Lung-resident alveolar macrophages regulate the timing of breast cancer metastasis.

Breast disseminated cancer cells (DCCs) can remain dormant in the lungs for extended periods, but the mechanisms limiting their expansion are not well understood. Research indicates that tissue-resident alveolar macrophages suppress breast cancer metastasis in lung alveoli by inducing dormancy. Through ligand-receptor mapping and intravital imaging, it was found that alveolar macrophages express transforming growth factor (TGF)-ß2. This expression, along with persistent macrophage-cancer cell interactions via the TGF-ßRIII receptor, maintains cancer cells in a dormant state. Depleting alveolar macrophages or losing the TGF-ß2 receptor in cancer cells triggers metastatic awakening. Aggressive breast cancer cells are either suppressed by alveolar macrophages or evade this suppression by avoiding interaction and downregulating the TGF-ß2 receptor. Restoring TGF-ßRIII in aggressive cells reinstates TGF-ß2-mediated macrophage growth suppression. Thus, alveolar macrophages act as a metastasis immune barrier, and downregulation of TGF-ß2 signaling allows cancer cells to overcome macrophage-mediated growth suppression.

Cytomegalovirus subverts macrophage identity.

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.

Autocrine, Paracrine, and Endocrine Signals That Can Alter Alveolar Macrophages Function.

Alveolar macrophages (AMs) are extremely versatile cells with complex functions involved in health or diseases such as pneumonia, asthma, and pulmonary alveolar proteinosis. In recent years, it has been widely identified that the different functions and states of macrophages are the results from the complex interplay between microenvironmental signals and macrophage lineage. Diverse and complicated signals to which AMs respond are mentioned when they are described individually or in a particular state of AMs. In this review, the microenvironmental signals are divided into autocrine, paracrine, and endocrine signals based on their secreting characteristics. This new perspective on classification provides a more comprehensive and systematic introduction to the complex signals around AMs and is helpful for understanding the roles of AMs affected by physiological environment. The existing possible treatments of AMs are also mentioned in it. The thorough understanding of AMs signals modulation may be contributed to the development of more effective therapies for AMs-related lung diseases.

Endogenous LXR signaling controls pulmonary surfactant homeostasis and prevents lung inflammation.

Lung type 2 pneumocytes (T2Ps) and alveolar macrophages (AMs) play crucial roles in the synthesis, recycling and catabolism of surfactant material, a lipid/protein fluid essential for respiratory function. The liver X receptors (LXR), LXRα and LXRß, are transcription factors important for lipid metabolism and inflammation. While LXR activation exerts anti-inflammatory actions in lung injury caused by lipopolysaccharide (LPS) and other inflammatory stimuli, the full extent of the endogenous LXR transcriptional activity in pulmonary homeostasis is incompletely understood. Here, using mice lacking LXRα and LXRß as experimental models, we describe how the loss of LXRs causes pulmonary lipidosis, pulmonary congestion, fibrosis and chronic inflammation due to defective de novo synthesis and recycling of surfactant material by T2Ps and defective phagocytosis and degradation of excess surfactant by AMs. LXR-deficient T2Ps display aberrant lamellar bodies and decreased expression of genes encoding for surfactant proteins and enzymes involved in cholesterol, fatty acids, and phospholipid metabolism. Moreover, LXR-deficient lungs accumulate foamy AMs with aberrant expression of cholesterol and phospholipid metabolism genes. Using a house dust mite aeroallergen-induced mouse model of asthma, we show that LXR-deficient mice exhibit a more pronounced airway reactivity to a methacholine challenge and greater pulmonary infiltration, indicating an altered physiology of LXR-deficient lungs. Moreover, pretreatment with LXR agonists ameliorated the airway reactivity in WT mice sensitized to house dust mite extracts, confirming that LXR plays an important role in lung physiology and suggesting that agonist pharmacology could be used to treat inflammatory lung diseases.

