Alveolar Macrophage Phenotype Research Articles

Effect of High Glucose on Human Alveolar Macrophage Phenotype and Phagocytosis of Mycobacteria.

Diabetes mellitus (DBM) reduces immunological activity and increases susceptibility to various infections, including tuberculosis (TB). Human alveolar macrophage (hAM) functions are altered in DBM. To mimic hyperglycemic conditions in the lung alveolus, we co-cultured a hAM cell line (Daisy cell line) with human umbilical vein endothelial cells for 48h in the presence of culture media alone, normal glucose (5mM), and high glucose (22mM). Using flow cytometry, immunophenotype characterization included cell surface markers CD 11c, CD14, CD16, CD86, CD163, CD169, CD206, CX3CR-1, CSF-1R, and matrix metalloproteinase-9 (MMP9). Phagocytic function was measured by immunofluorescence microscopy at 24h after inoculation of cells with GFP-expressing Mycobacterium smegmatis. Direct exposure of AMs to high glucose and exposure in the co-culture system yield different results for the same phenotypic markers. MMP9 expression was increased under both conditions. CD169 and CX3CR1 expressions were decreased when AMs were exposed directly to high glucose but increased under co-culture. Immunofluorescence assay revealed that phagocytosis decreased in AMs when directly exposed to increased glucose levels from 2.5mM to normal glucose (5mM), yet AMs under co-culture did not show decreased phagocytosis until concentrations were raised to 25mM. Alteration in the expression of certain receptors may contribute to defective sentinel function of AMs, promoting susceptibility to TB in a diabetic host. Variability in cell surface marker expression under direct glucose exposure compared to exposure via co-culture reveals that cell signaling between endothelial cells and AMs may play a crucial role in the phenotypic expression of AMs.

Transcriptional and functional diversity of human macrophage repolarization

Macrophage plasticity allows cells to adopt different phenotypes, a property with important implications in disorders such as cystic fibrosis (CF) and asthma. We sought to examine the transcriptional and functional significance of macrophage repolarization from an M1 to an M2 phenotype and assess the role of a common human genetic disorder (CF) and a prototypical allergic disease (asthma) in this transformation. Monocyte-derived macrophages were collected from healthy subjects and patients with CF and polarized to an M2 state by using IL-4, IL-10, glucocorticoids, apoptotic PMNs, or azithromycin. We performed transcriptional profiling and pathway analysis for each stimulus. We assessed the ability of M2-repolarized macrophages to respond to LPS rechallenge and clear apoptotic neutrophils and used murine models to determine conserved functional responses to IL-4 and IL-10. We investigated whether M2 signatures were associated with alveolar macrophage phenotypes in asthmatic patients. We found that macrophages exhibit highly diverse responses to distinct M2-polarizing stimuli. Specifically, IL-10 activated proinflammatory pathways and abrogated LPS tolerance, allowing rapid restoration of LPS responsiveness. In contrast, IL-4 enhanced LPS tolerance, dampening proinflammatory responses after repeat LPS challenge. Acommon theme observed across all M2 stimuli was suppression of interferon-associated pathways. We found that CF macrophages had intact reparative and transcriptional responses, suggesting that macrophage contributions to CF-related lung disease are primarily shaped by their environment. Finally, we leveraged invitro-derived signatures to show that allergen provocation induces distinct M2 state transcriptional patterns in alveolar macrophages. Our findings highlight the diversity of macrophage polarization, attribute functional consequences to different M2 stimuli, and provide a framework to phenotype macrophages in disease states.

