Immunology & Cell Biology

The cellular landscape of the paediatric respiratory system remains largely uncharacterised and as a result, the mechanisms of highly prevalent childhood respiratory diseases remain poorly understood. A major limitation in defining mechanisms of disease has been the availability of tissue samples collected in early life, as well as technologies that permit deep immune analysis from limited sample volumes. In this work, we developed new experimental methods and applied unsupervised analytical tools to profile the local (bronchoalveolar lavage) and systemic (whole blood) immune response in childhood respiratory disease. We quantified and comprehensively phenotyped immune cell populations across blood and lung compartments in young children (under 6 years of age), showed that inflammatory cells in the BAL express higher levels of activation and migration markers relative to their systemic counterparts, and applied new analytical tools to reveal novel tissue-resident macrophage and infiltrating monocyte populations in the paediatric lung. To our knowledge, this is the first description of the use of these methods for paediatric respiratory samples. Combined with matched analysis of the systemic immune cell profile, the application of these pipelines will increase our understanding of childhood lung disease with potential to identify clinically relevant disease biomarkers.

The extracellular matrix (ECM) forms the scaffold in which cells reside and interact. The composition of this scaffold guides the development of local immune responses and tissue function. With the advent of multiplexed spatial imaging methodologies, investigating the intricacies of cellular spatial organisation are more accessible than ever. However, the relationship between cellular organisation and ECM composition has been broadly overlooked. Using imaging mass cytometry, we investigated the association between cellular niches and their surrounding matrix environment during allergic airway inflammation in two commonly used mouse strains. By first classifying cells according to their canonical intracellular markers and then by developing a novel analysis pipeline to independently characterise a cells ECM environment, we integrated analysis of both intracellular and extracellular data. Applying this methodology to three distinct tissue regions we reveal disparate and restricted responses. Recruited neutrophils were dispersed within the alveolar parenchyma, alongside a loss of alveolar type I cells and an expansion of alveolar type II cells. This activated parenchyma was associated with increased proximity to hyaluronan and chondroitin sulphate. In contrast, infiltrating CD11b+ and MHCII+ cells accumulated in the adventitial cuff and aligned with an expansion of the subepithelial layer. This expanded subepithelial region was enriched for closely interacting stromal and CD11b+ immune cells which overlaid regions enriched for type-I and type-III collagen. The cell-cell and cell-matrix interactions identified here will provide a greater understanding of the mechanisms and regulation of allergic disease progression across different inbred mouse strains and provide specific pathways to target aspects of remodelling during allergic pathology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/623782v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@dcaf56org.highwire.dtl.DTLVardef@7b5d7forg.highwire.dtl.DTLVardef@1376d4eorg.highwire.dtl.DTLVardef@1e95801_HPS_FORMAT_FIGEXP M_FIG C_FIG

Persistent lung inflammation is a hallmark of Cystic fibrosis (CF) lung disease. Inflammation can lead to functional decline in hematopoietic stem cell (HSCs), tipping the balance towards myelopoiesis and contributing to chronic inflammation. However, its unknown whether the HSCs are dysfunctional in CF. We tested whether chronic lung inflammation impacts hematopoietic stem and progenitor cells (HSPCs) in a CF mouse model. Wild-type (WT) and Cftr-/- mice were nebulized with lipopolysaccharide (LPS) from Pseudomonas aeruginosa for 5 weeks. The mice were euthanized before the exposure (T0), 24 hours after the last LPS nebulization (T1), or 6 weeks (T2) after the last LPS nebulization. The bone marrow (BM) and lung tissue were collected for flow cytometry analysis of the HSPC population and immune cells in the lungs, respectively. Peripheral blood was collected for complete blood count analysis. At baseline, Cftr-/- mice show a larger HSPC population with a myeloid bias, indicated by higher frequencies of LSK, LT-HSC, CD41+ LT-HSC, GMPs, and MkPs. Following chronic LPS nebulization, Cftr-/- mice exhibit greater HSPC expansion and myeloid differentiation, alongside increased peripheral myeloid cell counts. Post-recovery, while most HSPC populations return to baseline, Cftr-/- mice retain elevated myeloid-biased LT-HSCs, suggesting a persistent myeloid bias. These findings underscore a prominent shift toward myeloid hematopoiesis in CF, which is accentuated by chronic inflammation and remains even after recovery. Further experiments are underway to assess maladaptive epigenetic changes in HSC as well as if chronic lung inflammation impacts HSC functionality.

