Alveolar Macrophage Phagocytosis Research Articles

Antibacterial peptide Reg4 ameliorates Pseudomonas aeruginosa-induced pulmonary inflammation and fibrosis.

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative facultative anaerobe that has become an important cause of severe infections in humans, particularly in patients with cystic fibrosis. The development of efficacious methods or mendicants against P. aeruginosa is still needed. We previously reported that regenerating islet-derived family member 4 (Reg4) has bactericidal activity against Salmonella Typhimurium, a Gram-negative flagellated bacterium. We herein explore whether Reg4 has bactericidal activity against P. aeruginosa. In the P. aeruginosa PAO1-chronic infection model, Reg4 significantly inhibits the colonization of PAO1 in the lung and subsequently ameliorates pulmonary inflammation and fibrosis. Reg4 recombinant protein suppresses the growth motility and biofilm formation capability of PAO1 in vitro. Mechanistically, Reg4 not only exerts bactericidal action via direct binding to the P. aeruginosa cell wall but also enhances the phagocytosis of alveolar macrophages in the host. Taken together, our study demonstrates that Reg4 may provide protection against P. aeruginosa-induced pulmonary inflammation and fibrosis via its antibacterial activity.IMPORTANCEChronic lung infection with Pseudomonas aeruginosa is a leading cause of morbidity and mortality in patients with cystic fibrosis. Due to the antibiotic resistance of Pseudomonas aeruginosa, antimicrobial peptides appear to be a potential alternative to combat its infection. In this study, we report an antimicrobial peptide, regenerating islet-derived 4 (Reg4), that showed killing activity against clinical strains of Pseudomonas aeruginosa PAO1 and ameliorated PAO1-induced pulmonary inflammation and fibrosis. Experimental data also showed Reg4 directly bound to the bacterial cell membrane and enhanced the phagocytosis of host alveolar macrophages. Our presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Alveolar macrophage phagocytosis-evading inhaled microgels incorporating nintedanib-PLGA nanoparticles and pirfenidone-liposomes for improved treatment of pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory and fibrotic response-driven lung disease that is difficult to cure because it manifests excessive profibrotic cytokines (e.g., TGF-β), activated myofibroblasts, and accumulated extracellular matrix (ECM). In an attempt to develop an inhalation formulation with enhanced antifibrotic efficacy, we sought to fabricate unique aerosolizable inhaled microgels (μGel) that contain nintedanib-poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs; n-PN) and pirfenidone-liposomes (p-LP). The aero-μGel was ∼12 μm, resisted phagocytosis by alveolar macrophages in vitro and in vivo, and protected inner-entrapped n-PN and p-LP. The n-PN/p-LP@aero-μGel caused enhanced/extended antifibrotic efficacy in a bleomycin-induced pulmonary fibrosis mouse presumably due to prolonged lung residence. Consequently, the results obtained by intratracheal aerosol insufflation of our n-PN/p-LP@aero-μGel twice a week were much better than those by as many as seven doses of single or mixed applications of n-PN or p-LP. The antifibrotic/pharmacokinetic results for the n-PN/p-LP@aero-μGel included reduced fibrosis progression, restored lung physiological functions, deactivated myofibroblasts, inhibited TGF-β progression, and suppressed ECM component production (collagen I and α-SMA) along with prolonged lung retention time. We believe that our n-PN/p-LP@aero-μGel increased the local availability of both nintedanib and pirfenidone due to evasion of alveolar macrophage phagocytosis and prolonged lung retention with reduced systemic distribution. Through this approach, our inhalation formulation subsequently attenuated fibrosis progression and improved lung function. Importantly, these results hold profound implications in the therapeutic potential of our n-PN/p-LP@aero-μGel to serve as a clinically promising platform, providing significant advancements for improved treatment of many respiratory diseases including IFP.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway: Erratum.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway: Erratum.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

Rosiglitazone improves alveolar macrophage mitochondrial derangements induced by chronic alcohol consumption and infection with Klebsiella pneumoniae.

