Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

Yongjian Wu,Dandan Li,Lianqiang Feng,Kang Chen,Wenting Qu,Minhao Wu,Xi Huang,Ngiambudulu M Francisco,Yi Wang,Xi Liu,Yuanqing Zhang

doi:10.3389/fimmu.2018.00211

Abstract

Beta-defensins 2 and 3 (BD2 and BD3) are inducible peptides present at the sites of infection, and they are well characterized for their antimicrobial activities and immune-regulatory functions. However, no study has thoroughly investigated their immunomodulatory effects on macrophage-mediated immune responses against Pseudomonas aeruginosa (PA). Here, we use THP-1 and RAW264.7 cell lines and demonstrate that BD2 and BD3 suppressed macrophage autophagy but enhanced the engulfment of PA and Zymosan bioparticles as well as the formation of phagolysosomes, using immunofluorescence staining and confocal microscopy. Plate count assay showed that macrophage-mediated phagocytosis and intracellular killing of PA were promoted by BD2 and BD3. Furthermore, microarray and real-time PCR showed that the expression of two genes, early growth response gene-1 (EGR1) and c-FOS, was attenuated by BD2 and BD3. Western blot revealed that BD2 and BD3 inhibited the expression and nuclear translocation of EGR1 and c-FOS. Knockdown of EGR1 and c-FOS by siRNA transfection suppressed macrophage autophagy before and after PA infection; while overexpression of these two transcription factors enhanced autophagy but reversed the role of BD2 and BD3 on macrophage-mediated PA eradication. Together, these results demonstrate a novel immune defense activity of BD2 and BD3, which promotes clearance of PA by inhibiting macrophage autophagy through downregulation of EGR1 and c-FOS.

Highlights

Pseudomonas aeruginosa (PA) is an extracellular Gram-negative bacterium commonly found in the environment [1]
These data indicated that PA infection induced Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) expression in macrophage
We found that the internalization percentages of PA were raised to 30.6 and 33.3% in THP-1 macrophages pretreated with BD2 or BD3 peptides, respectively; which were higher than the unstimulated control (14.8%) (Figure S1 in Supplementary Material)

Summary

Introduction

Pseudomonas aeruginosa (PA) is an extracellular Gram-negative bacterium commonly found in the environment [1]. It is reported that BDs are capable of electrostatically interacting with negatively charged components in microbial cell walls, by increasing the permeability of cell walls and leading to a host cell death [9]. In addition to their antimicrobial function, BDs function as inflammatory mediators, with impact on the immune activation of epithelial cells and immunocytes [10, 11], production of inflammatory cytokines [12, 13], as well as induction of chemotaxis [7, 14]. Though the evidence implicating BDs in bacterial pathogenesis is mounting, little is known regarding the immunoregulatory mechanism of BD2 and BD3 in bacterial clearance

Methods

Results

Conclusion