Alveolar Macrophage Phenotype Research Articles

Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Cathepsin C from extracellular histone-induced M1 alveolar macrophages promotes NETosis during lung ischemia-reperfusion injury

Primary graft dysfunction (PGD) is a severe form of acute lung injury resulting from lung ischemia/reperfusion injury (I/R) in lung transplantation (LTx), associated with elevated post-transplant morbidity and mortality rates. Neutrophils infiltrating during reperfusion are identified as pivotal contributors to lung I/R injury by releasing excessive neutrophil extracellular traps (NETs) via NETosis. While alveolar macrophages (AMs) are involved in regulating neutrophil chemotaxis and infiltration, their role in NETosis during lung I/R remains inadequately elucidated. Extracellular histones constitute the main structure of NETs and can activate AMs. In this study, we confirmed the significant involvement of extracellular histone-induced M1 phenotype of AMs (M1-AMs) in driving NETosis during lung I/R. Using secretome analysis, public protein databases, and transwell co-culture models of AMs and neutrophils, we identified Cathepsin C (CTSC) derived from AMs as a major mediator in NETosis. Further elucidating the molecular mechanisms, we found that CTSC induced NETosis through a pathway dependent on NADPH oxidase-mediated production of reactive oxygen species (ROS). CTSC could significantly activate p38 MAPK, resulting in the phosphorylation of the NADPH oxidase subunit p47phox, thereby facilitating the trafficking of cytoplasmic subunits to the cell membrane and activating NADPH oxidase. Moreover, CTSC up-regulated and activated its substrate membrane proteinase 3 (mPR3), resulting in an increased release of NETosis-related inflammatory factors. Inhibiting CTSC revealed great potential in mitigating NETosis-related injury during lung I/R. These findings suggests that CTSC from AMs may be a crucial factor in mediating NETosis during lung I/R, and targeting CTSC inhition may represent a novel intervention for PGD in LTx.

Differential regulation of lung homeostasis and silicosis by the TAM receptors MerTk and Axl.

TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 μL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-β and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.

Estrogen contributes to sex differences in M2a macrophages during multi-walled carbon nanotube-induced respiratory inflammation.

Lung diseases characterized by type 2 inflammation are reported to occur with a female bias in prevalence/severity in both humans and mice. This includes previous work examining multi-walled carbon nanotube (MWCNT)-induced eosinophilic inflammation, in which a more exaggerated M2a phenotype was observed in female alveolar macrophages (AMs) compared to males. The mechanisms responsible for this sex difference in AM phenotype are still unclear, but estrogen receptor (ER) signaling is a likely contributor. Accordingly, male AMs downregulated ERα expression after MWCNT exposure while female AMs did not. Thus, ER antagonist Fulvestrant was administered prior to MWCNT instillation. In females, Fulvestrant significantly attenuated MWCNT-induced M2a gene expression and eosinophilia without affecting IL-33. In males, Fulvestrant did not affect eosinophil recruitment but reduced IL-33 and M2a genes compared to controls. Regulation of cholesterol efflux and oxysterol synthesis is a potential mechanism through which estrogen promotes the M2a phenotype. Levels of oxysterols 25-OHC and 7α,25-OHC were higher in the airways of MWCNT-exposed males compared to MWCNT-females, which corresponds with the lower IL-1β production and greater macrophage recruitment previously observed in males. Sex-based changes in cholesterol efflux transporters Abca1 and Abcg1 were also observed after MWCNT exposure with or without Fulvestrant. Invitro culture with estrogen decreased cellular cholesterol and increased the M2a response in female AMs, but did not affect cholesterol content in male AMs and reduced M2a polarization. These results reveal the modulation of (oxy)sterols as a potential mechanism through which estrogen signaling may regulate AM phenotype resulting in sex differences in downstream respiratory inflammation.