IPSC-Derived Macrophages Effectively Treat Pulmonary Alveolar Proteinosis in Csf2rb-Deficient Mice

SummaryInduced pluripotent stem cell (iPSC)-derived hematopoietic cells represent a highly attractive source for cell and gene therapy. Given the longevity, plasticity, and self-renewal potential of distinct macrophage subpopulations, iPSC-derived macrophages (iPSC-Mφ) appear of particular interest in this context. We here evaluated the airway residence, plasticity, and therapeutic efficacy of iPSC-Mφ in a murine model of hereditary pulmonary alveolar proteinosis (herPAP). We demonstrate that single pulmonary macrophage transplantation (PMT) of 2.5–4 × 106 iPSC-Mφ yields efficient airway residence with conversion of iPSC-Mφ to an alveolar macrophage (AMφ) phenotype characterized by a distinct surface marker and gene expression profile within 2 months. Moreover, PMT significantly improves alveolar protein deposition and other critical herPAP disease parameters. Thus, our data indicate iPSC-Mφ as a source of functional macrophages displaying substantial plasticity and therapeutic potential that upon pulmonary transplantation will integrate into the lung microenvironment, adopt an AMφ phenotype and gene expression pattern, and profoundly ameliorate pulmonary disease phenotypes.

Pulmonary Transplantation of Human Induced Pluripotent Stem Cell-derived Macrophages Ameliorates Pulmonary Alveolar Proteinosis.

Although the transplantation of induced pluripotent stem cell (iPSC)-derived cells harbors enormous potential for the treatment of pulmonary diseases, in vivo data demonstrating clear therapeutic benefits of human iPSC-derived cells in lung disease models are missing. We have tested the therapeutic potential of iPSC-derived macrophages in a humanized disease model of hereditary pulmonary alveolar proteinosis (PAP). Hereditary PAP is caused by a genetic defect of the GM-CSF (granulocyte-macrophage colony-stimulating factor) receptor, which leads to disturbed macrophage differentiation and protein/surfactant degradation in the lungs, subsequently resulting in severe respiratory insufficiency. Macrophages derived from human iPSCs underwent intrapulmonary transplantation into humanized PAP mice, and engraftment, in vivo differentiation, and therapeutic efficacy of the transplanted cells were analyzed. On intratracheal application, iPSC-derived macrophages engrafted in the lungs of humanized PAP mice. After 2 months, transplanted cells displayed the typical morphology, surface markers, functionality, and transcription profile of primary human alveolar macrophages. Alveolar proteinosis was significantly reduced as demonstrated by diminished protein content and surfactant protein D levels, decreased turbidity of the BAL fluid, and reduced surfactant deposition in the lungs of transplanted mice. We here demonstrate for the first time that pulmonary transplantation of human iPSC-derived macrophages leads to pulmonary engraftment, their in situ differentiation to an alveolar macrophage phenotype, and a reduction of alveolar proteinosis in a humanized PAP model. To our knowledge, this finding presents the first proof-of-concept for the therapeutic potential of human iPSC-derived cells in a pulmonary disease and may have profound implications beyond the rare disease of PAP.

Effects of static magnetic fields on natural or magnetized mesenchymal stromal cells: Repercussions for magnetic targeting

The magnetic targeting (MT) technique improves delivery of mesenchymal stromal cells (MSCs) to target sites. However, the moderate-intensity static magnetic fields (SMF) used for MT may exert adverse effects on MSCs. Thus, we aimed to evaluate the effects of SMF on MSCs in vitro. Cells were initially magnetized using citrate-coated magnetite nanoparticles. Then, control and magnetized MSCs were transferred to an in vitro MT system and exposed to 0.3–0.45 Tesla SMFs. MSC viability, morphology, ultrastructure, proliferation rates, differentiation, and immunomodulation were evaluated after 24 and 48 hours of exposure. MSCs temporarily lost viability and exhibited ultrastructural changes after exposure to SMFs, regardless of magnetization. Moreover, exposure to SMF reduced magnetized MSC proliferation rates. Nevertheless, MSCs remained functional (i.e., capable of differentiating, secreting repair mediators, and modulating alveolar macrophage phenotype). Thus, the experimental protocol tested in this experiment can be applied in future in vivo MT studies.

