Acanthamoeba castellanii cysteine protease 3 promotes M1 macrophage polarization through the TLR4/NF‑κB pathway

Background: Acanthamoeba spp., which are free-living protozoan parasites, are etiological agents for Acanthamoeba keratitis and granulomatous amoebic encephalitis. Macrophages participate in the host defense response to resist Acanthamoeba spp. This study examined the effect of Acanthamoeba castellanii cysteine protease 3 (AcCP3) on macrophage activation during inflammatory responses and explored the underlying mechanisms.

Methods: The effects of recombinant AcCP3 (rAc-CP3) stimulation on inflammatory factor levels and macrophage polarization were examined using murine macrophage cells (RAW264.7 cells). Western blotting assay was carried out for analyzing TLR4/NF‑κB pathway-related protein levels. In addition, phosphorylated NF-κB was examined for its nuclear transport using immunofluorescence. The effect of the NF-κB Inhibitor pyrrolidinedithiocarbamate ammonium (PDTC) on rAc-CP3-induced M1 polarization was analyzed. Furthermore, RAW264.7 cells were co-cultivated using AcCP3 knockdown trophozoites to examine indicators of M1 polarization and pathway-related protein levels.

Results: As revealed by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assays, treatment with rAc-CP3 upregulated the mRNA, protein, and secretion levels, respectively, of Il6, Il1b, Tnfa, and Ifng in macrophages. Flow cytometric analysis demonstrated that rAc-CP3 promoted Cd86⁺ cell (macrophage) proliferation. Additionally, rAc-CP3 upregulated Nos2 expression and nitric oxide (NO) production, indicating that rAc-CP3 promotes macrophage polarization toward an M1-like phenotype. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that the NF-κB pathway was among the top 20 significantly enriched pathways. Treatment with rAc-CP3 upregulated the levels of TLR4, p-Rela, and p-Nfkbia in RAW264.7 cells. Immunofluorescence analysis demonstrated the nuclear translocation of p-Rela. Pretreatment with the NF-κB Inhibitor PDTC downregulated the TLR4, p-Rela, and p-Nfkbia levels in rAc-CP3-treated cells. Additionally, PDTC significantly mitigated the rAc-CP3-induced upregulation of Nos2, NO and pro-inflammatory factor production. AcCP3 knockdown decreased the number of Cd86⁺ cells and suppressed Acanthamoeba trophozoite-induced Nos2 upregulation and NO production. Additionally, AcCP3 knockdown downregulated TLR4, p-Rela, and p-Nfkbia in RAW264.7 cells. PDTC and AcCP3 knockdown suppressed the rAc-CP3-induced M1 macrophage polarization.

Conclusions: AcCP3 promotes M1 macrophage polarization through the TLR4/NF-κB pathway and may exacerbate inflammation through upregulating pro-inflammatory cytokines.

Acanthamoeba castellanii; Cysteine protease 3; Inflammation; Macrophage polarization; TLR4/NF-κB pathway.