Distinct Redox Signalling following Macrophage Activation Influences Profibrotic Activity

Barbara K Kemp-Harper,Grant R Drummond,Antony Vinh,Chrishan S Samuel,Caitlin V Lewis,Henry Diep

doi:10.1155/2019/1278301

Abstract

Aims To date, the ROS-generating capacities of macrophages in different activation states have not been thoroughly compared. This study is aimed at determining the nature and levels of ROS generated following stimulation with common activators of M1 and M2 macrophages and investigating the potential for this to impact fibrosis. Results Human primary and THP-1 macrophages were treated with IFN-γ+LPS or IL-4-activating stimuli, and mRNA expression of established M1 (CXCL11, CCR7, IL-1β) and M2 (MRC-1, CCL18, CCL22) markers was used to confirm activation. Superoxide generation was assessed by L-012-enhanced chemiluminescence and was increased in both M(IFN-γ+LPS) and M(IL-4) macrophages, as compared to unpolarised macrophages (MΦ). This signal was attenuated with NOX2 siRNA. Increased expression of the p47phox and p67phox subunits of the NOX2 oxidase complex was evident in M(IFN-γ+LPS) and M(IL-4) macrophages, respectively. Amplex Red and DCF fluorescence assays detected increased hydrogen peroxide generation following stimulation with IL-4, but not IFN-γ+LPS. Coculture with human aortic adventitial fibroblasts revealed that M(IL-4), but not M(IFN-γ+LPS), enhanced fibroblast collagen 1 protein expression. Macrophage pretreatment with the hydrogen peroxide scavenger, PEG-catalase, attenuated this effect. Conclusion We show that superoxide generation is not only enhanced with stimuli associated with M1 macrophage activation but also with the M2 stimulus IL-4. Macrophages activated with IL-4 also exhibited enhanced hydrogen peroxide generation which in turn increased aortic fibroblast collagen production. Thus, M2 macrophage-derived ROS is identified as a potentially important contributor to aortic fibrosis.

Highlights

Macrophages are key cells of the innate immune system and their activation and function is important in tissue homeostasis, disease pathogenesis, and immune regulation [1]
The healing process becomes pathological when it is continuous, leading to remodeling of the extracellular matrix. Macrophages recruited for these processes, termed “M2” or “alternatively activated” macrophages, are commonly activated by Th2 cytokines, anti-inflammatory cytokines, and growth factors [1]. This is of particular relevance in the setting of hypertension in which we have demonstrated the accumulation of M2 macrophages, as indicated by expression of the marker CD206, in the vessel wall associated with aortic stiffening and increased collagen deposition [14]
Of particular relevance to aortic stiffening, an important process involved in the development and clinical consequences of hypertension [24, 25], we reveal a potential role for M2 macrophage-derived hydrogen peroxide in promoting profibrotic responses in aortic adventitial fibroblasts

Summary

Introduction

Macrophages are key cells of the innate immune system and their activation and function is important in tissue homeostasis, disease pathogenesis, and immune regulation [1]. Macrophages exist as a heterogeneous population, continually responding to local microenvironment cues [2]. Representing either ends of a spectrum of activation or “polarisation,” M1 and M2 macrophages are thought to play predominant roles in inflammation and tissue repair, respectively, this paradigm is considered overly simplified [1,2,3]. Understanding how macrophages respond to different cytokines and factors in the microenvironment can inform on their contribution to various disease states and identify new therapeutic targets. The reactive oxygen species (ROS)-generating capacities of macrophages in different activation states have not been thoroughly compared

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Conclusion