Mesenchymal Stromal Cell Infusion Might Improve Bronchiolitis Obliterans after Allogeneic Hematopoietic Stem Cell Transplantation and Modulate Alveolar Macrophages in Mice

Some studies suggested that mesenchymal stromal cell (MSC) infusion might improve bronchiolitis obliterans (BO) in patients who received allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the underlying mechanism is little known. In this study, we investigated whether MSC infusion could improve BO after allo-HSCT in mice, and sought to explore its effects on lung immune microenvironment. To construct a BO after allo-HSCT model, bone marrow cells and spleen cells were harvested from C57BL/6 mice. BALB/c mice were lethally irradiated and received 5 × 10 6 bone marrow cells (for BM mice) or 5 × 10 6 bone marrow cells with 10 6 spleen cells (for BO mice) transplantation via tail vein injection. At day 28 post-transplantation, narrowing of the small airway and collagen accumulation within the small airway walls were observed in the BO mice instead of BM mice, as shown by Masson and hematoxylin-eosin staining. Bronchoalveolar lavage fluid (BALF) from the BO mice contained more lymphocytes and neutrophils than the BM mice. Also, autoreactive IgG was observed in bronchial epithelial cells in the BO group only. These data suggests that our model shared similar histological characteristics with BO patients. Alveolar macrophages (AMs) are a major immunocyte subgroup in the lung. The AMs of the BO mice had increased size compared to the BM mice, similar to “foam cells” in atherosclerosis. The number of “form cells” increased by time in the BO group, but was not found in the BM group. A significantly increased the transcription level of CD36 and ABCA1, two key lipid transport genes, was observed in the BO group at day 21, 30, and 45 post-transplantation. Also, mRNA level of M2-like macrophage-related genes like TGF-β1, CD206, Ym1 and Arg1 was upregulated significantly in the BO group compared to the BM group, since day 21 post-transplantation, but not TNF-α, a typical M1-related marker. We next tested could MSC infusion improve the outcomes of BO mice and its influences on the phenotype of AMs. To test this hypothesis, recipients were divided into four groups (BM, BO, MSC1 and MSC2). MSCs were isolated and cultured from femur bone chips of C57BL/6 mice. Mice within the MSC1 group received 10 6 MSCs via tail vein injection at day 7 and day 21 post-transplantation. Mice within the MSC2 group received 10 6 MSCs via tail vein injection at day 21 and day 28 post-transplantation. Mice within the BM or BO group did not receive MSC injection. BALF was harvested at day 14, 35, and 49. The cGVHD symptom score of the MSC1 group reached peak at day 21 post-transplantation and showed a trend of decrease afterwards. On the other hand, although the MSC2 group also showed the same trend, but increased again after day 42. The MSC1 group have significantly lower cGVHD symptom score compared to the BO group at day 35, 42 and 49. Also, the MSC1 group showed a trend of increased survival compared to the BO group. As for AM phenotypes, MSC1 group had a trend of decreased proportion on both M1-like (CD206- CD86+ iNOS+) and M2-like (CD86- CD163+) AMs compared to the BO group at day 14. At day 49, the MSC 1 and MSC2 group both showed a trend of decreased M2-like AMs and regulatory T cells (CD25+ Foxp3+) proportion. Taking together, we found that AMs of BO mice trended a M2-like phenotype. MSC infusion could improve the outcomes of BO mice, and modulate the phenotype of AMs.

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.

PROMOTING MITOCHONDRIAL DYNAMIC EQUILIBRIUM ATTENUATES SEPSIS-INDUCED ACUTE LUNG INJURY BY INHIBITING PROINFLAMMATORY POLARIZATION OF ALVEOLAR MACROPHAGES.

Sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by widespread pulmonary inflammation and immune response, in which proinflammatory polarization of alveolar macrophages (AMs) plays an important role. Mitochondria are the key intracellular signaling platforms regulating immune cell responses. Moreover, accumulating evidence suggests that the mitochondrial dynamics of macrophages are imbalanced in sepsis and severe ALI/ARDS. However, the functional significance of mitochondrial dynamics of AMs in septic ALI/ARDS remains largely unknown, and whether it regulates the polarized phenotype of AMs is also unclear. Here, we demonstrated that the mitochondrial dynamics of AMs are imbalanced, manifested by impaired mitochondrial fusion, increased fission and mitochondrial cristae remodeling, both in septic models and ARDS patients. However, suppressing excessive mitochondrial fission with Mdivi-1 or promoting mitochondrial fusion with PM1 to maintain mitochondrial dynamic equilibrium in AMs could inhibit the polarization of AMs into proinflammatory phenotype and attenuate sepsis-induced ALI. These data suggest that mitochondrial dynamic imbalance mediates altered polarization of AMs and exacerbates sepsis-induced ALI. This study provides new insights into the underlying mechanisms of sepsis-induced ALI, suggesting the possibility of identifying future drug targets from the perspective of mitochondrial dynamics in AMs.

Shaping of the alveolar landscape by respiratory infections and long-term consequences for lung immunity.

Respiratory infections and especially viral infections, along with other extrinsic environmental factors, have been shown to profoundly affect macrophage populations in the lung. In particular, alveolar macrophages (AMs) are important sentinels during respiratory infections and their disappearance opens a niche for recruited monocytes (MOs) to differentiate into resident macrophages. Although this topic is still the focus of intense debate, the phenotype and function of AMs that recolonize the niche after an inflammatory insult, such as an infection, appear to be dictated in part by their origin, but also by local and/or systemic changes that may be imprinted at the epigenetic level. Phenotypic alterations following respiratory infections have the potential to shape lung immunity for the long-term, leading to beneficial responses such as protection against allergic airway inflammation or against other infections, but also to detrimental responses when associated with the development of immunopathologies. This review reports the persistence of virus-induced functional alterations in lung macrophages, and discusses the importance of this imprinting in explaining inter-individual and lifetime immune variation.

Interstitial Macrophages Lead Early Stages of Bleomycin-Induced Lung Fibrosis and Induce Fibroblasts Activation.

A progressive fibrosing phenotype is critical in several lung diseases. It is irreversible and associated with early patient mortality. Growing evidence has revealed pulmonary macrophages' role as modulators of the fibrotic processes. The proportion, phenotype, and function of alveolar (AM) and interstitial macrophages (IM) at the early stages of bleomycin-induced pulmonary fibrosis have not been clearly described. In this way, our study aimed to characterize these macrophage populations and investigate the effect on fibroblast activation. C57BL/6 mice were intratracheally injected with bleomycin and were sacrificed at day 3, 5, and 7 for the performance of flow cytometry and fluorescent-activated cell sorting analysis for protein and gene expression quantification. After bleomycin administration, the proportion of IM was significantly higher than that of AM, which showed a decay during the inflammatory phase, and peaked at day 7. At day 7 of the inflammatory phase, AM started shifting their phenotype from M1-like towards M2, while IM showed a M2-like phenotype. Conditioned medium derived from IM sorted at day 7 induced fibroblast activation and differentiation in myofibroblasts in vitro. Our findings indicate that IM are the largest macrophage population at the early stages of experimental pulmonary fibrosis and are secreted mediators able to activate fibroblasts, pointing to macrophage modulation as a potential therapeutic strategy to restrain progressive fibrosing lung disorders.

M1-like, but not M0- or M2-like, macrophages, reduce RSV infection of primary bronchial epithelial cells in a media-dependent fashion.

Respiratory syncytial virus (RSV) is a common childhood infection that in young infants can progress into severe bronchiolitis and pneumonia. Disease pathogenesis results from both viral mediated and host immune processes of which alveolar macrophages play an important part. Here, we investigated the role of different types of alveolar macrophages on RSV infection using an in vitro co-culture model involving primary tissue-derived human bronchial epithelial cells (HBECs) and human blood monocyte-derived M0-like, M1-like, or M2-like macrophages. It was hypothesized that the in vitro model would recapitulate previous in vivo findings of a protective effect of macrophages against RSV infection. It was found that macrophages maintained their phenotype for the 72-hour co-culture time period and the bronchial epithelial cells were unaffected by the macrophage media. HBEC infection with RSV was decreased by M1-like macrophages but enhanced by M0- or M2-like macrophages. The medium used during the co-culture also impacted the outcome of the infection. This work demonstrates that alveolar macrophage phenotypes may have differential roles during epithelial RSV infection, and demonstrates that an in vitro co-culture model could be used to further investigate the roles of macrophages during bronchial viral infection.