Abstract Objective: People with alcohol use disorders (AUDs) are 2–4 times more likely to develop lung infections, sepsis, and acute respiratory distress syndrome. Chronic alcohol misuse blunts alveolar macrophage (AM) immunity, in part, due to decreased peroxisome proliferator-activated receptor (PPAR)γ which increases cellular oxidative stress. Mitochondrial (MT) oxidative stress may impair AM phagocytosis; thus, we posited that chronic alcohol plus acute Klebsiella pneumoniae(Kp) dysregulates MT and treatment with rosiglitazone (RSG), a PPARγ ligand, reverses these effects to improve AM phagocytosis of Kp. Methods: AMs were isolated from 8–10-month-old male C57BL/6J mice fed ± ethanol (EtOH, 20% wt/vol in drinking water) for 12 wks ± intratracheal 2 × 10 4CFU of Kpin saline vehicle for the last 24 h of water or EtOH ± RSG (10 mg/kg/d in methylcellulose vehicle) via oral gavage 4 h prior to sacrifice (n=5/group). MT O 2•−and H 2O 2were assessed by fluorescence microscopy. mRNA and protein levels of PPARγ and MT dynamics mediators, transcription factor A mitochondrial (TFAM), dynamin-related protein 1 (DRP1), and mitofusin-2 (MFN2) were measured by qRT-PCR and cytoimmunostaining. Kpclearance by AMs was determined by plating lysed AMs on MacConkey agar. Results: EtOH feeding to mice increased MT oxidative stress; decreased PPARγ, TFAM, and MFN2 levels; and increased DRP1 levels in AMs. Kpexacerbated EtOH’s MT derangements by ~51–63% and impaired Kpclearance in AMs by 57.3 ± 11.2%. RSG treatment in vivoreversed EtOH- and Kp-induced MT derangements in AMs and restored AM clearance of Kp. Conclusion: RSG may provide a novel therapeutic strategy for mitigating AM MT derangements and immune dysfunction in AUD individuals infected with Kp. NIAAA R01-AA026086

Pharmacological reversal of post-transcriptional alterations implicated in alcohol-induced alveolar macrophage dysfunction.

Alcohol use disorders (AUD) cause alveolar macrophage (AM) immune dysfunction and increase risk of lung infections. Excessive alcohol use causes AM oxidative stress, which impairs AM phagocytosis and pathogen clearance from the alveolar space. Alcohol induces expression of NADPH oxidases (Noxes), primary sources of oxidative stress in AM. In contrast, alcohol decreases AM peroxisome proliferator-activated receptor gamma (PPARγ), a critical regulator of AM immune function. To explore the underlying molecular mechanisms for these effects of alcohol, we hypothesized that ethanol promotes CCAAT/enhancer-binding protein beta (C/EBPβ)-mediated suppression of Nox-related microRNAs (miRs), in turn enhancing AM Nox expression, oxidative stress, and phagocytic dysfunction. We also hypothesized that PPARγ activation with pioglitazone (PIO) would reverse alcohol-induced C/EBPβ expression and attenuate AM oxidative stress and phagocytic dysfunction. Cells from the mouse AM cell line (MH-S) were exposed to ethanol invitro or primary AM were isolated from mice fed ethanol invivo. Ethanol enhanced C/EBPβ expression, decreased Nox 1-related miR-1264 and Nox 2-related miR-107 levels, and increased Nox1, Nox2, and Nox 4 expression in MH-S cells invitro and mouse AM invivo. These alcohol-induced AM derangements were abrogated by loss of C/EBPβ, overexpression of miRs-1264 or -107, or PIO treatment. These findings identify C/EBPβ and Nox-related miRs as novel therapeutic targets for PPARγ ligands, which could provide a translatable strategy to mitigate susceptibility to lung infections in people with a history of AUD. These studies further clarify the molecular underpinnings for a previous clinical trial using short-term PIO treatment to improve AM immunity in AUD individuals.