Purpose and appropriate sample typesThis 25-parameter, 22-colour full spectrum flow cytometry panel was designed and optimised for the comprehensive enumeration and functional characterisation of innate lymphoid cell (ILC) subsets in mouse tissues (Table 1). The panel presented here allows the discrimination of ILC progenitors (ILCP), ILC1, ILC2, NCR+ ILC3, NCR- ILC3, CCR6+ lymphoid tissue-inducer (LTi)-like ILC3 and mature natural killer (NK) cell populations. Further characterisation of ILC and NK cell functional profiles in response to stimulation is provided by the inclusion of subset-specific cytokine markers, and proliferation markers. Development and optimisation of this panel was performed on freshly isolated cells from adult BALB/c lungs and small intestine lamina propria, and ex vivo stimulation with phorbol 12-myrisate 13-acetate, ionomycin and pro-ILC activating cytokines. O_TBL View this table: org.highwire.dtl.DTLVardef@1b1415forg.highwire.dtl.DTLVardef@3adc6forg.highwire.dtl.DTLVardef@5e0aeaorg.highwire.dtl.DTLVardef@1e9e61org.highwire.dtl.DTLVardef@2ff463_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSummary Table C_TABLECAPTION C_TBL Ethical compliance statementAll mouse experiments were performed in accordance with the recommendations in the Guide for the Use of Laboratory Animals of Imperial College London, with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All animal procedures and care conformed strictly to the UK Home Office Guidelines under the Animals (Scientific Procedures) Act 1986, and the protocols were approved by the Home Office of Great Britain.

Tissue resident host responses to microbial infections in the respiratory tract are highly dynamic in space and time and rely on the interaction of a multitude of cell types. In an attempt to model these multicellular responses reliably in cell culture, we compare here the global transcriptional antimicrobial response to infection with influenza A virus (IAV) in precision cult lung slices (PCLS), volume defined organ discs largely maintaining the cellular composition and 3D architecture of the donor lung. To permit a fair comparison of host responses in an isogenic background we first challenged mice in vivo and murine PCLS (mPCLS) and assess host transciptomic changes by unbiased RNAseq. While core antiviral responses overlapped substantially, mPCLS lacked certain features--such as type II interferon expression--likely due to the absence of infiltrating immune cells responses. Importantly, when expanding our findings to immune experienced human precision cut lung slices (hPCLS), we find a much broader antiviral response after IAV challenge, including type I, II and III interferons, suggesting the presence of responsive tissue resident lymphocytes. To prove specificity of this response we infected hPCLS with Streptococcus pneumoniae. Ex vivo tissues responded with a distinct proinflammatory gene profile including IL1A, IL1B and IL17 expression. Blocking of IL-1 signaling partially inhibited the proinflammatory response, suggesting cellular cross-talk and a complex and specific antimicrobial reaction in this ex vivo model. In conclusion diversified tissue resident immune cell compartment distinguishes the human ex vivo model, making it an ideal system for microbiological and immunological research. ImportancePathogen interactions with the lung are very dynamic processes. In biomedical research it is paramount to model these processes in the laboratory as accurately as possible. Influenza A virus has been extensively studied in epithelial cell culture models, including advanced organoids and organ on a chip systems. We use here ex vivo cultured PCLS and use transcriptomics to assess the global tissue resident host response to viral and bacterial challenge. Our data show 1) that murine PCLS faithfully reflect core responses to viral infection, while missing proinflammatory responses linked to infiltrating immune cells and 2) that human PCLS show a highly diversified tissue resident immune response to viral infection due to previous exposures of the host to this pathogen. These responses are clearly distinct from antibacterial gene profiles. Our data advertise PCLS as a complex and realistic model to study tissue resident immune responses to microbes in a human system.