Flagellin-modulated inflammasome pathways characterize the human alveolar macrophage response to Burkholderia pseudomallei, a lung-tropic pathogen.

Melioidosis is an emerging tropical infection caused by inhalation, inoculation, or ingestion of the flagellated, facultatively intracellular pathogen Burkholderia pseudomallei. The melioidosis case fatality rate is often high, and pneumonia, the most common presentation, doubles the risk of death. The alveolar macrophage is a sentinel pulmonary host defense cell, but the human alveolar macrophage in B. pseudomallei infection has never been studied. The objective of this study was to investigate the host-pathogen interaction of B. pseudomallei infection with the human alveolar macrophage and to determine the role of flagellin in modulating inflammasome-mediated pathways. We found that B. pseudomallei infects primary human alveolar macrophages but is gradually restricted in the setting of concurrent cell death. Electron microscopy revealed cytosolic bacteria undergoing division, indicating that B. pseudomallei likely escapes the alveolar macrophage phagosome and may replicate in the cytosol, where it triggers immune responses. In paired human blood monocytes, uptake and intracellular restriction of B. pseudomallei are similar to those observed in alveolar macrophages, but cell death is reduced. The alveolar macrophage cytokine response to B. pseudomallei is characterized by marked interleukin (IL)-18 secretion compared to monocytes. Both cytotoxicity and IL-18 secretion in alveolar macrophages are partially flagellin dependent. However, the proportion of IL-18 release that is driven by flagellin is greater in alveolar macrophages than in monocytes. These findings suggest differential flagellin-mediated inflammasome pathway activation in the human alveolar macrophage response to B. pseudomallei infection and expand our understanding of intracellular pathogen recognition by this unique innate immune lung cell.

RSV enhances Staphylococcus aureus bacterial growth in the lung.

Patients coinfected with respiratory syncytial virus (RSV) and bacteria have longer hospital stays, higher risk of intensive care unit admission, and worse outcomes. We describe a model of RSV line 19F/methicillin-resistant Staphylococcus aureus (MRSA) USA300 coinfection that does not impair viral clearance, but prior RSV infection enhances USA300 MRSA bacterial growth in the lung. The increased bacterial burden post-RSV correlates with reduced accumulation of neutrophils and impaired bacterial killing by alveolar macrophages. Surprisingly, reduced neutrophil accumulation is likely not explained by reductions in phagocyte-recruiting chemokines or alterations in proinflammatory cytokine production compared with mice infected with S. aureus alone. Neutrophils from RSV-infected mice retain their ability to migrate toward chemokine signals, and neutrophils from the RSV-infected lung are better able to phagocytize and kill S. aureus ex vivo on a per cell basis. In contrast, while alveolar macrophages could ingest USA300 post-RSV, intracellular bacterial killing was impaired. The RSV/S. aureus coinfected lung promotes a state of overactivation in neutrophils, demonstrated by increased production of reactive oxygen species (ROS) that can drive formation of neutrophil extracellular traps (NETs), resulting in cell death. Mice with RSV/S. aureus coinfection had increased extracellular DNA and protein in bronchoalveolar lavage fluid and histological evidence confirmed NETosis in vivo. Taken together, these data highlight that prior RSV infection can prime the overactivation of neutrophils leading to cell death that impairs neutrophil accumulation in the lung. Additionally, alveolar macrophage killing of bacteria is impaired post-RSV. Together, these defects enhance USA300 MRSA bacterial growth in the lung post-RSV.

Therapeutic characteristics of alveolar-like macrophages in mouse models of hyperoxia and LPS-induced lung inflammation.

