LPS-treated Alveolar Macrophages Research Articles

Small Extracellular Vesicles Secreted by iPSC-Derived MSCs Ameliorate Pulmonary Inflammation and Lung Injury Induced by Sepsis through Delivery of miR-125b-5p.

Sepsis-induced acute lung injury is a common critical illness in intensive care units with no effective treatment is currently available. Small extracellular vesicles, secreted by mesenchymal stem cells (MSCs), derived from human-induced pluripotent stem cells (iMSC-sEV), possess striking advantages when incorporated MSCs and iPSCs, which are considered extremely promising cell-free therapeutic agents. However, no studies have yet been conducted to systemically examine the effects and underlying mechanisms of iMSC-sEV application on attenuated lung injury under sepsis conditions. iMSC-sEV were intraperitoneally administered in a rat septic lung injury model induced by cecal ligation and puncture (CLP). The efficacy of iMSC-sEV was assessed by histology, immunohistochemistry, and pro-inflammatory cytokines of bronchoalveolar lavage fluid. We also evaluated the in vitro effects of iMSC-sEV on the activation of the inflammatory response in alveolar macrophages (AMs). Small RNA sequencing was utilized to detect changes in the miRNA expression profile in lipopolysaccharide (LPS)-treated AMs after iMSC-sEV administration. The effects of miR-125b-5p on the function of AMs were studied. iMSC-sEV were able to attenuate pulmonary inflammation and lung injury following CLP-induced lung injury. iMSC-sEV were internalized by AMs and alleviated the release of inflammatory factors by inactivating the NF-κB signaling pathway. Moreover, miR-125b-5p showed a fold-change in LPS-treated AMs after iMSC-sEV administration and was enriched in iMSC-sEV. Mechanistically, iMSC-sEV transmitted miR-125b-5p into LPS-treated AMs to target TRAF6. Our findings demonstrated that iMSC-sEV treatment protects against septic lung injury and exerts anti-inflammatory effects on AMs at least partially through miR-125b-5p, suggesting that iMSC-sEV may provide a novel cell-free strategy for the treatment of septic lung injury.

LincRNA-p21 promotes classical macrophage activation in acute respiratory distress syndrome by activating NF-κB

Background: Previous studies have revealed the important role of alveolar macrophages (AMs) in the pathogenesis of acute respiratory distress syndrome (ARDS) and potential anti-inflammatory properties of lincRNA-p21. This study aims to study the association between lincRNA-p21 and active AMs to understand the molecular mechanisms of AMs-mediated inflammatory responses in ARDS.Methods: This study was mainly investigated in mice with the intratracheal instillation of lipopolysaccharide (LPS) or LPS-treated AMs. The expression of lincRNA-p21 and classical macrophage markers, IL-12β and iNOS, was detected by quantitative RT-PCR, while NF-κB p65 translocation was measured by western blotting analysis. And, NF-κB activity was analyzed through luciferase report assays. Gain- and loss-of-function studies were also performed for further investigations.Results: Elevated lincRNA-p21 levels were observed in both LPS-induced ARDS mice and LPS-treated AMs, with upregulated expression of IL-12β and iNOS, namely M1 activation, and p65 nuclear translocation. Further in vitro studies showed that LPS-induced M1 activation could be counteracted by both lincRNA-p21 inhibition and inhibited NF-κB activation. Moreover, both p65 nuclear translocation and NF-κB activity were promoted by lincRNA-p21 overexpression, while lincRNA-p21 inhibition showed a negative effect on LPS-induced p65 nuclear translocation and increase of NF-κB activity. Additionally, LPS-induced lung injuries could be attenuated by lincRNA-p21 inhibition in vivo.Conclusion: This study revealed elevated lincRNA-p21 levels in LPS-induced ARDS and investigated the potential role of lincRNA-p21 in LPS-induced pro-inflammatory response via NF-κB/p65 mediated pathways, suggesting the potential application of lincRNA-p21 for ADRS therapy.

