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An off switch for helminth immunityGroup 2 innate lymphoid cells (ILC2s) are involved in responses to helminths, viruses, and allergens. Moriyama et al. found that ILC2s interact with the nervous system to modulate helminth immunity. ILC2s from the small intestine expressed the β2-adrenergic receptor (β2AR), which normally interacts with the neurotransmitter epinephrine. Inactivating β2AR resulted in lower helminth burden and more ILC2s, eosinophils, and type 2 cytokine production in mice. Conversely, treatment of helminth-infected mice with a β2AR agonist enhanced worm burden and reduced proliferation of ILC2s. Thus, β2AR negatively regulates ILC2-driven protective immunity.Science, this issue p. 1056The type 2 inflammatory response is induced by various environmental and infectious stimuli. Although recent studies identified group 2 innate lymphoid cells (ILC2s) as potent sources of type 2 cytokines, the molecular pathways controlling ILC2 responses are incompletely defined. Here we demonstrate that murine ILC2s express the β2-adrenergic receptor (β2AR) and colocalize with adrenergic neurons in the intestine. β2AR deficiency resulted in exaggerated ILC2 responses and type 2 inflammation in intestinal and lung tissues. Conversely, β2AR agonist treatment was associated with impaired ILC2 responses and reduced inflammation in vivo. Mechanistically, we demonstrate that the β2AR pathway is a cell-intrinsic negative regulator of ILC2 responses through inhibition of cell proliferation and effector function. Collectively, these data provide the first evidence of a neuronal-derived regulatory circuit that limits ILC2-dependent type 2 inflammation.

Inducible genetic labelling of haematopoietic stem cells (HSCs) and linked mathematical modelling show that at least 30% of all HSCs are productive, and that adult haematopoiesis is largely sustained by ‘short-term’ downstream stem cells that operate near self-renewal in the steady state; HSC fate mapping provides a quantitative model for better understanding of HSC functions in health and disease. Following haematopoiesis in vivo Most of what we know of the properties of haematopoietic stem cells (HSCs) is derived from transplantation and reconstitution of an emptied blood and immune system. Relatively little is known about how HSCs behave under physiological conditions. It was reported recently that normal haematopoeisis in adults is driven by thousands of long-lived progenitors rather than classic HSCs. Hans-Reimer Rodewald and colleagues have used inducible genetic labelling of primitive HSCs in a mouse model, combined with mathematical modelling, to show that although HSCs participate in establishment of the blood system in early life, steady-state haematopoiesis depends mainly on progenitors that are able to self-renew but also receive rare input from long-term HSCs. This input is increased following physiological challenges. Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation 1 , 2 , 3 , 4 , 5 . Chromosomal marking 6 , viral integration 7 , 8 , 9 and barcoding 10 , 11 , 12 of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2 + HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse’s life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated ‘short-term’ stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.

Genetic defects that accumulate in haematopoietic stem cells (HSCs) are thought to be responsible for age-related changes in haematopoiesis that include a decline in lymphopoiesis and skewing towards the myeloid lineage. This HSC-centric view is based largely on studies showing that HSCs from aged mice exhibit these lineage biases following transplantation into irradiated young recipient mice. In this Opinion article, we make the case that the reliance on this approach has led to inaccurate conclusions regarding the effects of ageing on blood-forming stem cells; we suggest instead that changes in the environment contribute to haematopoietic system ageing. We propose that a complete understanding of how ageing affects haematopoiesis depends on the analysis of blood cell production in unperturbed mice. We describe how this can be achieved using in situ fate mapping. This approach indicates that changes in downstream progenitors, in addition to any HSC defects, may explain the reduced lymphopoiesis and sustained myelopoiesis that occur during ageing.As we age, haematopoiesis becomes skewed towards myelopoiesis. Studies of haematopoietic stem cells (HSCs) transplanted into irradiated recipient mice imply that HSC defects are responsible for this ageing effect. Here, the authors urge caution when using irradiated mice to study haematopoiesis ageing, and propose instead that age-related changes in the bone marrow environment and in downstream progenitors, not just HSCs, may also be responsible for myeloid skewing.

Thymic stromal lymphopoietin (TSLP) is a critical upstream cytokine inducing type 2 inflammation in various diseases, including asthma and atopic dermatitis. Accumulating evidence suggests that TSLP can directly stimulate a variety of immune cells, such as dendritic cells (DCs), basophils, T cells, and group 2 innate lymphoid cells (ILC2s). However, which cell types directly respond to TSLP in vivo and how TSLP initiates type 2 inflammation has remained controversial. To define the precise role of TSLP in vivo, for the first time we generated multiple cell lineage-specific TSLP receptor-deficient mice to systematically dissect the cell-intrinsic requirements for TSLP responsiveness in type 2 inflammation in the lung. In papain-induced innate immune-mediated type 2 airway inflammation, TSLP directly stimulated ILC2s, but not basophils, leading to enhanced type 2 inflammation. On the other hand, in OVA-induced adaptive immune-mediated type 2 airway inflammation, TSLP principally acted on DCs and CD4 + T cells during the sensitization phase, but not basophils or ILC2s, and facilitated the development of Th2 cell-mediated airway inflammation. Together, these findings reveal that TSLP activates distinct immune cell cascades in the context of innate and adaptive immune-mediated type 2 inflammation.

