miRNA let-7-5p present in the extracellular vesicles of Trichinella spiralis newborn larvae inhibits the function of M1-type RAW264.7 macrophages by targeting C/EBPδ.

BACKGROUND

Trichinella spiralis, in its newborn larva (NBL) stage, invades the host bloodstream and disseminates throughout the body. Concurrently, M1 macrophages undergo transformation into M2 macrophages. In our previous studies, we demonstrated that extracellular vesicles secreted by NBL (NBL-EVs) significantly express the microRNA (miRNA) cel-let-7-5p. In this study, we investigated the immunomodulatory effects and mechanisms of action of EVs derived from T. spiralis NBL and the influence of their key miRNA, cel-let-7-5p, on M1 macrophages.

METHODS

This study investigates the impact of T. spiralis NBL-EVs and cel-let-7-5p on RAW264.7 macrophages through in vitro co-culture, followed by a dual luciferase assay to confirm C/EBPδ as the target of cel-let-7-5p. M1-polarized RAW264.7 cells were subsequently transfected with various agents, including NBL-EVs, cel-let-7-5p mimic, C/EBPδ small interfering RNA (siRNA), and so forth. The cell functions, surface molecule expression, transcription, and cytokine release were analyzed using flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) to elucidate the regulatory mechanisms of NBL-EVs and cel-let-7-5p on macrophage polarization.

RESULTS

Results show that cel-let-7-5p transported by T. spiralis NBL-EVs inhibited the functional activity of M1 RAW264.7 macrophages by targeting C/EBPδ. This inhibition was validated by reduced CD86 and increased CD206 expression, along with decreased nitric oxide (NO) synthesis and downregulation of the M1 marker genes interleukin-12 (IL-12) and inducible nitric oxide synthase (iNOS). In contrast, the messenger RNA (mRNA) levels of IL-10 and arginase-1 (Arg1), which are M2 characteristic genes, were significantly enhanced. However, the release of M1 pro-inflammatory cytokines, such as IL-6, tumor necrosis factor-alpha (TNF-α), and IL-1β, was decreased proportionally. Notably, introducing a cel-let-7-5p inhibitor effectively reversed the suppressive effect of NBL-EVs on M1 macrophage function and partially mitigated their transition to the M2 phenotype, notably impacting Arg1 gene expression. However, no significant changes were observed in CD206 protein expression or IL-10 mRNA levels.

CONCLUSIONS

The findings of this study reveal that cel-let-7-5p in T. spiralis NBL-EVs can inhibit the function of M1-type RAW264.7 macrophages by targeting C/EBPδ.