Advanced age is associated with changes in alveolar macrophages and their responses to the stress of traumatic injury.

Alveolar macrophages (AMs) are tissue-resident cells of the lower airways that perform many homeostatic functions critical for pulmonary health and protection against pathogens. However, little is known about the factors that shape AMs during healthy aging. In these studies, we sought to characterize age-related changes in AM phenotype, function, and responses to a physiologic stressor, that is, distal injury. Age was associated with a wide range of changes in cell surface receptor and gene expression by AMs, reflecting a unique alternatively activated phenotype. AMs from aged mice also exhibited markers of cellular senescence along with down-regulation of genes involved in growth and cell cycle pathways relative to young controls. Furthermore, AMs from aged mice showed a stunted transcriptional response to distal injury compared with AMs from young mice. Many changes were found to involve glucocorticoid-regulated genes, and corticosteroid treatment of primary AMs ex vivo revealed diminished transcriptional responses in cells from aged animals. These results demonstrate that there is a complex age-dependent AM phenotype associated with dysregulated stress hormone signaling that may interfere with AM responses to physiologic stressors and could contribute to AM dysfunction and the decline of pulmonary immunity during healthy aging.

A forward genetic screen in alveolar macrophage-like cells highlights the essential role of peroxisome membrane regulation in hallmark alveolar macrophage phenotypes

Abstract Alveolar macrophages (AMs) are a tissue resident macrophage population within the lung that are critical to maintain lung homeostasis and respond to inhaled antigens. Mechanistic investigations of AM function employing genetic approaches like CRISPR-Cas9 are hindered by the inability to obtain large numbers or maintain the AM-likeness of these cells ex vivo. We recently developed fetal liver-derived alveolar-like macrophages (FLAMs), a model that maintains the expression profile and immune functions of AMs long-term ex vivo. Importantly, FLAMs are genetically tractable using CRISPR-Cas9, enabling previously impossible interrogation of AM functions. Leveraging this innovative tool, we generated a genome-wide knockout FLAM library and are now completing forward genetic screens to dissect the regulation of AM maintenance and function. One such screen identified genes that regulate the surface expression of the AM-specific protein, Siglec-F. Among the numerous AM-associated pathways we identified as positive regulators of Siglec-F, the most robust signature was for genes related to the peroxisome. Thus, we hypothesize that the peroxisome is an essential signaling platform for AM functions. To test this prediction further we are using chemical and genetic approaches to dissect peroxisome functions in AMs. We are examining how peroxisomes modulate gene expression and metabolic flux in AMs and determining how this alters AM responses to stimuli. Together, these results highlight the utility of our novel genetic screening platform in FLAMs to unlock our understanding of AM functional mechanisms like never before. This work was supported by the Rackham Research Endowment Award from Michigan State University.

Recruitment and training of alveolar macrophages after pneumococcal pneumonia.

Recovery from pneumococcal pneumonia remodels the pool of alveolar macrophages so that they exhibit new surface marker profiles, transcriptomes, metabolomes, and responses to infection. Mechanisms mediating alveolar macrophage phenotypes after pneumococcal pneumonia have not been delineated. IFN-γ and its receptor on alveolar macrophages were essential for certain, but not all, aspects of the remodeled alveolar macrophage phenotype. IFN-γ was produced by CD4+ T cells plus other cells, and CD4+ cell depletion did not prevent alveolar macrophage remodeling. In mice infected or recovering from pneumococcus, monocytes were recruited to the lungs, and the monocyte-derived macrophages developed characteristics of alveolar macrophages. CCR2 mediated the early monocyte recruitment but was not essential to the development of the remodeled alveolar macrophage phenotype. Lineage tracing demonstrated that recovery from pneumococcal pneumonias converted the pool of alveolar macrophages from being primarily of embryonic origin to being primarily of adult hematopoietic stem cell origin. Alveolar macrophages of either origin demonstrated similar remodeled phenotypes, suggesting that ontogeny did not dictate phenotype. Our data reveal that the remodeled alveolar macrophage phenotype in lungs recovered from pneumococcal pneumonia results from a combination of new recruitment plus training of both the original cells and the new recruits.

