Alveolar Macrophage Cell Research Articles

Lung immune tone via gut-lung axis: gut-derived LPS and short-chain fatty acids' immunometabolic regulation of lung IL-1β, FFAR2, and FFAR3 expression.

Microbial metabolites produced by the gut microbiome, e.g. short-chain fatty acids (SCFA), have been found to influence lung physiology and injury responses. However, how lung immune activity is regulated by SCFA is unknown. We examined fresh human lung tissue and observed the presence of SCFA with interindividual variability. In vitro, SCFA were capable of modifying the metabolic programming in LPS-exposed alveolar macrophages (AM). We hypothesized that lung immune tone could be defined by baseline detection of lung intracellular IL-1β. Therefore, we interrogated naïve mouse lungs with intact gut microbiota for IL-1β mRNA expression and localized its presence within alveolar spaces, specifically within AM subsets. We established that metabolically active gut microbiota, which produce SCFA, can transmit LPS and SCFA to the lung and thereby could create primed lung immunometabolic tone. To understand how murine lung cells sensed and upregulated IL-1β in response to gut microbiome-derived factors, we determined that, in vitro, AM and alveolar type II (AT2) cells expressed SCFA receptors, free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), and IL-1β but with distinct expression patterns and different responses to LPS. Finally, we observed that IL-1β, FFAR2, and FFAR3 were expressed in isolated human AM and AT2 cells ex vivo, but in fresh human lung sections in situ, only AM expressed IL-1β at rest and after LPS challenge. Together, this translational study using mouse and human lung tissue and cells point to an important role for the gut microbiome and their SCFA in establishing and regulating lung immune tone.

Glaesserella parasuis induces inflammatory response in 3D4/21 cells through activation of NLRP3 inflammasome signaling pathway via ROS

Glaesserella parasuis (G. parasuis) is an important pathogenic bacterium that can cause Glässer’s disease, and it has resulted in tremendous economic losses to the global swine industry. The intensive pulmonary inflammatory response caused by G. parasuis infection is the main cause of lung injury and death in pigs. However, the exact mechanism by which it causes severe pulmonary inflammation is not fully understood yet. In this study, severe pneumonia was observed in piglets infected with G. parasuis; and an infection cell model was established using porcine alveolar macrophages cell line 3D4/21, which was determined to be susceptible to G. parasuis infection in vitro. G. parasuis infection of 3D4/21 cells induced upregulation of proinflammatory cytokines TNF-α, IL-1β, IL-18 and production of intracellular reactive oxygen species (ROS). The expression of IL-1β related to activation of the NLRP3 inflammasome signaling pathway, which had not been shown before in G. parasuis infection. Furthermore, it was first found that release of intracellular ROS, which was mediated by NADPH oxidase in 3D4/21 cells, was found crucial for the activation of the NLRP3 signaling pathway and promoted the expression of proinflammatory cytokines, such as TNF-α and IL-1. In general, this study explored the specific mechanism of severe pulmonary inflammation caused by G. parasuis infection, and provides a foundation for further elucidating the pathogenic mechanism of G. parasuis.

Untargeted LC-MS based metabolomic profiling of iPAMs to investigate lipid metabolic pathways alternations induced by different Pseudorabies virus strains

Owing to viral recombination, interspecies transmission, and evolution, variant pseudorabies virus (PRV) strains exhibit different biological characteristics and pathogenicity. To improve the understanding of common and specific metabolic changes that occur upon infection by different PRV strains, we herein describe the comprehensive analysis of metabolites of PRV vaccine strain (Bartha K61), classical strain (EA) and variant strain (HNX) infection in immortalized porcine alveolar macrophage cells. Compared with uninfected cells, cells infected with Bartha K61, EA and HNX had 246, 225, and 272 differing metabolites, respectively. In the three types of PRV-strain-infected cells, lipids and lipid-like molecules accounted for over 50 % of the altered metabolites. As these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of the host metabolism. We analyzed the potential relationship between virus replication and the virus-mediated host metabolism. Our study resulted in the first reconstruction of the major lipid metabolic pathways involved in PRV infection, including those of glycerophospholipids, sphingolipids, glycerolipids, and fatty acyls. In addition, the metabolic perturbations caused by different PRV strain infections are consistent across many species, however, our results also revealed many specific metabolic alterations during HNX infection, such as the enrichment of phosphatidylinositol and 15R-PGE2 methyl ester 15-acetate, and the diminishment of phosphatidylethanolamine, phosphatidic acid, and ceramides. These strain-specific altered metabolites may be linked to the unique biological characteristics and pathogenicity of the HNX strain.

