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.

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.

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.

Bacteriophages Synergize with the Gut Microbial Community To Combat Salmonella.

Salmonella infection is one of the main causes of food-borne diarrheal diseases worldwide. Although most Salmonella infections can be cleared without treatment, some cause serious illnesses that require antibiotic treatment. In view of the growing emergence of antibiotic-resistant Salmonella strains, novel treatments are increasingly required. Furthermore, there is a striking paucity of data on how a balanced human gut microbiota responds to Salmonella infection. This study aimed to evaluate whether a balanced gut microbiota protects against Salmonella growth and to compare two antimicrobial approaches for managing Salmonella infection: bacteriophage (phage) treatment and antibiotic treatment. Anaerobically cultivated human intestinal microflora (ACHIM) is a feasible model for the human gut microbiota and naturally inhibits Salmonella infection. By mimicking Salmonella infection in vitro using ACHIM, we observed a large reduction of Salmonella growth by the ACHIM itself. Treatments with phage and antibiotic further inhibited Salmonella growth. However, phage treatment had less impact on the nontargeted bacteria in ACHIM than the antibiotic treatment did. Phage treatment has high specificity when combating Salmonella infection and offers a noninvasive alternative to antibiotic treatment. IMPORTANCE Antibiotic-resistant bacteria are a global threat. Therefore, alternative approaches for combatting bacteria, especially antibiotic-resistant bacteria, are urgently needed. Using a human gut microbiota model, we demonstrate that bacteriophages (phages) are able to substantially decrease pathogenic Salmonella without perturbing the microbiota. Conversely, antibiotic treatment leads to the eradication of close to all commensal bacteria, leaving only antibiotic-resistant bacteria. An unbalanced microbiota has been linked to many diseases both in the gastrointestinal tract or "nonintestinal" diseases. In our study, we show that the microbiota provides a protective effect against Salmonella. Since phage treatment preserves the healthy gut microbiota, it is a feasible superior alternative to antibiotic treatment. Furthermore, when combating infections caused by pathogenic bacteria, gut microbiota should be considered.

Microbiological diagnosis of adult tuberculous meningitis in a ten-year cohort in Indonesia.

We evaluated microbiological diagnosis of tuberculous (TB) meningitis in a referral hospital in Indonesia. Over a ten-year period, we examined cerebrospinal fluid (CSF) samples of 1180 adult meningitis suspects. Sensitivity of different methods was compared, and results were stratified for HIV status, disease severity, and CSF volume. TB meningitis was bacteriologically confirmed in 501 patients. Using clinical diagnosis as reference standard (n = 713), sensitivity of different methods was 12.2% (86/703) for microscopy, 42% (73/174) for Xpert MTB/RIF, 46.0% (163/354) for solid culture, 48.8% (332/680) for liquid culture, and 64.0% (212/331) for in-house PCR. Head to head comparisons in 654 patients showed a higher yield of in-house PCR (32.3%) compared to culture (15.6%, P < 0.01). Microscopic observation of drug susceptibility (MODS) culture more rapidly became positive compared to other culture methods. Yield of culture was lower in HIV-infected (39/105) than in HIV-negative patients (N = 316/585; P < 0.01). Molecular and culture methods gave higher yields in patients with more severe disease (P < 0.01). CSF volume of ≥6 ml increased the yield of culture (42.8% versus 12.1% for CSF <6 ml, P < 0.01) and ZN-microscopy (18.3% versus 1.9% for CSF <6 ml, P < 0.01). CSF centrifugation had no clear effect on sensitivity of Xpert MTB/RIF. ZN-microscopy lacks sensitivity for diagnosis of TB meningitis. For molecular assays, in-house IS6110-PCR is more sensitive than Xpert MTB/RIF. MODS culture has a clear advantage in terms of speed. Large CSF volumes are necessary for all tests. The effect of CSF processing for Xpert MTB/RIF needs further study.