Major histocompatibility complex class I (MHC I) molecules present endogenous peptides to CD8+ T-cells for immunosurveillance of infections and cancers. Recent studies revealed unexpected heterogeneity in MHC I expression among cells of different lineages. While respiratory diseases rank among the leading causes of mortality, studies in mice showed that lung epithelial cells (LECs) express lower MHC I levels than all other tested cell types. The present study aimed to evaluate MHC I expression in human LECs from parenchymal explants using single-cell RNA sequencing (scRNA-seq) and immunostaining of primary human LECs. After confirming the low constitutive MHC I expression in human LECs, we observed a significant upregulation of MHC I across three chronic respiratory diseases: chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and cystic fibrosis (CF). Additionally, we unveiled an unexpected sexual dimorphism in MHC I expression in both health and disease, with males exhibiting higher levels of MHC I under steady-state conditions. Gene expression analyses suggest that differential redox balance between sexes is instrumental in this dimorphism. Our study unveils the complex interplay between MHC I expression, sex, and respiratory diseases. Since, in other models, MHC I upregulation contributes to the development of immunopathologies, we propose that it might have a similar impact on chronic lung diseases. NEW & NOTEWORTHYThis study shows that MHC I expression is very low in healthy LECs but escalates significantly in three chronic respiratory diseases, potentially contributing to disease progression. Furthermore, sex-specific divergences in LEC MHC I levels hint at distinct susceptibilities to chronic lung inflammation between males and females. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/599895v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@44cfaborg.highwire.dtl.DTLVardef@19e1e29org.highwire.dtl.DTLVardef@1ce179aorg.highwire.dtl.DTLVardef@16f8063_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeLung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells of stable and ALAD patients to determine key cellular elements in dysfunctional lung allografts. Our particular focus here is on studying alveolar macrophages (AMs) as scRNAseq enables us to elucidate their heterogeneity and possible association with ALAD where our knowledge from cytometry-based assays is very limited. MethodsFresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (3044 {+/-} 1519 cells) and 3 undergoing an episode of ALAD (2593 {+/-} 904 cells) were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, pathway analysis, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. ResultsOur data revealed that AMs are highly heterogeneous (12 transcriptionally distinct subsets in stable). We identified two AM subsets uniquely represented in ALAD. Based on pathway analysis and the top differentially expressed genes in BAL we annotated them as pro-inflammatory interferon-stimulated genes (ISG) and metallothioneins-mediated inflammatory (MT). Pseudotime analysis suggested that ISG AMs represent an earlier stage of differentiation which may suggest them as monocyte drive macrophages. Our functional analysis on an independent set of BAL samples shows that ALAD samples have significantly higher expression of CXCL10, a marker of ISG AM, as we as higher secretion of pro-inflammatory cytokines. Single nucleotide variation calling algorithm has allowed us to identify macrophages of donor origin and demonstrated that donor AMs are lost with time post-transplant. ConclusionUsing scRNAseq, we observed AMs heterogeneity and identified specific subsets that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of AMs in allograft injury and dysfunction.

Neutrophils were once considered a homogenous population of transcriptionally static, pathogen-killing cells, however, recent models have demonstrated neutrophil functional and transcriptional plasticity. We performed transcriptomic analyses in a murine model of pneumococcal pneumonia to investigate neutrophil plasticity and demonstrate that neutrophils are highly dynamic, leading to three distinct alveolar neutrophil populations - one immature (early bronchoalveolar lavage neutrophils [BALN]) and two mature (late BALN). Early BALNs produce high levels of inflammatory cytokine transcripts, maturing into late BALNs, including a pro-degranulation and phagocytosis population (late-degranulating BALN) or a population specializing in translation machinery and inflammatory cytokine production (late-cytokine producing BALN). Neutrophil metabolism is also regulated in a stepwise manner - tricarboxylic acid (TCA) cycle and respiratory electron transport chain (ETC) genes are downregulated as neutrophils migrate from the vasculature to the interstitium, lipid and carbohydrate metabolism genes are downregulated during migration from interstitium to the airspace. These transitions may be regulated by aspects of the integrated stress response (ISR), as key regulators including Eif2ak2 are upregulated in interstitial neutrophils. Overall, we demonstrate that pneumonic neutrophils are transcriptionally plastic, developing through two distinct transcriptional phenotypes in the airspace, and are metabolically and transcriptionally rewired with potential points of regulation occurring in the interstitial space.