Elevated IL-33 promotes expression of MMP2 and MMP9 via activating STAT3 in alveolar macrophages during LPS-induced acute lung injury

BackgroundPulmonary inflammation and endothelial barrier permeability increase in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) induced by pro-inflammatory cytokines and matrix metalloproteinases (MMPs). However, the relationship between pro-inflammatory cytokines and MMPs in ALI/ARDS remains poorly understood.MethodsA lipopolysaccharide (LPS)-induced ALI rat model was established through intratracheal instillation. The wet/dry ratios of lung tissues were measured, and bronchoalveolar lavage fluid (BALF) was collected to test protein concentrations, total cell/macrophage numbers, and pro-inflammatory cytokine levels. LPS-treated alveolar macrophages were utilized in in vitro experiments. The expression and secretion of MMPs were respectively detected using quantitative PCR, Western blotting and ELISA assays.ResultsThe levels of IL-33 and MMP2/9 in BALF increased in all the ALI rats with severe lung injury. LPS-induced IL-33 autocrine upregulated the expression of MMP2 and MMP9 through activating STAT3. Neutralizing IL-33 in culture medium with specific antibodies suppressed the expression and secretion of MMP2 and MMP9 in LPS-treated alveolar macrophages. Consistently, eliminating IL-33 decreased the levels of MMP2 and MMP9 in BALF and alleviated lung injury in ALI rats.ConclusionThe IL-33/STAT3/MMP2/9 regulatory pathway is activated in alveolar macrophages during acute lung injury, which may exacerbate the pulmonary inflammation.

Deletion 101 residue at caveolin-1 scaffolding domain peptides impairs the ability of increasing heme oxygenase-1 activity

ObjectiveResident alveolar macrophages (AMs) are activated and release proinflammatory mediators and chemokines during acute lung injury. We have previous reported that caveolin-1 (Cav-1) scaffolding domain (CSD) peptide inhibited the proinflammatory cytokines expression by up-regulating heme oxygenase-1 (HO-1) activity. In this study, we aimed to investigate the effect of residue R101 in CSD peptide on the activity of HO-1 in AMs. MethodsThe binding mode between HO-1 and CSD peptides (WT CSD and Δ101 CSD truncation peptides) was analyzed and the free energy was calculated. The inflammatory genes and M1/M2macrophage polarization-associated genes expression were measured by real-time PCR. The activities of HO-1 were determined by the spectrophotometical method. Western blot analyzed the content of Cav-1, HO-1, IκB and MAPK signals (phosphorylated ERK, JNK and p38 MAPK). ResultsΔ101CSD peptide could bind to HO-1 protein and to disrupt the interaction of HO-1 and Cav-1. However, Δ101CSD peptide had lower activity of HO-1 in LPS-treated AMs compared with WT CSD. The expression of IL-1β and MCP-1 and NO content were decreased by WT CSD peptide in LPS treated AMs. However, only MCP-1 expression and NO content were downregulated byΔ101CSD peptide. Meanwhile, compared with those in LPS + hemin + WT CSD group, the mRNA expression of TNF-α, Cd86, IL-12b and NOS2 significantly increased while expression of IL10, Arg1 and CD163 significantly decreased in LPS + hemin + Δ101CSD group. The effect of WT CSD peptide on the inhibition of MAPK signaling pathway were stronger than Δ101 CSD peptide evidenced by the level of phosphorylated ERK, JNK and p38 MAPK. ConclusionDeletion of residue R101 impairs the ability of CSD peptide to increase HO-1 activity and to dampen inflammatory response in LPS-challenged rat AMs.

Caveolin-1 scaffolding domain peptides enhance anti-inflammatory effect of heme oxygenase-1 through interrupting its interact with caveolin-1.

