Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species

Agnieszka Mroczek,Aneta Manda-Handzlik,Małgorzata Wachowska,Angelika Muchowicz,Weronika Kuźmicka,Adrianna Cieloch

doi:10.3390/ijms21155194

Abstract

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils’ antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.

Highlights

In the last two decades, scientists have begun to pay attention to adaptive and to innate immunity as a key player in the outcome of various diseases
In order to investigate the role of autophagy/ATG5 in neutrophils functions, HL-60 cells were transduced with a lentiviral expression system
We employed a model based on a genetic modification of granulocytic-like cells

Summary

Introduction

In the last two decades, scientists have begun to pay attention to adaptive and to innate immunity as a key player in the outcome of various diseases. Neutrophils are directed by cytokines and other stimuli into infected tissues, where they eliminate invading microbes [1]. The role of these cells was underestimated for a long time, though recent research uncovered that neutrophils are sophisticated immune cells. They are able to precisely control enzymes in the granules, release chemokines and interact with several constituents of the immune system, including the adaptive branch. Neutrophils eliminate microbes through a variety of mechanisms such as phagocytosis, the production of reactive oxygen species (ROS), degranulation and neutrophil extracellular trap formation (NETs) [2]

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