Alveolar Macrophage Cell Line MH-S Research Articles

Analysis of particulate matter-induced alteration of genes and related signaling pathways in the respiratory system

Airborne particulate matter (PM) is a global environmental risk factor threatening human health and is a major cause of cardiovascular and respiratory disease-associated death. Current studies on PM exposure have been limited to large-scale cohort and epidemiological investigations, emphasizing the need for detailed individual-level studies to uncover specific differentially expressed genes and their associated signaling mechanisms. Herein, we revealed that PM exposure significantly upregulated inflammatory and immune responses, such as cytokine-mediated signaling pathways, complement system, and the activation and migration of immune cells in gene set enrichment analysis of our RNA sequencing (RNAseq) data. Remarkably, we discovered that the broad gene expression and signaling pathways mediated by macrophages were predominantly expressed in the respiratory system following PM exposure. Consistent with these observations, individual PMs, classified by aerodynamic size and origin, significantly promoted macrophage recruitment to the lungs in the mouse lung inflammation model. Additionally, we confirmed that RNAseq observations from the respiratory system were reproduced in murine bone marrow-derived macrophages and the alveolar macrophage cell line MH-S after individual PM exposure. Our findings demonstrated that PM exposure augmented broad inflammatory and immune responses in the respiratory system and suggested the reinforcement of global strategies for reducing particulate air pollution to prevent respiratory diseases and their exacerbation.

Visualization of Lipid Droplets in the Alveolar Macrophage Cell Line MH-S with Live-cell Imaging by 3D Holotomographic Microscopy (Nanolive).

Lipid droplets (LD), triglycerides and sterol esters among them, are well known for their capacity as lipid storage organelles. Recently, they have emerged as critical cytoplasmic structures involved in numerous biological functions. LD storage is generated de novo by the cell and provides an energy reserve, lipid precursors, and cell protection. Moreover, LD accumulation can be observed in some pathologies as obesity, atherosclerosis, or lung diseases. Fluorescence imaging techniques are the most widely used techniques to visualize cellular compartments in live cells, including LD. Nevertheless, presence of fluorophores can damage subcellular components and induce cytotoxicity, or even alter the dynamics of the organelles. As an alternative to fluorescence microscopy, label-free techniques such as stimulated Raman scattering and coherent anti-stokes Raman scattering microscopy offer a solution to avoid the undesirable effects caused by dyes and fluorescent proteins, but are expensive and complex. Here, we describe a label-free method using live-cell imaging by 3D holotomographic microscopy (Nanolive) to visualize LD accumulation in the MH-S alveolar macrophage cell line after treatment with oleic acid, a monounsaturated fatty acid that promotes lipid accumulation.

Antibiotic-Loaded Amphiphilic Chitosan Nanoparticles Target Macrophages and Kill an Intracellular Pathogen.

In this work, levofloxacin (LVX), a third-generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan-g-poly(methyl methacrylate) produced by self-assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non-crosslinked nanoparticles display a size of 29nm and a zeta-potential of +36mV, while the crosslinked counterparts display 45nm and +24mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH-S and the human bronchial epithelial cell line BEAS-2B in vitro. Internalization events are detected after 10min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX-loaded nanoformulations (50% w/w drug content) is assessed in MH-S and BEAS-2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.

Nicotine in E-Cigarettes Dysregulates Pulmonary Inflammation and MMP-12 Expression without Effecting Respiratory Syncytial Virus Virulence

The safety of electronic cigarettes (e-cigarettes) is a major topic of discussion. The key goals of this study were to examine the contents of e-cigarette vapor and determine if nicotine altered inflammatory responses against respiratory syncytial virus (RSV) infection. E-cigarette vapor was passed through a hollow 3D-model of an adult lung, and gas chromatography detected over 50 compounds passed through the 3D model, including nicotine, propylene glycol (PG), ethanol, methanol, and diacetyl. The murine alveolar macrophage cell line MH-S cells were exposed to nicotine and e-cigarette vapor with and without nicotine. Nicotine significantly induced the expression of matrix metalloprotease (Mmp) 12 and reduced expression of Ifnβ and Tnfα. To examine the role of nicotine in lung defense against RSV infection, A/J mice were exposed to PBS, e-cigarette vapor with and without nicotine for 2 months before RSV infection. E-cigarette vapor did not influence RSV infection-induced animal weight loss, RSV infectivity, airway hyperresponsiveness during methacholine challenge, or immune cell infiltration into the lungs. However, e-cigarette vapor containing nicotine enhanced obstruction and induced secretion of MMP12 and reduced levels of Ifnβ and TNFα. In conclusion, nicotine in vaping products modulates immune responses that may impact the lungs during a respiratory infection.

