Characterize the cellular and molecular events responsible for lipodystrophy in AGPAT2 deficient mice.

Lineage specification during development involves reprogramming of transcriptional states, but little is known about how this is regulated in vivo. The chromatin remodeler chomodomain helicase DNA-binding protein 1 (Chd1) promotes an elevated transcriptional output in mouse embryonic stem cells. Here we report that endothelial-specific deletion of Chd1 leads to loss of definitive hematopoietic progenitors, anemia, and lethality by embryonic day (E)15.5. Mutant embryos contain normal numbers of E10.5 intraaortic hematopoietic clusters that express Runx1 and Kit, but these clusters undergo apoptosis and fail to mature into blood lineages in vivo and in vitro. Hematopoietic progenitors emerging from the aorta have an elevated transcriptional output relative to structural endothelium, and this elevation is Chd1-dependent. In contrast, hematopoietic-specific deletion of Chd1 using Vav-Cre has no apparent phenotype. Our results reveal a new paradigm of regulation of a developmental transition by elevation of global transcriptional output that is critical for hemogenesis and may play roles in other contexts.

Interleukin-33 (IL-33) is a nuclear-associated cytokine of the IL-1 family originally described as a potent inducer of allergic type 2 immunity. IL-33 signals via the receptor ST2, which is highly expressed on group 2 innate lymphoid cells (ILC2s) and T helper 2 (Th2) cells, thus underpinning its association with helminth infection and allergic pathology. Recent studies have revealed ST2 expression on subsets of regulatory T cells, and for a role for IL-33 in tissue homeostasis and repair that suggests previously unrecognized interactions within these cellular networks. IL-33 can participate in pathologic fibrotic reactions, or, in the setting of microbial invasion, can cooperate with inflammatory cytokines to promote responses by cytotoxic NK cells, Th1 cells, and CD8(+) T cells. Here, we highlight the regulation and function of IL-33 and ST2 and review their roles in homeostasis, damage, and inflammation, suggesting a conceptual framework for future studies.

Group 2 innate lymphoid cells (ILC2s) are distributed systemically and produce type 2 cytokines in response to a variety of stimuli, including the epithelial cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP). Transcriptional profiling of ILC2s from different tissues, however, grouped ILC2s according to their tissue of origin, even in the setting of combined IL-25-, IL-33-receptor-, and TSLP-receptor-deficiency. Single-cell profiling confirmed a tissue-organizing transcriptome and identified ILC2 subsets expressing distinct activating receptors, including the major subset of skin ILC2s, which were activated preferentially by IL-18. Tissue ILC2 subsets were unaltered in number and expression in germ-free mice, suggesting that endogenous, tissue-derived signals drive the maturation of ILC2 subsets by controlling expression of distinct patterns of activating receptors, thus anticipating tissue-specific perturbations occurring later in life.

Eosinophils are specialized myeloid cells associated with allergy and helminth infections. Blood eosinophils demonstrate circadian cycling, as described over 80 years ago, and are abundant in the healthy gastrointestinal tract. Although a cytokine, interleukin (IL)-5, and chemokines such as eotaxins mediate eosinophil development and survival, and tissue recruitment, respectively, the processes underlying the basal regulation of these signals remain unknown. Here we show that serum IL-5 levels are maintained by long-lived type 2 innate lymphoid cells (ILC2) resident in peripheral tissues. ILC2 cells secrete IL-5 constitutively and are induced to co-express IL-13 during type 2 inflammation, resulting in localized eotaxin production and eosinophil accumulation. In the small intestine where eosinophils and eotaxin are constitutive, ILC2 cells co-express IL-5 and IL-13; this co-expression is enhanced after caloric intake. The circadian synchronizer vasoactive intestinal peptide also stimulates ILC2 cells through the VPAC2 receptor to release IL-5, linking eosinophil levels with metabolic cycling. Tissue ILC2 cells regulate basal eosinophilopoiesis and tissue eosinophil accumulation through constitutive and stimulated cytokine expression, and this dissociated regulation can be tuned by nutrient intake and central circadian rhythms.

Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility1. The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (TH2) cells within a distinct microanatomical niche. TH2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as TH2-interacting fascial fibroblasts (TIFFs), which expand in response to TH2 cytokines to form subcutaneous fibrous bands. Activation of the TH2-TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified TH2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life.

Chitin, a polysaccharide constituent of many allergens and parasites, initiates innate type 2 lung inflammation through incompletely defined pathways. We show that inhaled chitin induced expression of three epithelial cytokines, interleukin-25 (IL-25), IL-33, and thymic stromal lymphopoietin (TSLP), which nonredundantly activated resident innate lymphoid type 2 cells (ILC2s) to express IL-5 and IL-13 necessary for accumulation of eosinophils and alternatively activated macrophages (AAMs). In the absence of all three epithelial cytokines, ILC2s normally populated the lung but failed to increase IL-5 and IL-13. Although eosinophils and AAMs were attenuated, neutrophil influx remained normal without these epithelial cytokines. Genetic ablation of ILC2s, however, enhanced IL-1β, TNFα, and IL-23 expression, increased activation of IL-17A-producing γδ T cells, and prolonged neutrophil influx. Thus, chitin elicited patterns of innate cytokines that targeted distinct populations of resident lymphoid cells, revealing divergent but interacting pathways underlying the tissue accumulation of specific types of inflammatory myeloid cells.