Rate of recipient-derived alveolar macrophage development and major histocompatibility complex cross-decoration after lung transplantation in humans

Alveolar macrophages (AM) play critical roles in lung tissue homeostasis, host defense, and modulating lung injury. The rate of AM turnover (donor AM replacement by circulating monocytes) after transplantation has been incompletely characterized. Furthermore, the anatomic pattern of recipient-derived lung macrophages repopulation has not been reported, nor has their ability to accumulate and present donor major histocompatibility complex (a process we refer to as MHC cross-decoration). We longitudinally characterized the myeloid content of bronchoalveolar lavage (BAL) and biopsy specimens of lung transplant recipients and found a biphasic rate in AM turnover in the allograft, with a rapid turnover perioperatively, accelerated by both the type of induction immunosuppression and the presence of primary graft dysfunction. We found that recipient myeloid cells with cell surface AM phenotype repopulated the lung in a disorganized pattern, comprised mainly of large clusters of cells. Finally, we show that recipient AM take up and present donor peptide-MHC complexes yet are not able to independently induce an in vitro alloreactive response by circulating recipient T cells.

Gammaherpesvirus Alters Alveolar Macrophages According to the Host Genetic Background and Promotes Beneficial Inflammatory Control over Pneumovirus Infection.

Human respiratory syncytial virus (hRSV) infection brings a wide spectrum of clinical outcomes, from a mild cold to severe bronchiolitis or even acute interstitial pneumonia. Among the known factors influencing this clinical diversity, genetic background has often been mentioned. In parallel, recent evidence has also pointed out that an early infectious experience affects heterologous infections severity. Here, we analyzed the importance of these two host-related factors in shaping the immune response in pneumoviral disease. We show that a prior gammaherpesvirus infection improves, in a genetic background-dependent manner, the immune system response against a subsequent lethal dose of pneumovirus primary infection notably by inducing a systematic expansion of the CD8+ bystander cell pool and by modifying the resident alveolar macrophages (AMs) phenotype to induce immediate cyto/chemokinic responses upon pneumovirus exposure, thereby drastically attenuating the host inflammatory response without affecting viral replication. Moreover, we show that these AMs present similar rapid and increased production of neutrophil chemokines both in front of pneumoviral or bacterial challenge, confirming recent studies attributing a critical antibacterial role of primed AMs. These results corroborate other recent studies suggesting that the innate immunity cells are themselves capable of memory, a capacity hitherto reserved for acquired immunity.

SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

Nanoparticle-Induced Airway Eosinophilia Is Independent of ILC2 Signaling but Associated With Sex Differences in Macrophage Phenotype Development.

The majority of lung diseases occur with a sex bias in terms of prevalence and/or severity. Previous studies demonstrated that, compared with males, female mice develop greater eosinophilic inflammation in the airways after multiwalled carbon nanotube (MWCNT) exposure. However, the mechanism by which this sex bias occurs is unknown. Two immune cells that could account for the sex bias are type II innate lymphoid cells (ILC2s) and alveolar macrophages (AMs). In order to determine which immune cell type was responsible for MWCNT-induced airway eosinophil recruitment and subsequent sex differences in inflammation and disease, male and female C57BL/6 mice were exposed to MWCNTs (2 mg/kg) via oropharyngeal aspiration, and the respiratory immune response was assessed 7 d later. Greater eosinophilia and eotaxin 2 levels were observed in MWCNT-treated females and corresponded with greater changes in airway hyperresponsiveness than those in MWCNT-treated males. In MWCNT-treated females, there was a significant increase in the frequency of ILC2s within the lungs compared with control animals. However, depletion of ILC2s via α-CD90.2 administration did not decrease eosinophil recruitment 24 h and 7 d after MWCNT exposure. AMs isolated from control and MWCNT-treated animals demonstrated that M2a macrophage phenotype gene expression, ex vivo cytokine production, and activation of (p)STAT6 were upregulated to a significantly greater degree in MWCNT-treated females than in males. Our findings suggest that sex differences in AM phenotype development, not ILC2 signaling, are responsible for the observed female bias in eosinophilic inflammation after MWCNT inhalation.

