were determined by ELISA as described above.

Total RNA was isolated using TRIzol reagent following manufacturer’s instructions (Life Technologies, USA). RNA samples were reversely transcribed using the primescript reverse transcription kit (Takara, JPN). The expression of mouse miR-21, IL-17A, IL-17F, IFN-γ, IL-10, TGF-β, CCR-6, Ebi3, Gzmb and Prf1 was determined by quantitative RT-PCR using specific primers and the Applied Biosystems 7500 system. Relative levels of expression were determined using U6 (for miR-21) or GAPDH (for all others) as internal control. The primers for the reverse transcription and detection of miR-21 were supplied by RiboBio (Guangzhou, China). The primers for the detection of all other genes were synthesized by Invitrogen (USA). Primer sequences are as follows (forward and reverse): IL-17A, 5’-CTCCAGAAGGCCCTCAGACTAC-3’ and 5’-AGCTTTCCCTCCGCATTGACACAG-3’; IL-17F, 5’-CTGTTGATGTTGGGACTTGCC -3’ and 5’-TCACAGTGTTATCCTCCAGG -3’; IFN-γ, 5’-TTCTGGCTGTTACTGCCACGG-3’ and 5’-GCCTTGCTGTTGCTGAAGAAG-3’; IL-10, 5’-CTTACTGACTGGCATGAGGATCA-3’ and 5’- CCTGCATTAAGGAGTCGGTTAGC-3’; TGF-β, 5’- CTTCAATACGTCAGACATTCGG-3’ and 5’-AGCCACTCAGGCGTATCAGTG-3’; CCR-6, 5’-CCTGGGCAACATTATGGTGGT-3’ and 5’-CAGAACGGTAGGGTGAGGACA-3’; Prf1, 5’-AGCACAAGTTCGTGCCAGG-3’ and 5’-TGGCGTCTCTCATTAGGGAG-3’; Gzmb, 5’-CCACTCTCGACCCTACATGG-3’ and 5’-CCTTCACAGTGAGCAGCAGTC-3’; Ebi3, 5’-CTTACAGGCTCGGTGTGGC-3’ and 5’-GTGACATTTAGCATGTAGGGCA-3’; GAPDH, 5’-GGTCGGTGTGAACGGATTTGG-3’ and 5’-CCGTGAGTGGAGTCATACTGG-3’.

Mouse IL-10 3’-untranslated region (3’UTR) (+1 to +702) containing the miR-21-5p binding site was inserted into the NheI/XbaI site downstream of the luc2 gene of pmirGLO luciferase reporter plasmid (Promega, Madison, USA). Control and miR-21-5p agomir were purchased from Ruibo (Guangzhou, China). For dual-luciferase assay, CD4⁺CD8^-YFP⁺ Treg cells from the spleen of WT and Foxp3^Cre-YFPmiR-21^f/f mouse were flow sorted and transfected with mouse IL-10 3’UTR reporter plasmid alone or in combination with miR-21-5p agomir using Nucleofector Kit for mouse T cells (Lonza, Switzerland) per manufacturer’s instructions. Transfected cells were then cultured in the presence of 2 μg/ml anti-CD3, 2 μg/ml anti-CD28, 500 IU/ml IL-2, and 10 ng/mL TGF-β. After 48 h, the luciferase activities of the whole-cell lysate were analyzed using a dual-luciferase reporter assay system (Promega, USA). Data were normalized for transfection efficiency by dividing firefly luciferase activity by that of the Renilla luciferase.

Mice were intraperitoneally injected with 100 µl of 10 mg/ml BrdU. Administration of BrdU was repeated every 12 h for three consecutive days. Total cells were isolated from thymus, spleen and MLN 12 h after the last treatment. Cell surface staining was first performed with PE-anti-CD4 and PerCP-Cy5.5-anti-CD8. Cells were then stained with APC-anti-BrdU according to manufacturer’s instructions (BD Bioscience, USA) and analyzed by flow cytometry.