Natural oil blend formulation as an anti-African swine fever virus agent in in vitro primary porcine alveolar macrophage culture.

Background and Aim:African swine fever is one of the severe pathogens of swine. It has a significant impact on production and economics. So far, there are no known remedies, such as vaccines or drugs, reported working successfully. In the present study, the natural oil blend formulation’s (NOBF) efficacy was evaluated against ASFV in vitro using porcine alveolar macrophages (PAMs) cells of swine.Materials and Methods:The capacity of NOBF against the ASFV was tested in vitro. The NOBF combines Eucalyptus globulus, Pinus sylvestris, and Lavandula latifolia. We used a 2-fold serial dilution to test the NOBF formulation dose, that is, 105 HAD50/mL, against purified lethal dose of African swine in primary PAMs cells of swine. The PAM cells survival, real-time polymerase chain reaction (PCR) test, and hemadsorption (HAD) observation were performed to check the NOBF efficacy against ASFV.Results:The in vitro trial results demonstrated that NOBF up to dilution 13 or 0.000625 mL deactivates the lethal dose 105 HAD50 of ASFV. There was no HAD (Rosetta formation) up to dilution 12 or 0.00125 mL of NOBF. The Ct value obtained by running real-time PCR of the NOBF group at 96 h post-infection was the same as the initial value or lower (25), whereas the Ct value of positive controls increased several folds (17.84).Conclusion:The in vitro trial demonstrated that NOBF could deactivate the ASFV. The NOBF has the potential to act as anti-ASFV agent in the field. The next step is to conduct in vivo level trial to determine its efficacy.

Silencing of long noncoding RNA H19 alleviates pulmonary injury, inflammation, and fibrosis of acute respiratory distress syndrome through regulating the microRNA-423-5p/FOXA1 axis

Purpose This study aimed to explore the regulatory effects and mechanisms of long noncoding RNA H19 (H19) on pulmonary injury, inflammation, and fibrosis of acute respiratory distress syndrome (ARDS). Materials and Methods A rat model of ARDS was established by intratracheal instillation of 2 mg/kg lipopolysaccharide (LPS). qRT-PCR was performed to detect the expression of H19, miR-423-5p, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF). Histology score was assessed by hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of proinflammatory cytokines and the content of VEGF in bronchoalveolar lavage fluid (BALF). The lung fibrosis was evaluated using western blot and Masson’s trichrome staining. Dual-luciferase reporter gene assay was used for confirming the relationship between miR-423-5p and H19/FOXA1 in alveolar macrophage cells (MH-S) and alveolar epithelial cells (MLE-12). The regulatory effects of H19/miR-423-5p/FOXA1 axis on the inflammation and fibrosis were further analyzed in LPS-induced MH-S cells. Results The expression of H19 and FOXA1 was significantly up-regulated, while the expression of miR-423-5p was down-regulated in LPS-induced ARDS rats. Silencing of H19 decreased the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1, and VEGF, the contents of TNF-α, IL-1β, IL-6, and VEGF in BALF, and histology score in LPS-induced ARDS rats. H19 knockdown also reduced the fibrosis scores and the protein expression of vimentin and α-SMA, and elevated the protein expression of E-cadherin in LPS-induced ARDS rats. Furthermore, silencing of miR-423-5p and overexpression of FOXA1 reversed the inhibitory effects of si-H19 on the inflammation and fibrosis of LPS-induced MH-S cells. Conclusions Silencing of H19 relieved the pulmonary injury, inflammation and fibrosis of LPS-induced ARDS in rats. Silencing of H19 also alleviated the inflammation and fibrosis of LPS-induced MH-S cells through regulating the miR-423-5p/FOXA1 axis.