Challenges and Strategies to Enhance the Systemic Absorption of Inhaled Peptides and Proteins.

Proteins and peptides-based therapeutics are making substantial access to the market due to their obvious advantages of strong potency, high specificity and desirable safety profile. However, most clinical products are mainly delivered via parenteral route with inferior convenience. Lung is an attractive non-invasive alternative passage for systemic administration of biologics with numerous outstanding features, as examples of large absorptive surface area, extensive vascularization and mild local microenvironment. Even so, mucociliary clearance, alveolar macrophage phagocytosis, enzymatic metabolism, pulmonary surfactant adsorption and limited epithelium permeability constitute major obstacles affecting the systemic absorption of inhaled proteins and peptides. This article begins by giving a brief overview of challenges for the systemic absorption of inhaled proteins and peptides, and then goes on to a comprehensive review of possible strategies for enhanced pulmonary absorption, including chemical modification, addition of protease inhibitors, incorporation of absorption enhancers, modification with fusion proteins and development of particulate-based drug delivery systems. These strategies can provide enhanced transmembrane absorption capacity while avoiding pulmonary clearance, offering a valuable reference for designing pulmonary delivery systems of protein and peptide drugs.

Multiple Death Pathways of Neutrophils Regulate Alveolar Macrophage Proliferation.

Alveolar macrophage (AM) proliferation and self-renewal play an important role in the lung tissue microenvironment. However, the impact of immune cells, especially the neutrophils, on AM homeostasis or function is not well characterized. In this study, we induced in vivo migration of neutrophils into bronchoalveolar lavage (BAL) fluid and lung using CXCL1, and then co-cultured these with AMs in vitro. Neutrophils in the BAL (BAL-neutrophils), rather than neutrophils of bone marrow (BM-neutrophils), were found to inhibit AM proliferation. Analysis of publicly available data showed high heterogeneity of lung neutrophils with distinct molecular signatures of BM- and blood-neutrophils. Unexpectedly, BAL-neutrophils from influenza virus PR8-infected mice (PR8-neutrophils) did not inhibit the proliferation of AMs. Bulk RNA sequencing further revealed that co-culture of AMs with PR8-neutrophils induced IFN-α and -γ responses and inflammatory response, and AMs co-cultured with BAL-neutrophils showed higher expression of metabolism- and ROS-associated genes; in addition, BAL-neutrophils from PR8-infected mice modulated AM polarization and phagocytosis. BAL-neutrophil-mediated suppression of AM proliferation was abrogated by a combination of inhibitors of different neutrophil death pathways. Collectively, our findings suggest that multiple cell death pathways of neutrophils regulate the proliferation of AMs. Targeting neutrophil death may represent a potential therapeutic strategy for improving AM homeostasis during respiratory diseases.

Chlorogenic acid enhances alveolar macrophages phagocytosis in acute respiratory distress syndrome by activating G protein-coupled receptor 37 (GPR 37)

BackgroundImpaired alveolar macrophages phagocytosis can contribute to pathogenesis of acute respiratory distress syndrome (ARDS) and negatively impacts clinical outcomes. Chlorogenic acid (CGA) is a naturally occurring polyphenolic compound with potential anti-inflammatory and antioxidant bioactivities. Studies have shown that CGA plays a protective role in ARDS, however, the precise protective mechanism of CGA against ARDS, is still unclear. PurposeThe aim of this study was to investigate whether CGA enhances alveolar macrophages phagocytosis to attenuate lung injury during ARDS. MethodsRAW264.7 cells were stimulated with lipopolysaccharides (100 μg/ml for 24 h) and treated with CGA (100, 200, and 400 μM CGA for 1 h) to measure pro-inflammatory cytokine levels, GPR37 expression and macrophages phagocytosis. Mouse models of ARDS induced by cecal ligation and perforation (CLP) surgery were treated with CGA (100 or 200 mg/kg) to investigate lung inflammatory injury and alveolar macrophages phagocytosis. Computational modeling was performed to examine potential binding sites of G protein-coupled receptor 37 (GPR37) with CGA, and the results were validated by interfering with the binding sites. ResultIn vitro, CGA notably ameliorated inflammatory response and increased phagocytosis in lipopolysaccharides-induced RAW264.7 cells. In vivo, CGA administration significantly alleviated lung inflammatory injury, decreased the bacteria load in the lung, promoted alveolar macrophages phagocytosis and improved the survival rate in mice with CLP-induced ARDS. Moreover, CGA markedly upregulated the expression of GPR37 in vivo and in vitro. However, the protective effect of CGA against ARDS were reversed after silencing the expression of GPR37. ConclusionCGA has a protective effect against ARDS and may enhance alveolar macrophages phagocytosis and attenuate lung inflammatory injury by upregulating GPR37 expression.