ABSTRACTO_ST_ABSRationaleC_ST_ABSPatients with end stage lung diseases require lung transplantation (LTx) that can be impeded by ischemia-reperfusion injury (IRI) leading to subsequent chronic lung allograft dysfunction (CLAD) and inadequate outcomes. ObjectivesWe examined the undefined role of MerTK (receptor Mer tyrosine kinase) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis (phagocytosis of apoptotic cells) to facilitate resolution of lung IRI. MethodsSingle-cell RNA sequencing of lung tissue and BAL from post-LTx patients was analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with Balb/c (WT), cebpb-/- (MDSC-deficient), Mertk-/- or MerTK-CR (cleavage resistant) mice. Lung function, IRI (inflammatory cytokine and myeloperoxidase expression, immunohistology for neutrophil infiltration), and flow cytometry of lung tissue for efferocytosis of apoptotic neutrophils were assessed in mice. Measurements and Main ResultsA significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of CLAD patients compared to healthy subjects was observed. In the murine IRI model, significant increase in M-MDSCs, MerTK expression and efferocytosis was observed in WT mice during resolution phase that was absent in cebpb-/- Land Mertk-/- mice. Adoptive transfer of M-MDSCs in cebpb-/- mice significantly attenuated lung dysfunction, and inflammation leading to resolution of IRI. Additionally, in a preclinical murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. ConclusionsOur results suggest that MerTK-dependent efferocytosis by M-MDSCs can significantly contribute to the resolution of post-LTx IRI.

Although human lung macrophages are heterogenous and play key roles during health and disease, the mechanisms that govern their activation and function are unclear, particularly in type 2 settings. Our understanding of how human lung macrophages respond to inflammatory signals have predominantly relied on cell lines or peripheral blood derived cells, which have a limited capacity to reflect the complexity of tissue macrophage responses. Therefore, we isolated macrophages from resected human lung tissue and stimulated them ex vivo under type 2 (IL-4, IL-13, or IL-4 + IL-13) or type 1 (IFN{gamma} + LPS) conditions. Human lung macrophages stimulated with IL-4/13, alone or in combination, significantly upregulated expression of the chemokines CCL17, CCL18 and CCL22, along with the transglutaminase TGM2 and the lipoxygenase ALOX15. This type 2 activation profile was distinct from LPS + IFN{gamma} activated human lung macrophages, which upregulated IL6, IL8, IL1{beta}, TNF and CHI3L1 (YKL-40). Further, type 2 activated human lung macrophage products showed differential metabolic reliance for their induction, with IL-4/13 induced CCL22 being glycolytically controlled, while ALOX15 was regulated by fatty acid oxidation. These data clarify hallmarks of human lung macrophage activation and polarisation in addition to revealing novel metabolic regulation of type 2 markers.

Lung development begins in utero and reaches full maturity post birth. Dendritic cells (DC) play a key role in immune regulation in lungs. However, comprehensive exploration of DCs in these immature lungs has not been performed. Here we explored DCs from fetal to newborn mouse lungs phenotypically, ontogenetically, transcriptomically and functionally and found two DC subsets, resembling adult cDC1 and cDC2, but with key differences. Phenotypically, fetal-cDC1 lacks the classical-DC1 (cDC1) marker XCR1, while the fetal-cDC2 express both cDC-associated genes as well as monocyte-derived DC genes. Both DC subsets wane as lungs enter the alveolar stage, giving way to the more familiar adult cDC1 and cDC2. Both fetal-cDC1 and fetal-cDC2 derive from ED14.5 fetal liver Macrophage Dendritic Progenitors, not from monocytes or classic Precursor-cDC (Pre-cDC), indicating a unique ontogeny of first DCs in developing mouse lungs. Together we provide the first in depth exploration of first DCs in developing lungs.

