Phagocytic Activity Of Alveolar Macrophages Research Articles

Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ Tcell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

ADIPOSE-DERIVED STEM CELLS-DERIVED EXOSOMES ATTENUATE CIGARETTE SMOKE-INDUCED LUNG INFLAMMATION AND INJURY VIA DECREASING ALVEOLAR MACROPHAGES PYROPTOSIS

TYPE: Abstract TOPIC: Obstructive Lung Diseases PURPOSE: To investigate the protective roles of adipose-derived stem cells-derived exosomes (ADSCs-Exo) in cigarette smoke (CS)-induced COPD and explore the underlying mechanism by discovering the effects of ADSCs-Exo on alveolar macrophages (AMs) pyroptosis. METHODS: ADSCs were isolated from human adipose tissues, and then ADSCs-Exo were isolated. 24 8-week-old male C57/BL6 mice were exposed to CS for 4 weeks, followed by intratracheal administration of ADSCs-Exo or PBS. MH-S cells, derived from mouse AMs, were stimulated by 2% CS extract for 24 hours, followed by ADSCs-Exo or PBS treatment. Pulmonary inflammation was analyzed by detecting pro-inflammatory cells and mediators in the bronchoalveolar lavage fluid. Lung histology was assessed by HE staining. Mucus production was determined by PAS staining. The profile of AMs pyroptosis was evaluated by detecting the levels of pyroptosis-indicated proteins. The inflammatory response in AMs and the phagocytic activity of AMs were investigated. RESULTS: In mice with CS-induced COPD, the levels of pro-inflammatory cells and mediators were significantly were increased, mucus production was markedly increased and lung architecture was obviously disrupted. AMs pyroptosis was elevated and AMs phagocytosis was inhibited. However, the administration of ADSCs-Exo greatly reversed these alterations caused by CS exposure. Consistently, in MH-S cells with CS extract-induced COPD, the cellular inflammatory response was elevated, the pyroptotic activity was upregulated while the phagocytosis was decreased. Nonetheless, these abnormalities were remarkably alleviated by the treatment of ADSCs-Exo. CONCLUSIONS: ADSCs-Exo significantly attenuates CS-induced airway mucus overproduction, lung inflammation and injury by inhibiting AMs pyroptosis. CLINICAL IMPLICATIONS: ADSCs-Exo may be a promising cell-free therapeutic candidate for COPD. DISCLOSURE: Nothing to declare. KEYWORD: Chronic obstructive pulmonary disease

Exosomes derived from adipose-derived stem cells alleviate cigarette smoke-induced lung inflammation and injury by inhibiting alveolar macrophages pyroptosis

BackgroundChronic obstructive pulmonary disease (COPD) is a frequently encountered disease condition in clinical practice mainly caused by cigarette smoke (CS). The aim of this study was to investigate the protective roles of human adipose-derived stem cells-derived exosomes (ADSCs-Exo) in CS-induced lung inflammation and injury and explore the underlying mechanism by discovering the effects of ADSCs-Exo on alveolar macrophages (AMs) pyroptosis.MethodsADSCs were isolated from human adipose tissues harvested from three healthy donors, and then ADSCs-Exo were isolated. In vivo, 24 age-matched male C57BL/6 mice were exposed to CS for 4 weeks, followed by intratracheal administration of ADSCs-Exo or phosphate buffered saline. In vitro, MH-S cells, derived from mouse AMs, were stimulated by 2% CS extract (CSE) for 24 h, followed by the treatment of ADSCs-Exo or phosphate buffered saline. Pulmonary inflammation was analyzed by detecting pro-inflammatory cells and mediators in the bronchoalveolar lavage fluid. Lung histology was assessed by hematoxylin and eosin staining. Mucus production was determined by Alcian blue-periodic acid-Schiff staining. The profile of AMs pyroptosis was evaluated by detecting the levels of pyroptosis-indicated proteins. The inflammatory response in AMs and the phagocytic activity of AMs were also investigated.ResultsIn mice exposed to CS, the levels of pro-inflammatory cells and mediators were significantly increased, mucus production was markedly increased and lung architecture was obviously disrupted. AMs pyroptosis was elevated and AMs phagocytosis was inhibited. However, the administration of ADSCs-Exo greatly reversed these alterations caused by CS exposure. Consistently, in MH-S cells with CSE-induced properties modelling those found in COPD, the cellular inflammatory response was elevated, the pyroptotic activity was upregulated while the phagocytosis was decreased. Nonetheless, these abnormalities were remarkably alleviated by the treatment of ADSCs-Exo.ConclusionsADSCs-Exo effectively attenuate CS-induced airway mucus overproduction, lung inflammation and injury by inhibiting AMs pyroptosis. Therefore, hADSCs-Exo may be a promising cell-free therapeutic candidate for CS-induced lung inflammation and injury.

