Implication of Oxysterols in Infectious and Non-Communicable Inflammatory Diseases.

Oxysterols, derived from cholesterol oxidation, are formed either by autoxidation, via enzymes, or by both processes [...].

Single-cell RNA sequencing of human lung innate lymphoid cells in the vascular and tissue niche reveals molecular features of tissue adaptation.

Innate lymphoid cells (ILCs) are sentinels of healthy organ function, yet it is unknown how ILCs adapt to distinct anatomical niches within tissues. Here, we used a unique humanized mouse model, MISTRG mice transplanted with human hematopoietic stem and progenitor cells (HSPCs), to define the gene signatures of human ILCs in the vascular versus the tissue (extravascular) compartment of the lung. Single-cell RNA sequencing in combination with intravascular cell labeling demonstrated that heterogeneous populations of human ILCs and natural killer (NK) cells occupied the vascular and tissue niches in the lung of HSPC-engrafted MISTRG mice. Moreover, we discovered that niche-specific cues shape the molecular programs of human ILCs in the distinct sub-anatomical compartments of the lung. Specifically, extravasation of ILCs into the lung tissue was associated with the upregulation of genes involved in the acquisition of tissue residency, cell positioning within the lung, sensing of tissue-derived signals, cellular stress responses, nutrient uptake, and interaction with other tissue-resident immune cells. We also defined a core tissue signature shared between human ILCs and NK cells in the extravascular space of the lung, consistent with imprinting by signals from the local microenvironment. The molecular characterization of human ILCs and NK cells in the vascular and tissue niches of the lung provides new knowledge on the mechanisms of ILC tissue adaptation and represents a resource for further studies.

A single-cell map of vascular and tissue lymphocytes identifies proliferative TCF-1+ human innate lymphoid cells.

Innate lymphoid cells (ILCs) play important roles in tissue homeostasis and host defense, but the proliferative properties and migratory behavior of especially human ILCs remain poorly understood. Here we mapped at single-cell resolution the spatial distribution of quiescent and proliferative human ILCs within the vascular versus tissue compartment. For this purpose, we employed MISTRG humanized mice as an in-vivo model to study human ILCs. We uncovered subset-specific differences in the proliferative status between vascular and tissue ILCs within lymphoid and non-lymphoid organs. We also identified CD117-CRTH2-CD45RA+ ILCs in the spleen that were highly proliferative and expressed the transcription factor TCF-1. These proliferative ILCs were present during the neonatal period in human blood and emerged early during population of the human ILC compartment in MISTRG mice transplanted with human hematopoietic stem and progenitor cells (HSPCs). Single-cell RNA-sequencing combined with intravascular cell labeling suggested that proliferative ILCs actively migrated from the local vasculature into the spleen tissue. Collectively, our comprehensive map reveals the proliferative topography of human ILCs, linking cell migration and spatial compartmentalization with cell division.

CD116+ fetal precursors migrate to the perinatal lung and give rise to human alveolar macrophages.

Despite their importance in lung health and disease, it remains unknown how human alveolar macrophages develop early in life. Here we define the ontogeny of human alveolar macrophages from embryonic progenitors in vivo, using a humanized mouse model expressing human cytokines (MISTRG mice). We identified alveolar macrophage progenitors in human fetal liver that expressed the GM-CSF receptor CD116 and the transcription factor MYB. Transplantation experiments in MISTRG mice established a precursor-product relationship between CD34-CD116+ fetal liver cells and human alveolar macrophages in vivo. Moreover, we discovered circulating CD116+CD64-CD115+ macrophage precursors that migrated from the liver to the lung. Similar precursors were present in human fetal lung and expressed the chemokine receptor CX3CR1. Fetal CD116+CD64- macrophage precursors had a proliferative gene signature, outcompeted adult precursors in occupying the perinatal alveolar niche, and developed into functional alveolar macrophages. The discovery of the fetal alveolar macrophage progenitor advances our understanding of human macrophage origin and ontogeny.

