Mechanisms of epigenomic and functional convergence between glucocorticoid- and IL4-driven macrophage programming.

Macrophages adopt distinct phenotypes in response to environmental cues, with type-2 cytokine interleukin-4 promoting a tissue-repair homeostatic state (M2IL4). Glucocorticoids (GC), widely used anti-inflammatory therapeutics, reportedly impart a similar phenotype (M2GC), but how such disparate pathways may functionally converge is unknown. We show using integrative functional genomics that M2IL4 and M2GC transcriptomes share a striking overlap mirrored by a shift in chromatin landscape in both common and signal-specific gene subsets. This core homeostatic program is enacted by transcriptional effectors KLF4 and the glucocorticoid receptor, whose genome-wide occupancy and actions are integrated in a stimulus-specific manner by the nuclear receptor cofactor GRIP1. Indeed, many of the M2IL4:M2GC-shared transcriptomic changes were GRIP1-dependent. Consistently, GRIP1 loss attenuated phagocytic activity of both populations in vitro and macrophage tissue-repair properties in the murine colitis model in vivo. These findings provide a mechanistic framework for homeostatic macrophage programming by distinct signals, to better inform anti-inflammatory drug design.

Broad-spectrum antibiotics disrupt homeostatic efferocytosis.

The clearance of apoptotic cells, termed efferocytosis, is essential for tissue homeostasis and prevention of autoimmunity1. Although past studies have elucidated local molecular signals that regulate homeostatic efferocytosis in a tissue2,3, whether signals arising distally also regulate homeostatic efferocytosis remains elusive. Here, we show that large peritoneal macrophage (LPM) display impairs efferocytosis in broad-spectrum antibiotics (ABX)-treated, vancomycin-treated and germ-free mice in vivo, all of which have a depleted gut microbiota. Mechanistically, the microbiota-derived short-chain fatty acid butyrate directly boosts efferocytosis efficiency and capacity in mouse and human macrophages, and rescues ABX-induced LPM efferocytosis defects in vivo. Bulk messenger RNA sequencing of butyrate-treated macrophages in vitro and single-cell messenger RNA sequencing of LPMs isolated from ABX-treated and butyrate-rescued mice reveals regulation of efferocytosis-supportive transcriptional programmes. Specifically, we find that the efferocytosis receptor T cell immunoglobulin and mucin domain containing 4 (TIM-4, Timd4) is downregulated in LPMs of ABX-treated mice but rescued by oral butyrate. We show that TIM-4 is required for the butyrate-induced enhancement of LPM efferocytosis capacity and that LPM efferocytosis is impaired beyond withdrawal of ABX. ABX-treated mice exhibit significantly worse disease in a mouse model of lupus. Our results demonstrate that homeostatic efferocytosis relies on distal metabolic signals and suggest that defective homeostatic efferocytosis may explain the link between ABX use and inflammatory disease4-7.

Myeloid progenitor dysregulation fuels immunosuppressive macrophages in tumors.

Monocyte-derived macrophages (mo-macs) drive immunosuppression in the tumor microenvironment (TME) and tumor-enhanced myelopoiesis in the bone marrow (BM) fuels these populations. Here, we performed paired transcriptome and chromatin analysis over the continuum of BM myeloid progenitors, circulating monocytes, and tumor-infiltrating mo-macs in mice and in patients with lung cancer to identify myeloid progenitor programs that fuel pro-tumorigenic mo-macs. Analyzing chromatin accessibility and histone mark changes, we show that lung tumors prime accessibility for Nfe2l2 (NRF2) in BM myeloid progenitors as a cytoprotective response to oxidative stress. NRF2 activity is sustained and increased during monocyte differentiation into mo-macs in the lung TME to regulate oxidative stress, in turn promoting metabolic adaptation, resistance to cell death, and contributing to immunosuppressive phenotype. NRF2 genetic deletion and pharmacological inhibition significantly reduced mo-macs' survival and immunosuppression in the TME, enabling NK and T cell therapeutic antitumor immunity and synergizing with checkpoint blockade strategies. Altogether, our study identifies a targetable epigenetic node of myeloid progenitor dysregulation that sustains immunoregulatory mo-macs in the TME.

The potency of hematopoietic stem cell reprogramming for changing immune tone.