The effect of the imbalance of the neurotransmitter systems of the dorsal hippocampus on the activity of alveolar macrophages and lung surfactant

The purpose of the study. The paper analyzes the metabolism of surfactant lipids and phagocytic activity of alveolar macrophages with an imbalance of the neurotransmitter systems of the dorsal hippocampus. Materials and methods. Male rats were microinjected with L-glutamate and GABA into the dorsal hippocampus bilaterally by stereotactic coordinates. Studies included determination of surfactant lipid fractions (thin-layer chromatography method), total phospholipids and their surface-active properties (Wilhelmi method), phospholipase activity, evaluation of endopulmonary cytogram, phagocytic index, and phagocytic number. Results. It was found that the introduction of L-glutamate was accompanied by an increase in phospholipids in the surfactant due to «inert» fractions of phosphatidylserine, phosphatidylinositol, and a low-active fraction of phosphatidylethanolamine against the background of a decrease in the intensity of phospholipase hydrolysis and the ability of macrophages to phagocytosis. The introduction of GABA led to redistribution of phospholipid fractions toward a decrease in phosphatidylcholine surfactant and an increase in the proportion of lysophosphatidylcholine under conditions of phospholipase activation and macrophage phagocytic activity. Conclusions The imbalance of the glutamate and GABAergic systems of the hippocampus has a multidirectional effect on the composition of surfactant lipids, the ability of alveolar macrophages to phagocytosis, and is characterized by the deterioration of the surface-active properties of the lungs.

Transglutaminase 2 mediates lung inflammation and remodeling by transforming growth factor beta 1 via alveolar macrophage modulation

Transforming growth factor beta 1 (TGF-β1) induces pulmonary fibrosis by enhancing epithelial apoptosis and affects the enzymatic activity of transglutaminase 2 (TG2). The aim of this study was to determine the role of TG2 in TGF-β1-induced lung remodeling and alveolar macrophage modulation. We characterized the in vivo effects of TGF-β1 and TG2 on lung inflammation, fibrosis, and macrophage activity using transgenic C57BL/6 mice with wild and null TG2 loci. The effect of TG2 inhibition on in vitro TGF-β1-stimulated alveolar macrophages was assessed through mRNA analysis. TG2 was remarkably upregulated in the lungs of TGF-β1 transgenic (TGF-β1 Tg) mice, especially in alveolar macrophages and epithelial cells. In the absence of TG2, TGF-β1-induced inflammation was suppressed, decreasing the number of macrophages in the bronchoalveolar lavage fluid. In addition, the alveolar destruction and peribronchial fibrosis induced by TGF-β1 overexpression were significantly reduced, which correlated with decreases in the expression of fibroblast growth factor and matrix metallopeptidase 12, respectively. However, TG2 deficiency did not compromise the phagocytic activity of alveolar macrophages in TGF-β1 Tg mice. At the same time, TG2 contributed to the regulation of TGF-β1-induced macrophage activation. Inhibition of TG2 did not affect the TGF-β1-induced expression of CD86, an M1 marker, in macrophages, but it did reverse the TGF-β1-induced expression of CD206. This result suggests that TG2 mediates TGF-β1-induced M2-like polarization but does not contribute to TGF-β1-induced M1 polarization. In conclusion, TG2 regulates macrophage modulation and plays an important role in TGF-β1-induced lung inflammation, destruction, and fibrosis.

Cigarette Smoke Exposure Inhibits Early Phase of Antibody Production through Inhibition of Immune Functions in Alveolar Macrophage

Background::Cigarette smoke (CS) is inhaled into the lung. Alveolar macrophage (AM) is known to play an important role in the lung immune system. However, the relationship between AM functions and antibody production by CS is not fully investigated.Objective::Therefore, we investigated the effects of AM from CS exposed mice on antibody production. Mice were exposed to 20 cigarettes/day for 10 days. AM were obtained by broncho-alveolar lavage. Antibody production was analyzed by plaque-forming cell assay using seep red blood cell (SRBC) as antigen.Methods::B cell proliferation was analyzed by 3H-thymidine incorporation. Phagocytic activity using fluorescein isothiocyanate-labeled SRBC and expressions of surface antigens on AM were analyzed by flow cytometry. Cytokines and NF-κB mRNA expressions of AM were analyzed by RTPCR.Results and Discussion:Antibody production was decreased at the induction phase, but not at the expression phase by AM from smoked mice (SM) compared with non-smoked mice (NSM). B cell proliferation was decreased by cigarette extracts dose-dependently. Phagocytic activity of AM was decreased in SM compared with NSM. Expression of surface antigens on AM was decreased in SM compared with NSM. Cytokines or NF-κB mRNA expressions of AM were decreased in SM compared with NSM.Conclusion::These results suggest that the inhibition of antibody production by cigarette smoking is caused by the inhibition of phagocytosis and expressions of surface antigens associated with antigen presentation. Such inhibition of AM functions may increase the risk of bacterial and virus infections.

