MiR-29 controls innate and adaptive immune responses against intracellular bacterial infection by targeting IFN-γ Feng Ma 1,2,5, Sheng Xu 1,5, Xingguang Liu 1, Qian Zhang 1, Xiongfei Xu 1, Mofang Liu 3, Minmin Hua 3, Nan Li 1, Hangping Yao 2 & Xuetao Cao 1,2,4 Supplementary Figures Supplementary Fig. 1 Signature of in vitro differentiated helper T cells and sorted natural Tregs. (a) IL-4, IL-17A and Foxp3 mrna expression of different CD4 + T cells subsets and natural Tregs was measured by qpcr and normalized to β-actin mrna. Data are shown as mean ± s.e.m. (b) Intracellular cytokine staining for each helper T cell subsets after in vitro differentiation. Data are representative of 1
three independent experiments. 2
Supplementary Fig. 2 Analysis of mouse putative mir-29b-1-mir-29a promoter. (a) The diagram illustrates the location and length of the putative mir-29b-1-mir-29a promoter. Three YY1 and two NF-κB binding sites are located in the 1560bp putative promoter. The positions of the primers for ChIP are also indicated. (b) Two specific primers (primer#2, primer#3) for mir-29b-1-mir-29a promoter were used to detect the DNA bond by YY1. Data are shown as mean ± s.e.m of three independent experiments. (c) Human and mouse sequences of the two putative NF-κB binding sites (red) and their flanking regions in mir-29b-1-mir-29a promoter. 3
Supplementary Fig. 3 Regulation of IFN-γ production in T-bet-transfected EL4 cells by mir-29 mimics or inhibitor. (a) ELISA of IFN-γ in supernatants of EL4 cells transfected with T-bet vector and mir-29b mimics (left) or mir-29b inhibitor (right), 12 h (for mimics) or 24 h (for inhibitor) later, stimulated with PMA plus ionomycin for the indicated times. (b,c) Flow cytometry analysis of IFN-γ expression (b) and cell apoptosis (c) in EL4 cells transfected with T-bet vector and mir-29a mimics, 24 h later, stimulated with PMA plus ionomycin. Data are shown as mean ± s.e.m of three independent experiments (a) or are representative of three independent experiments (b-c). *P < 0.05. 4
Supplementary Fig. 4 Downregulation of IFN-γ mrna in T-bet-transfected EL4 cells by mir-29 mimics. Quantitative RT-PCR assay of IFN-γ mrna expression in EL4 cells transfected with T-bet vector and mir-29 mimics, 24 h later, stimulated with PMA plus ionomycin for the indicated times. Data are shown as mean ± s.e.m of three independent experiments. *P < 0.01. 5
Supplementary Fig. 5 Targets prediction of mir-29 and verification by reporter gene system. (a) The 3 UTRs of T-bet and Eomes mrna contain binding sites of mir-29 family, which are highly conserved in mammalian. (b-c) Luciferase activity in lysates of HEK293T cells transfected with T-bet (b) or Eomes (c) 3 UTR constructs, plus negative control (Ctrl), mir-29a, mir-29b, mir-29c mimics or inhibitors. Data are shown as mean ± s.e.m (b-c). *P < 0.05 and **P < 0.01. 6
Supplementary Fig. 6 Increased IFN-γ production in activated CD4 + T and CD8 + T cells transfected with mir-29a inhibitor. (a) Quantitative RT-PCR assay of mir-29a expression in CD4 + T cells from OT-II transgenic mice and CD8 + T cells from OT-I transgenic mice transfected with control inhibitor or mir-29a inhibitor, 12 h later, incubated with DC pulsed with OVA 323-339 (for CD4 + T cells), or OVA 257-264 (for CD8 + T cells) for 72 h. (b) ELISA of IFN-γ in supernatant of CD4 + T cells and CD8 + T cellsdescribed in (a). Data are shown as mean ± s.e.m of three independent experiments. *P < 0.01. 7
Supplementary Fig. 7 Effector and memory markers of CD4 + and CD8 + T cells transfected with LV-Ctrl and LV-29a. Flow cytometry analysis of CD25, CD69, CD44, CD127 and CD62L expression in CD4 + and CD8 + T cells transfected with LV-Ctrl or LV-29a lentivirus, and then activated with anti-cd3 plus anti-cd28 mab for 3 d. Data are representative of three independent experiments. 8
