Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State

Abstract

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression.

Keywords: dexamethasone; interleukin-10; miRNA; mmu-miR-223-3p; tolerogenic dendritic cells.

Figure 1

Immuno-phenotype of differentially tolerized APC populations. APC populations were generated as described in the Materials and Methods section, and surface expression of the DC lineage marker CD11c and of the activation markers MHCII and CD86 at unstimulated state and after stimulation with LPS were assessed by flow cytometry. Graphs shown are representative for 2 independent experiments each.

Figure 2

Differentially tolerized APCs at either state of activation largely differ from BMDCs in their miRNA expression signature, but show some overlap. In parallel settings, BMDCs and tolerogenic APC populations (DEX-APC, IL10-APC) were differentiated from the same bone marrow progenitors in the presence of GM-CSF, and the miRNA expression profiles of all APC populations at unstimulated state and after stimulation with LPS were assessed by array analysis in 2 independent experiments. (A) The numbers of miRNAs differentially expressed in tolerogenic APCs (DEX-APC, IL10-APC) as compared with BMDCs (iDC, mDC) are indicated (see Tables 1, 3, Tables S1, S2, S7, S8). miRNAs convergingly regulated in differentially tolerized APCs at unstimulated (see Table 1) and at stimulated state (see Table 3) as compared with the according control DC population (iDCs, mDCs) are listed in the assigned tables. (B) miRNAs convergingly regulated in differentially tolerized APCs as compared with the corresponding control DC population (iDCs, mDCs) at both states of stimulation. miRNAs commonly regulated by both APC populations at both states of stimulation are given in italics.

Figure 3

Validation of differential miRNA expression in tolerized APCs vs. BMDCs. Levels of mature miRNA expression in tolerized APC populations and BMDCs at (A) unstimulated and (B) LPS-stimulated state were assessed by real time PCR. To indicate stimulation-dependent alterations in miRNA expression in BMDCs, according levels in mDCs as compared with iDCs are given in (A). (A,B) Data indicate the mean ± SEM of 4 independent experiments performed in duplicate. Statistically significant differences: *vs. corresponding control DC population (iDC,mDC) (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4

Overexpression of mmu-miR-223-3p in BMDCs prevents stimulation-induced upregulation of MHCII and the acquisition of potent T cell stimulatory capacity. iDCs were transfected with a mmu-miR-223-3p mimick or a scrambled siRNA as a control, and aliquots were stimulated with LPS. (A) Relative expression levels of mmu-miR-223-3p and other miRNA species in mmu-miR-223-3p-transfected BMDC populations as compared with Ctrl sirRNA-transfectants were assessed by real time PCR analysis. Data indicate the mean ± SD of 2 independent experiments performed in duplicate. (B) Surface expression of MHCII (upper panel) and CD86 (lower panel) in differentially transfected BMDCs at either state of activation was monitored by flow cytometry. Data indicate the mean fluorescence intensity (MFI) ±SEM of 5 independent experiments each. (C) Differentially transfected BMDC populations were titrated (starting concentration: 2 × 10⁵/ml) and cocultured with allogeneic T cells (each 3 × 10⁶/ml) in triplicates at the indicated ratios for 4 days. Data indicate T cell proliferation quantitated as cpm due to genomic incorporation of ³H-thymidine applied for the last 16–18 h of coculture, and denote the mean ± SEM of 5 independent experiments performed in triplicate. (B,C) Statistically significant differences: *vs. Ctrl siRNA, and + vs. mmu-miR-223-3p transfected BMDC at unstimulated state (*^{, +}p < 0.05, **^{, ++}p < 0.01, ***^{, +++}p < 0.001).

Figure 5

Overexpression of mmu-miR-223-3p in DCs results in reduced mRNA expression of its genuine targets Rasa1 and Kras and of other mRNA species which may affect a network of immunologically relevant proteins. Database analysis of the mRNA expression pattern of DC-like XS52 cells stably transduced to overexpress mmu-miR-223-3p (see Table S11) predicted networks of the encoded proteins. The state of expression of corresponding mRNA species in mmu-miR-223-3p overexpressing XS52 cells as compared with the vector control transduced subline is indicated. The genuine (Kras, Rasa1, Cflar, Fbxl20) mRNA targets of mmu-miR-223-3p are accordingly labeled. Potentially interacting proteins that share immunological and gene regulatory functions are color-coded.