Novel role of CREB in regulating maturation of alveolar macrophages and thereby in maintaining lung‐fluid balance

Alveolar macrophages (AM) represent a specialized subpopulation of lung resident macrophages responsible for coordinating both the initiation and resolution of inflammatory lung injury. Analysis of the dynamics of lung resident macrophage populations using LyzM‐GFP mice (green myeloid cells) over the course of five days post‐injury by nebulized LPS showed a gradual decline in AM during injury phase but AM expanded and returned to baseline levels by 5th day post injury. While evidence suggests that recruited bone marrow monocytes can adopt AM phenotype following their differentiation into interstitial macrophage mechanisms reconstituting AM remains elusive. CREB, a cAMP‐Response Element Binding protein, is known to transcribe genes which regulate several cellular processes including cell differentiation, angiogenesis and anti‐inflammatory signaling. Mice lacking CREB rapidly die after birth primarily due to impairment of lung function. Thus, we generated mice lacking CREB in myeloid cells (CREBΔMye) by crossing CREBfl/fl mice with LyzM‐Cre mice to test the hypothesis that CREB regulates AM generation and thereby maintains lung‐fluid homeostasis. We show that in contrast to CREBfl/fl mice, CREBΔMye lungs developed lung edema basally. LPS induced significantly more edema formation at 4h in CREBΔMye than control mice. Whereas the edema resolved fully in control mice at 24h CREBΔMye lungs remained edematous. FACS analysis showed ~50% reduction in AM in CREBΔMye lungs (SiglecF+/CD11b‐/CD11c+ cells) basally and at 24 h following LPS‐induced lung injury. Intriguingly, we found increase in MHCII+/Siglec+/CD11clo lung cell population in CREBΔMye lungs suggesting loss of CREB in myeloid cells led to generation of incompetent pre‐AM/interstitial phenotype. Bone marrow derived macrophages from floxed and CREB lacking mice indicated dysregulation of several glycolytic enzymes, including hexokinase and pyruvate dehydrogenase. Further studies are being carried out to fully characterize the transcriptome of CREB lacking lung macrophages and whether CREB regulation of metabolic reprogramming is required for full differentiation of Pre‐AM/interstitial macrophages into AMs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Distinct Properties of Human M-CSF and GM-CSF Monocyte-Derived Macrophages to Simulate Pathological Lung Conditions In Vitro: Application to Systemic and Inflammatory Disorders with Pulmonary Involvement.

Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases (lung cancers, sarcoidosis and Systemic Sclerosis (SSc)-associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc.

Multiple-day binge ethanol intoxication is associated with an anti-inflammatory alveolar macrophage phenotype

Multiple-day binge ethanol intoxication is associated with an anti-inflammatory alveolar macrophage phenotype

Metabolic characterization and RNA profiling reveal glycolytic dependence of profibrotic phenotype of alveolar macrophages in lung fibrosis.

Metabolic reprogramming has been intrinsically linked to macrophage activation. Alveolar macrophages are known to play an important role in the pathogenesis of pulmonary fibrosis. However, systematic characterization of expression profile in these cells is still lacking. Furthermore, main metabolic programs and their regulation of cellular phenotype are completely unknown. In this study, we comprehensively analyzed the expression profile and main metabolic programs in alveolar macrophages from mice with or without experimental pulmonary fibrosis. We found that alveolar macrophages from both bleomycin and active TGF-β1-induced fibrotic mouse lungs demonstrated a primarily profibrotic M2-like profile that was distinct from the well-defined M1 or any of the M2 subtypes. More importantly, we found that fibrotic lung alveolar macrophages assumed augmented glycolysis, which was likely attributed to enhanced expression of multiple key glycolytic mediators. We also found that fatty acid oxidation was upregulated in these cells. However, the profibrotic M2-like profile of fibrotic lung alveolar macrophages was not dependent on fatty acid oxidation and synthesis or lipolysis, but instead on glycolysis, in contrast to the typical IL-4-induced macrophages M(IL-4). Additionally, glutaminolysis, a key metabolic program that has been implicated in numerous pathologies, was not required for the profibrotic M2-like phenotype of these macrophages. In summary, our study identifies a unique expression and metabolic profile in alveolar macrophages from fibrotic lungs and suggests glycolytic inhibition as an effective antifibrotic strategy in treating lung fibrosis.