RationaleTranscriptionally-defined populations of interstitial macrophages (IMs) and airspace macrophages (AMs) have recently been identified in the human lung. However, the anatomic locations occupied by these populations (i.e. alveoli, pleura, airways, or arteries) have not been fully defined. ObjectivesTo determine the distribution of transcriptionally-defined human macrophages in the major anatomical lung structures and to identify alterations in their distribution and programming induced by cigarette smoking. MethodsSingle-cell RNA sequencing was performed on lung tissue from eight human donors without pulmonary disease (four smokers and four nonsmokers). Microdissection was used to isolate distinct pulmonary anatomical structures from each lung: alveoli, pleura, airways, and arteries. Transcriptional profiles of subpopulations of interstitial macrophages (IMs) and alveolar macrophages (AMs) were analyzed based on their anatomical structure of origin and smoking status. Measurements and Main ResultsFive major IM and five AM subpopulations in human lungs are identified. We demonstrate significant differences in the accumulation patterns of each macrophage subset within anatomical structures, though each subset was detected in each. Immunofluorescent microscopy confirmed anatomical structure-specific accumulation patterns of IMs. ConclusionsIn this study, we highlight key differences in the accumulation of lung macrophage subpopulations in anatomical structures but find programming within macrophage subpopulations is largely conserved, regardless of structure of origin or smoking status. We also detect populations of inflammatory AMs and IMs which accumulate within the airways, but not the alveolar parenchyma, of human cigarette smokers. We introduce a novel three-tiered hierarchy nomenclature to distinguish transcriptionally defined human lung IM subsets as 1{degrees}) Monocyte-like vs Antigen Presenting, 2{degrees}) Quiescent vs Inflammatory, and 3{degrees}) FOLR2high vs FOLR2low. This study is the first to report the fractional accumulation of human lung macrophage subsets by lung anatomical structure. SummaryLung anatomical structure-specific single cell RNA sequencing is introduced to identify and determine the local composition of human lung leukocytes, including 5 populations of human interstitial macrophages.

The ontogenetic composition of tissue-resident macrophages following injury, environmental exposure, or experimental depletion can be altered upon re-establishment of homeostasis. However, the impact of altered resident macrophage ontogenetic milieu on subsequent immune responses is poorly understood. Hence, we assessed the effect of macrophage ontogeny alteration following return to homeostasis on subsequent allergic airway responses to house dust mites (HDM). Using lineage tracing, we confirmed alveolar and interstitial macrophage ontogeny and their replacement by bone marrow-derived macrophages following LPS exposure. This alteration in macrophage ontogenetic milieu reduced allergic airway responses to HDM challenge. In addition, we defined a distinct population of resident-derived interstitial macrophages expressing allergic airway disease genes, located adjacent to terminal bronchi, and reduced by prior LPS exposure. These findings support that the ontogenetic milieu of pulmonary macrophages is a central factor in allergic airway responses and has implications for how prior environmental exposures impact subsequent immune responses and the development of allergy.

Respiratory viral infections with SARS-CoV-2 or influenza viruses commonly induce a strong infiltration of immune cells into the lung, with potential detrimental effects on the integrity of the lung tissue. Despite comprising the largest fractions of circulating lymphocytes in the lung, little is known about how blood natural killer (NK) cells and T cell subsets are equipped for lung-homing in COVID-19 and influenza. Using 28-colour flow cytometry and re-analysis of published RNA-seq datasets, we provide a detailed comparative analysis of NK cells and T cells in peripheral blood from moderately sick COVID-19 and influenza patients, focusing on the expression of chemokine receptors known to be involved in leukocyte recruitment to the lung. The results reveal a predominant role for CXCR3, CXCR6, and CCR5 in COVID-19 and influenza patients, mirrored by scRNA-seq signatures in peripheral blood and bronchoalveolar lavage from publicly available datasets. NK cells and T cells expressing lung-homing receptors displayed stronger phenotypic signs of activation as compared to cells lacking lung-homing receptors, and activation was overall stronger in influenza as compared to COVID-19. Together, our results indicate migration of functionally competent CXCR3+, CXCR6+, and/or CCR5+ NK cells and T cells to the lungs in moderate COVID-19 and influenza patients, identifying potential common targets for future therapeutic interventions in respiratory viral infections. Author summaryThe composition of in particular CXCR3+ and/or CXCR6+ NK cells and T cells is altered in peripheral blood upon infection with SARS-CoV-2 or influenza virus in patients with moderate disease. Lung-homing receptor-expression is biased towards phenotypically activated NK cells and T cells, suggesting a functional role for these cells co-expressing in particular CXCR3 and/or CXCR6 upon homing towards the lung.