Treg cells (CD4⁺CD8^-YFP⁺) were flow sorted as described above and incubated with 4 μM eFluor-670 (Invitrogen, USA) at 37°C for 15 min. The reaction was stopped with the same volume of cold RPMI-1640 containing 10% heat-inactivated FBS. After washing three times with PBS, eFluor670-labeled cells were cultured at 2×10⁵ cells/well (96-well plate) and treated with 100 IU/ml IL-2, 2 ug/ml anti-CD3 and 2 ug/ml anti-CD28 for 72 h. Cell proliferation was then analyzed by flow cytometry.

Apoptosis detection were performed on freshly isolated Tregs or Tregs treated with α-CD3 (2 ug/ml) plus α-CD28 (2 ug/ml) in the presence of IL-2 (100 IU/ml), TNF-α (5 ng/ml), IL-1β (4 ng/ml) and IFN-γ (25 ng/ml) for 96 h. The percentage of apoptotic cells was determined by Annexin V/7AAD (7-Amino-Actinomycin D) staining according to manufacturer’s instructions (KeyGEN BioTECH, China). Briefly, Treg cells were stained with APC-anti-Annexin V and 7-AAD followed by analysis using flow cytometry (Beckman Coulter Inc, USA). Apoptotic cells were defined as Annexin V and 7-AAD double-positive cells.

Adenovirus over-expressing miR-21 was designed and supplied by Vigene Biosciences, China. Because of the color conflict, Treg cells (CD4⁺CD25⁺) from the spleen of C57BL/6 mice were isolated using EasySep Mouse CD4⁺CD25⁺ Treg Cell Isolation Kit (STEMCELL Technologies, Canada). Isolated Treg cells were first cultured in complete RPMI-1640 medium at a density of 2×10⁵/well in 96-well plate and stimulated with 100 IU/ml IL-2 in the presence of 2 ug/ml anti-CD3 plus 2 ug/ml anti-CD28 for 36~48 h. Then 9x10¹⁰ pfu adenovirus was added to the plate. After 12 h, replace culture medium with fresh complete RPMI-1640 medium in the presence of 2 μg/mL anti-CD3, 2 μg/mL anti-CD28, 100 IU/mL IL-2, and continue to culture for 36 h. Transduced cells were then cultured in the presence of 50 ng/ml IL-6, 20 ng/ml IL-1β, 20 ng/ml IL-23 for 72 h. Transduction efficiency was ~60-70% as determined by examination under a fluorescent microscope.

The data were analyzed using GraphPad Prism 8.0 and expressed as mean ± SD. The student’s t-test was used to compare the significance of the difference between two groups. ANOVA was utilized for multi-group comparison, with Tukey’s test for subsequent post-hoc comparisons between two groups. Differences were considered statistically significant at *P<0.05, **P<0.01, and ***P<0.001.

To study the functional significance of Treg-specific expression of miR-21 under physiological conditions, we generated mice with Treg specific deletion of miR-21 (Foxp3^Cre-YFPmiR-21^f/f). Deletion of miR-21 in both rested Treg (rTreg) and activated Treg (aTreg) was confirmed by RT-PCR, while miR-21 expression in freshly isolated and activated conventional T cells was not affected ( Figure 1A ). Thymic and peripheral T cell development was normal in Foxp3^Cre-YFPmiR-21^f/f mice ( Figure S1A ). The proportions ( Figure S1A ) and numbers ( Figure S1B ) of CD4⁺CD8^-YFP⁺ Treg cells from thymus, spleen and mesenteric lymph node (MLN) were comparable between Foxp3^Cre-YFPmiR-21^+/+ (WT) and Foxp3^Cre-YFPmiR-21^f/f mice. We further confirmed normal development of Foxp3^Cre-YFPmiR-21^f/f tTreg cells based on proportional expression of Nrp-1 ( Figure S1C ). The phenotype of Treg cells, characterized by the expression Foxp3, CD25, cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and glucocorticoid-induced TNFR-related protein (GITR), was also similar between WT and Foxp3^Cre-YFPmiR-21^f/f mice ( Figure S2A ). Furthermore, we observed a similar outcome for Treg cell phenotype when splenic Treg cells were activated and treated with pathogenic Th17-priming cytokines (IL-6+IL-1β+IL-23) or Th1-priming cytokine (IL-12) in vitro ( Figure S2B ). CD25 expression was not examined in this experimental setting because it is also a T cell activation marker.

