Intravital Microscopy for Imaging and Live Cell Tracking of Alveolar Macrophages in Real Time.

Classic in vitro coculture assays of pathogens with host cells have contributed significantly to our understanding of the intracellular lifestyle of several pathogens. Coculture assays with pathogens and eukaryotic cells can be analyzed through various techniques including plating for colony-forming units (CFU), confocal microscopy, and flow cytometry. However, findings from in vitro assays require validation in an in vivo model. Several physiological conditions can influence host-pathogen interactions, which cannot easily be mimicked in vitro. Intravital microscopy (IVM) is emerging as a powerful tool for studying host-pathogen interactions by enabling in vivo imaging of living organisms. As a result, IVM has significantly enhanced the understanding of infection mediated by diverse pathogens. The versatility of IVM has also allowed for the imaging of various organs as sites of local infection. This chapter specifically focuses on IVM conducted on the lung for elucidating pulmonary immune response, primarily involving alveolar macrophages, to pathogens. Additionally, in this chapter we outline the protocol for lung IVM that utilizes a thoracic suction window to stabilize the lung for acquiring stable images.

Protocol to develop human alveolar macrophage-like cells from mononuclear cells or purified monocytes for use in respiratory biology research.

Human alveolar macrophages are a unique myeloid subset critical for understanding pulmonary diseases and are difficult to access. Here, we present a protocol to generate human alveolar macrophage-like (AML) cells from fresh peripheral blood mononuclear cells or purified monocytes. We describe steps for cell isolation, incubation in a defined cocktail of pulmonary surfactant and lung-associated cytokines, phenotype analysis, and validation with human alveolar macrophages. We then detail procedures for quality control and technical readouts for monitoring microbial response. For complete details on the use and execution of this protocol, please refer to Pahari et al.1 and Neehus et al.2.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE.

Resident-tissue macrophages (RTMs) arise from embryonic precursors1,2, yet the developmental signals that shape their longevity remain largely unknown. Here we demonstrate in mice genetically deficient in 12-lipoxygenase and 15-lipoxygenase (Alox15-/- mice) that neonatal neutrophil-derived 12-HETE is required for self-renewal and maintenance of alveolar macrophages (AMs) during lung development. Although the seeding and differentiation of AM progenitors remained intact, the absence of 12-HETE led to a significant reduction in AMs in adult lungs and enhanced senescence owing to increased prostaglandin E2 production. A compromised AM compartment resulted in increased susceptibility to acute lung injury induced by lipopolysaccharide and to pulmonary infections with influenza A virus or SARS-CoV-2. Our results highlight the complexity of prenatal RTM programming and reveal their dependency on in trans eicosanoid production by neutrophils for lifelong self-renewal.

Picturing of the Lung Tumor Cellular Composition by Multispectral Flow Cytometry.

The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C-) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers.

The Competitive Endogenous RNA (ceRNA) Regulation in Porcine Alveolar Macrophages (3D4/21) Infected by Swine Influenza Virus (H1N1 and H3N2).

H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.

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.

Cytokine Cocktail Promotes Alveolar Macrophage Reconstitution and Functional Maturation in a Murine Model of Haploidentical Bone Marrow Transplantation.

Infectious pneumonia is one of the most common complications after bone marrow transplantation (BMT), which is considered to be associated with poor reconstitution and functional maturation of alveolar macrophages (AMs) post-transplantation. Here, we present evidence showing that lack of IL-13-secreting group 2 innate lymphoid cells (ILC2s) in the lungs may underlay poor AM reconstitution in a mouse model of haploidentical BMT (haplo-BMT). Recombinant murine IL-13 was able to potentiate monocyte-derived AM differentiation in vitro. When intranasally administered, a cocktail of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-13, and CCL2 not only promoted donor monocyte-derived AM reconstitution in haplo-BMT-recipient mice but also enhanced the innate immunity of the recipient animals against pulmonary bacterial infection. These results provide a useful clue for a clinical strategy to prevent pulmonary bacterial infection at the early stage of recipients post-BMT.

Alveolar macrophages rely on GM-CSF from alveolar epithelial type 2 cells before and after birth.

Programs defining tissue-resident macrophage identity depend on local environmental cues. For alveolar macrophages (AMs), these signals are provided by immune and nonimmune cells and include GM-CSF (CSF2). However, evidence to functionally link components of this intercellular cross talk remains scarce. We thus developed new transgenic mice to profile pulmonary GM-CSF expression, which we detected in both immune cells, including group 2 innate lymphoid cells and γδ T cells, as well as AT2s. AMs were unaffected by constitutive deletion of hematopoietic Csf2 and basophil depletion. Instead, AT2 lineage-specific constitutive and inducible Csf2 deletion revealed the nonredundant function of AT2-derived GM-CSF in instructing AM fate, establishing the postnatal AM compartment, and maintaining AMs in adult lungs. This AT2-AM relationship begins during embryogenesis, where nascent AT2s timely induce GM-CSF expression to support the proliferation and differentiation of fetal monocytes contemporaneously seeding the tissue, and persists into adulthood, when epithelial GM-CSF remains restricted to AT2s.