CD5 Surface Expression Marks Intravascular Human Innate Lymphoid Cells That Have a Distinct Ontogeny and Migrate to the Lung.

Innate lymphoid cells (ILCs) contribute to immune defense, yet it is poorly understood how ILCs develop and are strategically positioned in the lung. This applies especially to human ILCs due to the difficulty of studying them in vivo. Here we investigated the ontogeny and migration of human ILCs in vivo with a humanized mouse model ("MISTRG") expressing human cytokines. In addition to known tissue-resident ILC subsets, we discovered CD5-expressing ILCs that predominantly resided within the lung vasculature and in the circulation. CD5+ ILCs contained IFNγ-producing mature ILC1s as well as immature ILCs that produced ILC effector cytokines under polarizing conditions in vitro. CD5+ ILCs had a distinct ontogeny compared to conventional CD5- ILCs because they first appeared in the thymus, spleen and liver rather than in the bone marrow after transplantation of MISTRG mice with human CD34+ hematopoietic stem and progenitor cells. Due to their strategic location, human CD5+ ILCs could serve as blood-borne sentinels, ready to be recruited into the lung to respond to environmental challenges. This work emphasizes the uniqueness of human CD5+ ILCs in terms of their anatomical localization and developmental origin compared to well-studied CD5- ILCs.

Continuous human uterine NK cell differentiation in response to endometrial regeneration and pregnancy.

Immune cell differentiation is critical for adequate tissue-specific immune responses to occur. Here, we studied differentiation of human uterine natural killer cells (uNK cells). These cells reside in a tissue undergoing constant regeneration and represent the major leukocyte population at the maternal-fetal interface. However, their physiological response during the menstrual cycle and in pregnancy remains elusive. By surface proteome and transcriptome analysis as well as using humanized mice, we identify a differentiation pathway of uNK cells in vitro and in vivo with sequential acquisition of killer cell immunoglobulin-like receptors and CD39. uNK cell differentiation occurred continuously in response to the endometrial regeneration and was driven by interleukin-15. Differentiated uNK cells displayed reduced proliferative capacity and immunomodulatory function including enhanced angiogenic capacity. By studying human uterus transplantation and monozygotic twins, we found that the uNK cell niche could be replenished from circulation and that it was under genetic control. Together, our study uncovers a continuous differentiation pathway of human NK cells in the uterus that is coupled to profound functional changes in response to local tissue regeneration and pregnancy.

Distinct developmental pathways from blood monocytes generate human lung macrophage diversity.

The study of human macrophages and their ontogeny is an important unresolved issue. Here, we use a humanized mouse model expressing human cytokines to dissect the development of lung macrophages from human hematopoiesis in vivo. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) generated three macrophage populations, occupying separate anatomical niches in the lung. Intravascular cell labeling, cell transplantation, and fate-mapping studies established that classical CD14+ blood monocytes derived from HSPCs migrated into lung tissue and gave rise to human interstitial and alveolar macrophages. In contrast, non-classical CD16+ blood monocytes preferentially generated macrophages resident in the lung vasculature (pulmonary intravascular macrophages). Finally, single-cell RNA sequencing defined intermediate differentiation stages in human lung macrophage development from blood monocytes. This study identifies distinct developmental pathways from circulating monocytes to lung macrophages and reveals how cellular origin contributes to human macrophage identity, diversity, and localization in vivo.

Human macrophages and innate lymphoid cells: Tissue-resident innate immunity in humanized mice.