Supplementary Fig. 8 Endogenous mir-29 has no effect on T H 1 cell differentiation. (a) Induction efficiency of T H 1, T H 2 and T H 17 cells from LV-Ctrl, LV-miR-29a and LV-29sponge transfected CD4 + T cells, determined by flow cytometry. GFP positive cells were gated and analyzed. (b) Flow cytometry analysis of IFN-γ production in T H 1 cells cultured with 10 μg/ml anti-ifn-γ mab or isotype control. Percent of IFN-γ producing cells and their MFI were shown in the dot plot. Data are shown as mean ± s.e.m of three independent experiments (a) or representative of three independent experiments (b). *P < 0.05. 9
Supplementary Fig. 9 Construction and verification of GS29 mice. (a) UBC-29sponge expression vector contained GFP gene and a 3 UTR which consisted of 7 repeats of mir-29 sponge. (b) The working model of mir-29 sponge. UBC-29sponge expression vector with UBC promoter produced large amount of mir-29 sponge mrna which can absorb endogenous mir-29 efficiently. Transient mir-29 knockdown in cell culture and mir-29 knockdown in transgenic mice can be achieved by delivering UBC-29sponge expression vector into cultured cells and one-cell eggs, respectively. (c) Five GS29 founder mice (#26, #34, #56, #68, #85) were constructed (top panel), the F1 generation of three founder mice (#34, #56, #85) were generated by mating founder mice and wild type C57/BL6 mice (bottom panel). All the mice were identified by PCR assays of genomic DNA extracted from the tails of transgenic mice. GS29 is the characteristic fragment of mir-29 sponge expression vector. Data are representative of three independent experiments.
Supplementary Fig. 10 Primary and mature mir-29 mrna in the immune cells from GS29 mice. (a) Northern blot analysis of mature mir-29a in NK cells, CD4 + and CD8 + T cells from GS29 or littermate control mice. U6 mrna serves as a loading control. (b,c) Quantitative RT-PCR assay of mature (b) and primary (c) mir-29a mrna in NK cells, CD4 + T and CD8 + T cells. Data are representative of three independent experiments (a) or are shown as mean ± s.e.m of three independent experiments (b-c). *P < 0.01.
Supplementary Fig. 11 Development of T cell subsets and NK cells in GS29 mice. (a) The thymic TCRβ, CD4, CD8 profile in GS29 and littermate control mice at 6 weeks old. (b) Splenic CD4 + T and CD8 + T cell subsets and natural Treg frequencies. (c) Expression of CD44 and CD62L in splenic CD4 + T and CD8 + T cell from GS29 and littermate control mice. Percent of CD44 hi CD62L lo, CD44 hi CD62L hi and CD44 lo CD62L lo cells are indicated. (d) The number of live CD4 + T and CD8 + T cells isolated from GS29 and littermate control mice and stimulated with plate coated anti-cd3 plus anti-cd28 mab for 72 h. (e)the development of NK cells from GS29 and littermate control mice. Data are representative of three experiments (a,b,c,e) or are shown as mean ± s.e.m of three independent experiments (d).
Supplementary Fig. 12 mir-29 did not regulate the T-bet and Eomes expression. (a) Flow cytometry analysis of T-bet and Eomes expression in activated CD4 + T and CD8 + T cells transfected with LV-Ctrl or LV-29a lentivirus. (b) Flow cytometry analysis of T-bet and Eomes expression in activated CD4 + T and CD8 + T cells from control or GS29 mice. Data are representative of three independent experiments.
Supplementary Fig. 13 Expression of IFN-γ mrna and mir-29a in the immune cells from GS29 mice infected with intracellular pathogen. (a,b) Quantitative RT-PCR assay of IFN-γ and mir-29a mrna expression in NK cells from control and GS29 mice infected by LM for the indicated time. (c,d) Quantitative RT-PCR assay of IFN-γ and mir-29a mrna expression in CD4 + and CD8 + T cells from control and GS29 mice infected by BCG. Data are shown as mean ± s.e.m of three independent experiments. *P < 0.05.
