rFIP-nha activates macrophages towards a pro-inflammatory phenotype via AIM2 inflammasome modulation

Area of Science:

• Mycology
• Immunology
• Molecular Biology

Background:

• Fungal immunomodulatory proteins (FIPs) exhibit significant immunomodulatory effects.
• FIP-nha from *Nectria haematococca* shows thermostability and anti-tumour activity, but its macrophage interaction mechanism is unknown.
• Glycosylation sites in FIP-nha may influence its biological functions.

Purpose of the Study:

• To investigate the immunomodulatory activity of FIP-nha on macrophages.
• To elucidate the mechanism underlying FIP-nha-induced macrophage activation.
• To determine the role of glycosylation in FIP-nha's immunomodulatory functions.

Main Methods:

• Recombinant expression of FIP-nha glycan mutants (N5A, N39A, N5+39A) in *Pichia pastoris*.
• Assessment of THP-1 macrophage phagocytosis, cytokine secretion (IL-1β, IL-12, IL-10), and gene expression.
• Analysis of inflammasome (NLRP1, NLRP3, NLRC4, AIM2) activation and dependence on the ASC signaling pathway using THP1-KO-ASC cells and inhibitors.

Main Results:

• Recombinant FIP-nha and its glycan mutants reduced macrophage phagocytosis but significantly induced pro-inflammatory cytokine secretion (IL-1β, IL-12, IL-10).
• No significant differences in immunomodulatory activity were observed between wild-type FIP-nha and its glycan mutants, suggesting glycosylation-independent mechanisms.
• FIP-nha significantly upregulated AIM2 inflammasome transcription, and IL-1β release was dependent on the ASC signaling pathway.

Conclusions:

• FIP-nha activates THP-1 macrophages, promoting a pro-inflammatory response.
• The immunomodulatory activity of FIP-nha is independent of its glycosylation.
• FIP-nha activates macrophages through the AIM2 inflammasome and ASC signaling pathway.