In vitro polarized human neutrophils in pro and anti-inflammatory profiles show different patterns of epigenetic changes

Author(s)

Cícero José Luíz dos Ramos Almeida

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-07-02

Examining board members

Fabiani Gai Frantz; Luciane Alarcão Dias Melicio; Ester Silveira Ramos

• Advisor: Fabiani Gai Frantz

Abstract

Epigenetic changes are, by definition, inheritable changes to the functional genome that do not alter the nucleotide sequence of DNA. Among some events under epigenetic control, cell differentiation in macrophages and T cells are marked by changes such as histone methylation / acetylation to differentiate into M1, M2, Th1, Th2 and Th17, for example. The innate and adaptive immune system plasticity is under epigenetic control, when the infectious or inflammatory microenvironment defines the cell differentiation or polarization profile. In the tumor environment, polarized neutrophils were identified, associated with tumor control or susceptibility. Controversial roles have also been attributed to neutrophils in tuberculosis, even inflammatory, or anti-inflammatory. Our hypothesis is that the neutrophil polarization in these opposite profiles is under epigenetic regulation. Therefore, our objective was to investigate which epigenetic changes are involved the in vitro polarization of neutrophils, according to the inflammatory microenvironment. Therefore, neutrophils were isolated from the peripheral blood of 20 healthy participants. These cells were polarized in vitro with GM-CSF and IFN-γ to differentiate cells to the pro-inflammatory profile (NI) or with IL-4, IL-13 and TGF-β to differentiate cells to the anti-inflammatory profile (NonI). Neutrophils were characterized by phenotypic analysis and epigenetic signature of each cell profile. Phenotypic analysis consisted of morphological analysis, reactive oxygen species (ROS) and cytokine production, and cell surface receptor and cytokine gene expression. The epigenetic signature was performed through the analysis of the global DNA methylation profile, expression of epigenetic enzymes and histone methylation and acetylation. After 2h of polarization, NI have nuclear morphological changes, increase in cell size and increase in ROS production when compared to NonI. Additionally, NI have increased IL-8 production, as well as increased TNF-α, IL-10, TLR2 and TLR4 gene expression when compared to NonI. In addition, NonI have higher gene expression of ALOX15 compared to NI neutrophils. As for the epigenetic signature of cells, NI neutrophils have a lower percentage of global methylation when compared to other profiles, and NonI cells have greater gene expression of the DNMT3A enzyme when compared to NI neutrophils. The frequency of histone changes related to activation and repression of gene transcription also indicates possible modulation of the expression of the TNF-α, IL-6 e IL-1β genes. Thus, we suggest that the tissue microenvironment can influence the differentiation of the neutrophil into pro or anti-inflammatory profile and that epigenetic changes are involved in cellular plasticity. (AU)

• FAPESP's process: 18/12641-4 - Determination of the epigenetic signature related to human neutrophil polarization to N1 and N2 profiles
• Grantee: Cícero José Luíz dos Ramos Almeida
• Support Opportunities: Scholarships in Brazil - Master