Abstract
Macrophages orchestrate immune responses by sensing and responding to pathogen-associated molecules. These responses are modulated by prior conditioning with cytokines such as interferons (IFNs). Type I and II IFN have opposing functions in many biological scenarios, yet macrophages directly stimulated with Type I or II IFN activate highly overlapping gene expression programs. We hypothesized that a sequential conditioning-stimulation approach would reveal with greater specificity the differential effects of Type I and II IFN on human macrophages. By first conditioning with IFN then stimulating with toll-like receptor ligands and cytokines, followed by genome-wide RNA-seq analysis, we identified 713 genes whose expression was unaffected by IFN alone but showed potentiated or diminished responses to a stimulus after conditioning. For example, responses to the cytokine TNF were restricted by Type II IFN conditioning but potentiated by Type I IFN conditioning. We observed that the effects of IFN were not uniformly pro- or anti-inflammatory, but highly gene-specific and stimulus-specific. By assessing expression levels of key signal transducers and characterizing chromatin accessibility by ATAC-seq, we identify the likely molecular mechanisms underlying Type I and Type II-specific effects, distinguishing between modulation of cytoplasmic signaling networks and the nuclear epigenome that synergistically regulate macrophage immune responses.
Highlights
Macrophages play multiple crucial roles in initiating and coordinating healthy immune responses, and their dysregulation is associated with pathologic processes ranging from atherosclerosis to the cytokine storm seen in sepsis
On day 7, we stimulated the macrophages with the Tolllike receptor (TLR) ligands Pam3CSK, Lipid A (TLR4), and poly(I:C) (TLR3), and the cytokines TNFα and IFNβ in a time course over ten hours, and performed RNA-seq
Principal component analysis (PCA) revealed divergent gene expression patterns for the five stimuli (Fig. 1b), and using K-means clustering we identified nine distinct gene expression clusters based on stimulus-specificity (Fig. 1c)
Summary
Macrophages play multiple crucial roles in initiating and coordinating healthy immune responses, and their dysregulation is associated with pathologic processes ranging from atherosclerosis to the cytokine storm seen in sepsis. First described in the late 1990s, these M1/M2 polarization states are viewed as extremes of a wide spectrum of macrophage phenotypes that are defined by their exposure to diverse cytokine microenvironments[3,4]. In this model, cytokines “condition” macrophages, and the conditioning regimen can either “prime” or “tolerize” cells, respectively potentiating or diminishing their response to a subsequent stimulus. The Type I IFNs play a substantial role in regulating myeloid cell function[14,15]. A variety of mechanisms have been proposed for the opposing roles of Type I and II IFN, such as IFNβ leading to down-regulation of IL-12 and antimicrobial peptides through IL-10, or IFNβ suppression of IFNγ receptor expression[22,24,25]
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