Supplementary Fig. 14 GS29 mice exhibit more resistance to BCG infection. (a) H&E (left, 50) and immunohistochmical staining of IFN-γ (right, 400) of lung from littermate control and GS29 mice 4 weeks after i.v. infection with 5 10 6 BCG. (b) Intracellular IFN-γ staining of CD4 + T and CD8 + T lymphocytes isolated from lung in (a). Data are representative of three independent experiments.
Supplementary Fig. 15 GS29 mice exhibit more resistants to H37Rv infection. (a) Survival of GS29 and control mice after i.v. infection with virulent 5 10 5 H37Rv. (n=12 per genotype). P < 0.05 (Wilcoxon test). (b) H37Rv burden in lung from GS29 and control mice 21 days after i.v. infection with 1 10 5 H37Rv. Data are shown as mean ± s.e.m of three independent experiments. *P < 0.01.
Supplementary Fig. 16 Working model for the control of IFN-γ-mediated innate and adaptive immune responses by mir-29 via targeting IFN-γ
Supplementary methods RNA isolation, reverse transcrption and real time quantitative PCR (qpcr). Total RNA was extracted with TRIzol (Invitrogen) or mirneasy Mini Kit (Qiagen). qpcr analysis was performed by using LightCycer (Roche, Basel, Switherland) and SYBR RT-PCR kit (Toyobo, OSAKA, Japan). Primer pairs are listed below: gene primers sequences (5' to 3') mouse Forward GAACTGGCAAAAGGATGGTGA IFN-γ Reverse TGTGGGTTGTTGACCTCAAAC human Forward CTCTTGGCTGTTACTGCCAGG IFN-γ Reverse CTCCACACTCTTTTGGATGCT mouse Forward TCTGTGCCCACACTCCTGTA NPM1 Reverse TTGCCGTGTTCTTCAATCCCA mouse Forward AAGCCTGTAGCCCACGTCGTA TNF-α Reverse GGCACCACTAGTTGGTTGTCTTTG mouse Forward TAGTCCTTCCTACCCCAATTTCC IL-6 Reverse TTGGTCCTTAGCCACTCCTTC mouse Forward CTGCAGCACTTGGATCAGGAACCTG inos Reverse GGAGTAGCCTGTGTGCACCTGGAA mouse Forward ACTTGAGAGAGATCATCGGCA IL-4 Reverse AGCTCCATGAGAACACTAGAGTT mouse Forward ATGGTAATGTGGCCTACTCCT Rorc Reverse GCTGCTGTTGCAGTTGTTTCT mouse Forward CCCATCCCCAGGAGTCTTG Foxp3 Reverse ACCATGACTAGGGGCACTGTA mouse Forward AGTGTGACGTTGACATCCGT β-actin Reverse GCAGCTCAGTAACAGTCCGC human Forward CATGTACGTTGCTATCCAGGC β-actin Reverse CTCCTTAATGTCACGCACGAT RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTAACCG mir-29a Forward ATGCTAGCACCATCTGAAAT Reverse GTGCAGGGTCCGAGGT RT GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACACT mir-29b Forward ATGCTAGCACCATTTGAAATC Reverse GTGCAGGGTCCGAGGT RT AACGCTTCACGAATTTGCGT U6 Forward CTCGCTTCGGCAGCACA Reverse AACGCTTCACGAATTTGCGT
Flow cytometry and intracellular cytokine staining. All fluorescein-conjugated mabs and isotype controls were from BD PharMingen (San Diego, CA). For intracellular cytokine staining, cells were stimulated with 25 ng/ml Phorbol myristate acetate (PMA) and 500 ng/ml ionomycin (Sigma, St Louis, MO) or antigen peptide-pulsed DCs as indicated for 6hr at 37 C. 10 μg/ml Brefeldin A (ebioscience, San Diego, CA) was included for the last 4 hr of incubation. Cells were stained with the Cytofix/Cytoperm kit according to the manufacture s instructions (ebioscience). Human Th1 cell differentiation. Naïve CD4 + T cells were sorted by Dako MoFloTM XDP (DakoCytomation, Glostrup, Denmark) for CD45RA + and CD45RO - CD4 + T cells from PBMC. Cell purity was >97%. Then the cells were stimulated with plate-bound anti-cd3 (5 μg/ml) plus soluble anti-cd28 (2 μg/ml) in the presence of 10 μg/ml anti-il-4, 10ng/ml IL-12, and 50 U/ml