Влияние препарата природной двуспиральной РНК при интраназальном введении на факторы неспецифической резистентности дыхательных путей

В экспериментах на мышах проведено исследование влияния препарата дрожжевой двуспиральной РНК (дсРНК) при интраназальном введении на синтез интерферонов тканями респираторного тракта и активность альвеолярных макрофагов. Показано, что препарат в дозе 15 мг/кг обладает интерферон-индуцирующими свойствами, которые проявляются в повышении уровня интерферона альфа в ткани носоглотки и легких через 5 часов после 1 – 2-кратного введения, а также способностью модулировать фагоцитарную активность альвеолярных макрофагов. Полученные данные свидетельствуют о стимуляции под действием дсРНК факторов неспецифической резистентности дыхательных путей, играющих важную роль в формировании противовирусной защитной реакции.

Intrapulmonary (i.pulmon.) Pull Immunization With the Tuberculosis Subunit Vaccine Candidate H56/CAF01 After Intramuscular (i.m.) Priming Elicits a Distinct Innate Myeloid Response and Activation of Antigen-Presenting Cells Than i.m. or i.pulmon. Prime Immunization Alone.

Understanding the in vivo fate of vaccine antigens and adjuvants and their safety is crucial for the rational design of mucosal subunit vaccines. Prime and pull vaccination using the T helper 17-inducing adjuvant CAF01 administered parenterally and mucosally, respectively, has previously been suggested as a promising strategy to redirect immunity to mucosal tissues. Recently, we reported a promising tuberculosis (TB) vaccination strategy comprising of parenteral priming followed by intrapulmonary (i.pulmon.) mucosal pull immunization with the TB subunit vaccine candidate H56/CAF01, which resulted in the induction of lung-localized, H56-specific T cells and systemic as well as lung mucosal IgA responses. Here, we investigate the uptake of H56/CAF01 by mucosal and systemic innate myeloid cells, antigen-presenting cells (APCs), lung epithelial cells and endothelial cells in mice after parenteral prime combined with i.pulmon. pull immunization, and after parenteral or i.pulmon. prime immunization alone. We find that i.pulmon. pull immunization of mice with H56/CAF01, which are parenterally primed with H56/CAF01, substantially enhances vaccine uptake and presentation by pulmonary and splenic APCs, pulmonary endothelial cells and type I epithelial cells and induces stronger activation of dendritic cells in the lung-draining lymph nodes, compared with parenteral immunization alone, which suggests activation of both innate and memory responses. Using mass spectrometry imaging of lipid biomarkers, we further show that (i) airway mucosal immunization with H56/CAF01 neither induces apparent local tissue damage nor inflammation in the lungs, and (ii) the presence of CAF01 is accompanied by evidence of an altered phagocytic activity in alveolar macrophages, evident from co-localization of CAF01 with the biomarker bis(monoacylglycero)phosphate, which is expressed in the late endosomes and lysosomes of phagocytosing macrophages. Hence, our data demonstrate that innate myeloid responses differ after one and two immunizations, respectively, and the priming route and boosting route individually affect this outcome. These findings may have important implications for the design of mucosal vaccines intended for safe administration in the airways.

Phagocytosis of alveolar macrophages is suppressed in a mouse model of lipopolysaccharide-induced acute lung injury

To investigate the changes in phagocytic function of alveolar macrophages (AMs) in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and explore the possible mechanism. Kunming mice were randomly divided into normal control group and ALI (induced by LPS instillation in the airway) model group. AMs were obtained from bronchoalveolar lavage fluid in both groups, and phagocytosis of the AMs was observed using flow cytometry and fluorescence microscopy. Western blotting and ELISA were used to detect the expression and secretion of IL-33 in the lung tissue of the mice. We also detected the secretion of IL-33 by an alveolar epithelial cell line MLE-12 in response to stimulation with different concentrations of LPS. The AMs from the normal control mice were treated with different concentrations of LPS and IL-33, and the changes in the phagocytic activity of the cells were observed. Compared with those in normal control group, the percentage of AMs phagocytosing fluorescent microspheres was significantly decreased, and the expression of IL-33 in lung tissue and IL-33 level in the bronchoalveolar lavage fluid were significantly increased in ALI mice (P < 0.01). LPS (100-1000 ng/mL) obviously promoted the secretion of IL-33 in cultured MLE-12 cells (P < 0.01). Both LPS (10-500 ng/mL) and IL-33 (100 ng/mL) significantly inhibited the phagocytic activity of the AMs from normal control mice (P < 0.01). The phagocytic activity of AMs is weakened in ALI mice possibly due to direct LPS stimulation and the inhibitory effect of the alarmin IL-33 produced by LPS-stimulated alveolar epithelial cells.