Proteomic Analysis of ISGylation in Immortalized Porcine Alveolar Macrophage Cell Lines Induced by Type I Interferon.

Interferon-stimulated gene product 15 (ISG15), a ubiquitin-like molecule, can be conjugated to protein substrates through a reversible process known as ISGylation. ISG15 and ISGylation are both strongly upregulated by type I interferons and play putative key roles in host innate immunity against viral infection. However, the function of ISGylation and identities of ISGylation substrates are largely unknown. Here, a novel monoclonal antibody (Mab) that specifically recognizes porcine ISG15 (pISG15) was employed to capture ISG15-conjugated proteins from IFNs-stimulated porcine cell lysates. Next, Mab-captured conjugates were analyzed using proteomics-based tools to identify potential ISGylation protein targets in order to elucidate the roles of ISG15 and ISGylation in porcine cells. Subsequently, 190 putative ISGylation sites were detected within 98 identified ISGylation candidates; several candidates contained more than one ISGylation-modifiable lysine residue, including pISG15 itself. Motif enrichment analysis of confirmed ISGylation sites demonstrated a moderate bias towards certain sites with specific upstream amino acid residues. Meanwhile, results of Gene Ontology (GO)-based annotation and functional enrichment and protein-protein interaction (PPI) network analyses of porcine ISG15-conjugated substrate proteins indicated that these substrates were mainly associated with the host metabolism, especially nucleotide metabolic pathways that ultimately may participate in cellular antiviral defenses. Notably, several ISGs (MX1, IFIT1, OAS1, ISG15 and putative ISG15 E3 ligase Herc6) were also identified as putative ISGylation substrates within a regulatory loop involving ISGylation of ISGs themselves. Taken together, proteomics analysis of porcine ISGylation substrates revealed putative functional roles of ISG15 and novel host ISGylation targets that may ultimately be involved in cellular antiviral responses.

Effect of Pregnane X Receptor on CYP3A29 Expression in Porcine Alveolar Macrophages during Mycoplasma hyopneumoniae Infection.

Simple SummaryIn the currently intense production process, infection of swine with Mycoplasma pneumonia is common in pig farms around the world, and reduction in the feeding efficiency and the growth rate of sick pigs causes considerable economic losses to the pig-rearing industry. Our study aimed to determine the molecular mechanism by which Mycoplasma hyopneumoniae induces inflammation in pigs. Our study showed that Mycoplasma hyopneumoniae can regulate the expression of CYP3A29 by upregulating PXR during the inflammatory response induced in porcine alveolar macrophages. These findings may provide useful information for breeding pigs that are resistant to disease.Mycoplasma hyopneumoniae (M. hyopneumoniae, Mhp) is the causative agent of mycoplasma pneumonia of swine (MPS). M. hyopneumoniae infection causes inflammation in pigs and leads to considerable economic losses in the pig industry. Pregnane X receptor (PXR) is a pluripotent gene regulatory protein that plays an important role in regulating cytochrome P-450 (CYP) in pigs in the context of inflammatory responses, drug metabolism, homeostasis, etc. We previously reported that cytochrome P450 3A29 (CYP3A29) expression was significantly upregulated in pigs infected with M. hyopneumoniae compared with healthy control pigs. This experiment mainly focused on identifying the role of PXR in the regulation of CYP3A29 and inflammatory factors after M. hyopneumoniae infection by establishing pig alveolar macrophage (PAM) cells in which PXR was overexpressed or silenced. Our results showed that the overexpression of PXR could significantly improve the protein and the mRNA expression levels of CYP3A29 with and without M. hyopneumoniae infection in PAM cells. After the expression of PXR was inhibited, protein and mRNA expression levels of CYP3A29 were significantly reduced with and without M. hyopneumoniae infection in PAM cells. Moreover, PXR can regulate the mRNA expression levels of IL-6 and IL-8 during M. hyopneumoniae infection of PAM cells. In conclusion, these results suggest that PXR positively regulates CYP3A29 expression during the inflammatory response caused by M. hyopneumoniae infection.

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.

