TET Enzymes in the Immune System: From DNA Demethylation to Immunotherapy, Inflammation, and Cancer.

Ten-eleven translocation (TET) proteins are iron-dependent and α-ketoglutarate-dependent dioxygenases that sequentially oxidize the methyl group of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). All three epigenetic modifications are intermediates in DNA demethylation. TET proteins are recruited by transcription factors and by RNA polymerase II to modify 5mC at enhancers and gene bodies, thereby regulating gene expression during development, cell lineage specification, and cell activation. It is not yet clear, however, how the established biochemical activities of TET enzymes in oxidizing 5mC and mediating DNA demethylation relate to the known association of TET deficiency with inflammation, clonal hematopoiesis, and cancer. There are hints that the ability of TET deficiency to promote cell proliferation in a signal-dependent manner may be harnessed for cancer immunotherapy. In this review, we draw upon recent findings in cells of the immune system to illustrate established as well as emerging ideas of how TET proteins influence cellular function.

The Myeloid Cell Compartment-Cell by Cell.

Myeloid cells are a major cellular compartment of the immune system comprising monocytes, dendritic cells, tissue macrophages, and granulocytes. Models of cellular ontogeny, activation, differentiation, and tissue-specific functions of myeloid cells have been revisited during the last years with surprising results; for example, most tissue macrophages are yolk sac derived, monocytes and macrophages follow a multidimensional model of activation, and tissue signals have a significant impact on the functionality of all these cells. While these exciting results have brought these cells back to center stage, their enormous plasticity and heterogeneity, during both homeostasis and disease, are far from understood. At the same time, the ongoing revolution in single-cell genomics, with single-cell RNA sequencing (scRNA-seq) leading the way, promises to change this. Prevailing models of hematopoiesis with distinct intermediates are challenged by scRNA-seq data suggesting more continuous developmental trajectories in the myeloid cell compartment. Cell subset structures previously defined by protein marker expression need to be revised based on unbiased analyses of scRNA-seq data. Particularly in inflammatory conditions, myeloid cells exhibit substantially vaster heterogeneity than previously anticipated, and work performed within large international projects, such as the Human Cell Atlas, has already revealed novel tissue macrophage subsets. Based on these exciting developments, we propose the next steps to a full understanding of the myeloid cell compartment in health and diseases.

Purine Release, Metabolism, and Signaling in the Inflammatory Response.

ATP, NAD+, and nucleic acids are abundant purines that, in addition to having critical intracellular functions, have evolved extracellular roles as danger signals released in response to cell lysis, apoptosis, degranulation, or membrane pore formation. In general ATP and NAD+ have excitatory and adenosine has anti-inflammatory effects on immune cells. This review focuses on recent advances in our understanding of purine release mechanisms, ectoenzymes that metabolize purines (CD38, CD39, CD73, ENPP1, and ENPP2/autotaxin), and signaling by key P2 purinergic receptors (P2X7, P2Y2, and P2Y12). In addition to metabolizing ATP or NAD+, some purinergic ectoenzymes metabolize other inflammatory modulators, notably lysophosphatidic acid and cyclic GMP-AMP (cGAMP). Also discussed are extracellular signaling effects of NAD+ mediated by ADP-ribosylation, and epigenetic effects of intracellular adenosine mediated by modification of S-adenosylmethionine-dependent DNA methylation.

Gut Microbiota Regulation of T Cells During Inflammation and Autoimmunity.

The intestinal microbiota plays a crucial role in influencing the development of host immunity, and in turn the immune system also acts to regulate the microbiota through intestinal barrier maintenance and immune exclusion. Normally, these interactions are homeostatic, tightly controlled, and organized by both innate and adaptive immune responses. However, a combination of environmental exposures and genetic defects can result in a break in tolerance and intestinal homeostasis. The outcomes of these interactions at the mucosal interface have broad, systemic effects on host immunity and the development of chronic inflammatory or autoimmune disease. The underlying mechanisms and pathways the microbiota can utilize to regulate these diseases are just starting to emerge. Here, we discuss the recent evidence in this area describing the impact of microbiota-immune interactions during inflammation and autoimmunity, with a focus on barrier function and CD4+ T cell regulation.

Understanding Human Autoimmunity and Autoinflammation Through Transcriptomics.