Angiotensin II type-2 receptor activation in alveolar macrophages mediates protection against cigarette smoke-induced chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally. Cumulative evidence has implicated renin-angiotensin system (RAS) in the pathogenesis of COPD. Alveolar macrophages (AMs) are the first line immune defense in the respiratory system and play a critical role in the lung homeostasis. This study aimed to investigate the role of AMs in contributing to the protective effects of angiotensin II type-2 receptor (AT2R) activation in cigarette smoke (CS)-induced COPD. The AM polarization, phagocytosis and metabolism, and the underlying biochemical mechanisms of compound 21 (C21), a selective and potent non-peptide small molecule AT2R agonist, were evaluated in a two-week CS-induced COPD mouse model. C21 restored AM phagocytosis ability, reversing CS-induced AM phagocytosis impairment. CS exposure polarized AMs towards M1 phenotype, whereas, C21 skewed the CS-exposed AMs towards M2 phenotype. C21 reprogrammed CS-exposed AM metabolism from a high glycolysis-driven process to support inflammation energy demand to a high mitochondrial respiration process to limit inflammation. Besides, C21 upregulated AT2R and Mas receptor levels in CS-exposed AMs, favoring the anti-inflammatory Ang II/AT2R axis and Ang 1–7/Mas axis in the RAS. C21 restored the normal levels of sirtuin 1 (SIRT1) and MAPK phosphatase 1 (MKP1) in CS-exposed AMs, leading to the reduction of phospho-p38, phospho-ERK and p65 subunit of NF-κB levels in CS-exposed AMs. We report here for the first time that AT2R agonist C21 acts by boosting the protective functions of AMs against CS-induced COPD, and our results support the development of AT2R agonist for the treatment of COPD.

Protease-activated receptor 2 promotes clearance of Pseudomonas aeruginosa infection by inducing cAMP-Rac1 signaling in alveolar macrophages.

Efficient phagocytosis of pathogens by the innate immune system during infectious injury is vital for restoring tissue integrity. Impaired phagocytosis, such as in the case of infection with Pseudomonas aeruginosa, a broad-spectrum antibiotic-resistant Gram-negative bacterium, can lead to a life threatening lung disorder, acute lung injury (ALI). Evidence indicates that loss of protease-activated receptor 2 (PAR2) impaired Pseudomonas aeruginosa clearance leading to non-resolvable ALI, but the mechanism remains unclear. Here, we focused on the alveolar macrophages (AMs), the predominant population of lung-resident macrophages involved in sensing bacteria, to understand their role in PAR2-mediated phagocytosis of Pseudomonas aeruginosa. We found that upon binding Pseudomonas aeruginosa, PAR2-expressing but not PAR2-null AMs had increased cAMP levels, which activated Rac1 through protein kinase A. Activated Rac1 increased actin-rich protrusions to augment the phagocytosis of Pseudomonas aeruginosa. Administration of liposomes containing constitutively active Rac1 into PAR2-null mice lungs rescued phagocytosis and enhanced the survival of PAR2-null mice from pneumonia. These studies showed that PAR2 drives the cAMP-Rac1 signaling cascade that activates Pseudomonas aeruginosa phagocytosis in AMs, thereby preventing death from bacterial pneumonia.