MiR-23b Attenuates LPS-Induced Inflammatory Responses in Acute Lung Injury via Inhibition of HDAC2

Inflammatory responses play significant role in infectious etiology-induced acute lung injury (ALI). Histone deacetylase 2 is found to be essential and stimulated in lipopolysaccharide (LPS)-induced ALI by regulating proinflammatory cytokines. miR-23b has been demonstrated to be downregulated in LPS-induced inflammatory injury. In this study, we aimed to explore the interaction between miR-23b and HDAC2 and their function in LPS-induced ALI. LPS treatment was induced on murine alveolar macrophage cell line MH-S. Level of miR-23b and HDAC2 were determined by real-time PCR or Western blot. Proinflammatory cytokines expression and secretion were detected by real-time PCR and ELISA assay. The levels of miR-23b and HDAC2 were manipulated by transient transfection of miRNA mimics, shRNA or overexpression vector. The interaction between miR-23b and HDAC2 were tested by Luciferase reporter assay. LPS treatment inhibited miR-23b expression, while increased HDAC2 level in MH-S cells. Proinflammatory cytokines were stimulated by LPS treatment. Knockdown of HDAC2 or overexpression of miR-23b significantly repressed the expression of proinflammatory cytokines induced by LPS. miR-23b could suppress HDAC2 expression by directly targeting to its mRNA. LPS treatment stimulated the inflammatory responses in macrophages through inhibition of miR-23b, enhanced HDAC2 expression and inducing the expression of its downstream targets TNF-α, IL-6, and IL-1β. Overexpression of miR-23b was sufficient to suppress inflammatory responses by targeting HDAC2, making it a promising therapeutic target to ALI treatment.

Apoptosis signal-regulating kinase-1 promotes inflammasome priming in macrophages.

We previously showed that mice deficient in apoptosis signal-regulating kinase-1 (ASK1) were partially protected against ventilator-induced lung injury. Because ASK1 can promote both cell death and inflammation, we hypothesized that ASK1 activation regulates inflammasome-mediated inflammation. Mice deficient in ASK1 expression (ASK1-/-) exhibited significantly less inflammation and lung injury (as measured by neutrophil infiltration, IL-6, and IL-1β) in response to treatment with inhaled lipopolysaccharide (LPS) compared with wild-type (WT) mice. To determine whether this proinflammatory response was mediated by ASK1, we investigated inflammasome-mediated responses to LPS in primary macrophages and bone marrow-derived macrophages (BMDMs) from WT and ASK1-/- mice, as well as the mouse alveolar macrophage cell line MH-S. Cells were treated with LPS alone for priming or LPS followed by ATP for activation. When macrophages were stimulated with LPS followed by ATP to activate the inflammasome, we found a significant increase in secreted IL-1β from WT cells compared with ASK1-deficient cells. LPS priming stimulated an increase in NOD-like receptor 3 (NLRP3) and pro-IL-1β in WT BMDMs, but expression of NLRP3 was significantly decreased in ASK1-/- BMDMs. Subsequent ATP treatment stimulated an increase in cleaved caspase-1 and IL-1β in WT BMDMs compared with ASK1-/- BMDMs. Similarly, treatment of MH-S cells with LPS + ATP caused an increase in both cleaved caspase-1 and IL-1β that was diminished by the ASK-1 inhibitor NQDI1. These results demonstrate, for the first time, that ASK1 promotes inflammasome priming.