Because it has been reported that miR-21 inhibits cell apoptosis and promotes cell proliferation, we then assessed the apoptosis and proliferation of Treg cells from WT and Foxp3^Cre-YFPmiR-21^f/f mice. As shown in Figures S3A, B , the apoptosis ratio, as determined by the proportion of Annexin V⁺7AAD⁺ cells, was similar in fresh splenic Tregs and Tregs treated with inflammatory cytokine mix (TNF-α+IL-1β+IFN-γ) in vitro. Next we examined the proliferation of Tregs in vitro using fluorescent dye-based cell proliferation assay. Our results showed that miR-21-deficiency in Treg cell does not affect the proliferation of Tregs when cultured in vitro with IL-2 in the absence or presence of activation (anti-CD3 plus anti-CD28) ( Figures S4A, B ). In addition, we used labeling with the thymidine analog BrdU to assess Treg cell proliferation in vivo. We found that the proportions of BrdU-positive Treg cells in the thymus, spleen and MLN were also similar between WT and Foxp3^Cre-YFPmiR-21^f/f mice ( Figures S4C, D ). Taking together, these results indicate that miR-21-deficiency in Treg cells doesn’t affect their development, phenotype, apoptosis and proliferation.

Because Treg cells play a key role in the control of pathological immune responses, we next sought to determine whether miR-21-deficiency affects the immune-suppressive function of Tregs. MiR-21-deficiency in Tregs does not cause any apparent global immune abnormalities as reflected by no splenomegaly or lymphadenopathy was observed in any of the Foxp3^Cre-YFPmiR-21^f/f mice ( Figure S5 ). Fractions of splenic naïve and memory T cells in naïve mice were also comparable between WT and Foxp3^Cre-YFPmiR-21^f/f mice ( Figure S6 ). Next, we examined the in vitro suppressive activity of Treg cells. MiR-21-deficient splenic Treg cells proved as effective as WT Treg cells in inhibiting T cell proliferation ( Figures 1B, C ). It has been reported that Treg cells can suppress T cell proliferation indirectly by down-regulating co-stimulatory molecules on antigen presenting cells (such as dendritic cells) (31). We then examined the expression of CD80 and CD86 on bone-marrow derived dendritic cells (BMDCs) co-cultured with splenic Tregs from either WT or Foxp3^Cre-YFPmiR-21^f/f mice. Our results showed that Tregs from WT and Foxp3^Cre-YFPmiR-21^f/f mice exhibit no significant difference in reducing the expression of CD80 and CD86 on BMDCs ( Figure 1D ).

Secreting inhibitory cytokines, such as IL-10 and TGF-β, is one of the many effector pathways utilized by Treg cells to control immune response. On the other hand, inflammatory environments can cause the loss of Foxp3 expression and the generation of exTregs that gain an effector phenotype. Therefore, we examined re-programming of miR-21-deficient splenic Treg cells treated with anti-CD3 plus anti-CD28 in the presence of Th0, Th1 or pathogenic Th17 priming cytokines. Our results showed that under pathogenic Th17 priming condition, the levels of IL-17 mRNA and protein were significantly increased, while under Th1 priming condition, the levels of IFN-γ mRNA and protein were significantly increased ( Figures 2A, B ). Furthermore, we found that IFN-γ expression was comparable between WT and miR-21-deficient Treg cells upon above-mentioned destabilizing treatments. By contrast, the mRNA expression ( Figure 2A ) and secretion of IL-17A and IL-17F ( Figure 2B ) by miR-21-deficient Treg cells were significantly increased compared with WT Treg cells. In addition, we found that the mRNA and protein levels of IL-10, but not TGF-β, were significantly increased by miR-21-deficient Treg cells ( Figures 2A, B ). We further confirmed the up-regulation of IL-17 and IL-10 by miR-21-deficient Treg cells under pathogenic Th17-priming condition using flow cytometry ( Figures 2C, D ). However, Foxp3 was down-regulated to similar degrees in WT and miR-21-deficient Treg cells, as the proportion of exTregs (lost Foxp3 expression) and Tregs (maintained Foxp3 expression) were comparable between WT and miR-21-deficient Treg cells ( Figures 3A, B ). We further showed that the proportion of IL-17⁺ and IL-10⁺ cells were significantly increased by both exTregs and Tregs that lacking miR-21 ( Figures 3C, D ). It shall be noted that although ~50% of Tregs lost Foxp3 expression when cultured in vitro under Th17-inducing condition, these exTregs may not be the same as exTregs generated in vivo. For example, in vitro generated exTregs actually produce reduced IL-17 as shown in Figure 3C . We examined the expression of RORγT and found that, although in vitro generated exTregs lost Foxp3 expression, they exhibited reduced RORγT expression when compared with Tregs that retained Foxp3 expression ( Figure S7 ). Although this result is consistent with that of reduced IL-17 expression by exTregs generated in vitro, it remains elusive how RORγT expression was down-regulated in those Tregs.