Acute respiratory distress syndrome (ARDS) is a severe lung disease of high mortality (30-50%). Patients require lifesaving supplemental oxygen therapy; however, hyperoxia can induce pulmonary inflammation and cellular damage. Although alveolar macrophages (AMs) are essential for lung immune homeostasis, they become compromised during inflammatory lung injury. To combat this, stem cell-derived alveolar-like macrophages (ALMs) are a prospective therapeutic for lung diseases like ARDS. Using in vitro and in vivo approaches, we investigated the impact of hyperoxia on murine ALMs during acute inflammation. In vitro, ALMs retained their viability, growth, and antimicrobial abilities when cultured at 60% O2, whereas they die at 90% O2. In contrast, ALMs instilled in mouse lungs remained viable during exposure of mice to 90% O2. The ability of the delivered ALMs to phagocytose Pseudomonas aeruginosa was not impaired by exposure to 60 or 90% O2. Furthermore, ALMs remained immunologically stable in a murine model of LPS-induced lung inflammation when exposed to 60 and 90% O2 and effectively attenuated the accumulation of CD11b+ inflammatory cells in the airways. These results support the potential use of ALMs in patients with ARDS receiving supplemental oxygen therapy.NEW & NOTEWORTHY The current findings support the prospective use of stem cell-derived alveolar-like macrophages (ALMs) as a therapeutic for inflammatory lung disease such as acute respiratory distress syndrome (ARDS) during supplemental oxygen therapy where lungs are exposed to high levels of oxygen. Alveolar-like macrophages directly delivered to mouse lungs were found to remain viable, immunologically stable, phagocytic toward live Pseudomonas aeruginosa, and effective in reducing CD11b+ inflammatory cell numbers in LPS-challenged lungs during moderate and extreme hyperoxic exposure.

Alveolar macrophage-derived extracellular vesicles inhibit endosomal fusion of influenza virus.

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.

GDF15 ameliorates sepsis-induced lung injury via AMPK-mediated inhibition of glycolysis in alveolar macrophage.

Growth differentiation factor 15 (GDF15) as a stress response cytokine is involved in the development and progression of several diseases associated with metabolic disorders. However, the regulatory role and the underlying mechanisms of GDF15 in sepsis remain poorly defined. Our study analyzed the levels of GDF15 and its correlations with the clinical prognosis of patients with sepsis. In vivo and in vitro models of sepsis were applied to elucidate the role and mechanisms of GDF15 in sepsis-associated lung injury. We observed strong correlations of plasma GDF15 levels with the levels of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), and lactate as well as Sequential Organ Failure Assessment (SOFA) scores in patients with sepsis. In the mouse model of lipopolysaccharide-induced sepsis, recombinant GDF15 inhibited the proinflammatory responses and alleviated lung tissue injury. In addition, GDF15 decreased the levels of cytokines produced by alveolar macrophages (AMs). The anti-inflammatory effect of glycolysis inhibitor 2-DG on AMs during sepsis was mediated by GDF15 via inducing the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and the expression of activating transcription factor 4 (ATF4). Furthermore, we explored the mechanism underlying the beneficial effects of GDF15 and found that GDF15 inhibited glycolysis and mitogen-activated protein kinases (MAPK)/nuclear factor-κB (NF-κB) signaling via promoting AMPK phosphorylation. This study demonstrated that GDF15 inhibited glycolysis and NF-κB/MAPKs signaling via activating AMP-activated protein kinase (AMPK), thereby alleviating the inflammatory responses of AMs and sepsis-associated lung injury. Our findings provided new insights into novel therapeutic strategies for treating sepsis.