Aspergeillus Fumigatus: The Effects of Adiponectin on Inflammatory Cytokine Production

Background and Hypothesis: Although inflammatory cytokines are important for antifungal defenses, excessive production significantly increases host immune pathology. It is therefore important to identify host pathways that limit detrimental inflammation in invasive fungal infection. Our prior results showed that mice with invasive aspergillosis (IA) that were deficient in the metabolic cytokine production produced more of the cytokine Tumor Necrosis Factor (TNF) than alveolar in wild-type control mice. Therefore, we hypothesize that adiponectin inhibits antifungal cytokine secretion in alveolar macrophages. 
 Experimental Design or Project Methods: To test this hypothesis, the commonly used A. fumigatus strain Af293 was purchased from the Fungal Genetics Stock Center and grown on and harvested from agar. The alveolar macrophage cell line MH-S and cytokine ELISA kits was obtained from MilliporeSigma and ThermoFisher, respectively. MH-S cells were stimulated with swollen, fixed Af293 conidia for 24 hours in the presence or absence of recombinant mouse adiponectin. After 24 hours, supernatants and cells was were collected and assayed for ILs 1a, 6, and TNF protein and mRNA , by ELISA and quantitative RT-PCR, respectively. 
 Results: Although our preliminary results suggest possible inhibition of cytokine secretion by adiponectin in response to A. fumigatus, significant differences have thus far not been observed. 
 Conclusion and Potential Impact: We are therefore currently optimizing our experimental conditions to improve antifungal cytokine secretion. These studies may ultimately assist in the discovery of novel therapeutic targets and improve the prognosis of A. fumigatus infections

Alveolar Macrophage Innate Response to Mycobacterium immunogenum, the Etiological Agent of Hypersensitivity Pneumonitis: Role of JNK and p38 MAPK Pathways

Mycobacterium immunogenum is an emerging pathogen of the immune-mediated lung disease hypersensitivity pneumonitis (HP) reported in machinists occupationally exposed to contaminated metal working fluid (MWF). However, the mechanism of its interaction with the host lung is unclear. Considering that alveolar macrophages play a central role in host defense in the exposed lung, understanding their interaction with the pathogen could provide initial insights into the underlying immunopathogenesis events and mechanisms. In the current study, M. immunogenum 700506, a predominant genotype isolated from HP-linked fluids, was shown to multiply intracellularly, induce proinflammatory mediators (TNF-α, IL-1α, IL-1β, IL-6, GM-CSF, NO) and cause cytotoxicity/cell death in the cultured murine alveolar macrophage cell line MH-S in a dose- and time-dependent manner. The responses were detected as early as 3h post-infection. Comparison of this and four additional genotypes of M. immunogenum (MJY-3, MJY-4, MJY-12, MJY-14) using an effective dose-time combination (100 MOI for 24h) showed these macrophage responses in the following order (albeit with some variations for individual response indicators). Inflammatory: MJY-3 ≥ 700506 > MJY-4 ≥ MJY-14 ≥ MJY-12; Cytotoxic: 700506 ≥ MJY-3 > MJY-4 ≥ MJY-12 ≥ MJY-14. In general, 700506 and MJY-3 showed a more aggressive response than other genotypes. Chemical blocking of either p38 or JNK inhibited the induction of proinflammatory mediators (cytokines, NO) by 700506. However, the cellular responses showed a somewhat opposite effect. This is the first report on M. immunogenum interactions with alveolar macrophages and on the identification of JNK- and p38- mediated signaling and its role in mediating the proinflammatory responses during these interactions.

AMP-activated Protein Kinase Activation by 5-Aminoimidazole-4-carbox-amide-1-β-d-ribofuranoside (AICAR) Reduces Lipoteichoic Acid-induced Lung Inflammation

Adenosine monophosphate-activated protein (AMP)-activated kinase (AMPK) is a highly conserved kinase that plays a key role in energy homeostasis. Activation of AMPK was shown to reduce inflammation in response to lipolysaccharide in vitro and in vivo. 5-Aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR) is intracellularly converted to the AMP analog ZMP, which activates AMPK. Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria that can trigger inflammatory responses. In contrast to lipopolysaccharide, little is known on the effects of AMPK activation in LTA-triggered innate immune responses. Here, we studied the potency of AMPK activation to reduce LTA-induced inflammation in vitro and in lungs in vivo. Activation of AMPK in vitro reduced cytokine production in the alveolar macrophage cell line MH-S. In vivo, AMPK activation reduced LTA-induced neutrophil influx, as well as protein leak and cytokine/chemokine levels in the bronchoalveolar space. In conclusion, AMPK activation inhibits LTA-induced lung inflammation in mice.

