Gioacchino Natoli

Transcriptional regulation of macrophage polarization: Enabling diversity with identity

In terms of both phenotype and function, macrophages have remarkable heterogeneity, which reflects the specialization of tissue-resident macrophages in microenvironments as different as liver, brain and bone. Also, marked changes in the activity and gene expression programmes of macrophages can occur when they come into contact with invading microorganisms or injured tissues. Therefore, the macrophage lineage includes a remarkable diversity of cells with different functions and functional states that are specified by a complex interplay between microenvironmental signals and a hardwired differentiation programme that determines macrophage identity. In this Review, we summarize the current knowledge of transcriptional and chromatin-mediated control of macrophage polarization in physiology and disease.

The Histone H3 Lysine-27 Demethylase Jmjd3 Links Inflammation to Inhibition of Polycomb-Mediated Gene Silencing

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. Here we show that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation.

p38-dependent marking of inflammatory genes for increased NF-κB recruitment

We found that inflammatory stimuli induce p38 mitogen-activated protein kinase–dependent phosphorylation and phosphoacetylation of histone H3; this selectively occurred on the promoters of a subset of stimulus-induced cytokine and chemokine genes. p38 activity was required to enhance the accessibility of the cryptic NF-κB binding sites contained in H3 phosphorylated promoters, which indicated that p38-dependent H3 phosphorylation may mark promoters for increased NF-κB recruitment. These results show that p38 plays an additional role in the induction of the inflammatory and immune response: the regulation of NF-κB recruitment to selected chromatin targets.

A Large Fraction of Extragenic RNA Pol II Transcription Sites Overlap Enhancers

A substantial fraction of extragenic Pol II transcription sites coincides with transcriptional enhancers, which may be relevant for functional annotation of mammalian genomes.

Transcriptional regulation via the NF-κB signaling module

Stimulus-induced nuclear factor-κB (NF-κB) activity, the central mediator of inflammatory responses and immune function, comprises a family of dimeric transcription factors that regulate diverse gene expression programs consisting of hundreds of genes. A family of inhibitor of κB (IκB) proteins controls NF-κB DNA-binding activity and nuclear localization. IκB protein metabolism is intricately regulated through stimulus-induced degradation and feedback re-synthesis, which allows for dynamic control of NF-κB activity. This network of interactions has been termed the NF-κB signaling module. Here, we summarize the current understanding of the molecular structures and biochemical mechanisms that determine NF-κB dimer formation and the signal-processing characteristics of the signaling module. We identify NF-κB–κB site interaction specificities and dynamic control of NF-κB activity as mechanisms that generate specificity in transcriptional regulation. We discuss examples of gene regulation that illustrate how these mechanisms may interface with other transcription regulators and promoter-associated events, and how these mechanisms suggest regulatory principles for NF-κB-mediated gene activation.

Identification and Characterization of Enhancers Controlling the Inflammatory Gene Expression Program in Macrophages

Enhancers determine tissue-specific gene expression programs. Enhancers are marked by high histone H3 lysine 4 mono-methylation (H3K4me1) and by the acetyl-transferase p300, which has allowed genome-wide enhancer identification. However, the regulatory principles by which subsets of enhancers become active in specific developmental and/or environmental contexts are unknown. We exploited inducible p300 binding to chromatin to identify, and then mechanistically dissect, enhancers controlling endotoxin-stimulated gene expression in macrophages. In these enhancers, binding sites for the lineage-restricted and constitutive Ets protein PU.1 coexisted with those for ubiquitous stress-inducible transcription factors such as NF-kappaB, IRF, and AP-1. PU.1 was required for maintaining H3K4me1 at macrophage-specific enhancers. Reciprocally, ectopic expression of PU.1 reactivated these enhancers in fibroblasts. Thus, the combinatorial assembly of tissue- and signal-specific transcription factors determines the activity of a distinct group of enhancers. We suggest that this may represent a general paradigm in tissue-restricted and stimulus-responsive gene regulation.

Latent Enhancers Activated by Stimulation in Differentiated Cells

According to current models, once the cell has reached terminal differentiation, the enhancer repertoire is completely established and maintained by cooperatively acting lineage-specific transcription factors (TFs). TFs activated by extracellular stimuli operate within this predetermined repertoire, landing close to where master regulators are constitutively bound. Here, we describe latent enhancers, defined as regions of the genome that in terminally differentiated cells are unbound by TFs and lack the histone marks characteristic of enhancers but acquire these features in response to stimulation. Macrophage stimulation caused sequential binding of stimulus-activated and lineage-determining TFs to these regions, enabling deposition of enhancer marks. Once unveiled, many of these enhancers did not return to a latent state when stimulation ceased; instead, they persisted and mediated a faster and stronger response upon restimulation. We suggest that stimulus-specific expansion of the cis-regulatory repertoire provides an epigenomic memory of the exposure to environmental agents.

Tolerance and M2 (alternative) macrophage polarization are related processes orchestrated by p50 nuclear factor κB

Cells of the monocyte–macrophage lineage play a central role in the orchestration and resolution of inflammation. Plasticity is a hallmark of mononuclear phagocytes, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic or M1 and alternative or M2. NF-κB is a key regulator of inflammation and resolution, and its activation is subject to multiple levels of regulation, including inhibitory, which finely tune macrophage functions. Here we identify the p50 subunit of NF-κB as a key regulator of M2-driven inflammatory reactions in vitro and in vivo. p50 NF-κB inhibits NF-κB–driven, M1-polarizing, IFN-β production. Accordingly, p50-deficient mice show exacerbated M1-driven inflammation and defective capacity to mount allergy and helminth-driven M2-polarized inflammatory reactions. Thus, NF-κB p50 is a key component in the orchestration of M2-driven inflammatory reactions.