Influence of aging on the phenotype and function of alveolar macrophages

Abstract Aging is associated with a decline in immune function (immunosenescence), resulting in increased susceptibility to infectious diseases, malignancies, and autoimmunity. However, the immunosenescence paradigm does not explain increased circulating pro-inflammatory cytokines and increased tissue expression of pro-inflammatory genes with aging. These observations have led to a second paradigm, termed inflammaging, in which chronic, low grade inflammation develops with increasing age. Published studies have shown that inflammaging also occurs in the lung. However, the influence of inflammaging on alveolar macrophage (AM) phenotype and function is largely unexplored. In this study, we examined the phenotype and function of AMs isolated from young (3 months) and old (18 months) C57BL/6 mice. Phenotypic analysis of AMs by flow cytometry showed that in young mice there is a major AM population that is CD11c+CD11b− and a minor population that is CD11c+CD11b+. In young mice, this minor population represents 1.3% of AMs. However, in old mice the percentage of this minor population is significantly higher (4.9%, p <0.0001). The CD11c+CD11b+ AMs have higher expression of several cell surface markers, e.g. MHC II and CD64, indicating that these AMs are in a more prime or activated state. Functional studies of these AMs stimulated with LPS, PamCSK4, or infected with Mycobacterium tuberculosis show significant spontaneous production of IFN-β, CCL2, and IL-10 that is prolonged in old mice and increased further by stimulation with ligands or infection. Our studies indicate that inflammaging in the lung primes AMs, resulting in robust production of key cytokines upon stimulation that potentially alter host susceptibility.

Long‐Living Alveolar Macrophages Modulate Severity of Lung Injury and Postnatal Lung Development in Hyperoxia‐Induced Lung Injury Mouse Model

RationaleDespite advances in neonatology, bronchopulmonary dysplasia (BPD) continues to be a serious medical problem with long‐term consequences. Alveolar macrophages are long‐living cells which populate lung soon after birth and maintain their population via proliferation, without contribution from circulating monocytes. Normally alveolar macrophages play an essential role in maintaining lung homeostasis and limiting inflammatory response to the pathogens. We hypothesize that neonatal exposure to hyperoxia, via epigenetic mechanisms, may shape these long‐living alveolar macrophages into pro‐inflammatory cells, which contribute to the development of BPD. We sought to investigate the kinetic and phenotype of alveolar macrophages in a mouse model of BPD and to test whether depletion of hyperoxia‐primed alveolar macrophages and recruitment of the monocyte‐derived alveolar macrophages under the normoxic conditions may improve outcomes in mouse model of hyperoxia‐induced lung injury/BPD.MethodsNewborn mice were exposed to room air (normoxia) or 85% O2 (hyperoxia) for 14 days (P0–P14). Alveolar macrophages were depleted via intranasal instillation of clodronate‐loaded liposomes at P14, control group received PBS. Lungs were analyzed using histology, immunohistochemistry, and flow cytometry at P14 and P28. In addition, we performed gene expression profiling of the FACSorted alveolar macrophages, alveolar type 2 epithelial cells and endothelial cells at P14 and 28 via RNA‐seq.ResultsHyperoxia exposure affected lung development (increased mean alveolar area, decreased alveolar count, decreased number of small vessels at P14 and P28). At P14, the number of alveolar macrophages was decreased in hyperoxia‐exposed mice, and alveolar macrophages had an inflammatory phenotype. By P28, the number of alveolar macrophages was expanded in hyperoxia‐exposed mice. Intranasal administration of clodronate‐loaded liposomes at P14 depleted the preexisting pool of alveolar macrophags and induced recruitment of the circulating monocytes to repopulate the niche. This depletion of hyperoxia‐primed inflammatory alveolar macrophages resulted in partial reversal of hyperoxia‐induced lung injury/BPD phenotype (decrease in mean alveolar area, increase in mean alveolar count and number of small vessels). To unravel the contribution of alveolar macrophages to the development and amelioration of hyperoxia lung injury phenotype, we performed transcriptional profiling via RNA‐seq of the whole lung tissue, and FACSorted alveolar macrophages, alveolar type 2 cells and lung endothelial cells during the course of hyperoxia‐induced lung injury with and without treatment with clodronate‐loaded liposomes. We found that at P28 alveolar macrophages from mice exposed to hyperoxia from P0 to P14 had dramatically different gene expression profiles, with a prevalent pro‐inflammatory signature, which was attenuated after treatment with clodronate‐loaded liposomes.ConclusionNeonatal hyperoxia exposure can induce a long‐lasting pro‐inflammatory alveolar macrophage phenotype, most likely via epigenetic mechanisms, which can contribute to the development of hyperoxia‐induced lung injury. Depletion of hyperoxia‐primed alveolar macrophages and recruitment of the new alveolar macrophages under normoxia conditions resulted in a partial reversal of the lung injury/BPD phenotype in mouse.Support or Funding InformationThis work was supported by HL109478 for Dr. Farrow and ATS/Scleroderma Foundation Research Grant for Dr. Misharin.