Fusion expression of the two soluble viral receptors of porcine reproductive and respiratory syndrome virus with a single adeno-associated virus vector

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen affecting global swine industry. Our recent study has shown that the first four Ig-like domains of sialoadhesin (Sn4D) and the scavenger receptor cysteine-rich domains 5–9 (SRCR59) of CD163 can act as the soluble viral receptors (SVRs) of PRRSV. Co-injection with the two SVR-expressing recombinant adenovirus (rAd) vectors can protect pigs from the lethal challenge with three PRRSV strains. However, the in vivo expression of the two SVRs persists for only two weeks and thus their long-term anti-PRRSV effects remain to be improved. In this study, we fused the two SVRs with a flexible linker or self-cleaving peptide and expressed them with a single recombinant adeno-associated virus (rAAV) vector. The two rAAVs, namely rAAV-Sn4D-SRCR59-Fc and rAAV-SRCR59-Fc/Sn4D-Fc, were generated by using baculovirus-insect cell system. Western blotting analysis showed that the two SVR fusions were efficiently expressed in and secreted from the rAAV-transduced cells. Viral infection blocking assay showed that PRRSV titers in porcine alveolar macrophage (PAM) cells were reduced by 1.6–2.7 log10 after co-cultivation with rAAV-Sn4D-SRCR59-Fc-transduced cells or by 1.9–3.2 log10 after co-cultivation with rAAV-SRCR59-Fc/Sn4D-Fc-transduced cells. After single-dose injection of mice with the rAAV vectors, the expression of two SVR fusions persisted for at least 35 days, which was significantly longer than SRCR59-Fc expression in rAd-SRCR59-Fc-injected mice. Among the two SVR fusions expressed, both expression level and anti-PRRSV activity of SRCR59-Fc/Sn4D-Fc were higher than that of Sn4D-SRCR59-Fc. Therefore, rAAV-SRCR59-Fc/Sn4D-Fc generated can be developed as a novel anti-PRRSV reagent.

Exosomes derived from human-induced pluripotent mesenchymal stem cells inhibit the pyrolysis of alveolar macrophages

To investigate the effect and mechanism of exosomes derived from human-induced pluripotent mesenchymal stem cells (iMSC-Exos) on alveolar macrophages (AM) pyroptosis. The exosomes in the culture supernatant of human-induced pluripotent mesenchymal stem cells (iMSC) were extracted by rotating ultrafiltration, and the extracted exosomes were identified by transmission electron microscopy, Western blotting and high-resolution adjustable resistance pulse. The rat alveolar macrophage cells (NR8383 cells) were cultured in vitro and the logarithmic growth phase cells were divided into three groups: the control group was added with an equal volume of phosphate buffered saline (PBS) in the AM supernatant; in LPS/ATP group AM cells were stimulated with 500 μg/L LPS for 23 hours and then 5 mmol/L ATP was added for 1 hour to induce pyrolysis; iMSC-Exos group was incubated with AM and 100 mg/L iMSC-Exos for 3 hours before giving LPS and ATP. The cytotoxic activity was detected by cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) analysis, the apoptosis and the expression of caspase-1 were observed by immunofluorescence, the levels of inflammatory factors interleukins (IL-1β and IL-18) released by AM were detected by enzyme linked immunosorbent assay (ELISA), the NOD-like receptor protein 3 (NLRP3) inflammasome pathway and the expression level of pyroptosis related protein gasdermin D (GSDMD) were detected by Western blotting. The extracted exosomes were observed by transmission electron microscopy as round vesicles, expressing exosomal markers CD63 and CD9 showed by Western blotting, high-resolution adjustable resistance pulse showed the average diameter of the particles was 130 nm, and could be uptaken by AM. Compared with the control group, the cell activity decreased [(0.56±0.05)% vs. (1.06±0.07)%, P < 0.01], the release of necrotic substance LDH increased (U/L: 1 218.86±22.73 vs. 188.30±1.61, P < 0.01), the expression levels of inflammatory factors increased [IL-1β (ng/L): 958.91±32.78 vs. 194.63±5.14, IL-18 (ng/L): 870.89±21.86 vs. 288.85±24.48, both P < 0.01], and the apoptosis rate [(55.35±6.19)% vs. (12.01±1.32)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 41.06±3.65 vs. 2.80±0.54, P < 0.01) elevated in the AM after LPS/ATP stimulation, suggesting that LPS combined with ATP successfully induced alveolar pyroptosis. Compared with the LPS/ATP group, AM pretreated with iMSC-Exos showed increased cell viability [(0.81±0.05)% vs. (0.56±0.05)%, P < 0.01], decreased LDH secretion (U/L: 535.05±42.55 vs. 1 218.86±22.73, P < 0.01), decreased expression of inflammatory factors [IL-1β (ng/L): 381.82±19.50 vs. 958.91±32.78, IL-18 (ng/L): 533.77±31.54 vs. 870.89±21.86, both P < 0.01], and decreased apoptosis rate [(19.74±2.96)% vs. (55.35±6.19)%, P < 0.01] and caspase-1 expression (fluorescence intensity: 12.16±1.31 vs. 41.06±3.65, P < 0.01). At the same time, the expression of NLRP3 inflammasome pathway [NLRP3 protein (NLRP3/β-actin): 0.62±0.06 vs. 1.89±0.11; cleaved caspase-1 protein (cleaved caspase-1/β-actin): 0.42±0.07 vs. 1.22±0.17, both P < 0.01] and pyrolysis-related protein was significantly inhibited [GSDMD protein (GSDMD/β-actin): 0.57±0.05 vs. 1.22±0.05, P < 0.01]. iMSC-Exos successfully reversed the AM pyroptosis and inflammatory factor expression induced by LPS/ATP, which may be due to the targeted inhibition of NLRP3 inflammasome pathway, suggesting that iMSC-Exos can exert anti-inflammatory effects by inhibiting the pyrolysis of AM.

