Brain trauma elicits non-canonical macrophage activation states.

Charles C Kim,Christine L Hsieh,Mary C Nakamura

doi:10.1186/s12974-016-0581-z

Abstract

BackgroundMacrophage polarization programs, commonly referred to as “classical” and “alternative” activation, are widely considered as distinct states that are exclusive of one another and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. However, previous studies have not distinguished whether the simultaneous presence of both classical and alternative activation signatures represents the admixture of differentially polarized macrophages or if they have adopted a unique state characterized by components of both classical and alternative activation.MethodsWe analyzed the gene expression profiles of individual monocyte-derived brain macrophages responding to TBI using single-cell RNA sequencing. RNA flow cytometry was used as another single-cell analysis technique to validate the single-cell RNA sequencing results.ResultsThe analysis of signature polarization genes by single-cell RNA sequencing revealed the presence of diverse activation states, including M(IL4), M(IL10), and M(LPS, IFNγ). However, the expression of a given polarization marker was no more likely than at random to predict simultaneous expression or repression of markers of another polarization program within the same cell, suggesting a lack of exclusivity in macrophage polarization states in vivo in TBI. Also unexpectedly, individual TBI macrophages simultaneously expressed high levels of signature polarization genes across two or three different polarization states and in several distinct and seemingly incompatible combinations.ConclusionsSingle-cell gene expression profiling demonstrated that monocytic macrophages in TBI are not comprised of distinctly polarized subsets but are uniquely and broadly activated. TBI macrophage activation in vivo is deeply complex, with individual cells concurrently adopting both inflammatory and reparative features with a lack of exclusivity. These data provide physiologically relevant evidence that the early macrophage response to TBI is comprised of novel activation states that are discordant with the current paradigm of macrophage polarization—a key consideration for therapeutic modulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0581-z) contains supplementary material, which is available to authorized users.

Highlights

Macrophage polarization programs, commonly referred to as “classical” and “alternative” activation, are widely considered as distinct states that are exclusive of one another and are associated with different functions such as inflammation and wound healing, respectively
Validation of RNA sequencing (RNAseq) profiles of single monocytes responding to traumatic brain injury (TBI) Our previous approach of bulk-profiling-purified brain macrophages responding to experimental TBI demonstrated that macrophages expressed a mixture of signature polarization genes representing both classical and alternative activation [6]. It was unclear whether this mixed polarization signature reflected: (1) a mixture of classically and alternatively activated macrophage subsets, (2) an intermediate polarization state of macrophages that are transitioning between states, or (3) unusual subsets of macrophages adopting a stable state distinct from our current definitions of polarization
Cells (Fig. 1a) [20], which we demonstrated are a population of infiltrating macrophages that fail to increase in Ccr2−/− mice 1 day after TBI [21]

Summary

Introduction

Macrophage polarization programs, commonly referred to as “classical” and “alternative” activation, are widely considered as distinct states that are exclusive of one another and are associated with different functions such as inflammation and wound healing, respectively. In a number of disease contexts, such as traumatic brain injury (TBI), macrophage polarization influences the extent of pathogenesis, and efforts are underway to eliminate pathogenic subsets. In traumatic brain injury (TBI), which accounts for 30 % of deaths related to injury [1, 2] and impacts an estimated 2 % of the US population who live with persistent disabilities resulting from TBI [1,2,3], neuroinflammation has been recognized as a pathogenic factor and has garnered significant attention as a potential target for therapy [1, 3,4,5]. Identification of the precise molecular pathways and cellular subsets that impact pathology and/or cognitive recovery following TBI could lead to therapies that directly antagonize harmful mechanisms while preserving, or even enriching, beneficial mechanisms

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