Investigating neutrophil responses to stimuli: Comparative analysis of reactive species-dependent and independent mechanisms

Author(s)

Reis, Lorenna Rocha ; Nascimento, Rafaela Oliveira ; Massafera, Mariana Pereira ; Di Mascio, Paolo ; Ronsein, Graziella Eliza

• Document type: Journal article
• Source: REDOX BIOLOGY; v. 81, p. 11-pg., 2025-03-03.

Abstract

Neutrophils play a critical role in immune response, using mechanisms as degranulation, phagocytosis, and the release of extracellular DNA together with microbicidal proteins, the so-called neutrophil extracellular traps (NETs), to combat pathogens. Multiple mechanisms might be involved in neutrophil's response to stimuli, but the biochemical characterization of each different pathway is still lacking. In this study, we used superoxide measurements, live-imaging microscopy and high-resolution proteomics to provide a thorough biochemical characterization of the neutrophil's response following activation by two well-known stimuli, namely phorbol-12myristate-13-acetate (PMA), and ionomycin, a calcium ionophore. Our results demonstrated that although both stimuli induce extracellular DNA release, signals and mediators released by activated cells before this final event were distinct. Thus, PMA-treated neutrophils induce superoxide production, and degranulation of proteins from all granules, especially those derived from secretory vesicles and tertiary granules. On the other hand, ionomycin-treated neutrophils do not stimulate superoxide generation, but induce extensive protein citrullination (also known as arginine deimination), particularly modifying proteins related to actin cytoskeleton organization, nucleus stability, and the NADPH oxidase complex. Interestingly, many of the citrullinated proteins detected in this work were also found to act as autoantigens in autoimmune diseases such as rheumatoid arthritis. These striking differences show neutrophils' response to PMA and ionomycin are two distinct biochemical processes that point towards neutrophils diversification and plasticity responding to the environment. It also provides implications for understanding neutrophil-driven microbial response and potential roles in autoimmune diseases. (AU)

• FAPESP's process: 12/12663-1 - Singlet molecular oxygen and peroxides in chemical biology
• Grantee: Paolo Di Mascio
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 16/00696-3 - Proteomic as a tool to understand HDL function and composition
• Grantee: Graziella Eliza Ronsein
• Support Opportunities: Research Grants - Young Investigators Grants
• FAPESP's process: 23/00995-4 - Development of metrics of quantitative proteomics capable of capturing the functional and compositional complexity of high-density lipoprotein
• Grantee: Graziella Eliza Ronsein
• Support Opportunities: Regular Research Grants