Mesenchymal Stem Cells Enhance Pulmonary Antimicrobial Immunity and Prevent Following Bacterial Infection.

Background Immunosuppressants such as cyclophosphamide (CTX) have been employed to treat a wide array of autoimmune diseases. The most unfavourable side effects of these drugs are their suppression on the antimicrobial immunity and increasing the risk of infection. As a promising substitution/adjunct, mesenchymal stem cells (MSCs) are currently being tested in several clinical trials. However, their influence on the recipients' antimicrobial immunity remains unclear. Methods In this study, C57BL/6 mice were treated with either CTX or MSCs, and then both the innate and adaptive immunity of the lung were determined. To investigate the influence of CTX and MSCs on the immune defence against infection, the treated mice were intranasally infected with opportunistic pathogen Haemophilus influenzae (Hi). Bacterial clearance and antibacterial immune responses were analysed. Results Our data showed that CTX strongly inhibited the proliferation of lung immune cells, including alveolar macrophages (AMs) and T cells, whereas MSCs increased the numbers of these cells. CTX suppressed the phagocytic activity of AMs; on the contrary, MSCs enhanced it. Notably, infusion of MSCs led to a remarkable increase of regulatory T cells and Th1 cells in the lung. When infected by Hi, CTX did not significantly impair the elimination of invaded bacteria. However, MSC-treated mice exhibited accelerated bacterial clearance and moderate inflammation and tissue damage. Conclusion Our study reported that unlike traditional immunosuppressants, modulation of MSCs on the recipient's immune response is more elegant. It could preserve and even enhance the antimicrobial defence, suggesting that MSCs are better choice for patients with high risk of infection or those who need long-term immunosuppressive regimen.

Dexmedetomidine alleviates sleep-restriction-mediated exaggeration of postoperative immunosuppression via splenic TFF2 in aged mice

Major abdominal procedures could induce dysfunction in the immune system and lead to postoperative immunosuppression. Sleep dysfunction is associated with impaired immune activity. However, the effects of postoperative sleep dysfunction on postoperative immune function remain unclear. In this study, we found that sleep-restriction (SR) after surgery increased the spleen weight and the percentage of myeloid-derived suppressor cells (MDSCs) in the spleen, and inhibited splenic CD8+ T cells activity, which was via inhibiting subdiaphragmatic vagus nerve (SVN)-mediated trefoil factor 2 (TFF2) expression in the spleen of aged mice. Dexmedetomidine could alleviate SR-induced these changes via modulating gut microbiota, which acted through SVN. Moreover, we showed essential roles of splenic TFF2 in attenuating SR-induced reduced protective ability against Escherichia coli (E. coli) pneumonia, increased expression of IL-4 and IL-13 in the lung and M2 polarization of alveolar macrophages (AMs), and decreased phagocytic activity of AMs. Dexmedetomidine improved SR-induced reduced protective ability against E. coli pneumonia via splenic TFF2, and subsequently decreasing IL-4 and IL-13 expression in the lung via modulating gut microbiota/SVN, increasing the compromised phagocytic activity of AMs, and ultimately decreasing M2 polarization of AMs. Taken together, dexmedetomidine-induced increase in splenic TFF2 expresssion could alleviate SR-induced exaggeration of postoperative immunosuppression.

Aryl hydrocarbon receptor ligands enhance lung immunity through intestinal IKK\u03b2 pathways