Memory and flexibility of cytokine gene expression as separable properties of human T(H)1 and T(H)2 lymphocytes.

CD4+ T cell priming under T helper type I (TH1) or TH2 conditions gives rise to polarized cytokine gene expression. We found that in these conditions human naive T cells acquired stable histone hyperacetylation at either the Ifng or Il4 promoter. Effector memory T cells showed polarized cytokine gene acetylation patterns in vivo, whereas central memory T cells had hypoacetylated cytokine genes but acquired polarized acetylation and expression after appropriate stimulation. However, hypoacetylation of the nonexpressed cytokine gene did not lead to irreversible silencing because most TH1 and TH2 cells acetylated and expressed the alternative gene when stimulated under opposite TH conditions. Such cytokine flexibility was absent in a subset of TH2 cells that failed to up-regulate T-bet and to express interferon-γ when stimulated under TH1 conditions. Thus, most human CD4+ T cells retain both memory and flexibility of cytokine gene expression.

Trained immunity: A program of innate immune memory in health and disease

Training immune cells to remember Classical immunological memory, carried out by T and B lymphocytes, ensures that we feel the ill effects of many pathogens only once. Netea et al.review how cells of the innate immune system, which lack the antigen specificity, clonality, and longevity of T cell and B cells, have some capacity to remember, too. Termed “trained immunity,” the property allows macrophages, monocytes, and natural killer cells to show enhanced responsiveness when they reencounter pathogens. Epigenetic changes largely drive trained immunity, which is shorter lived and less specific than classical memory but probably still gives us a leg up during many infections. Science, this issue p. 10.1126/science.aaf1098 BACKGROUND Host immune responses are classically divided into innate immune responses, which react rapidly and nonspecifically upon encountering a pathogen, and adaptive immune responses, which are slower to develop but are specific and build up immunological memory. The dogma that only adaptive immunity can build immunological memory has recently been challenged by studies showing that innate immune responses in plants and invertebrates (organisms lacking adaptive immune responses) can mount resistance to reinfection. Furthermore, in certain mammalian models of vaccination, protection from reinfection has been shown to occur independently of T and B lymphocytes. These observations led to the hypothesis that innate immunity can display adaptive characteristics after challenge with pathogens or their products. This de facto immunological memory has been termed “trained immunity” or “innate immune memory.” ADVANCES In recent years, emerging evidence has shown that after infection or vaccination, prototypical innate immune cells (such as monocytes, macrophages, or natural killer cells) display long-term changes in their functional programs. These changes lead to increased responsiveness upon secondary stimulation by microbial pathogens, increased production of inflammatory mediators, and enhanced capacity to eliminate infection. Mechanistic studies have demonstrated that trained immunity is based on epigenetic reprogramming, which is broadly defined as sustained changes in transcription programs and cell physiology that do not involve permanent genetic changes, such as mutations and recombination. Histone modifications with chromatin reconfiguration have proven to be a central process for trained immunity, but other mechanisms—such as DNA methylation or modulation of microRNA and/or long noncoding RNA expression—are also expected to be involved. This leads to transcriptional programs that rewire the intracellular immune signaling of innate immune cells but also induce a shift of cellular metabolism from oxidative phosphorylation toward aerobic glycolysis, thus increasing the innate immune cells’ capacity to respond to stimulation. Trained immunity programs have evolved as adaptive states that enhance fitness of the host (e.g., protective effects after infection or vaccination, or induction of mucosal tolerance toward colonizing microorganisms). Proof-of-principle experimental studies support the hypothesis that trained immunity is one of the main immunological processes that mediate the nonspecific protective effects against infections induced by vaccines, such as bacillus Calmette-Guérin or measles vaccination. However, when inappropriately activated, trained immunity programs can become maladaptive, as in postsepsis immune paralysis or autoinflammatory diseases. OUTLOOK The discovery of trained immunity has revealed an important and previously unrecognized property of human immune responses. This advance opens the door for future research to explore trained immunity’s effect on disease, for both diseases with impaired host defense, such as postsepsis immune paralysis or cancers, and autoinflammatory diseases, in which there is inappropriate activation of inflammation. These findings have considerable potential for aiding in the design of new therapeutic strategies, such as new generations of vaccines that combine classical immunological memory and trained immunity, the activation of trained immunity for the treatment of postsepsis immune paralysis or other immune deficiency states, and modulation of exaggerated inflammation in autoinflammatory diseases. Innate immune activation by infections or vaccinations leads to histone modifications and functional reprogramming of cells (such as monocytes, macrophages, or NK cells) termed “trained immunity” or “innate immune memory.” Trained immunity evolved to lead to adaptive states that protect the host during microbial colonization or after infections. However, in certain situations, trained immunity may result in maladaptive states such as postsepsis immune paralysis or hyperinflammation. miRNA, microRNA. The general view that only adaptive immunity can build immunological memory has recently been challenged. In organisms lacking adaptive immunity, as well as in mammals, the innate immune system can mount resistance to reinfection, a phenomenon termed “trained immunity” or “innate immune memory.” Trained immunity is orchestrated by epigenetic reprogramming, broadly defined as sustained changes in gene expression and cell physiology that do not involve permanent genetic changes such as mutations and recombination, which are essential for adaptive immunity. The discovery of trained immunity may open the door for novel vaccine approaches, new therapeutic strategies for the treatment of immune deficiency states, and modulation of exaggerated inflammation in autoinflammatory diseases.