BackgroundType 2 cytokines such as IL-13 and IL-5 are important drivers of pathophysiology and exacerbation in asthma. Defining how these type 2 cytokine responses are regulated is a research priority. Epithelial cells promote type 2 responses by releasing alarmins including IL-25, IL-33 and TSLP, but much less is known about inhibitory factors. MethodsIL-13 release was measured from peripheral blood mononuclear cells (PBMC) cultured with Interleukin (IL)-2 for five days. Epithelial cell lines or human bronchial epithelial cells (HBEC) isolated from healthy or asthma donors were added to these PBMC cultured with IL-2 and release of IL-13 or IL-5 measured. To characterise the mechanisms, we assessed the effect of mechanical disruption of epithelial cells, addition of the COX inhibitor indomethacin and the G-protein inhibitor pertussis toxin. ResultsPBMC cultured with IL-2 secreted type 2 cytokines in a cell number and time dependent manner. Epithelial cell lines inhibited IL-13 and IL-5 release after co-culture with PBMC in the presence of IL-2, directly, across a transwell and using epithelial cell supernatant. Cells or supernatant from HBEC from healthy or asthma donors also inhibited the cytokine release. Trypsin treatment of conditioned media indicated that inhibitory factor(s) are trypsin insensitive. Mechanical disruption of epithelial cells or indomethacin treatment had no effect, but pertussis toxin reduced epithelial cell inhibition of IL-2 driven type 2 cytokine release. ConclusionEpithelial cells regulate cytokine release by soluble factor(s) and this could be an important immunoregulatory function of the airway epithelium.

Macrophage activation is underpinned by metabolic changes required to fight infection, resolve inflammation and enable effective wound healing. While metabolic control of macrophage activation is increasingly understood in culture systems in vitro, it remains poorly understood in more complex in vivo settings, like the lung. Here we applied novel flow cytometry based immunometabolic techniques to profile immune cell metabolism in the murine lung. We revealed a surprising role for glucose in naive alveolar macrophages (AMs) that was retained by AMs polarised in vivo with either interleukin-4 (IL-4) or lipopolysaccharide (LPS). We identified that naive interstitial macrophages (IMs) were dependent on mitochondrial and glucose metabolism, IMs polarised with IL-4 failed to induce metabolic alterations but displayed a glycolytic phenotype following LPS exposure as they adopted an M1 like metabolic profile. We also demonstrated that AMs were metabolically less responsive than IMs to intranasal delivery of LPS, but upregulated glycolysis and metabolic features of M2 polarisation (defined in vitro) in response to intranasal IL-4, including oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO) and arginine metabolism. Finally, we identified AM M2 polarisation as highly sensitive to glucose inhibition ex vivo. Thus, lung macrophage subsets display distinct metabolic responses to polarising stimuli in vivo. HighlightsO_LINaive alveolar macrophages require glucose metabolism despite residing in a low glucose environment. C_LIO_LIAlveolar macrophages are more responsive to IL-4 in vivo than LPS upregulating oxidative metabolism, lipid metabolism and glycolysis. C_LIO_LIInterstitial macrophages adopt a glycolytic phenotype characteristic of M1 BMDMs in vitro following in vivo LPS administration. C_LIO_LIAlternatively activated alveolar macrophages are extremely sensitive to glucose inhibition ex vivo. C_LI

It is now clear that group 2 innate lymphoid cells (ILC2) play crucial and sometimes opposing roles in the lung, such as restoring barrier function and integrity after viral infections or, on the contrary, exacerbating inflammation and tissue damage in allergic asthma. However, their role in lung cancer is still unclear. Here, we report that human non-small cell lung cancer patients bear increased frequencies of ILC2s in tumors, normal lung tissue and peripheral blood (PB) as compared to PB from healthy donors (HDs). Frequencies of Foxp3+ regulatory T cells were also increased in NSCLC patients, concomitantly with ILC2s. In mice bearing heterotopic lung cancer, adoptive transfer of ILC2s led to increased tumor growth and reduced survival. The frequencies of monocytic myeloid-derived suppressor cells (M-MDSCs) were found to be increased in the tumors of mice that received ILC2s as compared to controls. Overall, our results indicate that ILC2 cells play a pro-tumoral role in lung cancer potentially by recruiting immune-suppressive cells to the tumors.