BackgroundInfection by antibiotic-resistant microorganisms is common in intensive care units and has become a global problem. Here, we determined the effect of aryl hydrocarbon receptor (AhR) stimulation on antibiotics-induced systemic defense impairment and its mechanisms.MethodsC57BL/6 wild-type (WT) mice received combined antibiotics with or without Ahr ligands (tryptophan and indole), or dead Lactobacillus plantarum supplementation. The defense mechanisms against Pseudomonas aeruginosa infection in the lung were examined.ResultsAntibiotic treatments decreased the phagocytic activity, physiological activity, and the peroxynitrite production of alveolar macrophage (AMs). It also enhanced P. aeruginosa pneumonia-induced bacterial counts in the lung. Tryptophan and dead L. plantarum supplementation reversed antibiotic-induced intracellular adhesion molecule (ICAM) as well as IL-6 expression, and increased P. aeruginosa pneumonia-induced bacterial counts in the lung and increased phagocytic activity and peroxynitrite production of AMs. Moreover, these treatments reversed the antibiotics-induced reduction of Ahr expression, antibacterial proteins, reactive oxygen species (ROS) production, and NF-κB DNA binding activity of the intestinal mucosa and plasma IL-6 levels. P. aeruginosa counts increased and phagocytic activity of AMs and myeloperoxidase (MPO) activity decreased in intestinal IKKβ depleted mice. Antibiotics, antibiotic with tryptophan feeding, or antibiotic with dead L. plantarum feeding treatments did not change the phagocytic activity and peroxynitrite production of AMs, plasma IL-6 levels, and the expression of Ahr of intestine in intestinal IKKβ depleted mice.ConclusionAntibiotic treatment impairs lung immune defenses by decreasing Ahr expression in the intestine and peroyxnitrite production of the AMs. Ahr ligands reverses antibiotic-induced lung defense against bacterial infection through intestinal ROS production and NF-κB activation. The gut is critical in maintaining lung defense mechanism through the intestinal IKKβ pathways.

Increased leishmanicidal activity of alveolar macrophages from mature horses with mild equine asthma

ABSTRACT Alveolar macrophages (AMs) are an essential part of defense mechanisms within the lungs and their phagocytic activity is important for organ homeostasis. The phagocytic ability of AMs obtained from bronchoalveolar lavage from 17 mature mixed-breed pleasure horses (8 healthy and 9 diagnosed with mild equine asthma) was studied through assays with Leishmania (Viannia) braziliensis promastigotes, which enabled the calculation of a phagocytic index (PI) and a survival index (SI). Results indicate that phagocytic activity of AMs in asthma affected horses is similar to healthy horses, while leishmanicidal activity is significantly increased in horses with asthma.

Respiratory toxicity of TiO2 nanoparticles after intravenous instillation: an experimental study.

Nanomaterials are materials consisting of particles having one or more dimensions smaller than 100 nm. Nanoparticles (NP) have different properties and effects in comparison with the same particle materials of larger size. They can penetrate through various membranes and get from the bloodstream to other organs in the body. Therefore, in our experiment we have dealt with the impact of nanoparticles TiO2 instilled intravenously (i.v.) (to a tail vein of an animal) on the selected parameters of bronchoalveolar lavage (BAL). The aim of our study was to determine whether TiO2 nanoparticles do pass through the vascular system to the respiratory tract, and if so, how they affect the selected inflammatory and cytotoxic parameters of bronchoalveolar lavage. Wistar rats were intravenously given a suspension of TiO2 nanoparticles in saline solution. This suspension contained 10% volume of rat serum in dose: 1.0% from LD50 = 0.592 mg/kg of animal body weight. After the time intervals 1, 7, 14 and 28 days, the animals were sacrificed under anaesthesia; bronchoalveolar lavage was performed and the BAL cells were isolated. We have examined these markers: differential count of BAL cells - alveolar macrophages (AM), polymorphonuclear leukocytes (PMNL), lymphocytes (Ly); viability and phagocytic activity of AM; proportion of immature cells and cathepsin D enzyme levels. Regarding the respiratory toxicity of TiO2 nanoparticles we have found that TiO2 nanoparticles are relatively inert. BAL examined parameters (except the immature form of AM) were not significantly changed after 28 days of instillation compared to the control group. We found that the TiO2 nanoparticles used in our study were transferred from the bloodstream to the respiratory tract, but in a 28-day phase after i.v. instillation have been largely eliminated by the defence mechanism from the respiratory tract. We suggest low biopersistence and relatively rapid elimination of TiO2 nanoparticles from the lung under used experimental conditions.

Lactobacillus salivarius reverse antibiotic-induced lung defense impairment in a ventilator model

