Snake Venom Metalloproteinases Neuw-I and Leuc-a Downregulate Inflammasome-Related Genes and Pro-Inflammatory Cytokines in Staphylococcus aureus-Infected Macrophages

Staphylococcus aureus is an opportunistic Gram-positive pathogen that colonizes human skin and mucosa, causing local and systemic infections. Its virulence factors and antimicrobial resistance make it a major public health concern. Infection induces an inflammatory response characterized by TNF-α and IL-6 production and inflammasome activation. Thus, immunomodulatory agents may help control exacerbated inflammation. Snake venom metalloproteinases (SVMPs), major components of Bothrops venoms, exhibit antimicrobial and immunomodulatory activities. Here, we investigated Neuw-I (B. neuwiedi) and Leuc-a (B. leucurus) (SisGen AD998E4) during S. aureus infection. Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice (CEUA-UFMG 278/2025) received SVMPs (10 µg/mL) 1 h before or after infection with S. aureus ATCC 6538 (MOI 2). Neither SVMP was cytotoxic (MTT, LDH release, flow cytometry). In uninfected cells, Neuw-I induced TNF-α and IL-6, whereas Leuc-a did not. Both induced dose-dependent p38 MAPK phosphorylation (1–25 µg/mL), without altering ERK1/2 phosphorylation. Neuw-I also increased NF-κB p65 phosphorylation at 1 and 5 µg/mL. Atto 488-labeled SVMPs were internalized by RAW 264.7 macrophages. In infected cells, both SVMPs reduced TNF-α, IL-6, and IL-1β production at 18 h, without preventing cell death. Both reduced Casp1 expression at 4 and 18 h, regardless of treatment timing. Neuw-I also reduced Nlrp3 expression at both time points and Gsdmd expression following post-treatment. Leuc-a reduced Nlrp3 expression at 4 h following pre-treatment and Gsdmd at 18 h following post-treatment. Additionally, Leuc-a reduced bacterial load at 24 h, regardless of treatment timing. Flow cytometry revealed increased CD40 MFI following Leuc-a post-treatment and reduced CD80 MFI under both conditions. Neuw-I reduced CD80 MFI only following pre-treatment. Overall, Neuw-I and Leuc-a modulate macrophage responses to S. aureus, reducing pro-inflammatory cytokines and inflammasome-related gene expression. Leuc-a additionally decreases bacterial load, highlighting the immunomodulatory potential of these SVMPs.