In vitro primary porcine alveolar macrophage cell toxicity and African swine fever virus inactivation using five commercially supply compound disinfectants under various condition.

Efficacy of African swine fever virus (ASFV) inactivation using five commercially supply compound disinfectants was evaluated under various condition. Virucidal efficacy demonstrated that products A and E could inactivate at 1:800 within 1 min for both temperatures, while products B, C and D inactivated at 1:400. However, product D could inactivate at 1:800 when the exposure time was extended to 30 min and effected only 20°C. In addition, the cytotoxicity demonstrated that products A, B, C, D and E did not significantly affect to cell at 1:51,200, 1:12,800, 1:12,800, 1:25,600 and 1:12,800 dilution, respectively. In conclusion, these disinfectants could inactivate ASFV, however, the application of these products should be performed under safety precautions to prevent cytotoxicity in humans and animals.

Establishment of an in vitro model of alveolar macrophage cell efferocytosis dysfunction

To screen the time points of high survival rate and efferocytosis dysfunction of rat alveolar macrophages stimulated by cigarette smoke extract (CSE), establish an in vitro model of alveolar macrophage efferocytosis function, and study chronic respiratory diseases with chronic inflammatory reaction as the main pathological changes. (1) Time point screening experiment: rat alveolar macrophages (NR8383 cells) were cultured in vitro, and the cells in logarithmic growth phase were divided into blank control group (100 μL complete medium) and 5% CSE group (90 μL complete medium + 10 μL 100% CSE). Alma blue method was used to detect the effect of 5% CSE on the activity of NR8383 cells at 6, 12, 24 and 48 hours. (2) Apoptosis induction experiment: rat type II alveolar epithelial cells (RLE-6TN cells) were cultured in vitro as phagocytic target cells of NR8383 cells, and the cells in logarithmic growth phase were divided into blank control group and 10, 30 and 60 minutes groups after ultraviolet exposure (apoptosis was induced by 30 000 μJ/cm2 ultraviolet irradiation for 15 minutes). Flow cytometry was used to detect the apoptosis rate of RLE-6TN cells cultured for 10, 30 and 60 minutes after ultraviolet exposure. (3) Cell efferocytosis experiment: NR8383 cells in logarithmic phase were divided into blank control group and 5% CSE group. Two hours before NR8383 cells were stimulated by CSE for 6, 12 and 24 hours, RLE-6TN cells were exposed to ultraviolet to induce apoptosis, and the RLE-6TN cell suspension was added to NR8383 cells (the ratio of RLE-6TN cells to NR8383 cells was 5:1). Flow cytometry was used to detect the efferocytosis rate of NR8383 cells to RLE-6TN cells at different time points treated with 5% CSE. (1) Compared with the blank control group, the activity of NR8383 cells significantly decreased after treatment with 5% CSE for 48 hours [cell reduction rate: (68.5±4.1)% vs. (73.6±2.3)%, P < 0.05]. However, there were no significant differences when the activities of NR8383 cells treated with 5% CSE for 6, 12 and 24 hours were compared with the blank control group, so these three time points were selected for the subsequent establishment of alveolar macrophage cell efferocytosis dysfunction in vitro model experiment. (2) Compared with the blank control group, the apoptosis rate of RLE-6TN cells significantly