Dicer insufficiency and microRNA-155 overexpression in lupus regulatory T cells: an apparent paradox in the setting of an inflammatory milieu

Abstract

Systemic lupus erythematosus is a chronic autoimmune disease characterized by loss of tolerance to self-Ags and activation of autoreactive T cells. Regulatory T (Treg) cells play a critical role in controlling the activation of autoreactive T cells. In this study, we investigated mechanisms of potential Treg cell defects in systemic lupus erythematosus using MRL-Fas(lpr/lpr) (MRL/lpr) and MRL-Fas(+/+) mouse models. We found a significant increase in CD4(+)CD25(+)Foxp3(+) Treg cells, albeit with an altered phenotype (CD62L(-)CD69(+)) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared with non-autoimmune C3H/HeOuj mice. A search for mechanisms underlying the altered Treg cell phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs, including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a, and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155, that can target CD62L and may thus confer the altered Treg cell phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H/HeOuj) Treg cells reduced their CD62L expression, which mimics the altered Treg cell phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.

Figure 1. CD4⁺CD25⁺Foxp3⁺ Treg cells increase and exhibit altered phenotype in MRL/lpr mice

Splenocytes from MRL/lpr and C3H mice were analyzed for Treg cells, as indicated below. (a) CD4⁺CD25⁺ cells are indicated on dotplots as the proportion of gated small lymphocytes (left). Frequency and total numbers of CD4⁺CD25⁺ T cells are shown as the mean ± S.E. from 10 mice per group, all ≥12-wk-old (right). (b) CD4⁺ CD25⁺Foxp3⁺ T cells are indicated in right upper quadrants as the proportion of gated CD4⁺ cells (left). Frequency (as a proportion of splenocytes) and total numbers of CD4⁺CD25⁺Foxp3⁺ T cells are shown as the mean ± S.E. from 9 mice per group (right). (c) CD69 and CD62L expression on TCRβ⁺CD4⁺CD25⁺Foxp3⁺ T cells. (d) CD69⁺CD62L^– Treg cells are shown as the proportion of CD4⁺CD25⁺Foxp3⁺ cells from 6 mice per group. (e) CD62L expression on gated CD4⁺CD25⁺Foxp3⁺ T cells in 4-wk-old (pre-autoimmune) and 12-wk-old (early autoimmune, pre-clinical) MRL/lpr mice (left), 4-wk-old C3H and MRL/lpr mice (middle), and 11-week old C3H and MRL/lpr mice (right). Results represent two to five independent experiments.

Figure 2. Reduced suppression by Treg cells from MRL/lpr mice

Treg cells were purified from spleens of C3H, and MRL/lpr mice, and mixed with CFSE-labeled responder splenocytes from C3H (a) or MRL/lpr mice (b) in triplicates. On day 3, triplicate wells were pooled and stained with 7AAD to gate out dead cells, and proliferation of CD4⁺ T cells was analyzed. CFSE dilution at 1:2 Treg:responder cell ratio is shown. Gray shaded area – responder C3H (a) or MRL/lpr (b) splenocytes with no Treg cells; red line – responder cells + MRL/lpr Treg cells; and green line – responder cells + C3H Treg cells. (c) Percent suppression of proliferation of responder splenocytes in presence of Treg cells from C3H or MRL/lpr mice is shown as the mean ± S.E. (n = 17 C3H and 21 MRL/lpr mice in 8 independent experiments). (d) Histogram showing CD69 expression on CD4⁺CD25⁺Foxp3⁺ cells from C3H mice (gray line) and MRL/lpr mice that had no defect in Treg suppressive capacity (black line). Representative data from two independent experiments, each using two mice per group, are shown.

Figure 3. Expression of Dicer and miRNAs in MRL/lpr mice

(a) RNA was extracted from purified splenic Treg cells, as described in Methods, and qPCR was performed for Dicer expression. Relative Dicer expression normalized to Gapdh is shown as the mean ± S.E. in 12–16-wk-old C3H (n=13) and MRL/lpr (n=12, old) mice and in seven young, pre-autoimmune mice (4-wk-old, young). Results represent four independent experiments. (b) miRNA was extracted from purified splenic Treg cells and converted to cDNA, as described in Methods. miFinder PCR arrays were used to profile 88 miRNAs (Fig. S3). Raw threshold values for obtained and changes in miRNA expression were calculated using SABiosciences Web Analysis platform. ΔΔCt values were calculated from raw threshold cycle data. ΔΔCt values from three independent experiments, each using cells purified from one each of C3H and MRL/lpr mouse, were compared. miRNAs that are significantly different between C3H and MRL/lpr mice are shown (*p <0.05; **p<0.01; n = 3 each). (c, d) qPCR reactions for a few selected miRNAs were set up, as per manufacturer's instructions. Results are shown as the mean ± S.E. for miR-155 in 4–5-wk-old MRL/lpr (n=6), and 12–20-wk-old female C3H (n=4) and MRL/lpr (n=5) mice (c) and for miR-23a in 4 each of 12–20-wk-old female C3H and MRL/lpr mice (d). Results represent three (c) and two (d) independent experiments.

Figure 4. Overexpression of miR-155 in otherwise normal Treg cells alters their phenotype

Treg cells enriched from C3H mice were transfected with 40nM of miR-155, miR-23a or with HiPerfect reagent alone, as described in Methods. Representative histogram shows reduced CD62L expression in C3H cells transfected with miR-155 mimic. Similar results were obtained in 5 of 7 mice tested in three independent experiments