Evidence for reprogramming of monocytes into reparative alveolar macrophages in vivo by targeting PDE4b.

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

No association between pyrite content and lung cell responses to coal particles.

There has been an increase in the identification of cases of coal workers' pneumoconiosis (CWP) in recent years around the world. While there are a range of possible explanations for this, studies have implicated the pyrite content of coal as a key determinant of CWP risk. However, experimental studies to support this link are limited. The aim of this study was to assess the association between the pyrite content, and subsequent release of bioavailable iron, in coal particles and the response of lung cells involved in the pathogenesis of CWP (epithelial cells, macrophages and fibroblasts). Using real-world Australian coal samples, we found no evidence of an association between the pyrite content of the coal and the magnitude of the detrimental cell response. We did find evidence of an increase in IL-8 production by epithelial cells with increasing bioavailable iron (p = 0.01), however, this was not linked to the pyrite content of the coal (p = 0.75) and we did not see any evidence of a positive association in the other cell types. Given the lack of association between the pyrite content of real-world coal particles and lung cell cytotoxicity (epithelial cells and macrophages), inflammatory cytokine production (epithelial cells, macrophages and fibroblasts), and cell proliferation (fibroblasts) our data do not support the use of coal pyrite content as a predictor of CWP risk.

Discriminating mild from critical COVID-19 by innate and adaptive immune single-cell profiling of bronchoalveolar lavages.

How the innate and adaptive host immune system miscommunicate to worsen COVID-19 immunopathology has not been fully elucidated. Here, we perform single-cell deep-immune profiling of bronchoalveolar lavage (BAL) samples from 5 patients with mild and 26 with critical COVID-19 in comparison to BALs from non-COVID-19 pneumonia and normal lung. We use pseudotime inference to build T-cell and monocyte-to-macrophage trajectories and model gene expression changes along them. In mild COVID-19, CD8+ resident-memory (TRM) and CD4+ T-helper-17 (TH17) cells undergo active (presumably antigen-driven) expansion towards the end of the trajectory, and are characterized by good effector functions, while in critical COVID-19 they remain more naïve. Vice versa, CD4+ T-cells with T-helper-1 characteristics (TH1-like) and CD8+ T-cells expressing exhaustion markers (TEX-like) are enriched halfway their trajectories in mild COVID-19, where they also exhibit good effector functions, while in critical COVID-19 they show evidence of inflammation-associated stress at the end of their trajectories. Monocyte-to-macrophage trajectories show that chronic hyperinflammatory monocytes are enriched in critical COVID-19, while alveolar macrophages, otherwise characterized by anti-inflammatory and antigen-presenting characteristics, are depleted. In critical COVID-19, monocytes contribute to an ATP-purinergic signaling-inflammasome footprint that could enable COVID-19 associated fibrosis and worsen disease-severity. Finally, viral RNA-tracking reveals infected lung epithelial cells, and a significant proportion of neutrophils and macrophages that are involved in viral clearance.

Isolation and Functional Characterization of Myeloid-Derived Suppressor Cells in Infections Under High Containment.

The current absence of markers unique to MDSC, particularly those expanded during human infection, necessitate concurrent demonstration of their suppressive capacity to ensure unequivocal identification. This is further complicated by the array of heterogeneous markers used to characterize MDSC in various conditions and models. Standardization of phenotypic and functional characterization, as well as isolation, from infectious biological samples of patients, are critical for accurately reporting MDSC dynamics, function, organ abundance, and establishment of their therapeutic value in infectious diseases. To illustrate, we report on our established method for MDSC isolation from bronchoalveolar lavage fluid and peripheral blood of pulmonary TB patients, as well as functional impact on T cells by measuring T cell activation, proliferation, and cytokine production.

Neutrophilic asthma features increased airway classical monocytes.

BACKGROUND: Monocytes and macrophages are critical innate immune cells of the airways. Despite their differing functions, few clinical studies discriminate between them and little is known about their regulation in asthma. OBJECTIVE: We aimed to distinguish and quantify macrophages, monocytes and monocyte subsets in induced sputum and blood and examine their relationship with inflammatory and clinical features of asthma. METHODS: We applied flow cytometry to distinguish macrophages, monocytes and subsets in sputum and blood (n = 53; 45 asthma, 8 non-asthma) and a second asthma sputum cohort (n = 26). Monocyte subsets were identified by surface CD14/CD16 (CD14++ CD16- classical, CD14+ CD16+ intermediate and CD14+ CD16++ non-classical monocytes). Surface CD206, a marker of monocyte tissue differentiation, was measured in sputum. Relationship to airway inflammatory phenotype (neutrophilic n = 9, eosinophilic n = 14, paucigranulocytic n = 22) and asthma severity (severe n = 12, non-severe n = 33) was assessed. RESULTS: Flow cytometry- and microscope-quantified sputum differential cell proportions were significantly correlated. Sputum macrophage number was reduced (p = .036), while classical monocyte proportion was increased in asthma vs non-asthma (p = .032). Sputum classical monocyte number was significantly higher in neutrophilic vs paucigranulocytic asthma (p = .013). CD206- monocyte proportion and number were increased in neutrophilic vs eosinophilic asthma (p < .001, p = .013). Increased sputum classical and CD206- monocyte numbers in neutrophilic asthma were confirmed in the second cohort. Blood monocytes did not vary with airway inflammatory phenotype, but blood classical monocyte proportion and number were increased in severe vs non-severe asthma (p = .022, p = .011). CONCLUSION AND CLINICAL RELEVANCE: Flow cytometry allowed distinction of sputum macrophages, monocytes and subsets, revealing compartment-specific dysregulation of monocytes in asthma. We observed an increase in classical and CD206- monocytes in sputum in neutrophilic asthma, suggesting co-recruitment of monocytes and neutrophils to the airways in asthma. Our data suggest further investigation of how airway monocyte dysregulation impacts on asthma-related disease activity is merited.

