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Investigating Neutrophil Responses to Stimuli: Comparative Analysis of reactive species- dependent and independent mechanisms

ABSTRACT: Neutrophils play a critical role in immune response, using mechanisms as degranulation, and release of neutrophil extracellular traps (NETs) to combat pathogens. NETs are web-like structures composed of chromatin and microbicidal proteins, released by neutrophils in response to many different stimuli. Recent evidences suggest 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 careful biochemical characterization of the neutrophil’s response following activation by two commonly used, well characterized stimuli, namely phorbol-12-myristate-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 particularly in the 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 might exemplify distinct response to the environment. It also provides implications for understanding neutrophil-driven microbial response and potential roles in autoimmune diseases.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Blood

SUBMITTER: Rafaela Nascimento

LAB HEAD: Graziella Eliza Ronsein

PROVIDER: PXD057968 | Pride | 2025-03-03

REPOSITORIES: Pride

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			Action	DRS
	Combined.zip	Other
	Combined_lysate.zip	Other
	Ind1_220823_Lysate_Control_1_30min.raw	Raw
	Ind1_220823_Lysate_Control_1_90min.raw	Raw
	Ind1_220823_Lysate_Control_2_30min.raw	Raw

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Project description:Background: Our purpose was to obtain non-existing genome-wide expression data for the rabbit species on the responses of peripheral blood mononuclear cells (PBMCs) after in vitro stimulation by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) and ionomycin. This transcriptomic study was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome. Results: The LPS affected 15 to 20 less genes than PMA/Ionomycin after 4 hours (T4) or 24 hours (T24), of in vitro stimulation, by comparison with mock-stimulated PBMCs. LPS induced an inflammatory response as shown by a significant up-regulation of IL12A and CXCL11 at T4, followed by an increased transcription of IL6, IL1B, IL1A, IL36, IL37, TNF, CCL4 and SAA1 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24. PMA/ionomycin induced a very early expression of Th1, Th2, iTreg, and Th17 responses by PBMCs at T4. The Th1 response was increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines that significantly decreased from T4 to 24 (IL2, IL4, IL10, IL13, IL17A, CD69). The granulocyte-macrophage colony-stimulating factor (CSF2) was by far the most over-expressed gene at both T4 and T24 by comparison to mock-stimulated cells, confirming a major impact of PMA/ionomycin on cell growth and proliferation. A significant down-regulation of IL16 was observed at T4 and T24, in agreement with a role of IL16 in in PBMC apoptosis. Conclusion: We report new data on the responses of PBMCs to LPS and PMA/ionomycin for the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with highly performing genomic tools should pave the way for more intense genomic studies for this species known to be a very relevant animal model in immunology and physiology. To characterize gene expression changes in peripheral blood mononuclear cells (PBMCs), we collected blood from 4 adult rabbits. PBMCs were isolated and stimulated with LPS or a mixture of PMA/Ionomycin, versus a control group (C). Cells were harvested either 4h or 24h after activation.

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Investigating Neutrophil Responses to Stimuli: Comparative Analysis of reactive species- dependent and independent mechanisms

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