Protein profiling of whole cells can accurately define cell subsets specific to disease identity and/or severity. Preserving whole cells on cytospin slides is common practice but is often used only for differential counts. Here we ran two studies and successfully applied a high-plex protein panel using the NanoString GeoMx, quantifying up to 47 proteins (of immune, cell death, and MAPK signalling markers) in bronchoalveolar lavage (BAL) cytospins from the four major cell types: macrophages, neutrophils, type-2 granulocytes and lymphocytes. Despite the small sample size for this feasibility study, several significant differences between disease and controls within several BAL cell types for both the asthma cohort (n=21) and the COPD cohort (n=20) were found. Overall, we believe applying this method can maximise biomarker discover of any precious preserved cytospin clinical samples and that the significant disease specific cell subsets discovered during the method testing are worthy of future investigation.

The proliferation, differentiation and survival of cells of the macrophage lineage depends on signals from the macrophage colony-stimulating factor receptor (CSF1R). On a C57BL/6J background homozygous kinase-dead Csf1r mutation (Csf1rE631K/E631K - E631Km/m) causes perinatal lethality. Here we demonstrate that E631Km/m mice on a mixed genetic background (C57 x BALB/c F2) are osteopetrotic and growth retarded but viable as adults with no other gross developmental deficits. They lack osteoclasts, microglia and most peripheral tissue resident macrophages and exhibit perturbed hematopoiesis. Although CD169+ tissue resident macrophages in bone marrow are considered an essential component of the hematopoietic niche, CD169 is undetectable in E631Km/m marrow and F4/80+ macrophages are depleted. These changes are associated with expansion of mature and immature granulocytes and reduced B cells, whereas monocytes and stem and progenitor populations are unaffected as a proportion of total cells. Erythropoiesis in bone marrow is maintained in E631Km/m mice, associated with a residual population of CSF1R-independent CD169-ve/F4/80+ macrophages. Nevertheless, splenic extramedullary hematopoiesis in E631Km/m mice indicates a degree of bone marrow insufficiency. Red pulp macrophages are retained but CD169+ marginal metallophil macrophages are absent and CD209b+ (SIGNR1) macrophages are present but disorganized. Circulating white blood cell count is unchanged in E631Km/m mice, but the proportion of neutrophils is greatly increased whilst B cells and monocytes are reduced. This novel model reveals the essential roles of CSF1R-dependent macrophages in hematopoiesis and demonstrates that many developmental and homeostatic functions attributed to murine resident tissue macrophages are redundant and/or specific to inbred mouse strains.

Acquired immunity in mammals depends upon capture and presentation of antigens by specialised macrophage populations in splenic marginal zone and lymph node sinuses and follicular dendritic cells (FDC) within germinal centres. Cells referred to as FDC in chickens express CSF1R, the receptor for macrophage colony-stimulating factor (CSF1) and IL34. We utilised single cell RNA-seq on CSF1R+ cells from chicken spleen to identify monocytes and two distinct populations of macrophages. TIMD4/C1Q/MAFB+ macrophages were enriched for expression of genes involved in iron metabolism. A MARCO/VSIG4+ population expressed SPIC, a transcription factor associated with red pulp macrophages in mammals but also expressed receptors (CR2) and trophic factors (TNFSF13, CXCL13) associated with mammalian FDC. SPIC+ cells were located within follicles in spleen, caecal tonsil and bursa. We generated a CSF1R knockout in the chicken germ line. Mutant birds lack macrophages in the embryo. They were indistinguishable from wild type at hatch and behaved and fed normally but from day 5-6 post hatch they failed to thrive. Loss of CSF1R function in hatchlings led to monocytopenia and granulocytosis and the loss of macrophage subpopulations in lymphoid organs. Consistent with their expression of B cell trophic factors, the loss of follicular macrophages in the bursa was associated with involution and severe B cell deficiency in the circulation and spleen. In summary, lymphoid tissues of chickens contain specialised macrophage populations with distinct expression profiles. The details of regulation by CSF1R, specialised functions and underlying transcriptional regulation are quite different between birds and mammals.