To determine how miR-21 in Treg cells inhibits IL-17 expression, we first examined miR-21 expression in both freshly isolated and activated Treg cells in the absence or presence of pathogenic Th17-priming cytokines. Our results showed that while miR-21 expression in Treg cells was increased upon activation, it is further increased when Treg cells were activated in the presence pathogenic Th17-priming cytokines ( Figure 4A ). This is not surprising because pathogenic Th17-priming cytokines including IL-6, IL-1β and IL-23 can all activate Stat3, an important transcription factor that promotes miR-21 expression. Surprisingly, while phosphor-Stat3 (pStat3) level was increased in Treg cells upon treatment with pathogenic Th17-priming cytokines, miR-21-deficient Treg cells exhibit significantly elevated pStat3 level compared with WT Treg cells ( Figure 4B ). These results indicate that although Stat3 in Treg cells promotes miR-21 expression, miR-21 is able to down-regulate Stat3 activity in a negative feedback manner. We further confirmed this hypothesis by showing that adenoviral delivery of miR-21 into Treg cells is able to reduce pStat3 level ( Figure 4C ) and the proportion of IL-17⁺ cells ( Figures 4D, E ). To determine whether miR-21 reduces the proportion of IL-17⁺ cells through down-regulating Stat3 activity, we treated Treg cells with Stat3 inhibitor (Stattic) and then examined the proportion of IL-17⁺ cells upon treatment with pathogenic Th17-priming cytokines. Our results showed that the proportion of IL-17⁺ cells is no longer increased by miR-21-deficient Treg cells when they were treated with Stattic as determined by both intracellular staining ( Figure 4F ) and ELSIA ( Figure 4G ).

In Figure 2 we have shown that miR-21-deficient Treg cells produce significant more IL-10, this indicates that miR-21 in Treg cells may down-regulate IL-10 expression. We confirmed this hypothesis by showing that adenoviral delivery of miR-21 into Treg cells is able to reduce the proportion of IL-10⁺ cells ( Figures 5A, B ). To determine how miR-21 in Treg cells suppresses IL-10 expression, reporter plasmid of mouse IL-10 3’UTR together with control agomir or miR-21 agomir were electroporated into WT Tregs, and then luciferase activity was measured. Our results showed that luciferase activity was significantly lower when miR-21 level was increased in Treg cells ( Figure 5C ). Furthermore, reporter plasmid of mouse IL-10 3’UTR was electroporated into both WT and miR-21-deficient Treg cells, and then luciferase activity was measured. We found that luciferase activity was significantly higher in miR-21-deficient Treg cells ( Figure 5D ). Since it has previously been shown that the 3’UTR of IL-10 contains potential miR-21 binding site and mutagenesis analysis confirmed that IL-10 is a valid target of miR-21 (20), we believe that miR-21 in Treg cells may suppress IL-10 expression through direct targeting of IL-10.