Macrophages and innate lymphoid cells (ILCs) are tissue-resident cells that play important roles in organ homeostasis and tissue immunity. Their intricate relationship with the organs they reside in allows them to quickly respond to perturbations of organ homeostasis and environmental challenges, such as infection and tissue injury. Macrophages and ILCs have been extensively studied in mice, yet important species-specific differences exist regarding innate immunity between humans and mice. Complementary to ex-vivo studies with human cells, humanized mice (i.e. mice with a human immune system) offer the opportunity to study human macrophages and ILCs in vivo within their surrounding tissue microenvironments. In this review, we will discuss how humanized mice have helped gain new knowledge about the basic biology of these cells, as well as their function in infectious and malignant conditions. Furthermore, we will highlight active areas of investigation related to human macrophages and ILCs, such as their cellular heterogeneity, ontogeny, tissue residency, and plasticity. In the near future, we expect more fundamental discoveries in these areas through the combined use of improved humanized mouse models together with state-of-the-art technologies, such as single-cell RNA-sequencing and CRISPR/Cas9 genome editing.

Origin and ontogeny of lung macrophages: from mice to humans.

Macrophages are tissue-resident myeloid cells with essential roles in host defense, tissue repair, and organ homeostasis. The lung harbors a large number of macrophages that reside in alveoli. As a result of their strategic location, alveolar macrophages are critical sentinels of healthy lung function and barrier immunity. They phagocytose inhaled material and initiate protective immune responses to pathogens, while preventing excessive inflammatory responses and tissue damage. Apart from alveolar macrophages, other macrophage populations are found in the lung and recent single-cell RNA-sequencing studies indicate that lung macrophage heterogeneity is greater than previously appreciated. The cellular origin and development of mouse lung macrophages has been extensively studied, but little is known about the ontogeny of their human counterparts, despite the importance of macrophages for lung health. In this context, humanized mice (mice with a human immune system) can give new insights into the biology of human lung macrophages by allowing in vivo studies that are not possible in humans. In particular, we have created humanized mouse models that support the development of human lung macrophages in vivo. In this review, we will discuss the heterogeneity, development, and homeostasis of lung macrophages. Moreover, we will highlight the impact of age, the microbiota, and pathogen exposure on lung macrophage function. Altered macrophage function has been implicated in respiratory infections as well as in common allergic and inflammatory lung diseases. Therefore, understanding the functional heterogeneity and ontogeny of lung macrophages should help to develop future macrophage-based therapies for important lung diseases in humans.

Metabolite Sensing by Colonic ILC3s: How Far Is Too Ffar2 Go?

It is poorly understood how group 3 innate lymphoid cells (ILC3s) recognize metabolites produced by the gut microbiota. In this issue of Immunity, Chun et al. show that short-chain fatty acids sensed through the G protein-coupled receptor Ffar2 promote ILC3 function in the colon.

Metabolic Control of Innate Lymphoid Cell Migration.

Innate lymphoid cells (ILCs) are specialized immune cells that rapidly respond to environmental challenges, such as infection and tissue damage. ILCs play an important role in organ homeostasis, tissue repair, and host defense in the mucosal tissues intestine and lung. ILCs are sentinels of healthy tissue function, yet it is poorly understood how ILCs are recruited, strategically positioned, and maintained within tissues. Accordingly, ILC migration is an area that has recently come into focus and it is important to define the signals that control ILC migration to and within tissues. In this context, signals from the local tissue microenvironment are relevant. For example, ILCs in the intestine are exposed to an environment that is rich in dietary, microbial, and endogenous metabolites. It has been shown that the Vitamin A metabolite retinoic acid promotes ILC1 and ILC3 homing to the intestine. In addition, recent studies have discovered cholesterol metabolites (oxysterols) as a novel class of molecules that regulate ILC migration through the receptor GPR183. ILCs are considered to be largely tissue-resident cells, yet recent data indicate that ILCs actively migrate during inflammation. Furthermore, the discovery of circulating ILC precursors in humans and their presence within tissues has fueled the concept of local ILC-poiesis. However, it is unclear how circulating ILCs enter tissue during embryogenesis and inflammation and how they are directed to local tissue niches. In this review, I will discuss the metabolic signals that regulate ILC homing and their strategic positioning in healthy and inflamed tissues. It is becoming increasingly clear that ILC function is closely linked to their tissue localization. Therefore, understanding the tissue signals that control ILC migration could open new avenues for the treatment of chronic inflammatory diseases and cancer.

