The effects of abundant nematode-derived miRNAs on the transcriptome of mouse differentiating Th2 cells

Four microRNAs(miRNAs) highly abundantly secreted by the parasitic nematode Heligmosomoides polygyrus were synthesized (miRVana, Ambion) and transfected repeatedly into differentiating Th2 cells in vitro, individually or in combination. The effects of the different transfection regimens were assessed by RNA sequencing (Illumina). The spleen and lymph nodes from wild-type C57BL/6 female mice were used to isolate naïve CD4+ T cells (magnetic beads). Cells were put in culture and allowed to differentiate into Th2 cells over 4 days, in the presence of α-CD28 and α-CD3, IL-4, IL-2, and α-IFN-γ. Cells were transfected every 24 h with 10 nM miRNA(s), 4 times consecutively, or only once at experiment start (0 h), with 50 nM miRNA(s). Controls included the transfection reagent (Lipofectamine), no treatment, or a commercial universal negative control. After 4 days in culture, cells were restimulated with α-CD3 and total RNA was extracted for transcriptome analysis. The experiment was repeated 3 times independently.