MicroRNA-142-3p inhibits IFN-γ production via targeting of RICTOR in Aspergillus fumigatus activated CD4+ T cells

Abstract

Background: Aspergillus fumigatus (AFE) is a well-adapted, opportunistic fungus that causes a severe and commonly fatal disease, wherein IFN-γ is one of the most important protective cytokines. The aim of this study was to investigate the microRNA expression profile and explore the underlying mechanism during infection with AFE.

Methods: CD4⁺ T cells were activated by co-culturing with dendritic cells (DCs), which were pre-treated with AFE. Next, we performed microRNA microarray expression profiles of activated and control T cells, following which, miRNA-142-3P was selected. To explore the effect of miR-142-3P on T cell activation, miRNA-142-3P expression was disrupted by transient transfection with miR-142-3P mimic or inhibitor. Then, levels of RICTOR, phosphorylated AKT and IFN-γ were detected via Western blotting and qPCR respectively. We further used siRNA to decrease RICTOR expression and determined the role played by RICTOR in miR-142-3P mediated-IFN-γ expression by qPCR following AFE-mediated T cell activation.

Results: The heat-map of miRNA expression profiles showed that 54 microRNAs (miRNAs) were filtered, the levels of which, were significantly different between CD4⁺ T cells activated by AFE and control T cells, in which microRNA-142-3 was involved. Forced expression of miRNA-142-3P dramatically suppressed RICTOR levels, phosphorylated AKT and IFN-γ in AFE activated T cells. Conversely, loss of miRNA-142-3P elevated RICTOR levels, phosphorylated AKT and IFN-γ. Notably, RICTOR deficiency decreased AKT phosphorylation levels and IFN-γ secretion.

Conclusions: Observations indicated that down-regulation of microRNA-142-3p enhanced IFN-γ expression, and did so by promoting RICTOR expression in CD4⁺ T cells activated by AFE.

Keywords: Aspergillus fumigatus (AFE); IFN-γ; T cells; gastric cancer; miRNA-142-3p.

Figure 1

Elevated expression of IFN-γ in CD4⁺ T cells treated with AFE. CD14⁺ PBMCs were isolated, induced to immature DCs and then incubated with/without AFE for 24 h. DCs markers, CD83 (A) and CD80 (B), were detected by flow-cytometric analysis; (C) CD4⁺ T cells were co-cultured with mature DCs (AFE) or immature DCs (control) for 24 h. Expression of CD4 and CD69 were detected by flow-cytometric analysis; (D) CD4⁺ T cells were sorted from PBMC and co-cultured with mature DCs (AFE) for 0, 24, 48 h. The mRNA levels of inflammatory cytokines (IFN-γ, IL-4, IL-17 and IL-22) (normalized for Actin) were measured by qPCR. *, P<0.05; **, P<0.001. PBMC, peripheral blood mononuclear cell; DC, dendritic cell; AFE, Aspergillus fumigatus.

Figure 2

miRNA expression profiles in CD4⁺ T cells activated by AFE. (A) RNAs of AFE activated CD4⁺ T cells and inactivated CD4⁺ T cells were extracted and subjected to miRNA microarray analysis. Heat map represented miRNAs dramatically changed, with ≥ 2-fold differences and P<0.05 between activated CD4⁺ T cells and inactivated CD4⁺ T cells. (B) the levels of altered miRNAs (normalized for U6) by miRNA microarray analysis in CD4⁺ T cells were measured by qPCR. AFE, Aspergillus fumigatus; miRNA, microRNAs.

Figure 3

The miR-142-3p inhibitor enhanced the expression of RICTOR, and of phosphorylated AKT and IFN-γ. AFE activated CD4⁺ T cells were transfected with miR-142-3p mimic (A), miR-142-3p inhibitor (B) and corresponding control regents for 24 h. Levels of miR-142-3p were detected by qPCR; western blotting analyses were used to measure RICTOR and p-AKT expression in AFE activated CD4⁺ T cells with miR-142-3p mimic (C) and miR-142-3p inhibitor (D) treatment; IFN-γ mRNA levels in AFE activated CD4⁺ T cells with miR-142-3p mimic (E) and miR-142-3p inhibitor (F) treatment were detected by qPCR. AFE, Aspergillus fumigatus.

Figure 4

miRNA-142-3p suppresses IFN-γ expression and did so by stimulating RICTOR/AKT signaling AFE activated CD4⁺ T cells were transfected with control siRNA or RICTOR siRNAs for 24 h. The efficacy of RICTOR knockdown was measured by qPCR (A) and Western blotting (B) analysis; (C) p-473 AKT was detected by Western blotting; (D) the qPCR assay was implemented to measure IFN-γ expression in AFE activated CD4⁺ T cells that were transfected with control siRNA or RICTOR siRNAs. **, P<0.001 as compared with control. miRNA, microRNAs; AFE, Aspergillus fumigatus.