A novel therapeutic approach for IPF: Based on the “Autophagy - Apoptosis” balance regulation of Zukamu Granules in alveolar macrophages

Ethnopharmacological relevanceZukamu Granules (ZKMG) is one of the representative Uygur patent drugs widely used in China, which is included in the National Essential Drugs List (2018 edition). As the first choice for common cold treatment in Uygur medicine theory, it has unique anti-inflammatory and antitussive efficacy. Aim of the studyAccording to the recent inflammatory hypothesis, the abnormal proliferation, autophagy and apoptosis process of lung cells especially alveolar macrophages (AMs) may play an important role in the progress of idiopathic pulmonary fibrosis (IPF). Therefore, we came up with a novel treatment approach for IPF by regulating the balance of AMs “autophagy - apoptosis”, and took ZKMG as the sample drug for our research. Materials and methodsNetwork pharmacology approach was conducted to predict the active components and intersected targets between ZKMG and inflammation. PPI network, GO and KEGG enrichment analysis were screened and analyzed to predict the anti-inflammatory mechanism of ZKMG. Biological experiment adopted from 128 rats, and hematoxylin-eosin staining, flow cytometry and RT-PCR were performed to examine the pathological morphology, HYP contents in lung tissue, AMs counting, AMs apoptosis, AMs phagocytosis rate, mRNA relative quantity determination of 3 key factors associated with AMs “autophagy - apoptosis” and mRNA relative quantity determination of AMs surface receptor signaling pathway. ResultsThe predicted results showed that the mechanism of ZKMG in anti-inflammatory was related to the response and elimination of inflammatory stimuli, the intervention of apoptosis and surface receptor signaling pathways of cells. The verification experiments showed that excessive apoptosis and insufficient autophagy of AMs always existed in the progression of IPF. ZKMG could inhibit AMs proliferation, significantly reduce AMs apoptosis rate, intervene the binding of the Bcl-2 to Beclin 1, inhibit the Caspase 3 activation, stimulate the enhancement of AMs phagocytosis, and inhibit the high expression of TLR4/MyD88/NF-κB surface receptor signaling pathway, which may partly retard the fibrosis process. ConclusionBy inhibiting proliferation, enhancing phagocytosis, inhibiting the formation of Bcl-2 complex, and inhibiting the high expression of MYD88-dependent TLR4 signaling pathway, ZKMG can regulate the balance of AMs “autophagy - apoptosis” in the alveolitis stage to retard the fibrosis process partly. With a comprehensive strategy of “target prediction - experimental verification”, we have demonstrated that inhibiting the apoptosis and promoting autophagy activity of AMs may suggest a new perspective for IPF treatment, which would provide reference for the subsequent development.

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

Alcohol-Induced Glycolytic Shift in Alveolar Macrophages Is Mediated by Hypoxia-Inducible Factor-1 Alpha.