Impaired defenses of neonatal mouse alveolar macrophage with cftr deletion are modulated by glutathione and TGFβ1.

Our understanding of the intrinsic effects of cystic fibrosis (CF) transmembrane conductance regulator (cftr) deletion on resident neonatal alveolar macrophage (AM) remains limited. We previously demonstrated that diminished glutathione (GSH) or excessive AM transforming growth factor beta one (TGF β1) contributes to AM dysfunction in a variety of disease states. In this study, using a gut‐corrected cftr neonatal knockout (KO) mouse model and a siRNA‐manipulated macrophage‐like cell line (THP‐1 cell), we hypothesized (1) that cftr mutation alone increases neonatal AM oxidant stress and cellular TGF β1 signaling via altered GSH, thereby impairing cellular function, and (2) that exogenous GSH attenuates AM alterations and dysfunction in the KO AM. In neonatal KO mice, the baseline bronchoalveolar lavage fluid demonstrated a near doubling in mixed disulfides (P ≤ 0.05) and oxidized GSSG (P ≤ 0.05) without concurrent inflammation compared to WT littermates. KO AM demonstrated diminished AM thiols (P ≤ 0.05), increased AM mitochondrial ROS (P ≤ 0.05), increased AM TGF β1 (P ≤ 0.05) with increased TGF β1 signaling (P ≤ 0.05), and impaired phagocytosis (P ≤ 0.05). KO AM mitochondrial ROS was modulated by exogenous GSH (P ≤ 0.05). Conversely, TGF β1 was reduced (P ≤ 0.05) and impaired phagocytosis was rescued (P ≤ 0.05) by exogenous GSH in the KO AM. These results suggest that an altered neonatal AM phenotype may contribute to the initiation of lung inflammation/infection in the CF lung. Modulation of the AM in the neonatal CF lung may potentially alter progression of disease.

First-Breath-Induced Type 2 Pathways Shape the Lung Immune Environment

SummaryFrom birth onward, the lungs are exposed to the external environment and therefore harbor a complex immunological milieu to protect this organ from damage and infection. We investigated the homeostatic role of the epithelium-derived alarmin interleukin-33 (IL-33) in newborn mice and discovered the immediate upregulation of IL-33 from the first day of life, closely followed by a wave of IL-13-producing type 2 innate lymphoid cells (ILC2s), which coincided with the appearance of alveolar macrophages (AMs) and their early polarization to an IL-13-dependent anti-inflammatory M2 phenotype. ILC2s contributed to lung quiescence in homeostasis by polarizing tissue resident AMs and induced an M2 phenotype in transplanted macrophage progenitors. ILC2s continued to maintain the M2 AM phenotype during adult life at the cost of a delayed response to Streptococcus pneumoniae infection in mice. These data highlight the homeostatic role of ILC2s in setting the activation threshold in the lung and underline their implications in anti-bacterial defenses.