Transcriptomics, the high-throughput characterization of RNAs, has been instrumental in defining pathogenic signatures in human autoimmunity and autoinflammation. It enabled the identification of new therapeutic targets in IFN-, IL-1- and IL-17-mediated diseases. Applied to immunomonitoring, transcriptomics is starting to unravel diagnostic and prognostic signatures that stratify patients, track molecular changes associated with disease activity, define personalized treatment strategies, and generally inform clinical practice. Herein, we review the use of transcriptomics to define mechanistic, diagnostic, and predictive signatures in human autoimmunity and autoinflammation. We discuss some of the analytical approaches applied to extract biological knowledge from high-dimensional data sets. Finally, we touch upon emerging applications of transcriptomics to study eQTLs, B and T cell repertoire diversity, and isoform usage.

Maternal inflammation regulates fetal emergency myelopoiesis.

Neonates are highly susceptible to inflammation and infection. Here, we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPCs) respond to inflammation, testing the hypothesis that deficits in the engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical adult-like EM transcriptional program. Moreover, we find that fetal HSPCs can respond to EM-inducing inflammatory stimuli in vitro but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that the loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of fetal demise. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection.

Neutrophil trapping and nexocytosis, mast cell-mediated processes for inflammatory signal relay.

Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them. MCs release the attractant leukotriene B4 to re-route neutrophils toward them, thus exploiting a chemotactic system that neutrophils normally use for intercellular communication. After MC intracellular trap (MIT) formation, neutrophils die, but their undigested material remains inside MC vacuoles over days. MCs benefit from MIT formation, increasing their functional and metabolic fitness. Additionally, they are more pro-inflammatory and can exocytose active neutrophilic compounds with a time delay (nexocytosis), eliciting a type 1 interferon response in surrounding macrophages. Together, our study highlights neutrophil trapping and nexocytosis as MC-mediated processes, which may relay neutrophilic features over the course of chronic allergic inflammation.

Innate immune memory after brain injury drives inflammatory cardiac dysfunction.

The medical burden of stroke extends beyond the brain injury itself and is largely determined by chronic comorbidities that develop secondarily. We hypothesized that these comorbidities might share a common immunological cause, yet chronic effects post-stroke on systemic immunity are underexplored. Here, we identify myeloid innate immune memory as a cause of remote organ dysfunction after stroke. Single-cell sequencing revealed persistent pro-inflammatory changes in monocytes/macrophages in multiple organs up to 3 months after brain injury, notably in the heart, leading to cardiac fibrosis and dysfunction in both mice and stroke patients. IL-1ß was identified as a key driver of epigenetic changes in innate immune memory. These changes could be transplanted to naive mice, inducing cardiac dysfunction. By neutralizing post-stroke IL-1ß or blocking pro-inflammatory monocyte trafficking with a CCR2/5 inhibitor, we prevented post-stroke cardiac dysfunction. Such immune-targeted therapies could potentially prevent various IL-1ß-mediated comorbidities, offering a framework for secondary prevention immunotherapy.

Chemokines and Chemokine Receptors in Lymphoid Tissue Dynamics.

The continuous migration of immune cells between lymphoid and nonlymphoid organs is a key feature of the immune system, facilitating the distribution of effector cells within nearly all compartments of the body. Furthermore, reaching their correct position within primary, secondary, or tertiary lymphoid organs is a prerequisite to ensure immune cells' unimpaired differentiation, maturation, and selection, as well as their activation or functional silencing. The superfamilies of chemokines and chemokine receptors are of major importance in guiding immune cells to and within lymphoid and nonlymphoid tissues. In this review we focus on the role of the chemokine system in the migration dynamics of immune cells within lymphoid organs at the steady state and on how these dynamics are affected by infectious and inflammatory processes.

Mechanisms of Pediatric Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), including Crohn disease and ulcerative colitis, is characterized by chronic intestinal inflammation due to a complex interaction of genetic determinants, disruption of mucosal barriers, aberrant inflammatory signals, loss of tolerance, and environmental triggers. Importantly, the incidence of pediatric IBD is rising, particularly in children younger than 10 years. In this review, we discuss the clinical presentation of these patients and highlight environmental exposures that may affect disease risk, particularly among people with a background genetic risk. With regard to both children and adults, we review advancements in understanding the intestinal epithelium, the mucosal immune system, and the resident microbiota, describing how dysfunction at any level can lead to diseases like IBD. We conclude with future directions for applying advances in IBD genetics to better understand pathogenesis and develop therapeutics targeting key pathogenic nodes.

