Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages.

H.R Bao,F Li,J.L Chen,X.L Zeng,X.J Liu

doi:10.1590/1414-431x20209207

Abstract

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Highlights

Chronic obstructive pulmonary disease (COPD) poses a serious threat to human health and is showing a gradual rise in incidence and mortality rates [1]
After 72 h of transfection, enhanced green fluorescent protein (EGFP) could be observed on transfected cells under the inverted fluorescence microscope with transfection efficiencies 480% (Figure 1) and gene-silencing efficiencies of 82.1±2.1 and 81.5±2.3% for phosphoinositide 3-kinases (PI3K)-RNA interference (RNAi) and Mammalian target of rapamycin (mTOR)-RNAi, respectively
79.97±1.07 79.60±1.17 70.73±2.66aabb 87.72±1.58aabb aa Po0.01 compared with the blank group, bbPo0.01 compared with the negative control (ANOVA and LSD t-test)

Summary

Introduction

Chronic obstructive pulmonary disease (COPD) poses a serious threat to human health and is showing a gradual rise in incidence and mortality rates [1]. It is expected to rank third in the world in terms of incidence and mortality rates by 2020, imposing huge economic burdens [2]. COPD is a complex and heterogeneous disease at the clinical and cellular levels, with airflow obstruction and a progressive decline in lung function, including smallairway obstructions and emphysema [3]. Recent studies found that a decline in the phagocytic capacity of alveolar macrophages (AM) reduced their bacterial eradication rate and led to frequent acute exacerbations in patients with COPD and asthma [4,5]. It is important to investigate the exact mechanism underlying the weakened phagocytic capacity of AM, in order to understand the acute exacerbations of COPD

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