Regulatory effects of miR-155 and miR-146a on repolarization and inflammatory cytokine secretion in human alveolar macrophages in vitro

Macrophages play an important role in inflammatory responses; however, miRNA-mediated repolarization of macrophages is essential for fulfilling this function. To clarify a series of changes at the RNA level in alveolar macrophages under normal and inflammatory conditions, bronchial alveolar lavage liquid (BALF) was collected from healthy volunteers or patients with pneumonia. This approach, which differs from that used in previously, provides more accurate information about the states of macrophages in different lung microenvironments. In this study, the density plots of macrophage subtypes (M1 and M2) in the BALF of healthy volunteers differed from that of the patients with pneumonia. The M2 subtype dominated in healthy volunteers and was rapidly repolarized to M1 in response to miRNA-mediated gene regulation. Differential miRNA expression in the two macrophage subtypes revealed lower expression of miR-155 and MIR-146a in patients with pneumonia compared with healthy volunteers; this may be related to inflammation and the use of anti-inflammatory drugs. We also found increased TNF-α and IL-6 expression at the RNA level, while macrophage galactose-type C-type lectin 1 (MGL-1) expression decreased with downregulation of miR-155 and miR-146a expression. These results indicate that the gene regulation mediated by miR-155 and miR-146a contributes to human alveolar macrophage phenotype repolarization, thus leading to an early switch from pro-inflammatory to anti-inflammatory cytokine production.

Impaired Recognition of Mycobacterium tuberculosis by Alveolar Macrophages From Diabetic Mice.

Diabetes mellitus is associated with increased tuberculosis risk and severity. We previously reported that tuberculosis susceptibility in diabetic mice results from a delay in innate immune response to inhaled Mycobacterium tuberculosis, leading to delayed adaptive immune priming and, consequently, a higher plateau lung bacterial burden and greater immune pathology. We tested the capacity of alveolar macrophages from diabetic mice to phagocytose M. tuberculosis ex vivo and promote T-cell activation in vivo. Alveolar macrophages from diabetic mice had reduced expression of CD14 and macrophage receptor with collagenous structure (MARCO), which recognize the bacterial cell wall component trehalose 6,6'-dimycolate (TDM). Diabetic alveolar macrophages exhibited reduced phagocytosis of M. tuberculosis or TDM-coated latex beads. This alveolar macrophage phenotype was absent in peritoneal and bone marrow-derived macrophages. Transfer of infected alveolar macrophages from diabetic mice into nondiabetic recipients confirmed an intrinsic alveolar macrophage defect that hindered T-cell priming. The diabetic alveolar macrophage phenotype depended in part on expression of the receptor for advanced glycation end products. Reduced MARCO and CD14 expression contributes to defective sentinel function of alveolar macrophages, promoting tuberculosis susceptibility in diabetic hosts at a critical early step in the immune response to aerosol infection.

Identification of alveolar macrophage phenotypes predictive of disease progression in idiopathic pulmonary fibrosis

Identification of alveolar macrophage phenotypes predictive of disease progression in idiopathic pulmonary fibrosis

Update on the role of alternatively activated macrophages in asthma.

Lung macrophages link innate and adaptive immune responses during allergic airway inflammatory responses. Alveolar macrophages (AMs) and interstitial macrophages are two different phenotypes that differentially exert immunological function under physiological and pathological conditions. Exposure to pathogen induces polarization of AM cells into classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells). M1 cells dominantly express proinflammatory cytokines such as TNF-α and IL-1 β and induce lung inflammation and tissue damage. M2 cells are further divided into M2a and M2c subsets. M2a cells dominantly produce allergic cytokines IL-4 and IL-13, but M2c cells dominantly produce anti-inflammatory cytokine IL-10. M2a and M2c cells are differently involved in initiation, inflammation resolution, and tissue remodeling in the different stages of asthma. Microenvironment dynamically influences polarization of AM cells. Cytokines, chemokines, and immune-regulatory cells interplay and affect the balance between the polarization of M1 and M2 cells, subsequently influencing disease progression. Thus, modulation of AM phenotypes through molecular intervention has therapeutic potential in the treatment of asthma and other allergic inflammatory diseases. This review updated recent advances in polarization and functional specialization of these macrophage subtypes with emphasis on modulation of polarization of M2 cells in asthma of human subjects and animal models.

