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

## TL;DR

A fungal protein called rFIP-nha activates macrophages to behave pro-inflammatorily by modulating the AIM2 inflammasome.

## Contribution

The study reveals that rFIP-nha activates macrophages via the AIM2 inflammasome, independent of glycosylation.

## Key findings

- rFIP-nha and its glycan mutants induce pro-inflammatory cytokine secretion in THP-1 macrophages.
- AIM2 inflammasome is 10-fold upregulated and essential for IL-1β release induced by rFIP-nha.
- Glycosylation does not significantly affect the immunomodulatory activity of rFIP-nha.

## Abstract

Fungal immunomodulatory proteins (FIPs) are small proteins from fungi with considerable immunomodulatory activity. FIP-nha (Nectria haematococca) contains two glycosylation sites at positions N5 and N39, and displays a high thermostability and notable anti-tumour activity. However, FIP-nha’s immunomodulatory activity on macrophages and the associated mechanism remain unclear. In this study, three rFIP-nha glycan mutants (N5A, N39A, N5+39A) were recombinantly expressed in Pichia pastoris. To test the impact on FIP-nha’s immunomodulatory activity, the phagocytotic activity, cytokine secretion, and gene expression of THP-1 macrophages were investigated. rFIP-nha and its mutants reduced macrophage phagocytosis, and induced IL-1β, IL-12 and IL-10 cytokine secretion significantly, indicating that the protein confers a pro-inflammatory behaviour on THP-1 macrophages. However, there were no obvious differences among the different glycan mutants, indicating that the observed activation mechanisms are likely glycosylation-independent. Furthermore, to study the immunomodulatory mechanism, four kinds of inflammasomes (NLRP1, NLRP3, NLRC4 and AIM2) were tested at transcriptional level. AIM2 was found to be 10-fold upregulated. Then, THP1-KO-ASC cells and AIM2 related inhibitors showed that IL-1β release induced by rFIP-nha is ASC signalling pathway dependent. Taken together, these findings suggest that rFIP-nha activates THP-1 macrophages in a pro-inflammatory way by activating the AIM2 inflammasome.