increased at 10, 30 and 60 minutes after ultraviolet exposure [(66.87±8.63)%, (85.51±2.39)%, (96.13±2.74)% vs. (9.13±3.17)%, all P < 0.01] in a time-dependent manner. Considering that it taked about 50 minutes for RLE-6TN cells to be labeled with PKH26 membrane labeling probe, 10 minutes after ultraviolet exposure was selected to label RLE-6TN cells. (3) Compared with the blank control group, the efferocytosis function of NR8383 cells was significantly decreased after treatment with 5% CSE for 12 hours [cell efferocytosis rate: (33.64±1.30)% vs. (44.02±2.71)%, P < 0.01], but there was no significant effect on the efferocytosis function of NR8383 cells at 6 hours and 24 hours. CSE can induce alveolar macrophage cell efferocytosis dysfunction. Based on the test results of the effect of 5% CSE on NR8383 cell activity and cell efferocytosis function, 12 hours with high survival rate and weak efferocytosis effect of NR8383 cells can be selected as the in vitro model condition of alveolar macrophage cell efferocytosis dysfunction.

The potential anti-African swine fever virus effects of medium chain fatty acids on in vitro feed model: An evaluation study using epidemic ASFV straincirculating in Vietnam.

Background:African swine fever (ASF) is an important disease affecting swine and has a significant economic loss in both the developed and developing world.Aim:In this study, we evaluated the potential effects of medium-chain fatty acids (MCFAs) in individual and synergistic forms to prevent and/or reduce ASF virus (ASFV) infection using in vitro feed model.Methods:The cytotoxicity of MCFAs on porcine alveolar macrophages cells was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The potential effects of MCFAs, including C8 (caprylic acid), C8-C6-C10 (caprylic acid-caproic acid-capric acid; 1:1:1 ratio) and C8-C10-C12 (caprylic acid-capric acid-lauric acid; 1:1:1 ratio) against a field ASFV strain isolated in the capital Hanoi of Vietnam, were further examined by real-time PCR and haemadsorption assays in in vitro feed model.Results:Our results indicated that all tested products do not induce cytotoxicity at the dose of 100 μg/ml and are suitable for further in vitro examination. These products have shown a strong antiviral effect against ASFV infectivity at doses of 0.375% and 0.5%. Interestingly, the synergistic MCFAs have shown clearly their potential activities against ASFV in which at a lower dose of 0.25%, pre-treatment with product two and three induced significant increases at the level of Cq value when compared to positive control and/or product 1 (p < 0.05). However, the viral titre was not changed after 24 hours post-inoculation when compared to positive control. Our findings suggested that all tested products, both individual and synergistic forms of MCFAs, have possessed a strong anti-ASFV effect, and this effect is dose-dependence in in vitro feed model. Additionally, synergistic effects of MCFAs are more effective against ASFV when compared to individual forms.Conclusion:Together, the findings in this study indicate that MCFAs, both individual and synergistic forms, inhibit against a field ASFV strain in the feed model, which may support minimizing the risk of ASF transmission in the pig population. Further studies focusing on in vivo anti-ASFV effects of MCFAs are important to bring new insight into the mode of ASFV-reduced action by these compounds in swine feed.