Caveolin-1(Cav-1) scaffolding domain (CSD) peptides compete with the plasma membrane Cav-1, inhibit the interaction of the proteins and Cav-1, and re-store the functions of Cav-1 binding proteins. Heme oxygenase-1 (HO-1) binds to Cav-1 and its enzymatic activity was inhibited. In this study, we investigated the effect of CSD peptides on interaction between HO-1 and Cav-1, and on the HO-1 activity in vitro and in vivo. Our data showed that CSD peptides decreased the compartmentalization of HO-1 and Cav-1, and increased the HO-1 activity both in LPS-treated alveolar macrophages and in mice. Meanwhile, CSD peptides obviously ameliorated the pathology changes in mice and lowered the following injury indexes: the wet/dry ratio of lung tissues, total cell numbers in bronchoalveolar lavage fluid and lactate dehydrogenase activity in the serum. Mechanistically, it was firstly found that CSD peptides promoted alveolar macrophages polarization to M2 phenotype and inhibited the IκB degeneration. Furthermore, CSD peptides down-regulated the expression of IL-1β, IL-6, TNF-α, MCP-1, and iNOS in alveolar macrophages and in lung tissue. However, the protective role of CSD peptides on LPS-induced acute lung injury in mice could be abolished by zinc protoporphyrin IX (ZnPP, a HO-1 activity inhibitor). In summary, CSD peptides have beneficial anti-inflammatory effects by restoring the HO-1 activity suppressed by Cav-1 on plasma membrane.

MiR-132 inhibits lipopolysaccharide-induced inflammation in alveolar macrophages by the cholinergic anti-inflammatory pathway

ABSTRACTObjective: Although microRNA-132 (miR-132) has been shown to be involved in the inflammatory regulation, its role in sepsis-induced lung injury is unknown. We hypothesized that miR-132 attenuated lipopolysaccharide (LPS)-induced inflammation of alveolar macrophages by targeting acetylcholinesterase (AChE) and enhancing the acetylcholine (ACh)-mediated cholinergic anti-inflammatory response. Methods: The LPS-treated rat alveolar macrophage cell line NR8383 was used as the inflammatory model. To assess the effect of miR-132, alveolar macrophages were transfected with miR-132 mimic or inhibitor. Results: We found that miR-132 was upregulated in LPS-stimulated alveolar macrophages. Induction of AChE mRNA showed an inverse pattern with respect to AChE protein and activity, suggesting posttranscriptional regulation of AChE. Utilizing miR-132 mimic transfection, we found that overexpression of miR-132 enhanced the ACh-mediated cholinergic anti-inflammatory reaction by targeting AChE mRNA in LPS-treated alveolar macrophages. Blockage of miR-132 using miR-132 inhibitor reversed the Ach action upon LPS-induced release of inflammatory mediators and reduction in AchE protein/activity. Moreover, in the presence of ACh, upregulation of miR-132 suppressed LPS-induced nuclear translocation of NF-κB and production of STAT3 and phosphorylated STAT3, while downregulation of miR-132 enhanced the nuclear translocation of NF-κB. Conclusion: We propose that miR-132 functions as a negative regulator of the inflammatory response in alveolar macrophages by potentiating the cholinergic anti-inflammatory pathway, and represents a potential therapeutic leverage point in modulating inflammatory responses.

Antioxidant and anti-inflammatory activities of Radix Isatidis polysaccharide in murine alveolar macrophages

Radix Isatidis is an official herbal medicine for treatment of infection and inflammation in China. In this study, a novel heteropolysaccharide (RIWP) was isolated from R. Isatidis through DEAE Sepharose Fast Flow column and Sepharose CL-6B column. RIWP had a molecular weight of 57kDa and was mainly composed of glucose, galactose and arabinose with a relative molar ratio of 2.0:1.1:1.0. The cytoprotective, antioxidant and anti-inflammatory properties of RIWP in lipopolysaccharide (LPS) stimulated murine alveolar macrophages were first reported here. Pretreatment with RIWP was found to potently prolong cell survival and repress the generation of reactive oxygen species (ROS) and lipid peroxidation after LPS-stimulation in murine alveolar macrophages. Furthermore antioxidant status was significantly deteriorated in the LPS-treated alveolar macrophages, such as low superoxide dismutase (SOD) activity and G-SH content, which was effectively restored by RIWP supplementation. More importantly, RIWP significantly suppressed LPS-induced increase in nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production in murine alveolar macrophages. Additionally RIWP recovered mitochondrial membrane potential to normal conditions. All above response to LPS stimulation behaved in a concentration-dependent manner. This study provided evidences that RIWP appears to have the potential to prevent inflammatory disease in lung.

Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and Akt activity in human alveolar macrophages.