Destructive fibrotic teamwork: how both microenvironment stiffness and profibrotic interleukin 13 impair alveolar macrophage phenotype and function.

The pulmonary fibrotic microenvironment is characterized by increased stiffness of lung tissue and enhanced secretion of profibrotic soluble cues contributing to a feedback loop that leads to dysregulated wound healing and lung failure. Pinpointing the individual and tandem effects of profibrotic stimuli in impairing immune cell response remains difficult and is needed for improved therapeutic strategies. We utilized a statistical design of experiment (DOE) to investigate how microenvironment stiffness and interleukin 13 (IL13), a profibrotic soluble factor linked with disease severity, contribute to the impaired macrophage response commonly observed in pulmonary fibrosis. We used engineered bioinspired hydrogels of different stiffness, ranging from healthy to fibrotic lung tissue, and cultured murine alveolar macrophages (MH-S cells) with or without IL13 to quantify cell response and analyze independent and synergistic effects. We found that, while both stiffness and IL13 independently influence macrophage morphology, phenotype, phagocytosis and efferocytosis, these factors work synergistically to exacerbate impaired macrophage phenotype and efferocytosis. These unique findings provide insights into how macrophages in fibrotic conditions are not as effective in clearing debris, contributing to fibrosis initiation/progression, and more broadly inform how underlying drivers of fibrosis modulate immune cell response to facilitate therapeutic strategies.

P5‐7: Phagocytotic analysis of alveolar macrophage phenotypes in patients with Mycobacterium avium complex lung disease

P5‐7: Phagocytotic analysis of alveolar macrophage phenotypes in patients with <i>Mycobacterium avium</i> complex lung disease

Assessment of Alveolar Macrophage Dysfunction Using an in vitro Model of Acute Respiratory Distress Syndrome.

Background: Impaired alveolar macrophage (AM) efferocytosis may contribute to acute respiratory distress syndrome (ARDS) pathogenesis; however, studies are limited by the difficulty in obtaining primary AMs from patients with ARDS. Our objective was to determine whether an in vitro model of ARDS can recapitulate the same AM functional defect observed in vivo and be used to further investigate pathophysiological mechanisms.Methods: AMs were isolated from the lung tissue of patients undergoing lobectomy and then treated with pooled bronchoalveolar lavage (BAL) fluid previously collected from patients with ARDS. AM phenotype and effector functions (efferocytosis and phagocytosis) were assessed by flow cytometry. Rac1 gene expression was assessed using quantitative real-time PCR.Results: ARDS BAL treatment of AMs decreased efferocytosis (p = 0.0006) and Rac1 gene expression (p = 0.016); however, bacterial phagocytosis was preserved. Expression of AM efferocytosis receptors MerTK (p = 0.015) and CD206 (p = 0.006) increased, whereas expression of the antiefferocytosis receptor SIRPα decreased following ARDS BAL treatment (p = 0.036). Rho-associated kinase (ROCK) inhibition partially restored AM efferocytosis in an in vitro model of ARDS (p = 0.009).Conclusions: Treatment of lung resection tissue AMs with ARDS BAL fluid induces impairment in efferocytosis similar to that observed in patients with ARDS. However, AM phagocytosis is preserved following ARDS BAL treatment. This specific impairment in AM efferocytosis can be partially restored by inhibition of ROCK. This in vitro model of ARDS is a useful tool to investigate the mechanisms by which the inflammatory alveolar microenvironment of ARDS induces AM dysfunction.