Abstract

Background: During the early stages of Trichinella spiralis infection, macrophages predominantly undergo polarization to the M1-like phenotype, causing the host's inflammatory response and resistance against T. spiralis infection. As the disease progresses, the number of M2-type macrophages gradually increases, contributing to tissue repair processes within the host. While cysteine protease overexpression is typically associated with inflammation, the specific role of T. spiralis cathepsin L (TsCatL) in mediating macrophage polarization remains unknown. The aim of this study was to assess the killing effect of macrophage polarization mediated by recombinant T. spiralis cathepsin L domains (rTsCatL2) on newborn larvae (NBL).

Methods: rTsCatL2 was expressed in Escherichia coli strain BL21. Polarization of the rTsCatL2-induced RAW264.7 cells was analyzed by enzyme-linked immunosorbent assay (ELISA), quantitative PCR (qPCR), western blot, immunofluorescence and flow cytometry. The effect of JSH-23, an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), on rTsCatL2-induced M1 polarization investigated. Cytotoxic effects of polarized macrophages on NBL were observed using in vitro killing assays.

Results: Following the co-incubation of rTsCatL2 with RAW264.7 murine macrophage cells, qPCR and ELISA revealed increased transcription and secretion levels of inducible nitric oxide synthase (iNOS), interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α) in macrophages. Western blot analysis showed a significant increase in iNOS protein expression, while the expression level of arginase-1 protein remained unchanged. Flow cytometry revealed a substantial increase in the number of CD86-labeled macrophages. The western blot results also indicated that rTsCatL2 increased the expression levels of phospho-NF-κB and phospho-nuclear factor-κB inhibitor alpha (IκB-α) proteins in a dose-dependent manner, while immunofluorescence revealed that rTsCatL2 induced nuclear translocation of the p65 subunit of NF-κB (NF-κB p65) protein in macrophages. The inhibitory effect of JSH-23 suppressed and abrogated the effect of rTsCatL2 in promoting M1 macrophage polarization. rTsCatL2 mediated polarization of macrophages to the M1-like phenotype and enhanced macrophage adhesion and antibody-dependent cell-mediated cytotoxicity (ADCC) killing of NBL.

Conclusions: The results indicated that rTsCatL2 induces macrophage M1 polarization via the NF-κB pathway and enhances the ADCC killing of NBL. This study provides a further understanding of the interaction mechanism between T. spiralis and the host.