Changes in cell fate and identity are essential for endothelial-to-haematopoietic transition (EHT), an embryonic process that generates the first adult populations of haematopoietic stem cells (HSCs) from hemogenic endothelial cells. Dissecting EHT regulation is a critical step towards the production of in vitro derived HSCs. Yet, we do not know how distinct endothelial and haematopoietic fates are parsed during the transition. Here we show that genes required for arterial identity function later to repress haematopoietic fate. Tissue-specific, temporally controlled, genetic loss of arterial genes (Sox17 and Notch1) during EHT results in increased production of haematopoietic cells due to loss of Sox17-mediated repression of haematopoietic transcription factors (Runx1 and Gata2). However, the increase in EHT can be abrogated by increased Notch signalling. These findings demonstrate that the endothelial haematopoietic fate switch is actively repressed in a population of endothelial cells, and that derepression of these programs augments haematopoietic output.

Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Here, we identified persistent fibrotic scarring in the CNS following immune cell infiltration in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Using lineage tracing and single-cell sequencing in EAE, we determined that the majority of the fibrotic scar is derived from proliferative CNS fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibrotic cells using cell-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within the inflammatory lesions and a reduction in motor disability. We further identified that interferon-gamma pathway genes are enriched in CNS fibrotic cells, and the fibrotic cell-specific deletion of Ifngr1 resulted in reduced fibrotic scarring in EAE. These data delineate a framework for understanding the CNS fibrotic response.

Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.

The innate immune system plays essential roles in brain synaptic development, and immune dysregulation is implicated in neurodevelopmental diseases. Here we show that a subset of innate lymphocytes (group 2 innate lymphoid cells, ILC2s) is required for cortical inhibitory synapse maturation and adult social behavior. ILC2s expanded in the developing meninges and produced a surge of their canonical cytokine Interleukin-13 (IL-13) between postnatal days 5-15. Loss of ILC2s decreased cortical inhibitory synapse numbers in the postnatal period where as ILC2 transplant was sufficient to increase inhibitory synapse numbers. Deletion of the IL-4/IL-13 receptor ( Il4ra ) from inhibitory neurons phenocopied the reduction inhibitory synapses. Both ILC2 deficient and neuronal Il4ra deficient animals had similar and selective impairments in adult social behavior. These data define a type 2 immune circuit in early life that shapes adult brain function.

Type 2 lymphocytes promote both physiologic tissue remodeling and allergic pathology, yet their physical tissue niches are poorly described. Here, we used quantitative imaging to define the tissue niches of group 2 innate lymphoid cells (ILC2s), which are critical instigators of type 2 immunity. We identified a dominant adventitial niche around lung bronchi and larger vessels in multiple tissues, where ILC2s localized with subsets of dendritic and regulatory T cells. However, ILC2s were most intimately associated with adventitial stromal cells (ASCs), a mesenchymal fibroblast-like subset that expresses interleukin-33 (IL-33) and thymic stromal lymphopoietin (TSLP). In vitro, ASCs produced TSLP that supported ILC2 accumulation and activation. ILC2s and IL-13 drove reciprocal ASC expansion and IL-33 expression. During helminth infection, ASC depletion impaired lung ILC2 and Th2 cell accumulation and function, which are in part dependent on ASC-derived IL-33. These data indicate that adventitial niches are conserved sites where ASCs regulate type 2 lymphocyte expansion and function.

Type 2 innate lymphoid cells (ILC2s), an innate source of the type 2 cytokines interleukin (IL)-5 and -13, participate in the maintenance of tissue homeostasis. Although type 2 immunity is critically important for mediating metabolic adaptations to environmental cold, the functions of ILC2s in beige or brown fat development are poorly defined. We report here that activation of ILC2s by IL-33 is sufficient to promote the growth of functional beige fat in thermoneutral mice. Mechanistically, ILC2 activation results in the proliferation of bipotential adipocyte precursors (APs) and their subsequent commitment to the beige fat lineage. Loss- and gain-of-function studies reveal that ILC2- and eosinophil-derived type 2 cytokines stimulate signaling via the IL-4Rα in PDGFRα(+) APs to promote beige fat biogenesis. Together, our results highlight a critical role for ILC2s and type 2 cytokines in the regulation of adipocyte precursor numbers and fate, and as a consequence, adipose tissue homeostasis. PAPERCLIP:

Eosinophils in visceral adipose tissue (VAT) have been implicated in metabolic homeostasis and the maintenance of alternatively activated macrophages (AAMs). The absence of eosinophils can lead to adiposity and systemic insulin resistance in experimental animals, but what maintains eosinophils in adipose tissue is unknown. We show that interleukin-5 (IL-5) deficiency profoundly impairs VAT eosinophil accumulation and results in increased adiposity and insulin resistance when animals are placed on a high-fat diet. Innate lymphoid type 2 cells (ILC2s) are resident in VAT and are the major source of IL-5 and IL-13, which promote the accumulation of eosinophils and AAM. Deletion of ILC2s causes significant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associated with increased adipose ILC2 cytokine production and enhanced insulin sensitivity. Further, IL-33, a cytokine previously shown to promote cytokine production by ILC2s, leads to rapid ILC2-dependent increases in VAT eosinophils and AAMs. Thus, ILC2s are resident in VAT and promote eosinophils and AAM implicated in metabolic homeostasis, and this axis is enhanced during Th2-associated immune stimulation.