BackgroundWidespread use of antibiotics in the intensive care unit is a potential cause of the emergence of hospital-acquired pneumonia. This study determined whether Lactobacillus salivarius feeding could reverse antibiotic-induced lung defense impairment in a ventilator model.MethodsC57BL/6 wild-type (WT) mice received mechanical ventilation for 3 h after intramuscular antibiotic treatment for 6 days. Treatment with dead Lactobacillus salivarius and fructo-oligosaccharides (FOS) feeding were used to stimulate antibacterial protein expression in the intestine. Reactive oxygen species (ROS) in the intestinal mucosa was detected using 2ʹ7ʹ-dichlorofluorescein diacetate. The peroxynitrite production of alveolar macrophages (AMs) was measured using dihydrorhodamine 123 oxidation assay. N-acetylcysteine (NAC), an ROS scavenger, was orally administered to mice receiving antibiotics with FOS feeding.ResultsAntibiotic treatment decreased Pseudomonas aeruginosa (PA) phagocytic activity and activity of AMs and protein expression of regenerating islet-derived protein 3β (Reg3β) as well as Toll-like receptor 4 (TLR4) in the intestinal mucosa in the ventilator model. Antibiotic treatment also decreased ROS production in the intestinal mucosa, peroxynitrite production of AMs, and RELMβ expression as well as NF-κB DNA binding activity of the intestinal mucosa in WT mice but not in MyD88−/− mice. Treatment with dead L. salivarius or FOS feeding increased ROS production, bacterial killing activity, and protein expression of Reg3β as well as TLR4 in the intestinal mucosa and reversed the inhibitory effects of antibiotics on PA phagocytic activity of AMs.ConclusionTaken together with the finding that ablation of FOS-induced intestinal ROS using NAC decreased peroxynitrite production as well as PA phagocytic activity of AMs and protein expression of CRP-ductin, IL-17, Reg3β, and RELMβ in the intestinal mucosa, we conclude that commensal microflora plays a key role in stimulating lung immunity. Intestinal ROS plays a role as a predictive indicator and modulator of pulmonary defense mechanisms. Antibiotic treatment reduces lung defense against PA infection through the decrease in intestinal Reg3β and TLR4 expression. Treatment with dead L. salivarius or FOS feeding reverses the antibiotic-induced lung defense impairment through the intestinal ROS/MyD88 pathways.

Abstract 5738: Hypoxia-inducible factor-1 in myeloid cells is required to protect the irradiated lung from metastasis

Abstract Recent evidence suggests that metastasis is proceeded by premetastatic niche formation where bone marrow-derived cells (BMDC), particularly those of myeloid cells (monocytes and macrophages), form clusters preparing for the arrival of cancer cells in the metastasis-prone organs such as the lung. Many molecules involved in the premetastatic niche formation has been identified, some of which include vascular endothelial growth factor (VEGF), lysyl oxidase (LOX), and S100A8. Since VEGF, LOX, and S100A8 are downstream targets of hypoxia-inducible factor (HIF), a major transcription factor stabilized under hypoxic conditions, we investigated whether HIF-1 in myeloid cells would affect the premetastatic niche formation hence metastasis to the lung. To do this, we used our novel strain of myeloid specific Hif-1α knockout (KO) mice using human S100A8, an intracellular calcium-binding protein as the myeloid promoter. We first performed bone marrow (BM) transplantation from GFP-expressing myeloid specific Hif-1α KO (hereafter denoted as Hif-1α KO) mice or GFP-expressing littermate control mice to lethally irradiated wild-type (WT) mice. Two weeks later, these mice were subcutaneously implanted for Lewis lung carcinoma (LLC) primary tumors followed by 20 Gy chest irradiation to promote the premetastatic niche formation. Primary tumors were then removed and luciferase-expressing LLC cells were intravenously injected to induce the lung metastasis. We observed to our surprise that mice receiving Hif-1α KO BM had 10-fold increase in the lung metastasis, indicating that HIF-1α in myeloid cells is required to protect the lung from the metastasis. While the total number of GFP-expressing BMDC was similar, there were significantly decreased alveolar macrophages (GFP+F4/80+CD11c+CD11b-) in the irradiated lung of mice receiving BM of Hif-1α KO BM than the littermate control mice. Because alveolar macrophages are specialized immune cells involved in phagocytosis of numerous airborne antigens, we hypothesized that HIF-1α-deficient alveolar macrophages are deficient in phagocytosis and this may be responsible for the increase in the lung metastasis. Indeed, we observed that alveolar macrophages isolated from bronchioalveolar lavage fluid of Hif-1α KO mice demonstrated a decreased phagocytic activity. We are currently investigating whether we could pharmacologically modulate phagocytic activities of alveolar macrophages to regulate the lung metastasis. In conclusion, we demonstrate that HIF-1 in alveolar macrophages is required to protect the irradiated lung from the lung metastasis, possibly in a mechanism involving phagocytosis. Citation Format: Hoibin Jeong, Beom-Ju Hong, Hyung-Seok Choi, Jung-Min Oh, G-One Ahn. Hypoxia-inducible factor-1 in myeloid cells is required to protect the irradiated lung from metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5738.

Bone marrow mesenchymal stem cells ameliorate lung injury through anti-inflammatory and antibacterial effect in COPD mice.