Antiasthmatic effect of atorvastatin via modulation of macrophage activation

Purpose: Asthma is a chronic airway inflammatory disorder and is associated with macrophages. Statin, a well-known lipid-lowering agent, has recently been noted for its anti-inflammatory effect on macrophage. This study was designed to evaluate the antiasth matic effect of atorvastatin via modulation of macrophage activation by using an animal model of allergic asthma. Methods: Atorvastatin 40 mg/kg was given by gavage once a day for 3 days before challenge of ovalbumin (OVA); airway hyperre -sponsiveness (AHR), airway inflammatory cells, and cytokines were evaluated in the murine asthma model. The direct effect of ator vastatin on the activation of macrophages in vitro was determined using the alveolar macrophage cell line CRL-2456. Results: Administration of atorvastatin reduced the numbers of total inflammatory cells, macrophages, and eosinophils as well as lung histology enhanced in the murine asthma model. AHR measured by enhanced pause was significantly reduced after atorvas tatin administration in the murine asthma model (P<0.05). Atorvastatin administration resulted in the reduction in serum OVA-spe cific IgE levels and the increase in serum OVA-specific IgG2a levels (P<0.05). The mRNA levels of Ccr3, Il-17, and Muc5ac enhanced by OVA challenge were decreased by treatment with atorvastatin (P<0.05). Along with these improvement in allergic inflammatory changes, the population of CD11c-CD206+ macrophages as well as the expression of Ym-1 and Relm-α in the lungs were reduced with atorvastatin (P<0.05). In vitro test with CRL-2456 showed that atorvastatin reduced the expression of Cd206, Arg-1, and Fgf-2 in duced by IL-4 stimulation (P<0.05). Conclusion: This study highlighted the antiasthmatic effect of atorvastatin on the suppression of M2 macrophage activation in al lergic asthma. (Allergy Asthma Respir Dis 2021;9:27-35)

Therapeutic effect of atorvastatin on interleukin-13-induced lung pathology

Purpose: Asthma is a common chronic lung disease, in which interleukin (IL)-13 is implicated as a central regulator of IgE synthesis, mucus hypersecretion, airway hyperresponsiveness (AHR), and fibrosis. This study was designed to determine the anti-inflammatory effect of atorvastatin, a widely used lipid-lowering agent, on the IL-13-induced lung pathology through the modulation of macro phages. Methods: Atorvastatin (40 mg/kg) was given to transgenic mice overexpressing IL-13 (IL-13 TG mice) and their wild type littermates by oral gavage for 2 weeks. AHR, numbers of inflammatory cells in the airway, and cytokine levels in IL-13 TG mice were measured. Using the alveolar macrophage cell line CRL-2456, the direct effect of atorvastatin on macrophages activated by recombinant IL-13 was assessed. Results: Significant reduction in total leukocytes and alleviation of AHR were observed with administration of atorvastatin in IL-13 TG mice compared to those without atorvastatin treatment (P<0.05). Atorvastatin administration resulted in upregulation of IL-10 in the lungs of IL-13 TG mice (P<0.05). In addition, mRNA expression of connective tissue growth factor, fibronectin, and type III col lagen as well as chord length enhanced by IL-13 overexpression were reduced by atorvastatin administration (P<0.05). M2 macro phage markers, such as Ym-1 and CD206, were decreased, while M1 macrophage marker, inducible nitric oxide synthase, was increased upon atorvastatin treatment (P<0.05). Administration of atorvastatin resulted in improved removal of apoptotic cells (P<0.05). Conclusion: The results of this study reveal a potential of atorvastatin as an effective antiasthmatic agent by reducing IL-13-induced lung inflammation via the modulation of macrophage polarization. (Allergy Asthma Respir Dis 2021;9:76-83)

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Alcohol induces mitochondrial derangements in alveolar macrophages by upregulating NADPH oxidase 4.