Metastasis constitutes the primary cause of cancer-related deaths, with the lung being a commonly affected organ. We found that activation of lung-resident group 2 innate lymphoid cells (ILC2s) orchestrated suppression of natural killer (NK) cell-mediated innate antitumor immunity, leading to increased lung metastases and mortality. Using multiple models of lung metastasis, we show that interleukin (IL)-33-dependent ILC2 activation in the lung is involved centrally in promoting tumor burden. ILC2-driven innate type 2 inflammation is accompanied by profound local suppression of interferon-γ production and cytotoxic function of lung NK cells. ILC2-dependent suppression of NK cells is elaborated via an innate regulatory mechanism, which is reliant on IL-5-induced lung eosinophilia, ultimately limiting the metabolic fitness of NK cells. Therapeutic targeting of IL-33 or IL-5 reversed NK cell suppression and alleviated cancer burden. Thus, we reveal an important function of IL-33 and ILC2s in promoting tumor metastasis via their capacity to suppress innate type 1 immunity. Pathological group 2 innate lymphoid cells (ILC2s) have mainly been implicated in allergy. Halim and colleagues demonstrate that ILC2s orchestrate a prometastatic pathway via the recruitment of eosinophils that suppress NK cell function.

The next generation of genetically engineered mouse models of pancreatic cancer involving a new inducible dual-recombinase system that combines Flp- FRT and Cre- loxP . Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre- loxP –based models. We have developed an inducible dual-recombinase system by combining flippase- FRT (Flp- FRT ) and Cre- loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell–autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.

The cell-intrinsic pathways controlling the function of innate lymphoid cells are poorly defined. Artis and colleagues demonstrate that ILC2s selectively express arginase 1 and that this is critical for their bioenergetics, proliferation and function. Group 2 innate lymphoid cells (ILC2s) regulate tissue inflammation and repair after activation by cell-extrinsic factors such as host-derived cytokines. However, the cell-intrinsic metabolic pathways that control ILC2 function are undefined. Here we demonstrate that expression of the enzyme arginase-1 (Arg1) during acute or chronic lung inflammation is a conserved trait of mouse and human ILC2s. Deletion of mouse ILC-intrinsic Arg1 abrogated type 2 lung inflammation by restraining ILC2 proliferation and dampening cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and reducing aerobic glycolysis. These data identify Arg1 as a key regulator of ILC2 bioenergetics that controls proliferative capacity and proinflammatory functions promoting type 2 inflammation.

Fate mapping is a powerful genetic tool for linking stem or progenitor cells with their progeny, and hence for defining cell lineages in vivo. The resolution of fate mapping depends on the numbers of distinct markers that are introduced in the beginning into stem or progenitor cells; ideally, numbers should be sufficiently large to allow the tracing of output from individual cells. Highly diverse genetic barcodes can serve this purpose. We recently developed an endogenous genetic barcoding system, termed Polylox . In Polylox , random DNA recombination can be induced by transient activity of Cre recombinase in a 2.1-kb-long artificial recombination substrate that has been introduced into a defined locus in mice ( Rosa26 Polylox reporter mice). Here, we provide a step-by-step protocol for the use of Polylox , including barcode induction and estimation of induction efficiency, barcode retrieval with single-molecule real-time (SMRT) DNA sequencing followed by computational barcode identification, and the calculation of barcode-generation probabilities, which is key for estimations of single-cell labeling for a given number of stem cells. Thus, Polylox barcoding enables high-resolution fate mapping in essentially all tissues in mice for which inducible Cre driver lines are available. Alternative methods include ex vivo cell barcoding, inducible transposon insertion and CRISPR–Cas9-based barcoding; Polylox currently allows combining non-invasive and cell-type-specific labeling with high label diversity. The execution time of this protocol is ~2–3 weeks for experimental data generation and typically <2 d for computational Polylox decoding and downstream analysis. Pei et al. describe a genetic barcoding system termed Polylox . Induced by transient activity of Cre recombinase in specific mouse tissues, random DNA recombination in the Polylox substrate produces unique barcodes that can be used for fate mapping.

B lineage development requires the transcription factors E2A, EBF1, Foxo1 and Ikaros. Murre and colleagues show that these factors gain access to lineage-specific enhancer sites by the action of the chromatin remodeler Brg1. Early B cell development is orchestrated by the combined activities of the transcriptional regulators E2A, EBF1, Foxo1 and Ikaros. However, how the genome-wide binding patterns of these regulators are modulated during B lineage development remains to be determined. Here we found that in lymphoid progenitor cells, the chromatin remodeler Brg1 specified the B cell fate. In committed pro-B cells, Brg1 regulated contraction of the locus encoding the immunoglobulin heavy chain ( Igh ) and controlled expression of the gene encoding the transcription factor c-Myc ( Myc ) to modulate the expression of genes encoding products that regulate ribosome biogenesis. In committed pro-B cells, Brg1 suppressed a pre-B lineage–specific pattern of gene expression. Finally, we found that Brg1 acted mechanistically to establish B cell fate and modulate cell growth by facilitating access of lineage-specific transcription factors to enhancer repertoires.

Type-2 innate lymphoid cells (ILC2) are a prominent source of type II cytokines and are found constitutively at mucosal surfaces and in visceral adipose tissue. Despite their role in limiting obesity, how ILC2s respond to high fat feeding is poorly understood, and their direct influence on the development of atherosclerosis has not been explored. Here, we show that ILC2 are present in para-aortic adipose tissue and lymph nodes and display an inflammatory-like phenotype atypical of adipose resident ILC2. High fat feeding alters both the number of ILC2 and their type II cytokine production. Selective genetic ablation of ILC2 in Ldlr −/− mice accelerates the development of atherosclerosis, which is prevented by reconstitution with wild type but not Il5 −/− or Il13 −/− ILC2. We conclude that ILC2 represent a major innate cell source of IL-5 and IL-13 required for mounting atheroprotective immunity, which can be altered by high fat diet. Type-2 innate lymphoid cells (ILC2) affect adipose tissue metabolism and function. Here the authors show that the ILC2 are present in para-aortic adipose tissue and represent a major source of IL-5 and IL-13 required for mounting atheroprotective immunity, which can be altered by high fat diet.