IL-2. Immunoblot, RNA-Binding protein immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP). Cells were lysated using complete RNA lysis buffer provided by Magna RIP kit (Millipore). Protein concentration of the extracts were measured with a BCA assay (Pierce) and equalized with the extraction reagent. Equal amount of the extracts was loaded and subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and then blotted. RIP experiments were performed according to the protocol provided by the kit (Millipore, catalogue 17-701). ChIP
experiments were performed according to the protocol provided by the kit (Millipore, catalogue 17-408). The primer pairs for mir-29b-1-mir-29a promoter ChIP assay were listed as below: Primer#1 Primer#2 Primer#3 Forward: 5 -TTTCAGTTGGTGGCTTGATC-3 Reverse: 5 -AGCTCTTCAGTGGGAGACTTT-3 Forward: 5 -AGAAATGAATAGCCGCAGATT-3 Reverse: 5 -AACTACCACCTACTCACCCAGA-3 Forward: 5 -TGGCGTGTCATCTGGATTGG-3 Reverse: 5 -TGAGAAAGGACGGCTGTTGG-3 Plsmids and primers. The primer pair for amplifying mir-29b-1/mir-29a promoter was 5 - CTACTCCGAAGTTGTCGATTGC-3 (forward) and 5 -ATCCACGGCTCA AGTTGCTGAA-3 (reverse). The mir-29b-1-mir-29a promoter reporter vector was indicated as normal. The vector mut-yy1 was converted from normal by deleting all the CCAT core elements of the three YY1 binding sites. The vector mut-nf-κb (1) was constructed by deleting the GGGTC of the (1) NF-κB binding site, while mut-nf-κb (2) was constructed by deleting the GGTGG of the (2) NF-κB binding site. The vector mut-nf-κb was constructed by mutating both of NF-κB binding sites. GFP primer and GS-29 primer were used for identification of GS29 mice, their sequences were 5 -GTGACCACCCTGACCTACGG-3 (forward) and 5 -CTGCTTGTCGGCC ATGATAT-3 (reverse) for GFP primer, 5 -GAGCAAAGACCCCAACGAGA
AG-3 (forward) and 5 -TCTACAAATGTGGTATGGCTGAT-3 (reverse) for GS-29 primer. Luciferase reporter assays in TLR-triggered RAW264.7 cells. The RAW264.7 cells were plated in the 96-well plate (10000/well) overnight, followed to be cotransfected with TK vector and reporter vector (normal or mutant mir-29b-1-mir-29a promoter reporter), according to the protocol provided by Jet-ENDO transfection reagents (Polyplus-transfection, Illrich, France). 12h later, the transfected cells were stimulated with 100 ng/ml LPS or 0.1 μm CpG ODN. In some experiments, PDTC (30 μm) was added 30 min before stimulation to block NF-kB pathway. 24h after stimulation, the firefly luciferase activity was measured and normalized to Renilla luciferase activity. Northern Blot. For northern blot analysis, total RNA was loaded onto 15% acrylamide, 8M urea TBE (Tris & borate & EDTA) gels. After electrophoresis, RNA was transferred to a nylon membrane (PerkinElmer) and pre-hybridized overnight at 50 C in hybridization buffer (modified Church-Gilbert hyb-mix). The in vitro transcribed and radiolabeled RNA, which was reverse complementary to mir-29a, was used as probes, and hybridized to the membranes for 12 hours at 50 C. After three washing in NSWS (Non-Stringent Wash Solution) for 30 minutes at 37 C, and followed by 5 minutes washing in SWS (Stringent Wash Solution) at 37 C. Kept the membrane in the film cassette and put the phosphor screen on it overnight, then
scanned the phosphor screen. The membrane was stripped and followed to detect the U6 RNA.