It has been reported that, although Stat3 is a critical transcription factor for Th17 differentiation, Treg cells suppresses Th17-specific immune response upon activation of Stat3 (32, 33). Since we have shown that Stat3 activity is increased in miR-21-deficient Treg cells, it is possible that those Treg cells may display improved function in suppressing Th17 response. IL-10 is implicated in Stat3-dependent promotion of Treg cell function. However, we have shown in Figure 4 that miR-21-deficient Treg cells actually produce significant more IL-10. We further demonstrated that, although Stat3 inhibition leads to the down-regulation of IL-10, miR-21-deficient Treg cells still produce significant more IL-10 ( Figure 5E ). We next examined the expression of CCR6, Prf1, Gzmb, and Ebi3, all of which are also implicated in Stat3-dependent promotion of Treg cell function. Our results showed that none of them displayed significant difference between WT and miR-21-deficient Treg cells ( Figure S8 ).

Since Treg cells that are defective in inhibiting effector T cells in vivo may display normal in vitro suppressive activity, we next examined whether miR-21 affects Treg function in vivo using three mouse models of autoimmune disease, all of which are closely related to Th17-mediated inflammatory immune pathogenesis.

Experimental Autoimmune Uveitis (EAU) is the animal model of human uveitis that most closely resembles sympathetic ophthalmia. IRBP-induced EAU model was established in both WT and Foxp3^Cre-YFPmiR-21^f/f mice. Fundoscopic exam was used to assess the degree of fundus inflammation at different time-points after immunization, and we found no significant difference between WT and Foxp3^Cre-YFPmiR-21^f/f mice ( Figure 6A ). In consistency with the phenotypical profiles, there is no significant difference in the concentration of IL-17A, IL-17F, IFN-γ and TNF-α in the eye between WT and Foxp3^Cre-YFPmiR-21^f/f mice ( Figure 6B ).

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used rodent model of multiple sclerosis (MS). MOG peptide-induced EAE model was established in both WT and Foxp3^Cre-YFPmiR-21^f/f mice. There is no significant difference in clinical scores except that Foxp3^Cre-YFPmiR-21^f/f mice exhibit a slightly increased severity during the induction phase ( Figure 6C ). When the concentration of inflammatory cytokines in the brain was examined, we found no significant difference in the concentration of IFN-γ and TNF-α between WT and Foxp3^Cre-YFPmiR-21^f/f mice. However, the concentration of IL-17 was increased in Foxp3^Cre-YFPmiR-21^f/f mice ( Figure 6D ).

The collagen-induced arthritis (CIA) mouse model is the most commonly studied autoimmune model of rheumatoid arthritis. Collagen II-induced CIA model was established in both WT and Foxp3^Cre-YFPmiR-21^f/f mice. Mice were examined physically every other day and four-point arthritis indexes were assessed. As shown in Figure 6E , Foxp3^Cre-YFPmiR-21^f/f mice exhibit a slightly increased but significant disease severity than WT mice. In consistency with the phenotypical profiles, the concentrations of inflammatory cytokines including IL-17A, IL-17F, IFN-γ and TNF-α were all significantly increased in the paw of Foxp3^Cre-YFPmiR-21^f/f mice ( Figure 6F ).

Taking together, these data indicate that although Foxp3^Cre-YFPmiR-21^f/f mice exhibit a slightly increased RA severity than WT mice, miR-21 in Treg cells is largely dispensable for the development of autoimmune disease.

Since we have shown that IL-17 and IL-10 production was increased by miR-21-deficient Treg cells upon treatment in vitro with pathogenic Th17-priming cytokines, we then examined whether they also produce more IL-17 and IL-10 after experiencing inflammatory environment in vivo. In all three autoimmune disease models studied, the proportion of CD4⁺CD8^-YFP⁺ cells in the draining lymph node was comparable between WT and Foxp3^Cre-YFPmiR-21^f/f mice ( Figures 7A, B ). In consistency with the in vitro results from Figure 4 , we found that the mRNA ( Figure 7C ) and protein levels ( Figure 7D ) of IL-17A, IL-17F, and IL-10 were significant increased by Treg cells from Foxp3^Cre-YFPmiR-21^f/f mice in all three autoimmune disease models studied.