Lethal eosinophilic crystalline pneumonia in mice expressing a stabilized Csf2 mRNA.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the proliferation and differentiation of granulocyte and macrophage precursors. The mouse gene-encoding GM-CSF, Csf2, is regulated at both transcriptional and post-transcriptional levels. An adenine-uridine-rich element (ARE) within the 3'-untranslated region of Csf2 mRNA was shown in cell transfection studies to confer instability on this transcript. To explore the physiological importance of this element in an intact animal, we generated mice with a knock-in deletion of the 75-nucleotide ARE. Mice heterozygous for this ARE deletion developed severe respiratory distress and death within about 12 weeks of age. There was dense infiltration of lung alveolar spaces by crystal-containing macrophages. Increased stability of Csf2 mRNA was confirmed in bone marrow-derived macrophages, and elevated GM-CSF levels were observed in serum and lung. These mice did not exhibit notable abnormalities in blood or bone marrow, and transplantation of bone marrow from mutant mice into lethally irradiated WT mice did not confer the pulmonary phenotype. Mice with a conditional deletion of the ARE restricted to lung type II alveolar cells exhibited an essentially identical lethal lung phenotype at the same ages as the mice with the whole-body deletion. In contrast, mice with the same conditional ARE deletion in myeloid cells, including macrophages, exhibited lesser degrees of macrophage infiltration into alveolar spaces much later in life, at approximately 9 months of age. Post-transcriptional Csf2 mRNA stability regulation in pulmonary alveolar epithelial cells appears to be essential for normal physiological GM-CSF secretion and pulmonary macrophage homeostasis.

Lactate produced by alveolar type II cells suppresses inflammatory alveolar macrophages in acute lung injury.

Alveolar inflammation is a hallmark of acute lung injury (ALI), and its clinical correlate is acute respiratory distress syndrome-and it is as a result of interactions between alveolar type II cells (ATII) and alveolar macrophages (AM). In the setting of acute injury, the microenvironment of the intra-alveolar space is determined in part by metabolites and cytokines and is known to shape the AM phenotype. In response to ALI, increased glycolysis is observed in AT II cells, mediated by the transcription factor hypoxia-inducible factor (HIF) 1α, which has been shown to decrease inflammation. We hypothesized that in acute lung injury, lactate, the end product of glycolysis, produced by ATII cells shifts AMs toward an anti-inflammatory phenotype, thus mitigating ALI. We found that local intratracheal delivery of lactate improved ALI in two different mouse models. Lactate shifted cytokine expression of murine AMs toward increased IL-10, while decreasing IL-1 and IL-6 expression. Mice with ATII-specific deletion of Hif1a and mice treated with an inhibitor of lactate dehydrogenase displayed exacerbated ALI and increased inflammation with decreased levels of lactate in the bronchoalveolar lavage fluid; however, all those parameters improved with intratracheal lactate. When exposed to LPS (to recapitulate an inflammatory stimulus as it occurs in ALI), human primary AMs co-cultured with alveolar epithelial cells had reduced inflammatory responses. Taken together, these studies reveal an innate protective pathway, in which lactate produced by ATII cells shifts AMs toward an anti-inflammatory phenotype and dampens excessive inflammation in ALI.

Biomarkers of genotoxic damage in pulmonary alveolar macrophages: a review.

DNA damage is one of the primary mechanisms underlying cancer and other chronic degenerative diseases. Early evaluation of this damage in the affected cells and tissues is crucial for understanding pathogenesis and implementing effective prevention strategies. However, isolating target cells from affected organs, such as the lungs, can be challenging. Therefore, an alternative approach is to evaluate genotoxic damage in surrogate cells. Pulmonary alveolar macrophages are ideally suited for this purpose because they are in close contact with the target cells of the bronchial and alveolar epithelium, share the exact mechanisms and levels of exposure, and are easily recoverable in large numbers. This review comprehensively lists all studies using alveolar macrophages as surrogate cells to show genotoxic lung damage in humans or laboratory animals. These investigations provide fundamental information on the mechanisms of DNA damage in the lung and allow for better assessment and management of risk following exposure to inhalable genotoxic agents. Furthermore, they may be a valuable tool in cancer chemoprevention, helping the right choice of agents for clinical trials.

The suppression of nuclear factor kappa B/microRNA 222 axis alleviates lipopolysaccharide-induced acute lung injury through increasing the alkylglyceronephosphate synthase expression.