In utero nicotine exposure promotes M2 activation in neonatal mouse alveolar macrophages

BackgroundMaternal smoking in utero has been associated with adverse health outcomes including lower respiratory tract infections in infants and children, but the mechanisms underlying these associations continue to be investigated. We hypothesized that nicotine plays a significant role in mediating the effects of maternal tobacco smoke on neonatal alveolar macrophage (AM) function, the resident immune cell in the neonatal lung.MethodsPrimary AMs were isolated at postnatal day 7 from a murine model of in utero nicotine exposure. The murine AM cell line MH-S was used for additional in vitro studies.ResultsIn utero nicotine increased IL-13 and transforming growth factor beta one (TGFβ1) in the neonatal lung. Nicotine-exposed AMs demonstrated increased TGFβ1 and increased markers of alternative activation with diminished phagocytic function. However, AMs from mice deficient in the α7 nicotinic acetylcholine receptor (α7 nAChR) had less TGFβ1, reduced alternative activation and improved phagocytic functioning despite similar in utero nicotine exposure.ConclusionIn utero nicotine exposure, mediated in part via the α7 nAChR, may increase the risk of lower respiratory tract infections in neonates by changing the resting state of AM towards alternative activation. These findings have important implications for immune responses in the nicotine-exposed neonatal lung.

Burkholderia mallei cellular interactions in a respiratory cell model

Burkholderia mallei is a facultative intracellular pathogen that survives and replicates in phagocytic cell lines. The bacterial burden recovered from naïve BALB/c mice infected by intranasal delivery indicated that B. mallei persists in the lower respiratory system. To address whether B. mallei invades respiratory non-professional phagocytes, this study utilized A549 and LA-4 respiratory epithelial cells and demonstrated that B. mallei possesses the capacity to adhere poorly to, but not to invade, these cells. Furthermore, it was found that B. mallei was taken up by the murine alveolar macrophage cell line MH-S following serum coating, an attribute suggestive of complement- or Fc receptor-mediated uptake. Invasion/intracellular survival assays of B. mallei-infected MH-S cells demonstrated decreased intracellular survival, whilst a type III secretion system effector bopA mutant strain survived longer than the wild-type. Evaluation of the potential mechanism(s) responsible for efficient clearing of intracellular organisms demonstrated comparable levels of caspase-3 in both the wild-type and bopA mutant with characteristics consistent with apoptosis of infected MH-S cells. Furthermore, challenge of BALB/c mice with the bopA mutant by the intranasal route resulted in increased survival. Overall, these data suggest that B. mallei induces apoptotic cell death, whilst the BopA effector protein participates in intracellular survival.

Chemical structure and immunobiological activity of lipid A from Serratia marcescens LPS

The chemical structure and immunobiological activities of Serratia marcescens lipid A, an active centre of LPS, were investigated. LPS preparations of S. marcescens were extracted using a hot phenol/water method, after which purified lipid A specimens were prepared by weak acid hydrolysis, followed by normal phase and gel filtration chromatographic separation. The lipid A structure was determined by MS to be a diglucosamine backbone with diphosphates and five C(14) normal chain acyl groups, including two acyloxyacyl groups at the 2 and 3 positions of the non-reducing side. S. marcescens lipid A and Escherichia coli-type synthetic lipid A (compound 506) exhibited definite reactivity in Limulus amoebocyte lysate assays. The lethal toxicity of S. marcescens lipid A was nearly comparable to that of compound 506, and both induced nuclear factor-kappaB activation in murine cells via Toll-like receptor (TLR)4/MD-2 but not TLR2, as well as various inflammatory cytokines in peritoneal macrophages of C3H/HeN mice but not C3H/HeJ mice. Furthermore, S. marcescens lipid A induced nearly the same amounts of tumour necrosis factor alpha, interleukin-6, and nitric oxide production by the murine alveolar macrophage cell line MH-S as compared with compound 506. These results indicate that S. marcescens possesses a penta-acylated lipid A, which is nearly identical to E. coli lipid A in regard to biological activities, while it also may be a crucial virulence factor of the bacterium.

