Opposing tumor-cell-intrinsic and -extrinsic roles of the IRF1 transcription factor in antitumor immunity.

Type I interferon (IFN-I) and IFN-γ foster antitumor immunity by facilitating T cell responses. Paradoxically, IFNs may promote T cell exhaustion by activating immune checkpoints. The downstream regulators of these disparate responses are incompletely understood. Here, we describe how interferon regulatory factor 1 (IRF1) orchestrates these opposing effects of IFNs. IRF1 expression in tumor cells blocks Toll-like receptor- and IFN-I-dependent host antitumor immunity by preventing interferon-stimulated gene (ISG) and effector programs in immune cells. In contrast, expression of IRF1 in the host is required for antitumor immunity. Mechanistically, IRF1 binds distinctly or together with STAT1 at promoters of immunosuppressive but not immunostimulatory ISGs in tumor cells. Overexpression of programmed cell death ligand 1 (PD-L1) in Irf1-/- tumors only partially restores tumor growth, suggesting multifactorial effects of IRF1 on antitumor immunity. Thus, we identify that IRF1 expression in tumor cells opposes host IFN-I- and IRF1-dependent antitumor immunity to facilitate immune escape and tumor growth.

Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of stem cell maintenance and neuronal differentiation.

Development of embryonic stem cells (ESCs) into neurons requires intricate regulation of transcription, splicing, and translation, but how these processes interconnect is not understood. We found that polypyrimidine tract binding protein 1 (PTBP1) controls splicing of DPF2, a subunit of BRG1/BRM-associated factor (BAF) chromatin remodeling complexes. Dpf2 exon 7 splicing is inhibited by PTBP1 to produce the DPF2-S isoform early in development. During neuronal differentiation, loss of PTBP1 allows exon 7 inclusion and DPF2-L expression. Different cellular phenotypes and gene expression programs were induced by these alternative DPF2 isoforms. We identified chromatin binding sites enriched for each DPF2 isoform, as well as sites bound by both. In ESC, DPF2-S preferential sites were bound by pluripotency factors. In neuronal progenitors, DPF2-S sites were bound by nuclear factor I (NFI), while DPF2-L sites were bound by CCCTC-binding factor (CTCF). DPF2-S sites exhibited enhancer modifications, while DPF2-L sites showed promoter modifications. Thus, alternative splicing redirects BAF complex targeting to impact chromatin organization during neuronal development.

The transcription factor NF-κB orchestrates nucleosome remodeling during the primary response to Toll-like receptor 4 signaling.

Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regions was conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.

IL-10 constrains sphingolipid metabolism to limit inflammation.

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.

IL-10 constrains sphingolipid metabolism via fatty acid desaturation to limit inflammation.

Unchecked chronic inflammation is the underlying cause of many diseases, ranging from inflammatory bowel disease to obesity and neurodegeneration. Given the deleterious nature of unregulated inflammation, it is not surprising that cells have acquired a diverse arsenal of tactics to limit inflammation. IL-10 is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types; however, the exact mechanism by which IL-10 signaling subdues inflammation remains unclear. Here, we find that IL-10 signaling constrains sphingolipid metabolism. Specifically, we find increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10-deficient macrophages. Genetic deletion of CerS2, the enzyme responsible for VLC ceramide production, limited exacerbated inflammatory gene expression associated with IL-10 deficiency both in vitro and in vivo , indicating that "metabolic correction" is able to reduce inflammation in the absence of IL-10. Surprisingly, accumulation of saturated VLC ceramides was regulated by flux through the de novo mono-unsaturated fatty acid (MUFA) synthesis pathway, where addition of exogenous MUFAs could limit both saturated VLC ceramide production and inflammatory gene expression in the absence of IL-10 signaling. Together, these studies mechanistically define how IL-10 signaling manipulates fatty acid metabolism as part of its molecular anti-inflammatory strategy and could lead to novel and inexpensive approaches to regulate aberrant inflammation.

Cold Spring Harbor Laboratory 2022: emerging insights and viewpoints in immunology.

Some of the current and former organizers of the Cold Spring Harbor Laboratory (CSHL) 'Gene Expression and Signaling in the Immune System' (GESIS) meeting offer opinions on emerging questions in immunology, discussing the strong value of this recurring scientific meeting in the field.

Hepatic GATA4 regulates cholesterol and triglyceride homeostasis in collaboration with LXRs.