BACKGROUND: Acute lung injury (ALI) is a serious and rapidly progressing pulmonary disorder with a high mortality rate. In this study, we aimed to investigate the relationship between miR-222 and NF-κB (p65) activation in ALI. METHODS: ALI was induced in mice using lipopolysaccharide (LPS). Lung tissues and bronchoalveolar lavage fluid were collected for analysis. MH-S cell lines were used as an ALI model. Various techniques including histopathology, molecular analysis, and cell culture assays were employed. RESULTS: Increased miR-222 levels were observed in the LPS-induced ALI mouse model. ALI mice exhibited severe lung pathology, inflammatory cell infiltration, edema, elevated W/D ratio, MPO activity, and increased TNFα, IL1, and IL6 levels, which were reversed by miR-222 antagomir, confirming miR-222's exacerbation of LPS-induced ALI. miR-222 directly targeted the 3'-UTR of alkylglyceronephosphate synthase (AGPS) mRNA, reducing its expression. AGPS is crucial for plasmalogen synthesis, which protects against oxidative stress. NF-κB (p-p65) levels were increased in ALI models, and LPS promoted the enrichment of the miR-222 promoter region, suggesting NF-κB (p65) involvement in miR-222 transcriptional regulation. The NF-κB/miR-222/AGPS axis played a significant role in ALI progression. CONCLUSIONS: The present study indicates that NF-κB (p65) activates miR-222 transcription by enriching its promoter region, leading to increased miR-222 expression. Elevated miR-222 levels downregulate AGPS, thereby accelerating the progression of ALI. Targeting the NF-κB/miR-222/AGPS axis may hold promise as a therapeutic approach for ALI, although further research is needed to fully understand its significance.

Targeting the TRAF3-ULK1-NLRP3 regulatory axis to control alveolar macrophage pyroptosis in acute lung injury.

Acute lung injury (ALI) is a serious condition characterized by damage to the lungs. Recent research has revealed that activation of the NLRP3 inflammasome in alveolar macrophages, a type of immune cell in the lungs, plays a key role in the development of ALI. This process, known as pyroptosis, contributes significantly to ALI pathogenesis. Researchers have conducted comprehensive bioinformatics analyses and identified 15 key genes associated with alveolar macrophage pyroptosis in ALI. Among these, NLRP3 has emerged as a crucial regulator. This study further reveal that the ULK1 protein diminishes the expression of NLRP3, thereby reducing the immune response of alveolar macrophages and mitigating ALI. Conversely, TRAF3, another protein, is found to inhibit ULK1 through a process called ubiquitination, leading to increased activation of the NLRP3 inflammasome and exacerbation of ALI. This TRAF3-mediated suppression of ULK1 and subsequent activation of NLRP3 are confirmed through various in vitro and in vivo experiments. The presence of abundant M0 and M1 alveolar macrophages in the ALI tissue samples further support these findings. This research highlights the TRAF3-ULK1-NLRP3 regulatory axis as a pivotal pathway in ALI development and suggests that targeting this axis could be an effective therapeutic strategy for ALI treatment.

Pulmonary and Nonpulmonary Sepsis Differentially Modulate Lung Immunity toward Secondary Bacterial Pneumonia: A Critical Role for Alveolar Macrophages.

Clinical observations suggest that the source of primary infection accounts for a major determinant of further nosocomial pneumonia in critically ill patients with sepsis. Here we addressed the impact of primary nonpulmonary or pulmonary septic insults on lung immunity using relevant double-hit animal models. C57BL/6J mice were first subjected to polymicrobial peritonitis induced by cecal ligation and puncture (CLP) or bacterial pneumonia induced by intratracheal challenge with Escherichia coli. Seven days later, postseptic mice received ab intratracheal challenge with Pseudomonas aeruginosa. Compared with controls, post-CLP mice became highly susceptible to P. aeruginosa pneumonia, as demonstrated by defective lung bacterial clearance and increased mortality rate. In contrast, all postpneumonia mice survived the P. aeruginosa challenge and even exhibited improved bacterial clearance. Nonpulmonary and pulmonary sepsis differentially modulated the amounts and some important immune functions of alveolar macrophages. Additionally, we observed a Toll-like receptor 2 (TLR2)-dependent increase in regulatory T cells (Tregs) in lungs from post-CLP mice. Antibody-mediated Treg depletion restored the numbers and functions of alveolar macrophages in post-CLP mice. Furthermore, post-CLP TLR2-deficient mice were found resistant to secondary P. aeruginosa pneumonia. In conclusion, polymicrobial peritonitis and bacterial pneumonia conferred susceptibility or resistance to secondary gram-negative pulmonary infection, respectively. Immune patterns in post-CLP lungs argue for a TLR2-dependent cross-talk between Tregs and alveolar macrophages as an important regulatory mechanism in postseptic lung defense.

