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.

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.

Highlights of the ERS Lung Science Conference 2022.

This article presents the highlights of the ERS Lung Science Conference 2022, including a session organised by the Early Career Member Committee (ECMC) dedicated to career development https://bit.ly/3tarCXc.

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.

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.

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.