A Proteolytic Axis Linking NLRP3 and MMP-9 Drives IL-1β Processing, NET Formation, and Cell Death in Human Neutrophils

Vinícius Nunes Cordeiro Leal1, Milena Mary de Souza Andrade2, Julia Silva Cantoni1, Maria Notomi Sato2, Alessandra Pontillo1

1. ICB/USP, Laboratory of Immunogenetics, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo.; São Paulo, SP, Brazil.
2. FMUSP, Laboratório de Investigação Médica 56, Faculdade de Medicina da Universidade de São Paulo; São Paulo, SP, Brazil.

Background: Over the past five years, our group has demonstrated that the NLRP3 inflammasome is expressed and activated in neutrophils in response to canonical stimuli and during SARS-CoV-2 infection. We identified that NLRP3 presents unique regulatory features in neutrophils, with MMP-9 acting as a key protease involved in pro-IL-1β processing and NET release. Given these novel insights, we investigated NLRP3/MMP-9 axis activity in neutrophils from people living with HIV (PLWH), a group characterized by chronic inflammation, neutropenia, and altered granulopoiesis.
Methods: Peripheral blood was collected from PLWH (n=50) and matched healthy donors (HD, n=50). NLRP3 activation was assessed ex vivo and in vitro by immunofluorescence, confocal microscopy, immunoblotting, flow cytometry (FC), and ELISA. MMP-9 involvement was studied using a selective inhibitor (JNJ0906) and gelatinolytic assays. Granule content and morphology were evaluated by FC, TEM, and ELISA. NETs were measured by IF of CitH3 and MPO, and cfDNA release by adapted PicoGreen fluorometric assay. HL-60-derived neutrophil-like cells were used for siRNA silencing of NLRP3.
Results: PLWH had neutropenia with increased spontaneous LDH release, NET formation, CD62L shedding, and CD11b expression. MMP-9 was elevated in neutrophils and plasma, confirmed by IF, enzymatic assays, and TEM revealing more tertiary granules. Increased ASC speck formation and NLRP3/ASC colocalization were seen in PLWH, which was enhanced by LPS + Nigericin and inhibited by MCC950 or JNJ0966, indicating a functional NLRP3/MMP-9 axis. HL-60 assays confirmed these findings. Systemic cytokines and clinical parameters correlated with the inflammatory neutrophil phenotype.
Conclusion: Neutrophils from PLWH show dysregulated NLRP3 inflammasome activation associated with MMP-9 activity, contributing to excessive NET formation and IL-1β release. These findings suggest that the NLRP3/MMP-9 axis could serve as a potential biomarker or therapeutic target in chronic inflammatory conditions like HIV.

Agradecimentos: CNPq: 446201/2024-3; 314798/2025-0; FAPESP: 2020/15323-3; 2021/13049-4.