IL-33-ST2 pathway regulates AECII transdifferentiation by targeting alveolar macrophage in a bronchopulmonary dysplasia mouse model.

Evidence points to the indispensable function of alveolar macrophages (AMs) in normal lung development and tissue homeostasis. However, the importance of AMs in bronchopulmonary dysplasia (BPD) has not been elucidated. Here, we identified a significant role of abnormal AM proliferation and polarization in alveolar dysplasia during BPD, which is closely related to the activation of the IL-33-ST2 pathway. Compared with the control BPD group, AMs depletion partially abolished the epithelialmesenchymal transition process of AECII and alleviated pulmonary differentiation arrest. In addition, IL-33 or ST2 knockdown has protective effects against lung injury after hyperoxia, which is associated with reduced AM polarization and proliferation. The protective effect disappeared following reconstitution of AMs in injured IL-33 knockdown mice, and the differentiation of lung epithelium was blocked again. In conclusion, the IL-33-ST2 pathway regulates AECII transdifferentiation by targeting AMs proliferation and polarization in BPD, which shows a novel strategy for manipulating the IL-33-ST2-AMs axis for the diagnosis and intervention of BPD.

Pharmacokinetics of SPR206 in Plasma, Pulmonary Epithelial Lining Fluid, and Alveolar Macrophages following Intravenous Administration to Healthy Adult Subjects.

SPR206 is a next-generation polymyxin being developed for the treatment of multidrug-resistant (MDR) Gram-negative infections. This Phase 1 bronchoalveolar lavage (BAL) study was conducted to evaluate SPR206's safety and pharmacokinetics in plasma, pulmonary epithelial lining fluid (ELF), and alveolar macrophages (AM) in healthy volunteers. Subjects received a 100 mg intravenous (IV) dose of SPR206 infused over 1 h every 8 h for 3 consecutive doses. Each subject underwent 1 bronchoscopy with BAL at 2, 3, 4, 6, or 8 h after the start of the third IV infusion. SPR206 concentrations in plasma, BAL, and cell pellet were measured with a validated LC-MS/MS assay. Thirty-four subjects completed the study and 30 completed bronchoscopies. Mean SPR206 peak concentrations (Cmax) in plasma, ELF, and AM were 4395.0, 735.5, and 860.6 ng/mL, respectively. Mean area under the concentration-time curve (AUC0-8) for SPR206 in plasma, ELF, and AM was 20120.7, 4859.8, and 6026.4 ng*h/mL, respectively. The mean ELF to unbound plasma concentration ratio was 0.264, and mean AM to unbound plasma concentration ratio was 0.328. Mean SPR206 concentrations in ELF achieved lung exposures above the MIC for target Gram-negative pathogens for the entire 8-h dosing interval. Overall, SPR206 was well tolerated; 22 subjects (64.7%) reported at least 1 treatment-emergent adverse event (TEAE). Of the 40 reported TEAEs, 34 (85.0%) were reported as mild in severity. The most frequent TEAEs were oral paresthesia (10 subjects [29.4%]) and nausea (2 subjects [5.9%]). This study demonstrates pulmonary penetration of SPR206 and supports further development of SPR206 for the treatment of patients with serious infections caused by MDR Gram-negative pathogens.

Identification of a FGF18-expressing alveolar myofibroblast that is developmentally cleared during alveologenesis.