The anti-inflammatory and antibacterial mechanisms of bone marrow mesenchymal stem cells (MSCs) ameliorating lung injury in chronic obstructive pulmonary disease (COPD) mice induced by cigarette smoke and Haemophilus Parainfluenza (HPi) were studied. The experiment was divided into four groups in vivo: control group, COPD group, COPD+HPi group, and COPD+HPi+MSCs group. The indexes of emphysematous changes, inflammatory reaction and lung injury score, and antibacterial effects were evaluated in all groups. As compared with control group, emphysematous changes were significantly aggravated in COPD group, COPD+HPi group and COPD+HPi+MSCs group (P<0.01), the expression of necrosis factor-kappaB (NF-κB) signal pathway and proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) were increased (P<0.01), and the phagocytic activity of alveolar macrophages was downregulated (P<0.01). As compared with COPD group, lung injury score, inflammatory cells and proinflammatory cytokines were significantly increased in the BALF of COPD+HPi group and COPD+HPi+MSCs group (P<0.01). As compared with COPD+HPi group, the expression of tumor necrosis factor-α stimulated protein/gene 6 (TSG-6) was increased, the NF-κB signal pathway was depressed, proinflammatory cytokine was significantly reduced, the anti-inflammatory cytokine IL-10 was increased, and lung injury score was significantly reduced in COPD+HPi+MSCs group. Meanwhile, the phagocytic activity of alveolar macrophages was significantly enhanced and bacterial counts in the lung were decreased. The results indicated cigarette smoke caused emphysematous changes in mice and the phagocytic activity of alveolar macrophages was decreased. The lung injury of acute exacerbation of COPD mice induced by cigarette smoke and HPi was alleviated through MSCs transplantation, which may be attributed to the fact that MSCs could promote macrophages into anti-inflammatory phenotype through secreting TSG-6, inhibit NF-кB signaling pathway, and reduce inflammatory response through reducing proinflammatory cytokines and promoting the expression of the anti-inflammatory cytokine. Simultaneously, MSCs could enhance phagocytic activity of macrophages and bacterial clearance. Meanwhile, we detected anti-inflammatory and antibacterial activity of macrophages regulated by MSCs in vitro. As compared with RAW264.7+HPi+CSE group, the expression of NF-кB p65, IL-1β, IL-6 and TNF-α was significantly reduced, and the phagocytic activity of macrophages was significantly increased in RAW264.7+HPi+CSE+MSCs group (P<0.01). The result indicated the macrophages co-cultured with MSCs may inhibit NF-кB signaling pathway and promote phagocytosis by paracrine mechanism.

Hypercapnic Conditions After Experimental Blunt Chest Trauma Increase Efferocytosis of Alveolar Macrophages and Reduce Local Inflammation.

Blunt chest trauma induces severe local and systemic inflammatory alterations and an accumulation of apoptotic polymorphonuclear granulocytes (aPMN) in the lungs, frequently followed by bacterial infection. Alveolar macrophages (AM) represent one of the main actors for their clearance. However, little is known regarding regulatory and influencing factors of AM efferocytic and phagocytic activities. In this context, we investigated the influence of impaired gas exchange on AM activity.Male rats underwent blunt chest trauma or sham procedure and aPMN or Escherichia coli (E. coli) were instilled. Subsequently, the efferocytic and phagocytic activities were assessed by analyzing AM obtained from bronchoalveolar lavage fluids at three time points. To determine whether efferocytic and phagocytic activities of AM are affected by shifting gas concentrations, AM were subjected in vitro to hypoxic and hypercapnic conditions.Trauma significantly upregulated the capacity of AM to ingest E. coli starting 24 h after trauma, whereas the aPMN uptake rate remained virtually unchanged. In vitro, AM reacted to hypercapnic conditions by enhanced efferocytosis associated with increased release of anti-inflammatory cytokines. Additionally, phagocytosis and the pro-inflammatory reaction of AM after trauma appeared to be impaired. In contrast, hypoxic conditions displayed no regulatory effect on AM.In conclusion, blunt chest trauma enhances phagocytic activity of AM. On the other hand, hypercapnic conditions in the lungs may significantly contribute to the clearance of aPMN. The application of CO2 in clinical settings must be properly assessed, with the benefits of CO2 balanced against the detrimental effects of impaired bacterial clearance.