Endothelial cells transduce mechanical forces from blood flow into intracellular signals required for vascular homeostasis. Here we show that endothelial NOTCH1 is responsive to shear stress, and is necessary for the maintenance of junctional integrity, cell elongation, and suppression of proliferation, phenotypes induced by laminar shear stress. NOTCH1 receptor localizes downstream of flow and canonical NOTCH signaling scales with the magnitude of fluid shear stress. Reduction of NOTCH1 destabilizes cellular junctions and triggers endothelial proliferation. NOTCH1 suppression results in changes in expression of genes involved in the regulation of intracellular calcium and proliferation, and preventing the increase of calcium signaling rescues the cell-cell junctional defects. Furthermore, loss of Notch1 in adult endothelium increases hypercholesterolemia-induced atherosclerosis in the descending aorta. We propose that NOTCH1 is atheroprotective and acts as a mechanosensor in adult arteries, where it integrates responses to laminar shear stress and regulates junctional integrity through modulation of calcium signaling.

To restrict infection by Legionella pneumophila, mouse macrophages require Naip5, a member of the nucleotide-binding oligomerization domain leucine-rich repeat family of pattern recognition receptors, which detect cytoplasmic microbial products. We report that mouse macrophages restricted L. pneumophila replication and initiated a proinflammatory program of cell death when flagellin contaminated their cytosol. Nuclear condensation, membrane permeability, and interleukin-1beta secretion were triggered by type IV secretion-competent bacteria that encode flagellin. The macrophage response to L. pneumophila was independent of Toll-like receptor signaling but correlated with Naip5 function and required caspase 1 activity. The L. pneumophila type IV secretion system provided only pore-forming activity because listeriolysin O of Listeria monocytogenes could substitute for its contribution. Flagellin monomers appeared to trigger the macrophage response from perforated phagosomes: once heated to disassemble filaments, flagellin triggered cell death but native flagellar preparations did not. Flagellin made L. pneumophila vulnerable to innate immune mechanisms because Naip5+ macrophages restricted the growth of virulent microbes, but flagellin mutants replicated freely. Likewise, after intratracheal inoculation of Naip5+ mice, the yield of L. pneumophila in the lungs declined, whereas the burden of flagellin mutants increased. Accordingly, macrophages respond to cytosolic flagellin by a mechanism that requires Naip5 and caspase 1 to restrict bacterial replication and release proinflammatory cytokines that control L. pneumophila infection.

The emergence of haematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium results in the formation of sizeable HSPC clusters attached to the vascular wall. We evaluate the cell cycle and proliferation of HSPCs involved in cluster formation, as well as the molecular signatures from their initial appearance to the point when cluster cells are capable of adult engraftment (definitive HSCs). We uncover a non-clonal origin of HSPC clusters with differing cell cycle, migration, and cell signaling attributes. In addition, we find that the complement cascade is highly enriched in mature HSPC clusters, possibly delineating a new role for this pathway in engraftment.

Acute kidney injury (AKI) can be fatal and is a well-defined risk factor for the development of chronic kidney disease. Group 2 innate lymphoid cells (ILC2s) are innate producers of type-2 cytokines and are critical regulators of homeostasis in peripheral organs. However, our knowledge of their function in the kidney is relatively limited. Recent evidence suggests that increasing ILC2 numbers by systemic administration of recombinant interleukin (IL)-25 or IL-33 protects against renal injury. Whilst ILC2s can be induced to protect against ischemic- or chemical-induced AKI, the impact of ILC2 deficiency or depletion on the severity of renal injury is unknown. Firstly, the phenotype and location of ILC2s in the kidney was assessed under homeostatic conditions. Kidney ILC2s constitutively expressed high levels of IL-5 and were located in close proximity to the renal vasculature. To test the functional role of ILC2s in the kidney, an experimental model of renal ischemia-reperfusion injury (IRI) was used and the severity of injury was assessed in wild-type, ILC2-reduced, ILC2-deficient, and ILC2-depleted mice. Surprisingly, there were no differences in histopathology, collagen deposition or mRNA expression of injury-associated (Lcn2), inflammatory (Cxcl1, Cxcl2, and Tnf) or extracellular matrix (Col1a1, Fn1) factors following IRI in the absence of ILC2s. These data suggest the absence of ILC2s does not alter the severity of renal injury, suggesting possible redundancy. Therefore, other mechanisms of type 2-mediated immune cell activation likely compensate in the absence of ILC2s. Hence, a loss of ILC2s is unlikely to increase susceptibility to, or severity of AKI.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvβ5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.