Stromal cells in chronic inflammation and tertiary lymphoid organ formation.

Inflammation is an unstable state. It either resolves or persists. Why inflammation persists and the factors that define tissue tropism remain obscure. Increasing evidence suggests that tissue-resident stromal cells not only provide positional memory but also actively regulate the differential accumulation of inflammatory cells within inflamed tissues. Furthermore, at many sites of chronic inflammation, structures that mimic secondary lymphoid tissues are observed, suggesting that chronic inflammation and lymphoid tissue formation share common activation programs. Similarly, blood and lymphatic endothelial cells contribute to tissue homeostasis and disease persistence in chronic inflammation. This review highlights our increasing understanding of the role of stromal cells in inflammation and summarizes the novel immunological role that stromal cells exert in the persistence of inflammatory diseases.

Endoplasmic reticulum stress in immunity.

Immune responses occur in the midst of a variety of cellular stresses that can severely perturb endoplasmic reticulum (ER) function. The unfolded protein response is a three-pronged signaling axis dedicated to preserving ER homeostasis. In this review, we highlight many important and emerging functional roles for ER stress in immunity, focusing on how the bidirectional cross talk between immunological processes and basic cell biology leads to pleiotropic signaling outcomes and enhanced sensitivity to inflammatory stimuli. We also discuss how dysregulated ER stress responses can provoke many diseases, including autoimmunity, firmly positioning the unfolded protein response as a major therapeutic target in human disease.

Molecular mechanisms in genetically defined autoinflammatory diseases: disorders of amplified danger signaling.

Patients with autoinflammatory diseases present with noninfectious fever flares and systemic and/or disease-specific organ inflammation. Their excessive proinflammatory cytokine and chemokine responses can be life threatening and lead to organ damage over time. Studying such patients has revealed genetic defects that have helped unravel key innate immune pathways, including excessive IL-1 signaling, constitutive NF-κB activation, and, more recently, chronic type I IFN signaling. Discoveries of monogenic defects that lead to activation of proinflammatory cytokines have inspired the use of anticytokine-directed treatment approaches that have been life changing for many patients and have led to the approval of IL-1-blocking agents for a number of autoinflammatory conditions. In this review, we describe the genetically characterized autoinflammatory diseases, we summarize our understanding of the molecular pathways that drive clinical phenotypes and that continue to inspire the search for novel treatment targets, and we provide a conceptual framework for classification.

Arid1a-dependent canonical BAF complex suppresses inflammatory programs to drive efficient germinal center B cell responses.

The mammalian Brg1/Brm-associated factor (BAF) complexes are major regulators of nucleosomal remodeling that are commonly mutated in several cancers, including germinal center (GC)-derived B cell lymphomas. However, the specific roles of different BAF complexes in GC B cell biology are not well understood. Here we show that the AT-rich interaction domain 1a (Arid1a) containing canonical BAF (cBAF) complex is required for maintenance of GCs and high-affinity antibody responses. While Arid1a-deficient B cells undergo initial activation, they fail to sustain the GC program. Arid1a establishes permissive chromatin landscapes for B cell activation and is concomitantly required to suppress inflammatory gene programs. The inflammatory signatures instigated by Arid1a deficiency promoted the recruitment of neutrophils and inflammatory monocytes. Dampening of inflammatory cues through interleukin-1ß blockade or glucocorticoid receptor agonist partially rescued Arid1a-deficient GCs, highlighting a critical role for inflammation in impeding GCs. Our work reveals essential functions of Arid1a-dependent cBAF in promoting efficient GC responses.

Coordinated chemokine expression defines macrophage subsets across tissues.

Lung-resident macrophages, which include alveolar macrophages and interstitial macrophages (IMs), exhibit a high degree of diversity, generally attributed to different activation states, and often complicated by the influx of monocytes into the pool of tissue-resident macrophages. To gain a deeper insight into the functional diversity of IMs, here we perform comprehensive transcriptional profiling of resident IMs and reveal ten distinct chemokine-expressing IM subsets at steady state and during inflammation. Similar IM subsets that exhibited coordinated chemokine signatures and differentially expressed genes were observed across various tissues and species, indicating conserved specialized functional roles. Other macrophage types shared specific IM chemokine profiles, while also presenting their own unique chemokine signatures. Depletion of CD206hi IMs in Pf4creR26EYFP+DTR and Pf4creR26EYFPCx3cr1DTR mice led to diminished inflammatory cell recruitment, reduced tertiary lymphoid structure formation and fewer germinal center B cells in models of allergen- and infection-driven inflammation. These observations highlight the specialized roles of IMs, defined by their coordinated chemokine production, in regulating immune cell influx and organizing tertiary lymphoid tissue architecture.