The Application of Flow Cytometry for Simultaneous and Multi-parametric Analysis of Heterogenous Cell Populations in Basic and Clinical Research.

The use of flow cytometry allows simultaneous measurement and multiparametric analysis of single cells in a heterogenous solution. The purpose of flow cytometry can vary depending on the use of antibodies and dyes targeted for specific cell molecules. The method of immune-phenotyping with fluorescently conjugated antibodies to label cell proteins or DNA works in tandem with fluidic, optic, and electrical systems present in the flow cytometer. Some flow cytometers can detect numerous fluorescent molecules on a single cell, allowing the measurement of more than 30 parameters. This ability to detect, measure, and quantitate multiple fluorescent markers on a single cell makes the flow cytometer a useful tool for analyzing various aspects of cell phenotype and function. Here we describe a standardized protocol for surface and intracellular immune-phenotyping of murine lungs, beginning with the building of an optimal antibody panel and ending with data analysis and representation, including sample gating strategies for innate and adaptive immune responses.

Cellular and Biochemical Analysis of Bronchoalveolar Lavage Fluid from Murine Lungs.

Bronchoalveolar lavage (BAL) is a technique used to collect the contents of the airways. The fluid recovered, called BAL fluid (BALF), serves as a dynamic tool to identify various disease pathologies ranging from asthma to infectious diseases to cancer in the lungs. A wide array of tests can be performed with BALF, including total and differential leukocyte counts (DLC), enzyme-linked immunosorbent assays (ELISA) or flow-cytometric quantitation of inflammatory mediators, such as cytokines, chemokines and adhesion molecules, and assessment of nitrate and nitrite content for estimation of nitric oxide synthase (NOS) activity. Here, we describe a detailed procedure for the collection of BALF for a variety of downstream usages, including DLC by cytological and flow-cytometry-based methods, multiplex cytokine analysis by flow cytometry, and NOS activity analysis by determining nitrate and nitrite levels.

Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation.

Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis adenylate cyclase toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

GM-CSF and IL-33 Orchestrate Polynucleation and Polyploidy of Resident Murine Alveolar Macrophages in a Murine Model of Allergic Asthma.

Allergic asthma is a chronical pulmonary disease with high prevalence. It manifests as a maladaptive immune response to common airborne allergens and is characterized by airway hyperresponsiveness, eosinophilia, type 2 cytokine-associated inflammation, and mucus overproduction. Alveolar macrophages (AMs), although contributing to lung homeostasis and tolerance to allergens at steady state, have attracted less attention compared to professional antigen-presenting and adaptive immune cells in their contributions. Using an acute model of house dust mite-driven allergic asthma in mice, we showed that a fraction of resident tissue-associated AMs, while polarizing to the alternatively activated M2 phenotype, exhibited signs of polynucleation and polyploidy. Mechanistically, in vitro assays showed that only Granulocyte-Macrophage Colony Stimulating Factor and interleukins IL-13 and IL-33, but not IL-4 or IL-5, participate in the establishment of this phenotype, which resulted from division defects and not cell-cell fusion as shown by microscopy. Intriguingly, mRNA analysis of AMs isolated from allergic asthmatic lungs failed to show changes in the expression of genes involved in DNA damage control except for MafB. Altogether, our data support the idea that upon allergic inflammation, AMs undergo DNA damage-induced stresses, which may provide new unconventional therapeutical approaches to treat allergic asthma.

Teaching Old Dogs New Tricks? The Plasticity of Lung Alveolar Macrophage Subsets.

Alveolar macrophages (AMs) are highly abundant lung cells with important roles in homeostasis and immunity. Their function influences the outcome of lung infections, lung cancer, and chronic inflammatory disease. Recent findings reveal functional heterogeneity of AMs. Following lung insult, resident AMs can either remain unchanged, acquire new functionality, or be replaced by monocyte-derived AMs. Evidence from mouse models correlates AM function with their embryonic or monocyte origin. We hypothesize that resident AMs are terminally differentiated cells with low responsiveness and limited plasticity, while recruited, monocyte-derived AMs are initially highly immunoreactive but more plastic, able to change their function in response to environmental cues. Understanding cell-intrinsic and -extrinsic mechanisms determining AM function may provide opportunities for intervention in lung disease.