Lung Tissue Macrophages Research Articles

Huaxian formula prevents the progression of radiation-induced pulmonary fibrosis by inhibiting the pro-fibrotic effects of macrophages

Ethnopharmacological relevanceThe Huaxian formula (HXF), a traditional Chinese medicine (TCM) remedy, specifically targets the pathological factors of “heat toxicity” and “phlegm stasis” induced by radiation in radiation-induced pulmonary fibrosis (RIPF). It works by clearing heat and invigorating the blood, addressing these key factors in the development of RIPF. Aim of the studyThe HXF has demonstrated potential in preventing RIPF, although its underlying mechanisms remain unclear. This study aims to investigate the efficacy, molecular targets, and mechanisms of action of HXF. Materials and methodsThe major constituents of the HXF were identified by ultra performance liquid chromatography and tandem mass spectrometry (UPLC-MS). C57BL/6j mice were divided into four groups: control (Ctrl), HXF alone (HXF), 17Gy-irradiation alone (IR), and irradiation plus HXF (IR + HXF). Lung damage and fibrosis were assessed by histopathological staining, and the flow cytometry and immunohistochemistry (IHC) were used to detect the macrophages phenotype of lung tissues in vivo at 16 weeks post-irradiation. Transcriptomic sequencing and bioinformatics analyses identified key genes modulated by HXF. In vitro assays included flow cytometry, western bolt, and quantitative PCR (qPCR) explored the impact of HXF on macrophage polarization and fibrotic activity, while co-culture experiments of the macrophage conditional medium and mouse embryo fibroblast NIH/3T3 investigated macrophage-fibroblast interactions. Results20 major constituents of HXF were identified. And the in vivo results revealed significant lung damage and fibrosis in the IR group, which were notably mitigated in the IR + HXF group. And HXF has been shown to significantly inhibit the infiltration of M2-type macrophages in lung tissues. Transcriptomic analysis identified differentially expressed genes (DEGs) such as Arg1, Mmp10, and Fgf23. Bioinformatics enrichment analysis indicated that these DEGs are involved in pathways related to the inhibition of extracellular matrix formation and inflammation. In vitro, HXF-containing serum reduced M2-type macrophage polarization and decreased the secretion of Arginase1 and TGFβ1. Conditioned medium from HXF-treated macrophages suppressed fibroblast activation. ConclusionHXF's preventive effects on RIPF involve multiple targets and mechanisms, including the modulation of Arg1, Mmp10, and Fgf23 expression. By inhibiting the pro-fibrotic capacity of macrophages, HXF suppresses fibroblast activation and collagen production, thereby alleviating lung fibrosis. These findings underscore the potential of HXF as a preventive strategy in managing RIPF.

The effect of adrenalectomy on bleomycin-induced pulmonary fibrosis in mice.

Progressive lung fibrosis is often fatal and has limited treatment options. Though the mechanisms are poorly understood, fibrosis is increasingly linked with catecholamines such as adrenaline (AD) and noradrenaline (NA), and hormones such as aldosterone (ALD). The essential functions of the adrenal glands include the production of catecholamines and numerous hormones, but the contribution of adrenal glands to lung fibrosis remains less well studied. Here, we characterized the impact of surgical adrenal ablation in the bleomycin model of lung fibrosis. Wild type mice underwent surgical adrenalectomy or sham surgery followed by bleomycin administration. We found that while bleomycin-induced collagen over-deposition in the lung was not affected by adrenalectomy, histologic indices of lung remodeling were ameliorated. These findings were accompanied by a decrease of lymphocytes in bronchoalveolar lavage (BAL) and macrophages in lung tissues, along with concomitant reductions in alpha smooth muscle actin (⍺SMA) and fibronectin. Surgical adrenalectomy completely abrogated AD, not NA, detection in all compartments. Systemic ALD levels were reduced after adrenalectomy while ALD levels in lung tissues remained unaffected. Taken together, these results support the presence of a pulmonary-adrenal axis in lung fibrosis and suggest that adrenalectomy is protective in this disease. Further investigation will be needed to better understand this observation and aid in the development of novel therapeutic strategies.

Shengmai Powder regulates alveolar macrophage PPAR-γ and improves the chronic inflammatory state of chronic obstructive pulmonary disease.

This study examines the therapeutic effects of Shengmai Powder (SMP) on both in vitro and in vivo models of chronic obstructive pulmonary disease (COPD) and the underlying mechanisms. Cigarette smoke and cigarette extracts were used to create in vitro and in vivo models of COPD. ELISA was used to measure the levels of pro-inflammatory factors (IL-6, TNF-α, and IL-1β) in mouse lung tissue and alveolar macrophages. Flow cytometry assessed the phagocytic capacity of alveolar macrophage. Western blotting was used to analyze the expression of RhoA, PPARγ, IκBα, p-IκBα, P65, and p-P65 in alveolar. The results show that SMP reversed the increased levels of pro-inflammatory factors (IL-6, TNF-α, and IL-1β) in mouse lung tissue and alveolar macrophages induced by cigarette smoke and cigarette extract. SMP also restored the decreased fluorescence intensity and RhoA levels in alveolar macrophages caused by cigarette extract. Additionally, SMP increased PPARγ expression and decreased IκBα and P65 phosphorylation in alveolar macrophages exposed to cigarette extract. Also, the effects of SMP were reversed by PPARγ inhibitors. The study concluded that SMP regulates alveolar macrophage phagocytic function through the PPAR-γ/NF-κB pathway, thereby improving the chronic inflammatory state of COPD.

