Investigation of the intrinsic mechanisms to NETs release through proteomics

Author(s)

Lorenna Rocha Reis

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2023-06-01

Examining board members

Graziella Eliza Ronsein; Flavia Carla Meotti; André Zelanis Palitot Pereira

• Advisor: Graziella Eliza Ronsein

Abstract

Neutrophil extracellular traps (NETs) are webs of DNA and cytotoxic proteins released by neutrophils to kill pathogens through a process called NETosis. Studies have shown that the mechanisms of NETosis might be stimulus-dependent. Thus, the stimulus phorbol-12-myristate-13-acetate (PMA) lead to NETs release through activation of the NADPH oxidase, while NETosis caused by calcium ionophores such as ionomycin, occurs without NADPH oxidase activation. Regardless the stimulus, a series of biochemical and structural changes need to occur in neutrophils before the event of NETs release, including chromatin decondensation, and degranulation of cytotoxic proteins. These events are not fully understood, but recent studies have shown cytotoxic proteins associated with NETs can aggravate pathologies and cause vascular damage. Here, we used proteomics studies, live imaging, and fixed microscopy to investigate the early events of NETosis from two different angles. First, an investigation of the biochemical changes that occur upon ionomycin treatment before NETs release was undertaken. Thus, live imaging microscopy showed ionomycin-treated neutrophils rapidly lose their polymorphic nucleus. Moreover, cell fractionation together with proteomic studies pointed to profound biochemical changes seen in and around neutrophil nucleus after 2 minutes of ionomycin treatment, such as cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Second, a comparison of the neutrophils response to three different stimuli, namely PMA, ionomycin, and the peptide N-formylmethionyl-leucyl-phenylalanine (fMLP) was undertaken. Interestingly, the release of NETs occurred after 30 minutes of ionomycin treatment, and after 90 minutes of PMA stimulation. We monitored the cells for up to 120 minutes and the chemotactic peptide fMLP did not induce NETs release. Moreover, only fMLP- and PMA-treated neutrophils activated NADPH oxidase. The analysis of neutrophils secreted proteins after each distinct treatment showed that PMA and fMLP-stimulated neutrophils released more proteins related to neutrophil adhesion and migration, and positive regulation of superoxide activity. In contrast, ionomycin-treated neutrophils had a more cytotoxic secretome, given the enrichment of proteins from azurophilic granules. In conclusion, by exploring the events that happen before NETs release, our studies revealed biochemical mediators involved in this process, and showed that despite leading to the same final outcome, PMA and ionomycin trigger different responses in stimulated neutrophils. (AU)

• FAPESP's process: 17/17270-1 - Study of the effect of HDL on activated neutrophils
• Grantee: Lorenna Rocha Reis
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)