Excessive alcohol use increases the risk of developing respiratory infections partially due to impaired alveolar macrophage (AM) phagocytic capacity. Previously, we showed that chronic ethanol (EtOH) exposure led to mitochondrial derangements and diminished oxidative phosphorylation in AM. Since oxidative phosphorylation is needed to meet the energy demands of phagocytosis, EtOH mediated decreases in oxidative phosphorylation likely contribute to impaired AM phagocytosis. Treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, pioglitazone (PIO), improved EtOH-mediated decreases in oxidative phosphorylation. In other models, hypoxia-inducible factor-1 alpha (HIF-1α) has been shown to mediate the switch from oxidative phosphorylation to glycolysis; however, the role of HIF-1α in chronic EtOH mediated derangements in AM has not been explored. We hypothesize that AM undergo a metabolic shift from oxidative phosphorylation to a glycolytic phenotype in response to chronic EtOH exposure. Further, we speculate that HIF-1α is a critical mediator of this metabolic switch. To test these hypotheses, primary mouse AM (mAM) were isolated from a mouse model of chronic EtOH consumption and a mouse AM cell line (MH-S) were exposed to EtOH in vitro. Expression of HIF-1α, glucose transporters (Glut1 and 4), and components of the glycolytic pathway (Pfkfb3 and PKM2), were measured by qRT-PCR and western blot. Lactate levels (lactate assay), cell energy phenotype (extracellular flux analyzer), glycolysis stress tests (extracellular flux analyzer), and phagocytic function (fluorescent microscopy) were conducted. EtOH exposure increased expression of HIF-1α, Glut1, Glut4, Pfkfb3, and PKM2 and shifted AM to a glycolytic phenotype. Pharmacological stabilization of HIF-1α via cobalt chloride treatment in vitro mimicked EtOH-induced AM derangements (increased glycolysis and diminished phagocytic capacity). Further, PIO treatment diminished HIF-1α levels and reversed glycolytic shift following EtOH exposure. These studies support a critical role for HIF-1α in mediating the glycolytic shift in energy metabolism of AM during excessive alcohol use.

House Dust Mite Aeroallergen Suppresses Leukocyte Phagocytosis and Netosis Initiated by Pneumococcal Lung Infection.

Asthmatics are highly susceptible to developing lower respiratory tract infections caused by Streptococcus pneumoniae (SPN, the pneumococcus). It has recently emerged that underlying allergic airway disease creates a lung microenvironment that is defective in controlling pneumococcal lung infections. In the present study, we examined how house dust mite (HDM) aeroallergen exposure altered immunity to acute pneumococcal lung infection. Alveolar macrophage (AM) isolated from HDM-exposed mice expressed alternatively activated macrophage (AAM) markers including YM1, FIZZ1, IL-10, and ARG-1. In vivo, prior HDM exposure resulted in accumulation of AAMs in the lungs and 2-log higher bacterial titres in the bronchoalveolar (BAL) fluid of SPN-infected mice (Day 2). Acute pneumococcal infection further increased the expression of IL-10 and ARG1 in the lungs of HDM-exposed mice. Moreover, prior HDM exposure attenuated neutrophil extracellular traps (NETs) formation in the lungs and dsDNA levels in the BAL fluid of SPN-infected mice. In addition, HDM-SPN infected animals had significantly increased BAL fluid cellularity driven by an influx of macrophages/monocytes, neutrophils, and eosinophils. Increased lung inflammation and mucus production was also evident in HDM-sensitised mice following acute pneumococcal infection, which was associated with exacerbated airway hyperresponsiveness. Of note, PCV13 vaccination modestly reduced pneumococcal titres in the BAL fluid of HDM-exposed animals and did not prevent BAL inflammation. Our findings provide new insights on the relationship between pneumococcal lung infections and allergic airways disease, where defective AM phagocytosis and NETosis are implicated in increased susceptibility to pneumococcal infection.

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.

Selenium donor restricts the intracellular growth of Mycobacterium tuberculosis through the induction of c-Jun-mediated both canonical autophagy and LC3-associated phagocytosis of alveolar macrophages

The relationship between selenium and Mycobacterium tuberculosis (MTB) infection has been reported previously; however, the specific mechanism is still not clear. In this study, selenium levels decreased in the serum of patients with pulmonary tuberculosis (PTB) compared with the healthy controls; they were associated with the treatment outcome of such patients. The qRT-PCR assay revealed that selenium might function through proinflammatory and autophagy pathways. The treatment with methylseleninic acid (MSeA), a selenium donor, blocked the M1 polarization of MTB-infected macrophages through the induction of both canonical autophagy and LC3-associated phagocytosis (LAP). c-Jun is vital in mediating the MSeA-triggered canonical autophagy and LAP process, thus displaying a restricting function against intracellular MTB. An in vivo study confirmed that the activity of MSeA was shown through enhancing macrophage autophagy related pathway. The results showed that selenium had a restricting function against intracellular MTB by regulating autophagy in macrophages. The findings might provide a novel direction for PTB therapy in the future.