Antigen-presenting ILC3 regulate T cell-dependent IgA responses to colonic mucosal bacteria.

Intestinal immune homeostasis is dependent upon tightly regulated and dynamic host interactions with the commensal microbiota. Immunoglobulin A (IgA) produced by mucosal B cells dictates the composition of commensal bacteria residing within the intestine. While emerging evidence suggests the majority of IgA is produced innately and may be polyreactive, mucosal-dwelling species can also elicit IgA via T cell-dependent mechanisms. However, the mechanisms that modulate the magnitude and quality of T cell-dependent IgA responses remain incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3) regulate steady state interactions between T follicular helper cells (TfH) and B cells to limit mucosal IgA responses. ILC3 used conserved migratory cues to establish residence within the interfollicular regions of the intestinal draining lymph nodes, where they act to limit TfH responses and B cell class switching through antigen presentation. The absence of ILC3-intrinsic antigen presentation resulted in increased and selective IgA coating of bacteria residing within the colonic mucosa. Together these findings implicate lymph node resident, antigen-presenting ILC3 as a critical regulatory checkpoint in the generation of T cell-dependent colonic IgA and suggest ILC3 act to maintain tissue homeostasis and mutualism with the mucosal-dwelling commensal microbiota.

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation.

Group 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.

Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.

Prolonged T-cell receptor (TCR) signaling is required for the proliferation of T lymphocytes. Ligation of the TCR activates signaling, but also causes internalization of the TCR from the cell surface. How TCR signaling is sustained for many hours despite lower surface expression is unknown. Using genetic inhibition of endocytosis, we show here that TCR internalization promotes continued TCR signaling and T-lymphocyte proliferation. T-cell-specific deletion of dynamin 2, an essential component of endocytosis, resulted in reduced TCR signaling strength, impaired homeostatic proliferation, and the inability to undergo clonal expansion in vivo. Blocking endocytosis resulted in a failure to maintain mammalian target of rapamycin (mTOR) activity and to stably induce the transcription factor myelocytomatosis oncogene (c-Myc), which led to metabolic stress and a defect in cell growth. Our results support the concept that the TCR can continue to signal after it is internalized from the cell surface, thereby enabling sustained signaling and cell proliferation.

Dynamin 2-dependent endocytosis is required for sustained S1PR1 signaling.

Sphingosine-1-phosphate (S1P) receptor 1 (S1PR1) is critical for lymphocyte egress from lymphoid organs. Lymphocytes encounter low S1P concentrations near exit sites before transmigration, yet S1PR1 signaling is rapidly terminated after exposure to S1P. How lymphocytes maintain S1PR1 signaling in a low S1P environment near egress sites is unknown. Here we identify dynamin 2, an essential component of endocytosis, as a novel regulator of T cell egress. Mice with T cell-specific dynamin 2 deficiency had profound lymphopenia and impaired egress from lymphoid organs. Dynamin 2 deficiency caused impaired egress through regulation of S1PR1 signaling, and transgenic S1PR1 overexpression rescued egress in dynamin 2 knockout mice. In low S1P concentrations, dynamin 2 was essential for S1PR1 internalization, which enabled continuous S1PR1 signaling and promoted egress from both thymus and lymph nodes. In contrast, dynamin 2-deficient cells were only capable of a pulse of S1PR1 signaling, which was insufficient for egress. Our results suggest a possible mechanism by which T lymphocytes positioned at exit portals sense low S1P concentrations, promoting their egress into circulatory fluids.

Development and function of human innate immune cells in a humanized mouse model.

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

ESCaping rejection: A step forward for embryonic-stem-cell-based regenerative medicine.

The use of human embryonic stem cells (hESCs) for regenerative medicine currently faces several hurdles, including immune rejection of transplanted cells. Now in Cell Stem Cell, Rong et al. (2014) describe a strategy to protect hESCs from immune rejection while avoiding systemic immunosuppression, potentially facilitating clinical implementation of hESC-based therapies.

Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.