Alveologenesis is an essential developmental process that increases the surface area of the lung through the formation of septal ridges. In the mouse, septation occurs postnatally and is thought to require the alveolar myofibroblast (AMF). Though abundant during alveologenesis, markers for AMFs are minimally detected in the adult. After septation, the alveolar walls thin to allow efficient gas exchange. Both loss of AMFs or retention and differentiation into another cell type during septal thinning have been proposed. Using a novel Fgf18:CreERT2 allele to lineage trace AMFs, we demonstrate that most AMFs are developmentally cleared during alveologenesis. Lung mesenchyme also contains other poorly described cell types, including alveolar lipofibroblasts (ALF). We show that Gli1:CreERT2 marks both AMFs as well as ALFs, and lineage tracing shows that ALFs are retained in adult alveoli while AMFs are lost. We further show that multiple immune cell populations contain lineage-labeled particles, suggesting a phagocytic role in the clearance of AMFs. The demonstration that the AMF lineage is depleted during septal thinning through a phagocytic process provides a mechanism for the clearance of a transient developmental cell population.

Self-replicating murine ex vivo cultured alveolar macrophages as a model for toxicological studies of particle-induced inflammation.

Alveolar macrophages (AM) are integral to maintaining homeostasis within the lungs following exposure to inhaled particles. However, due to the high animal number requirements for in vitro research with primary AM, there remains a need for validated cell models that replicate alveolar macrophages in form and function to better understand the mechanisms that contribute to particle-induced inflammation and disease. A novel, easily adaptable, culture model that facilitates the continued expansion of murine alveolar macrophages for several months, termed murine ex vivo cultured AM (mexAM) has been recently described. Therefore, the present work evaluated the use of mexAMs as a suitable model for primary AM interactions with nano- and micro-sized particles. mexAM displayed a comparable profile of functional phenotype gene expression as primary AM and similar particle uptake capabilities. The NLRP3 inflammasome-driven IL-1ß inflammatory response to crystalline silica and various nanoparticles was also assessed, as well as the effects of cationic amphiphilic drugs to block particle-induced inflammation. For all endpoints, mexAM showed a comparable response to primary AM. Altogether, the present work supports the use of mexAM as a validated replacement for primary AM cultures thereby reducing animal numbers and serving as an effective model for mechanistic investigation of inflammatory pathways in particle-induced respiratory disease.

Profiling alveolar macrophage responses to inhaled compounds using in vitro high content image analysis.

One of the main hurdles in the development of new inhaled medicines is the frequent observation of foamy macrophage (FM) responses in non-clinical studies in experimental animals, which raises safety concerns and hinders progress into clinical trials. We have investigated the potential of a novel multi-parameter high content image analysis (HCIA) assay as an in vitro safety screening tool to predict drug induced FM. Rat (NR8383) and human U937-derived alveolar macrophages were exposed in vitro to a panel of model compounds with different biological activity, including inhaled bronchodilators, inhaled corticosteroids (ICS), phospholipidosis inducers and proapoptotic agents. An HCIA was utilized to produce drug-induced cell response profiles based on individual cell health, morphology and lipid content parameters. The profiles of both rat and human macrophage cell lines differentiated between cell responses to marketed inhaled drugs and compounds known to induce phospholipidosis and apoptosis. Hierarchical clustering of the aggregated data allowed identification of distinct cell profiles in response to exposure to phospholipidosis and apoptosis inducers. Additionally, in NR8383 cell responses formed two distinct clusters, associated with increased vacuolation with or without lipid accumulation. U937 cells presented a similar trend but appeared less sensitive to drug exposure and presented a narrower range of responses. These results indicate that our multi-parameter HCIA assay is suitable to generate characteristic drug-induced macrophage response profiles, thus enabling differentiation of foamy macrophage phenotypes associated with phospholipidosis and apoptosis. This approach shows great potential as pre-clinical in vitro screening tool for safety assessment of candidate inhaled medicines.