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.

Basil Polysaccharide Reverses Development of Experimental Model of Sepsis-Induced Secondary Staphylococcus aureus Pneumonia.

Background Basil polysaccharide (BPS) represents a main active ingredient extracted from basil (Ocimum basilicum L.), which can regulate secondary bacterial pneumonia development in the process of sepsis-mediated immunosuppression. Methods In this study, a dual model of sepsis-induced secondary pneumonia with cecal ligation and puncture and intratracheal instillation of Staphylococcus aureus or Pseudomonas aeruginosa was constructed. Results The results indicated that BPS-treated mice undergoing CLP showed resistance to secondary S. aureus pneumonia. Compared with the IgG-treated group, BPS-treated mice exhibited better survival rate along with a higher bacterial clearance rate. Additionally, BPS treatment attenuated cell apoptosis, enhanced lymphocyte and macrophage recruitment to the lung, promoted pulmonary cytokine production, and significantly enhanced CC receptor ligand 4 (CCL4). Notably, recombinant CCL4 protein could enhance the protective effect on S. aureus-induced secondary pulmonary infection of septic mice, which indicated that BPS-induced CCL4 partially mediated resistance to secondary bacterial pneumonia. In addition, BPS priming markedly promoted the phagocytosis of alveolar macrophages while killing S. aureus in vitro, which was related to the enhanced p38MAPK signal transduction pathway activation. Moreover, BPS also played a protective role in sepsis-induced secondary S. aureus pneumonia by inducing Treg cell differentiation. Conclusions Collectively, these results shed novel lights on the BPS treatment mechanism in sepsis-induced secondary S. aureus pneumonia in mice.

Pioglitazone improves alcohol-induced alveolar macrophage phagocytic dysfunction via increases in microRNAs-1264 and -107.

Abstract Objective: People with a history of alcohol use disorders (AUD) are 2–4 times more likely to develop lung infections, in part due to impaired alveolar macrophage (AM) immune function. Ethanol (EtOH) induces oxidative stress via increases in NAPDH oxidase (Nox)1 and Nox2 and decreases in peroxisome proliferator-activated receptor (PPAR)γ, leading to AM phagocytic dysfunction. Since PPARγ activation with its ligand pioglitazone (PIO) attenuated acute lung injury, we hypothesized that PIO upregulates Nox1-related microRNA (miR)-1264 and Nox2-related miR-107, thereby lowering Nox1 and Nox2 expression and oxidative stress and improving AM phagocytic function. Methods: AM were isolated from C57BL/6J mice fed EtOH (20% w/v) in the drinking water for 12 wks ± PIO (10 mg/kg/day by oral gavage during week 12). In parallel, a mouse AM cell line, MH-S cells, were transfected ± 50 nM miR-1264/-107 mimics and treated ± 0.08% EtOH for 3 d ± 10 μM PIO on day 3. miR-1264 and -107 were assessed by qRT-PCR. Nox1 and Nox2 mRNA and protein levels were measured by qRT-PCR and cytoimmunostaining. Oxidative stress was assessed with DCFH-DA and Amplex Red assays. AM phagocytosis was evaluated by S. aureus internalization. Results: In vivo and in vitro, EtOH: decreased miR-1264/-107 levels, increased Nox1 and Nox2 expression, enhanced oxidative stress, and impaired phagocytosis. miR-1264/-107 mimics or PIO reversed these EtOH-induced AM derangements. Conclusions: PIO rapidly upregulated miRs-1264/-107 and attenuated EtOH-induced AM oxidative stress and phagocytic dysfunction. Our studies suggest PIO as a clinically relevant intervention to mitigate AM immune derangements and may decrease susceptibility of lung infections in people with a history of AUD.