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide. Because ceramide exposure induces apoptosis in other cell types and the PI 3-kinase pathway is known to inhibit apoptosis, we determined the relationship between LPS-induced ceramide and PI 3-kinase activation in alveolar macrophages. We found that ceramide exposure activated PI 3-kinase and Akt. When we blocked LPS-induced ceramide with the inhibitor D609, we blocked LPS-induced PI 3-kinase and Akt activation. Evaluating cell survival after ceramide or LPS exposure, we found that blocking PI 3-kinase induced a significant increase in cell death. Because these effects of PI 3-kinase inhibition were more pronounced in ceramide- vs LPS-treated alveolar macrophages, we also evaluated NF-kappaB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-kappaB translocation to the nucleus. As a composite, these studies suggest that the effects of ceramide exposure in alveolar macrophages may be very different from the effects described for other cell types. We believe that LPS induction of ceramide results in PI 3-kinase activation and represents a novel effector mechanism that promotes survival of human alveolar macrophages in the setting of pulmonary sepsis.

A Phosphatidylcholine-Specific Phospholipase C Regulates Activation of p42/44 Mitogen-Activated Protein Kinases in Lipopolysaccharide-Stimulated Human Alveolar Macrophages

This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC zeta, and this activation was inhibited by D609. LPS-activated PKC zeta phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.

Lipopolysaccharides stimulate Na-dependent transport in alveolar cells and protect against oxidant injury.

We have evaluated the effect of lipopolysaccharides (LPS), endotoxins from gram negative bacteria, on sodium-coupled amino acid and phosphate transport by alveolar epithelial type II cells and on their alteration induced by oxidants. Alveolar type II cells were obtained by enzymatic digestion of rat lung and grown for 24 h prior to incubation with LPS and then exposed or not exposed to H2O2 (2.5 mM; 20 min). LPS (10 micrograms/ml, 24 h) induced a significant increase in the Na-dependent component of alanine and phosphate uptake while they decreased Na,K-ATPase activity measured by ouabain-sensitive 86Rb influx. We showed that this stimulatory effect i) was independent from macrophage products since it was not mimicked either by supernatant of LPS-treated alveolar macrophages or by pretreatment with tumor necrosis factor and/or interleukin 1 and ii) was dependent on protein synthesis since it was abolished by protein synthesis inhibitors cycloheximide and actinomycin D. Moreover, LPS blunted H2O2-induced decrease of Na-dependent alanine and phosphate uptake. This protective effect of LPS against H2O2 injury i) was independent of macrophage products, ii) was abolished by cycloheximide, and iii) was not associated with either changes in extracellular H2O2 clearance or catalase and glutathione peroxidase activities. We conclude that, in alveolar type II cells, LPS stimulate sodium-coupled transport by a process involving protein synthesis and partially prevent H2O2-induced decrease of Na-coupled transport without discernible change in antioxidant activities.

Regulation of Human Alveolar Macrophage- and Blood Monocyte-derived Interleukin-8 by Prostaglandin E2 and Dexamethasone

Mononuclear phagocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities, monocytes/macrophages produce a vast array of regulatory and chemotactic cytokines. Interleukin-8 (IL-8), a potent neutrophil-activating and chemotactic peptide, is produced in large quantities by mononuclear phagocytes and may be an important mediator of local and systemic inflammatory events. In this investigation, we describe the effects of prostaglandin E2 (PGE2) and dexamethasone (Dex) on IL-8 mRNA and protein expression from lipopolysaccharide (LPS)-treated human peripheral blood monocytes (PBM) and alveolar macrophages (AM). We demonstrate the dose-dependent suppression of IL-8 from LPS-stimulated PBM by PGE2. Treatment of stimulated PBM with 10(-6) M PGE2 resulted in maximal inhibition, causing 60% suppression of both IL-8 mRNA and extracellular protein levels. In contrast, PGE2 (10(-6) to 10(-8) M) did not significantly alter IL-8 mRNA or protein expression from LPS-treated AM. Treatment of LPS-stimulated PBM and AM with Dex (10(-6) to 10(-8) M) resulted in 75% decline in IL-8 mRNA and extracellular protein from either cell population. Pretreatment of PBM with PGE2 or Dex 1 or 2 h before LPS stimulation caused a significant suppression of steady-state IL-8 mRNA levels; however, administration of either of these modulators 1 or 2 h after LPS stimulation failed to have an inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)