Transcriptional profiling identifies novel regulators of macrophage polarization.

Kimberline Y Gerrick,Elias R Gerrick,Sarah J Wheelan,Elizabeth M Jaffee,Srinivasan Yegnasubramanian,Anuj Gupta,Manjula Karpurapu

doi:10.1371/journal.pone.0208602

Abstract

Macrophages are key inflammatory immune cells that display dynamic phenotypes and functions in response to their local microenvironment. Major advances have occurred in understanding the transcriptional, epigenetic, and functional differences in various macrophage subsets by in vitro modeling and gene expression and epigenetic profiling for biomarker discovery. However, there is still no standardized protocol for macrophage polarization largely due to the lack of thorough validation of macrophage phenotypes following polarization. In addition, transcriptional regulation is recognized as a major mechanism governing differential macrophage polarization programs and as such, many genes have been identified to be associated with each macrophage subset. However, the functional role of many of these genes in macrophage polarization is still unknown. Moreover, the role of other regulatory mechanisms, such as DNA methylation, in macrophage polarization remains poorly understood. Here, we employed an optimized model of human M1 and M2 macrophage polarization which we used for large-scale transcriptional and DNA methylation profiling. We were unable to demonstrate a role for DNA methylation in macrophage polarization, as no significant changes were identified. However, we observed significant changes in the transcriptomes of M1 and M2 macrophages. Additionally, we identified numerous novel differentially regulated genes involved in macrophage polarization, including CYBB and DHCR7 which we show as important regulators of M1 and M2 macrophage polarization, respectively. Taken together, our improved in vitro human M1 and M2 macrophage model provides new understandings of the regulation of macrophage polarization and candidate macrophage biomarkers.

Highlights

Macrophages serve critical roles as first responders in acute insults where they mediate innate inflammatory responses and influence adaptive immunity
In the process of validating previously published protocols for M1 and M2 macrophage polarization, we found that while the traditional polarization conditions produced M1 macrophages with expected cytokine, cell surface, and gene expression profiles (Fig 1) and M2 macrophages with expected cell surface and gene expression profiles (Fig 1B and 1C), M2 macrophages lacked enhanced secretion of the hallmark M2 cytokines IL-10 and CCL17 when compared to M1 macrophages (Fig 1A)
There was a complete absence of IL-10 secretion from M2 macrophages, which was surprising as IL-10 is a major cytokine for M2 function [25,26,27]

Summary

Introduction

Macrophages serve critical roles as first responders in acute insults (bacterial and viral infections, and early cancerous changes) where they mediate innate inflammatory responses and influence adaptive immunity. They are extremely plastic cells that display heterogeneous phenotypes and functions depending on their environmental cues. They are often simplified into two broad polarization states that mimic the dichotomous Th1/Th2 nomenclature, termed M1 and M2 macrophages, that are the extreme opposites in terms of their phenotypes and functions. Due to the important roles these macrophage subsets serve in homeostatic and disease immunity, it is imperative to understand the underlying molecular and genetic differences for reliable and comprehensive biomarker identification

Methods

Results

Conclusion