Excessive alcohol users have increased risk of developing respiratory infections in part due to oxidative stress-induced alveolar macrophage (AM) phagocytic dysfunction. Chronic ethanol exposure increases cellular oxidative stress in AMs via upregulation of NADPH oxidase (Nox) 4, and treatment with the peroxisome proliferator-activated receptor gamma (PPARγ) ligand, rosiglitazone, decreases ethanol-induced Nox4. However, the mechanism by which ethanol induces Nox4 expression and the PPARγ ligand reverses this defect has not been elucidated. Since microRNA (miR)-92a has been predicted to target Nox4 for destabilization, we hypothesized that ethanol exposure decreases miR-92a expression and leads to Nox4 upregulation. Previous studies have implicated mitochondrial-derived oxidative stress in AM dysfunction. We further hypothesized that ethanol increases mitochondrial-derived AM oxidative stress and dysfunction via miR-92a, and that treatment with the PPARγ ligand, pioglitazone, could reverse these derangements. To test these hypotheses, a mouse AM cell line, MH-S cells, was exposed to ethanol invitro, and primary AMs were isolated from a mouse model of chronic ethanol consumption to measure Nox4, mitochondrial target mRNA (qRT-PCR) and protein levels (confocal microscopy), mitochondria-derived reactive oxygen species (confocal immunofluorescence), mitochondrial fission (electron microscopy), and mitochondrial bioenergetics (extracellular flux analyzer). Ethanol exposure increased Nox4, enhanced mitochondria-derived oxidative stress, augmented mitochondrial fission, and impaired mitochondrial bioenergetics. Transfection with a miR-92a mimic invitro or pioglitazone treatment invivo diminished Nox4 levels, resulting in improvements in these ethanol-mediated derangements. These findings demonstrate that pioglitazone may provide a novel therapeutic approach to mitigate ethanol-induced AM mitochondrial derangements.

Autophagy alleviates mitochondrial DAMP-induced acute lung injury by inhibiting NLRP3 inflammasome

AimAcute lung injury (ALI) is characterized by alveolar macrophage overactivation and uncontrolled pulmonary inflammation. Mitochondrial damage-associated molecular patterns (MTDs), one type of damage-associated molecular patterns (DAMPs) released from ruptured mitochondrial, can induce inflammation which participates in the pathogenesis of ALI. Despite the critical role of autophagy in inflammatory response, little is known about its function in MTDs-induced ALI. Herein we have studied how autophagy attenuates MTDs-induced ALI in vitro and in vivo. Main methodsExogenous MTDs were injected into mice through tail vein injection or directly treated with cultured alveolar macrophage cell lines to construct MTDs-induced ALI models. Rapamycin and 3-MA were used to regulate autophagy in vivo and in vitro. The expressions of Caspase-1, IL-1β, and their precursor were measured. Inhibition the activation of NLRP3 inflammasome to discover the candidate targets and potential molecular pathways involved in autophagy mitigating the MTDs-induced ALI. Key findingsAfter treatment with MTDs the expression levels of inflammatory cytokines and NLRP3 inflammasome-associated proteins were gradually increased in vitro and in vivo. Most importantly, with autophagy enhanced by rapamycin, all the secretion of inflammation cytokine, the level of lung injury, and the expression level of NLRP3 inflammasome-associated proteins were greatly decreased in MTDs-induced mouse model. MTDs-induced inflammation and lung injury were alleviated by autophagy enhancement. Autophagy can function as an effective way to alleviate inflammation in MTDs-induced ALI by inhibiting NLRP3 inflammasome and may represent a therapeutic target in modulating MTDs-induced inflammatory response.

Establishment of an in vitro model using the rat alveolar macrophage cell line NR8383.

To establish an in vitro model of radiation-induced lung injury using rat lung alveolar macrophages (NR8383). Using a medical electronic linear accelerator, cells were irradiated with either 0 Gy or 6 Gy X-rays. At 6, 12, 24, 30 and 48 h, the DNA damage index (8-OHdG) and lipid damage index (MDA) were measured in the two groups. We also determined the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β). The levels of 8-OHdG and MDA in the 6 Gy irradiation group were higher than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. The levels reached the highest value -6 h after irradiation, and then gradually decreased. The levels of the inflammatory factors TNF-α, TGF-β and IL-6 were higher in the 6 Gy irradiation group than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. Six Gy X-ray irradiated NR8383 cells can be used to establish an in-vitro model of radiation-induced lung injury. The levels of 8-OHdG, MDA, TNF-α, TGF-β and IL-6 can be used as effective evaluation indicators.