P121: Morphological and functional changes in the population of alveolar macrophages of mice with induced carcinogenesis in the lung

Background Alveolar macrophages (AM) are pulmonary residents of the bone marrow-derived mononuclear phagocyte system that play a critical role in several diverse lung functions and in lung host defense. The role of AM in lung cancer is multifaceted. AM secretion of proinflammatory cytokines has been found to enhance antitumor functions, cytostasis and cytotoxicity. AM have also demonstrated to have protumor functions resulting in tumor progression. Materials and methods 24-week-old male A/f mice were kept in plastic cages under standard laboratory conditions and received food and water ad libitum. Animals were randomly assigned to the following experimental groups: I – control, II – radiation only, III – sulphur dioxide only, IV – sulphur dioxide + radiation. Animals were subjected to total irradiation at dose 1.0 Gy (dose density of 16.6 cGy/min, using a 60Co gamma equipment). After irradiation mice were inhaled air with sulphur dioxide (20 mg/m 3 for 1 h) in separate exposure chamber. At the 20th week of experiment the 20 mice per each group were subjected to autopsy. Excised lungs were fixed in neutral buffered 10% formalin. To obtain an index of tumor incidence, the percentage of tumor – bearing mice per total number of mice in each group was calculated. Tumor multiplicity was defined as the average number of tumors per mice, obtained by dividing the total number of tumors by the total number of mice per group, including non-tumor bearing animals. Bronchoalveolar lavage (BAL) of lungs was performed on 6 mice (animals were anesthetized by an ip injection of sodium thiopental) from each study group at the 1, 7, 15 and 30 days after irradiation and SO2 inhalation. Total cells of BAL were counted using hemocytometer chamber and an optical microscope with a 400× zoom. BAL differential cell (macrophages, lymphocytes, eosinophiles and neutrophils) counts were performed on slides (Romanowsky stain). Phagocytic activity of AM was counted on slides. AM morphometric study was done by measuring the area of each cell (square of cell) and the area of nuclear of each cell (square of nuclear) with use of the original image analyzer computer system. Results Results of the combined effects of radiation and inhalation of sulfur dioxide showed the growth of tumor quantity in the lung: the frequency of adenomas/mouse was more than 42% higher than that of control group. Synergism coefficient was 1.1 in this case. Irradiation at a dose of 1.0 Gy modifies the reaction of free cells of lung on the action of sulfur dioxide, which is manifested in the change of the number of cells in the BAL fluid, the phagocytic activity of the AM, as well as of the changes the morphometric parameters of their cells. Conclusion Thus, if the amount of radiation exposure of BAL cells was significantly reduced by the 1st and 7th days, and after inhalation of sulfur dioxide – tended to increase, then the combined action on the 7th day it was almost half the theoretical (hypothetical) the sum of the separate effects of the studied factors. Exposure to sulfur dioxide significantly slowed the activation of cell count. It was found, that after combined action of the investigated factors the morphological characteristics of macrophages (reducing the square of cells and their nuclei) have changed (7th and 15th day). The absorption activity of phagocytes was significantly reduced (7 day).

Beryllium increases the CD14(dim)CD16+ subset in the lung of chronic beryllium disease.

CD14dimCD16+ and CD14brightCD16+ cells, which compose a minor population of monocytes in human peripheral blood mononuclear cells (PBMC), have been implicated in several inflammatory diseases. The aim of this study was to investigate whether this phenotype was present as a subset of lung infiltrative alveolar macrophages (AMs) in the granulomatous lung disease, chronic beryllium disease (CBD). The monocytes subsets was determined from PBMC cells and bronchoalveolar lavage (BAL) cells from CBD, beryllium sensitized Non-smoker (BeS-NS) and healthy subjects (HS) using flow cytometry. The impact of smoking on the AMs cell phenotype was determined by using BAL cells from BeS smokers (BeS-S). In comparison with the other monocyte subpopulations, CD14dimCD16+ cells were at decreased frequency in PBMCs of both BeS-NS and CBD and showed higher HLA-DR expression, compared to HS. The AMs from CBD and BeS-NS demonstrated a CD14dimCD16+phenotype, while CD14brightCD16+ cells were found at increased frequency in AMs of BeS, compared to HS. Fresh AMs from BeS-NS and CBD demonstrated significantly greater CD16, CD40, CD86 and HLA-DR than HS and BeS-S. The expression of CD16 on AMs from both CBD and BeS-NS was downregulated significantly after 10μM BeSO4 stimulation. The phagocytic activity of AMs decreased after 10μM BeSO4 treatment in both BeS-NS and CBD, although was altered or reduced in HS and BeS-S. These results suggest that Be increases the CD14dimCD16+ subsets in the lung of CBD subjects. We speculate that Be-stimulates the compartmentalization of a more mature CD16+ macrophage phenotype and that in turn these macrophages are a source of Th1 cytokines and chemokines that perpetuate the Be immune response in CBD. The protective effect of cigarette smoking in BeS-S may be due to the low expression of co-stimulatory markers on AMs from smokers as well as the decreased phagocytic function.