Ascaris Lumbricoides Cystatin Impairs IL-1β Maturation and CD14 Expression in Human Monocytes.

Abstract

Ascaris lumbricoides cystatin (Al-CPI), a cysteine protease inhibitor, has shown anti-inflammatory effects in models of house dust mite-induced respiratory allergy and experimental colitis. Since monocytes are primary targets of cystatins, and helminth-derived products can induce immunoregulatory monocytes that mitigate autoimmune and inflammatory diseases in murine models, we aimed to investigate the immunoregulatory effects of Al-CPI on human monocytes and its mechanisms. Monocytes were isolated from healthy donors using CD14+ magnetic beads and treated with recombinant Al-CPI (rAl-CPI) before activation with LPS. Surface marker expression and caspase-1 activity were analysed by flow cytometry, while cytokine levels were quantified using bead-based assays. Caspase-8 inhibition was measured by fluorescence, and pro-IL-1β expression was evaluated by Western blot. RNA-seq and differential gene expression analyses were performed using DESeq2 and GSEA. rAl-CPI reduced cell surface expression of HLA-DR and CD14 as well as IL-1β levels in cell culture supernatants. Transcriptomic analysis identified 30 differentially expressed genes (12 upregulated and 18 downregulated; padj < 0.05 and fold change [FC] < |1.5|), including the downregulation of CD14 and SLAMF1, both involved in LPS-mediated signalling through the TLR4-TRIF pathway. GSEA analysis showed that rAl-CPI plus LPS induced an enrichment of the IFN signalling pathway compared with LPS (NES = 1.88; padj < 0.0001). Further analysis of IL-1β production and maturation showed that rAl-CPI did not alter pro-IL-1β levels, but reduced caspase-1 activation. In conclusion, rAl-CPI may impair IL-1β maturation and release by modulating the alternative inflammasome activation, potentially through downregulation of CD14 and other key regulatory molecules involved in this pathway.