GATA4 is a transcription factor known for its crucial role in the development of many tissues, including the liver; however, its role in adult liver metabolism is unknown. Here, using high-throughput sequencing technologies, we identified GATA4 as a transcriptional regulator of metabolism in the liver. GATA4 expression is elevated in response to refeeding, and its occupancy is increased at enhancers of genes linked to fatty acid and lipoprotein metabolism. Knocking out GATA4 in the adult liver (Gata4LKO) decreased transcriptional activity at GATA4 binding sites, especially during feeding. Gata4LKO mice have reduced plasma HDL cholesterol and increased liver triglyceride levels. The expression of a panel of GATA4 binding genes involved in hepatic cholesterol export and triglyceride hydrolysis was down-regulated in Gata4LKO mice. We further demonstrate that GATA4 collaborates with LXR nuclear receptors in the liver. GATA4 and LXRs share a number of binding sites, and GATA4 was required for the full transcriptional response to LXR activation. Collectively, these results show that hepatic GATA4 contributes to the transcriptional control of hepatic and systemic lipid homeostasis.

Oct4:Sox2 binding is essential for establishing but not maintaining active and silent states of dynamically regulated genes in pluripotent cells.

Much has been learned about the mechanisms of action of pluripotency factors Oct4 and Sox2. However, as with other regulators of cell identity, little is known about the impact of disrupting their binding motifs in a native environment or the characteristics of genes they regulate. By quantitatively examining dynamic ranges of gene expression instead of focusing on conventional measures of differential expression, we found that Oct4 and Sox2 enhancer binding is strongly enriched near genes subject to large dynamic ranges of expression among cell types, with binding sites near these genes usually within superenhancers. Mutagenesis of representative Oct4:Sox2 motifs near such active, dynamically regulated genes revealed critical roles in transcriptional activation during reprogramming, with more limited roles in transcriptional maintenance in the pluripotent state. Furthermore, representative motifs near silent genes were critical for establishing but not maintaining the fully silent state, while genes whose transcript levels varied by smaller magnitudes among cell types were unaffected by nearby Oct4:Sox2 motifs. These results suggest that Oct4 and Sox2 directly establish both active and silent transcriptional states in pluripotent cells at a large number of genes subject to dynamic regulation during mammalian development, but are less important than expected for maintaining transcriptional states.

Allogeneic hematopoietic stem cell transplantation: lessons learned by a molecular immunologist/transplant patient.

Much has been learned about the genes and pathways that contribute to a diverse array of hematopoietic malignancies and other hematopoietic diseases. However, for many of these diseases, an allogeneic hematopoietic stem cell (HSC) transplant remains the preferred treatment option. This opinion article provides the perspective of a molecular immunologist who became a transplant patient after many years studying basic mechanisms of blood cell development. Among many lessons learned were the magnitude of racial and ethnic disparities in donor registries, the substantial improvement in outcomes over time that were due to the collective impact of numerous advances, the benefits and limitations of genetic and clinical data, and the remarkably intricate balance between promoting graft-versus-disease activity of donor cells while suppressing graft-versus-host disease (GVHD).

Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2.

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.

Species-Specific Relationships between DNA and Chromatin Properties of CpG Islands in Embryonic Stem Cells and Differentiated Cells.

CpG islands often exhibit low DNA methylation, high histone H3 lysine 4 trimethylation, low nucleosome density, and high DNase I hypersensitivity, yet the rules by which CpG islands are sensed remain poorly understood. In this study, we first evaluated the relationships between the DNA and the chromatin properties of CpG islands in embryonic stem cells using modified bacterial artificial chromosomes. Then, using a bioinformatic approach, we identified strict CpG-island density and length thresholds in mouse embryonic stem and differentiated cells that consistently specify low DNA methylation levels. Surprisingly, the human genome exhibited a dramatically different relationship between DNA properties and DNA methylation levels of CpG islands. Further analysis allowed speculation that this difference is accommodated in part by evolutionary changes in the nucleotide composition of orthologous promoters. Thus, a change in the rules by which CpG-island properties are sensed may have co-evolved with compensatory genome adaptation events during mammalian evolution.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.

Toll-Like Receptors Induce Signal-Specific Reprogramming of the Macrophage Lipidome.

Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems-level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary "shotgun" and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. "Shotgun" lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity.

Single cell analysis reveals immune cell-adipocyte crosstalk regulating the transcription of thermogenic adipocytes.

Immune cells are vital constituents of the adipose microenvironment that influence both local and systemic lipid metabolism. Mice lacking IL10 have enhanced thermogenesis, but the roles of specific cell types in the metabolic response to IL10 remain to be defined. We demonstrate here that selective loss of IL10 receptor α in adipocytes recapitulates the beneficial effects of global IL10 deletion, and that local crosstalk between IL10-producing immune cells and adipocytes is a determinant of thermogenesis and systemic energy balance. Single Nuclei Adipocyte RNA-sequencing (SNAP-seq) of subcutaneous adipose tissue defined a metabolically-active mature adipocyte subtype characterized by robust expression of genes involved in thermogenesis whose transcriptome was selectively responsive to IL10Rα deletion. Furthermore, single-cell transcriptomic analysis of adipose stromal populations identified lymphocytes as a key source of IL10 production in response to thermogenic stimuli. These findings implicate adaptive immune cell-adipocyte communication in the maintenance of adipose subtype identity and function.