Direct presentation of inflammation-associated self-antigens by thymic innate-like T cells induces elimination of autoreactive CD8+ thymocytes.

Upregulation of diverse self-antigens that constitute components of the inflammatory response overlaps spatially and temporally with the emergence of pathogen-derived foreign antigens. Therefore, discrimination between these inflammation-associated self-antigens and pathogen-derived molecules represents a unique challenge for the adaptive immune system. Here, we demonstrate that CD8+ T cell tolerance to T cell-derived inflammation-associated self-antigens is efficiently induced in the thymus and supported by redundancy in cell types expressing these molecules. In addition to thymic epithelial cells, this included thymic eosinophils and innate-like T cells, a population that expressed molecules characteristic for all major activated T cell subsets. We show that direct T cell-to-T cell antigen presentation by minute numbers of innate-like T cells was sufficient to eliminate autoreactive CD8+ thymocytes. Tolerance to such effector molecules was of critical importance, as its breach caused by decreased thymic abundance of a single model inflammation-associated self-antigen resulted in autoimmune elimination of an entire class of effector T cells.

Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

BACH2 regulates diversification of regulatory and proinflammatory chromatin states in TH17 cells.

Interleukin-17 (IL-17)-producing helper T (TH17) cells are heterogenous and consist of nonpathogenic TH17 (npTH17) cells that contribute to tissue homeostasis and pathogenic TH17 (pTH17) cells that mediate tissue inflammation. Here, we characterize regulatory pathways underlying TH17 heterogeneity and discover substantial differences in the chromatin landscape of npTH17 and pTH17 cells both in vitro and in vivo. Compared to other CD4+ T cell subsets, npTH17 cells share accessible chromatin configurations with regulatory T cells, whereas pTH17 cells exhibit features of both npTH17 cells and type 1 helper T (TH1) cells. Integrating single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq), we infer self-reinforcing and mutually exclusive regulatory networks controlling different cell states and predicted transcription factors regulating TH17 cell pathogenicity. We validate that BACH2 promotes immunomodulatory npTH17 programs and restrains proinflammatory TH1-like programs in TH17 cells in vitro and in vivo. Furthermore, human genetics implicate BACH2 in multiple sclerosis. Overall, our work identifies regulators of TH17 heterogeneity as potential targets to mitigate autoimmunity.

Nociceptor-immune interactomes reveal insult-specific immune signatures of pain.

Inflammatory pain results from the heightened sensitivity and reduced threshold of nociceptor sensory neurons due to exposure to inflammatory mediators. However, the cellular and transcriptional diversity of immune cell and sensory neuron types makes it challenging to decipher the immune mechanisms underlying pain. Here we used single-cell transcriptomics to determine the immune gene signatures associated with pain development in three skin inflammatory pain models in mice: zymosan injection, skin incision and ultraviolet burn. We found that macrophage and neutrophil recruitment closely mirrored the kinetics of pain development and identified cell-type-specific transcriptional programs associated with pain and its resolution. Using a comprehensive list of potential interactions mediated by receptors, ligands, ion channels and metabolites to generate injury-specific neuroimmune interactomes, we also uncovered that thrombospondin-1 upregulated by immune cells upon injury inhibited nociceptor sensitization. This study lays the groundwork for identifying the neuroimmune axes that modulate pain in diverse disease contexts.

Chromatin contributions to the regulation of innate immunity.

A fundamental property of cells of the innate immune system is their ability to elicit a transcriptional response to a microbial stimulus or danger signal with a high degree of cell type and stimulus specificity. The selective response activates effector pathways to control the insult and plays a central role in regulating adaptive immunity through the differential regulation of cytokine genes. Selectivity is dictated by signaling pathways and their transcription factor targets. However, a growing body of evidence supports models in which different subsets of genes exhibit distinct chromatin features that play active roles in shaping the response. Chromatin also participates in innate memory mechanisms that can promote tolerance to a stimulus or prime cells for a more robust response. These findings have generated interest in the capacity to modulate chromatin regulators with small-molecule compounds for the treatment of diseases associated with innate or adaptive immunity.