Figure 1.
miRNAs miR-133b and miR-206 are syntenic to the IL-17A/IL-17F locus and are specifically expressed in Th17 cells polarized in vitro.
(A) Schematic representation of the Il17a/f locus. Il17a and Il17f are syntenically linked in approx. 44 kB distance. The two miRNAs miR-133b and miR-206 are located in close proximity upstream to the Il17a/f locus. (B) VISTA plot of the mouse Il17a/f locus in which mouse sequence is shown on the x axis and percent similarity to human on the y axis. (C) Sequence comparison of mature miR-133b and miR-206 in H.s., M.m. R.n. and P.t. (D), (E) Spleen and peripheral lymph node cells were isolated from DO11.10 mice, co-cultured with sex-matched BALB/c irradiated feeder cells and polarized for the different T helper cell subset conditions (Th0, Th1, Th2, Th17 and Treg). Expression levels of miR-133b (D) and miR-206 (E) were analyzed by qRT-PCR. Values are plotted as fold difference compared to non-polarized cells (Th0). Error bars show ±SEM of n≥3 experiments with 2–3 mice per experiment.
Figure 2.
IL-17 producing in vivo polarized CD4+ Th17 cells express elevated amounts of miR-133b and miR-206.
(A) CD4+ T cells from C57BL/6 mice were sorted into IL-17A+ and IL-17A− populations and analyzed for expression of miR-133b and miR-206 by qRT-PCR. Dot plots show post-sort analysis of one representative experiment. (B) CD4+ T cells were sorted into GFP+ (Rorγt+) and GFP− (Rorγt−) populations from heterozygous Rorγt reporter mice and analyzed as in (A). Dot plots show post-sort analysis of one representative experiment. Values are plotted as fold increase ( = ratio) compared to the respective negative population. The graphs show representative experiments from n = 3 independent experiments with similar results. Error bars represent SD values of triplicates from one experiment with 4–6 mice.
Figure 3.
Correlation of IL-17A production and miR-206/133b expression in γδ T cells.
(A) γδ T cells from Tcrd-H2BeGFP mice were sorted into a CCR6+ and CCR6− populations and analyzed for expression of miR-133b and miR-206 by qRT-PCR. Dot plots show post-sort analysis of one representative experiment. (B) γδ T cells were sorted into GFP+ (Rorγt+) and GFP− (Rorγt−) populations from heterozygous Rorγt reporter mice and analyzed as in (A). (C) γδ T cells from Tcrd-H2BeGFP mice were sorted into CD27− and CD27+ populations and analyzed as in (A). Dot plots show post-sort analysis of one representative experiment. Values are plotted as fold increase ( = ratio) compared to the respective negative population. The graphs show representative experiments from n = 3 independent experiments with similar results. Error bars represent SD values of triplicates from one experiment with 4–6 mice.
Figure 4.
The Th17 polarizing cytokine IL-23 promotes expression of miR-206/133b as well as secretion of IL-17A.
(A, B) Spleen and peripheral lymph node cells were isolated from DO11.10 mice, cocultered with sex-matched BALB/c irradiated feeder cells and polarized to either Th1 or treated with TGF-β, IL-6, IL-23, IL-1β, IL-21 and TNF-α in various combinations (4cytokines = TGF-β, IL-6, IL-23 and IL-1β). Values show fold increase ( = ratio) compared to cells cultured only with Ova323–329/antibodies. Expression levels of miR-133b (A) and miR-206 (B) were analyzed by qRT-PCR. (C) Secreted IL-17A was determined in culture supernatants of each condition from the cells in (A) and (B) by ELISA. Values show absolute amounts of IL-17A in the cell culture supernatant in ng/ml. Error bars show ±SEM of n = 3 experiments with 2–3 mice per experiment. (D), (E) Scatter plot of IL-17A protein concentration versus relative expression of miR-133b (D) and miR-206 (E) and correlation coefficient for ELISA compared to qRT-PCR.
Figure 5.
IL-23R signalling is not essential for the induction of miR-133b and mir-206.
IL-23R+ (GFP+) or IL-23R− (GFP−) γδ and αβ T cells from either homozygous (IL-23R−/−) or heterozygous IL-23Rgfp.KI mice were compared for their miR-133b and miR-206 expression in (A) and (B). GFP+ γδ and αβ T cells from heterozygous or GFP+ γδ and αβ T cells from homozygous IL-23Rgfp.KI mice were compared for their miR-133b and miR-206 expression in (C) and (D). Error bars show ±SEM of n = 3 experiments with cells pooled from 10 mice per group.
Figure 6.
Primary human Th17 cells, but not Th1 cells, express miR-133b and miR-206.
CD4+ T cells were isolated from peripheral blood of 3 human healthy donors, enriched using magnetic beads and then FACS sorted into 3 different populations: IL-17A+, IL-17A− IFNγ+ and IL-17A−IFN-γ−. Expression levels of miR-133b and miR-206 were analyzed by qRT-PCR. Values are plotted as fold increase compared to the respective IL-17A− IFN-γ− population. P-value for miR-206 in IL-17A+ compared to IFN-γ+ expressing cells was 0.0645. Error bars show ±SEM for the 3 different donors.
Figure 7.
Functional outcome of ectopically expressed miR-133b and miR-206 in vitro and in vivo.
(A) In vitro assay. MACS-enriched CD4+ T cells from TCR-transgenic DO.11.10 mice were retrovirally transduced with miR-133b or miR-206 or with both and stimulated under Th17 polarization inducing conditions. Frequency of IL-17 producing CD4+ T cells among transduced (GFP+) and non-transduced (GFP−) cells of the same well was compared. One representative of two independent experiments with similar results is shown. (B, C) Frequency of IL-17 producing cells overexpressing miR-133b or miR-206 in vivo. Lineage negative bone marrow was transduced with miR-133b or miR-206 or with the empty vector MDH1-PGK-GFP2.0 and served to reconstitute lethally irradiated C57BL/6 wild type mice. After 8–10 weeks, chimeras were analyzed for the occurrence and frequency of IL-17 producing cells within the transduced GFP+ and non-transduced GFP− lymphocytes from peripheral lymph nodes and spleen. (B) Representative gating strategy after excluding autofluorescent and B220+ cells. (C) Frequency of IL-17 producing CD4+ and CD4− cells, respectively. At least 5 chimeric mice were individually analyzed for each condition.