Dissection of acute stimulus-inducible nucleosome remodeling in mammalian cells.

Accessibility of the genomic regulatory information is largely controlled by the nucleosome-organizing activity of transcription factors (TFs). While stimulus-induced TFs bind to genomic regions that are maintained accessible by lineage-determining TFs, they also increase accessibility of thousands of cis-regulatory elements. Nucleosome remodeling events underlying such changes and their interplay with basal positioning are unknown. Here, we devised a novel quantitative framework discriminating different types of nucleosome remodeling events in micrococcal nuclease ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data sets and used it to analyze nucleosome dynamics at stimulus-regulated cis-regulatory elements. At enhancers, remodeling preferentially affected poorly positioned nucleosomes while sparing well-positioned nucleosomes flanking the enhancer core, indicating that inducible TFs do not suffice to overrule basal nucleosomal organization maintained by lineage-determining TFs. Remodeling events appeared to be combinatorially driven by multiple TFs, with distinct TFs showing, however, different remodeling efficiencies. Overall, these data provide a systematic view of the impact of stimulation on nucleosome organization and genome accessibility in mammalian cells.

Comparison of reprogramming factor targets reveals both species-specific and conserved mechanisms in early iPSC reprogramming.

BACKGROUND: Both human and mouse fibroblasts can be reprogrammed to pluripotency with Oct4, Sox2, Klf4, and c-Myc (OSKM) transcription factors. While both systems generate pluripotency, human reprogramming takes considerably longer than mouse. RESULTS: To assess additional similarities and differences, we sought to compare the binding of the reprogramming factors between the two systems. In human fibroblasts, the OSK factors initially target many more closed chromatin sites compared to mouse. Despite this difference, the intra- and intergenic distribution of target sites, target genes, primary binding motifs, and combinatorial binding patterns between the reprogramming factors are largely shared. However, while many OSKM binding events in early mouse cell reprogramming occur in syntenic regions, only a limited number is conserved in human. CONCLUSIONS: Our findings suggest similar general effects of OSKM binding across these two species, even though the detailed regulatory networks have diverged significantly.

IL-10 Signaling Remodels Adipose Chromatin Architecture to Limit Thermogenesis and Energy Expenditure.

Signaling pathways that promote adipose tissue thermogenesis are well characterized, but the limiters of energy expenditure are largely unknown. Here, we show that ablation of the anti-inflammatory cytokine IL-10 improves insulin sensitivity, protects against diet-induced obesity, and elicits the browning of white adipose tissue. Mechanistic studies define bone marrow cells as the source of the IL-10 signal and adipocytes as the target cell type mediating these effects. IL-10 receptor alpha is highly enriched in mature adipocytes and is induced in response to differentiation, obesity, and aging. Assay for transposase-accessible chromatin sequencing (ATAC-seq), ChIP-seq, and RNA-seq reveal that IL-10 represses the transcription of thermogenic genes in adipocytes by altering chromatin accessibility and inhibiting ATF and C/EBPß recruitment to key enhancer regions. These findings expand our understanding of the relationship between inflammatory signaling pathways and adipose tissue function and provide insight into the physiological control of thermogenesis that could inform future therapy.

Defined Sensing Mechanisms and Signaling Pathways Contribute to the Global Inflammatory Gene Expression Output Elicited by Ionizing Radiation.

Environmental insults are often detected by multiple sensors that activate diverse signaling pathways and transcriptional regulators, leading to a tailored transcriptional output. To understand how a tailored response is coordinated, we examined the inflammatory response elicited in mouse macrophages by ionizing radiation (IR). RNA-sequencing studies revealed that most radiation-induced genes were strongly dependent on only one of a small number of sensors and signaling pathways, notably the DNA damage-induced kinase ATM, which regulated many IR-response genes, including interferon response genes, via an atypical IRF1-dependent, STING-independent mechanism. Moreover, small, defined sets of genes activated by p53 and NRF2 accounted for the selective response to radiation in comparison to a microbial inducer of inflammation. Our findings reveal that genes comprising an environmental response are activated by defined sensing mechanisms with a high degree of selectivity, and they identify distinct components of the radiation response that might be susceptible to therapeutic perturbation.