LIPOPOLYSACCHARIDE O-ANTIGEN PROMOTES PERSISTENT MURINE BACTEREMIA

Bacteremia is a common complication of pneumonia with Klebsiella pneumoniae. In the previous work, we have shown that the lipopolysaccharide (LPS) O-antigen in K. pneumoniae O1:K2 contributes to lethality during pneumonia in part by promoting bacteremia. In the current work, we studied an O-antigen-deficient K. pneumoniae strain to further evaluate this polysaccharide's role in bloodstream infection. Cultured macrophage and murine bacteremia models were studied. In vitro, O-antigen-deficient bacteria, compared with wild-type organisms, were stronger activators of the murine alveolar macrophage cell line MH-S as assessed by nuclear localization of RelA/p65 and by secretion of cytokines and chemokines. O-antigen-deficient Klebsiellae were also more susceptible to killing by murine neutrophils. In vivo, the absence of O-antigen allowed more rapid and complete clearance of bacteria from the bloodstream, liver, and spleen after intravenous injection in mice. Survival was also greater among animals infected with bacteria missing the O-antigen. Gene expression profiling (via reverse transcriptase-polymerase chain reaction of 84 inflammatory mediator complementary DNA) revealed that by 24 h postinfection, the livers and spleens of animals infected with O-antigen-deficient organisms had significantly downregulated cytokine and chemokine expression compared with wild-type infected animals. The O-antigen surface carbohydrate of O1:K2 serotype K. pneumoniae appears to contribute to bacterial virulence by lessening the activation of macrophages, conveying resistance to killing by neutrophils, and by promoting persistent infection in the blood, liver, and spleen after the onset of bacteremia.

DNA damage, redox changes, and associated stress-inducible signaling events underlying the apoptosis and cytotoxicity in murine alveolar macrophage cell line MH-S by methanol-extracted Stachybotrys chartarum toxins

Spore-extracted toxins of the indoor mold Stachybotrys chartarum (SC) caused cytotoxicity (release of lactate dehydrogenase), inhibition of cell proliferation, and cell death in murine alveolar macrophage cell line MH-S in a dose- and time-dependent manner. Apoptotic cell death, confirmed based on morphological changes, DNA ladder formation, and caspase 3/7 activation, was detectable as early as at 3 h during treatment with a toxin concentration of 1 spore equivalent/macrophage and was preceded by DNA damage beginning at 15 min, as evidenced by DNA comet formation in single cell gel electrophoresis assay. The apoptotic dose of SC toxins did not induce detectable nitric oxide and pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) but showed exacerbated cytotoxicity in presence of a non-apoptotic dose of the known pro-inflammatory agent LPS (10 ng/ml). Intracellular reduced glutathione (GSH) level showed a significant decrease beginning at 9 h of the toxin treatment whereas oxidized glutathione (GSSG) showed a corresponding significant increase, indicating a delayed onset of oxidative stress in the apoptosis process. The toxin-treated macrophages accumulated p53, an indicator of DNA damage response, and showed activation of the stress-inducible MAP kinases, JNK, and p38, in a time-dependent manner. Chemical blocking of either p38 or p53 inhibited in part the SC toxin-induced apoptosis whereas blocking of JNK did not show any such effect. This study constitutes the first report on induction of DNA damage and associated p53 activation by SC toxins, and demonstrates the involvement of p38- and p53-mediated signaling events in SC toxin-induced apoptosis of alveolar macrophages.

Biochemical function of msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv: biosynthesis of monomethyl branched unsaturated fatty acids.

We show that the disruption of one of the mycocerosic acid synthase (mas)-like genes, msl5 (pks8 plus pks17) in Mycobacterium tuberculosis H37Rv generates a mutant incapable of producing monomethyl branched unsaturated C(16) to C(20) fatty acids that are minor constituents of acyltrehaloses and sulfolipids. The msl5 mutation did not cause any significant change in the acyl lipid composition and also did not affect growth in culture, in mouse alveolar macrophage cell line MH-S, or in the murine lung.

The largest open reading frame (pks12) in the Mycobacterium tuberculosis genome is involved in pathogenesis and dimycocerosyl phthiocerol synthesis.

The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-(14)C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.