Single-cell analysis reveals differential regulation of the alveolar macrophage actin cytoskeleton by surfactant proteins A1 and A2: implications of sex and aging.

BackgroundSurfactant protein A (SP-A) contributes to lung immunity by regulating inflammation and responses to microorganisms invading the lung. The huge genetic variability of SP-A in humans implies that this protein is highly important in tightly regulating the lung immune response. Proteomic studies have demonstrated that there are differential responses of the macrophages to SP-A1 and SP-A2 and that there are sex differences implicated in these responses.MethodsPurified SP-A variants were used for administration to alveolar macrophages from SP-A knockout (KO) mice for in vitro studies, and alveolar macrophages from humanized SP-A transgenic mice were isolated for ex vivo studies. The actin cytoskeleton was examined by fluorescence and confocal microscopy, and the macrophages were categorized according to the distribution of polymerized actin.ResultsIn accordance with previous data, we report that there are sex differences in the response of alveolar macrophages to SP-A1 and SP-A2. The cell size and F-actin content of the alveolar macrophages are sex- and age-dependent. Importantly, there are different subpopulations of cells with differential distribution of polymerized actin. In vitro, SP-A2 destabilizes actin in female, but not male, mice, and the same tendency is observed by SP-A1 in cells from male mice. Similarly, there are differences in the distribution of AM subpopulations isolated from SP-A transgenic mice depending on sex and age.ConclusionsThere are marked sex- and age-related differences in the alveolar macrophage phenotype as illustrated by F-actin staining between SP-A1 and SP-A2. Importantly, the phenotypic switch caused by the different SP-A variants is subtle, and pertains to the frequency of the observed subpopulations, demonstrating the need for single-cell analysis approaches. The differential responses of alveolar macrophages to SP-A1 and SP-A2 highlight the importance of genotype in immune regulation and the susceptibility to lung disease and the need for development of individualized treatment options.

Pulmonary resident neutrophils regulate the production of GM-CSF and alveolar macrophages.

Alveolar macrophages exist in the lung airspaces, and their differentiation and function are considerably regulated by the microenvironment. In this study, we examine the important role of resident neutrophil/IL-23/granulocyte/macrophage colony-stimulating factor (GM-CSF) axis in the development and preferential phenotype of alveolar macrophages under physiological conditions. Using CD18-deficient (CD18(-/-) ) mice, we show a correlation between increased granulopoiesis and enhanced alveolar macrophage development in an IL-23- and GM-CSF-dependent manner. The apoptotic neutrophils could inhibit the secretion of IL-23 from alveolar macrophages, which is important for the production of GM-CSF, and depletion of neutrophils disrupted the regulation of IL-23 and GM-CSF. This study reveals a mechanism for the regulation of the local alveolar macrophage population and function by neutrophil apoptosis in the circulatory system.

Acute Exposure to Crystalline Silica Reduces Macrophage Activation in Response to Bacterial Lipoproteins.

Numerous studies have examined the relationship between alveolar macrophages (AMs) and crystalline silica (SiO2) using in vitro and in vivo immunotoxicity models; however, exactly how exposure to SiO2 alters the functionality of AM and the potential consequences for immunity to respiratory pathogens remains largely unknown. Because recognition and clearance of inhaled particulates and microbes are largely mediated by pattern recognition receptors (PRRs) on the surface of AM, we hypothesized that exposure to SiO2 limits the ability of AM to respond to bacterial challenge by altering PRR expression. Alveolar and bone marrow-derived macrophages downregulate TLR2 expression following acute SiO2 exposure (e.g., 4 h). Interestingly, these responses were dependent on interactions between SiO2 and the class A scavenger receptor CD204, but not MARCO. Furthermore, SiO2 exposure decreased uptake of fluorescently labeled Pam2CSK4 and Pam3CSK4, resulting in reduced secretion of IL-1β, but not IL-6. Collectively, our data suggest that SiO2 exposure alters AM phenotype, which in turn affects their ability to uptake and respond to bacterial lipoproteins.