Innate immune memory endows innate immune cells with antigen independent heightened responsiveness to subsequent challenges. The durability of this response can be mediated by inflammation induced epigenetic and metabolic reprogramming in hematopoietic stem and progenitor cells (HSPCs) that are maintained through differentiation to mature immune progeny. Understanding the mechanisms and extent of trained immunity induction by pathogens and vaccines, such as BCG, in HSPC remains a critical area of exploration with important implications for health and disease. Here we review these concepts and present new analysis to highlight how inflammatory reprogramming of HSPC can potently alter immune tone, including to enhance specific anti-tumor responses. New findings in the field pave the way for novel HSPC targeting therapeutic strategies in cancer and other contexts of immune modulation. Future studies are expected to unravel diverse and extensive effects of infections, vaccines, microbiota, and sterile inflammation on hematopoietic progenitor cells and begin to illuminate the broad spectrum of immunologic tuning that can be established through altering HSPC phenotypes. The purpose of this review is to draw attention to emerging and speculative topics in this field where we posit that focused study of HSPC in the framework of trained immunity holds significant promise.

Microbial cancer immunotherapy reprograms hematopoietic stem cells to enhance anti-tumor immunity.

Mycobacterium bovis BCG is the vaccine against tuberculosis and an immunotherapy for bladder cancer. When administered intravenously, BCG reprograms bone marrow hematopoietic stem and progenitor cells (HSPCs), leading to heterologous protection against infections. Whether HSPC-reprogramming contributes to the anti-tumor effects of BCG administered into the bladder is unknown. We demonstrate that BCG administered in the bladder in both mice and humans reprograms HSPCs to amplify myelopoiesis and functionally enhance myeloid cell antigen presentation pathways. Reconstitution of naive mice with HSPCs from bladder BCG-treated mice enhances anti-tumor immunity and tumor control, increases intratumor dendritic cell infiltration, reprograms pro-tumorigenic neutrophils, and synergizes with checkpoint blockade. We conclude that bladder BCG acts systemically, reprogramming HSPC-encoded innate immunity, highlighting the broad potential of modulating HSPC phenotypes to improve tumor immunity.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Transcriptional Activation of Regenerative Hematopoiesis via Vascular Niche Sensing.

Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine.

Histone variant H3.3 maintains adult haematopoietic stem cell homeostasis by enforcing chromatin adaptability.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability.

HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies.

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

Histone H3.3 phosphorylation amplifies stimulation-induced transcription.

Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1-3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4-7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in 'canonical' H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and 'ejects' the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate the connections between inflammatory signaling pathways and chromatin through genomic footprinting of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

Greater Than the Sum of Parts: Complexity of the Dynamic Epigenome.

Information encoded in DNA is interpreted, modified, and propagated as chromatin. The diversity of inputs encountered by eukaryotic genomes demands a matching capacity for transcriptional outcomes provided by the combinatorial and dynamic nature of epigenetic processes. Advances in genome editing, visualization technology, and genome-wide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to long-standing questions and presenting new challenges. Here, we review recent findings, exemplified by the emerging understanding of crossregulatory interactions within chromatin, and emphasize the pathologic outcomes of epigenetic misregulation in cancer.

Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification.

Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to preaccessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3-bound Treg cell enhancers lacking in Foxp3(-) CD4(+) cells became accessible upon T cell receptor activation prior to Foxp3 expression, and only a small subset associated with several functionally important genes were exclusively Treg cell specific. Thus, in a late cellular differentiation process, Foxp3 defines Treg cell functionality in an "opportunistic" manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.

Regulatory T cells: mechanisms of differentiation and function.

The immune system has evolved to mount an effective defense against pathogens and to minimize deleterious immune-mediated inflammation caused by commensal microorganisms, immune responses against self and environmental antigens, and metabolic inflammatory disorders. Regulatory T (Treg) cell-mediated suppression serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and autoinflammatory disorders, allergy, acute and chronic infections, cancer, and metabolic inflammation. The discovery that Foxp3 is the transcription factor that specifies the Treg cell lineage facilitated recent progress in understanding the biology of regulatory T cells. In this review, we discuss cellular and molecular mechanisms in the differentiation and function of these cells.