Research progress on the relationship between phenotype and signaling pathways of pulmonary macrophages and asthma

Objective The purpose of this study is to systematically investigate the different phenotypes and functional analyses of macrophages in lung tissue. Data sources A search was performed using three databases (Web of Science, Science Direct, and MEDLINE) for all relevant studies published from January 1, 2019, to December 31, 2023. Study selections This systematic review complied with the PRlSMA document’s requirements, including studies related to the signaling pathway relationship between pulmonary macrophages and asthma phenotype. The search includedstudies published in English or French lanquage, and was based on title, abstract, and complete textDocuments not meeting inclusion requirements were excluded Results We have identified studies published within the past five years that meet the criteria for inclusion in this review. We found that asthma is a heterogeneous chronic inflammatory lung disease, and lung tissue macrophages are important immune cells in the respiratory tract. Pulmonary macrophages are also heterogeneous, as they have different subgroups with varying effector functions depending on the environment. They have different phenotypes and biological functions in different disease environments. The phenotypic changes of pulmonary macrophages occur during asthma, and the study of the different phenotypes and functions of macrophages in lung tissue is of great significance for treatment. Conclusions This review summarizes current literature and provides a detailed introduction to the role of macrophages as key inflammatory mediators in the pathogenesis of asthma, as well as existing knowledge gaps. In addition, we propose that regulatory macrophages may prevent the development of asthma by producing IL-10, and regulating the polarization of pulmonary macrophages may be a new direction for asthma treatment.

ATP-induced fibrogenic pathway in circulating cells obtained by idiopathic pulmonary fibrotic (IPF) patients is not blocked by nintedanib and pirfenidone

Idiopathic pulmonary fibrosis (IPF) is a severe disability due to progressive lung dysfunction. IPF has long been viewed as a non-immune form of pulmonary fibrosis, but nowadays it is accepted that a chronic inflammatory response can exacerbate fibrotic patterns. IL-1-like cytokines and ATP are highly detected in the lung and broncho-alveolar lavage fluid of IPF patients. Because ATP binds the purinergic receptor P2RX7 involved in the release of IL-1-like cytokines, we aimed to understand the role of P2RX7 in IPF. PBMCs from IPF patients were treated with nintedanib or pirfenidone in the presence of ATP. Under these conditions, PBMCs still released IL-1-like cytokines and the pro-fibrotic TGFβ. Bulk and scRNAseq demonstrated that lung tissues of IPF patients had higher levels of P2RX7, especially on macrophages, which were correlated to T cell activity and inflammatory response with a TGFBI and IL-10 signature. A subcluster of macrophages in IPF lung tissues had 2055 genes that were not in common with the other subclusters, and that were involved in metabolic and PDGF, FGF and VEGF associated pathways. These data confirmed what observed on circulating cells that, although treated with anti-fibrotic agents, nintedanib or pirfenidone, they were still able to release IL-1 cytokines and the fibrogenic TGFβ. In conclusion, these data imply that because nintedanib and pirfenidone do not block ATP-induced IL-1-like cytokines and TGFβ induced during P2RX7 activation, it is plausible to consider P2RX7 on circulating cells and/or tissue biopsies as potential pharmacological tool for IPF patients.

IPF-related new macrophage subpopulations and diagnostic biomarker identification - combine machine learning with single-cell analysis

sIdiopathic pulmonary fibrosis (IPF) is a chronic disease of unknown etiology that lacks a specific treatment. In IPF, macrophages play a key regulatory role as a major component of the lung immune system, especially during inflammation and fibrosis. However, our understanding of the cellular heterogeneity and molecular characterization of macrophages in IPF, as well as their relevance in the clinical setting, is relatively limited. In this study, we analyzed in-depth single-cell transcriptome sequencing (scRNA-seq) data from lung tissues of IPF patients, identified macrophage subpopulations in IPF, and probed their molecular characteristics and biological functions. hdWGCNA identified co-expressed gene modules of a subpopulation of IPF-associated macrophages (IPF-MΦ), and probed the IPF-MΦ by a machine-learning approach. hdWGCNA identified a subpopulation of IPF-associated macrophage subpopulations and probed the IPF-MΦ signature gene (IRMG) for its prognostic value, and a prediction model was developed on this basis. In addition, IPF-MΦ was obtained after recluster analysis of macrophages in IPF lung tissues. Coexpressed gene modules of IPF-MΦ were identified by hdWGCNA. Then, a machine learning approach was utilized to reveal the characteristic genes of IPF-MΦ, and a prediction model was built on this basis. In addition, we discovered a type of macrophage unique to IPF lung tissue named ATP5-MΦ. Its characteristic gene encodes a subunit of the mitochondrial ATP synthase complex, which is closely related to oxidative phosphorylation and proton transmembrane transport, suggesting that ATP5-MΦ may have higher ATP synthesis capacity in IPF lung tissue. This study provides new insights into the pathogenesis of IPF and provides a basis for evaluating disease prognosis and predictive medicine in IPF patients.Graphical

Hydrogen therapy promotes macrophage polarization to the M2 subtype in radiation lung injury by inhibiting the NF-κB signalling pathway

BackgroundRadiotherapy has become a standard treatment for chest tumors, but a common complication of radiotherapy is radiation lung injury. Currently, there is still a lack of effective treatment for radiation lung injury. MethodsA mouse model of radioactive lung injury (RILI) was constructed and then treated with different cycles of hydrogen inhalation. Lung function tests were performed to detect changes in lung function.HE staining was used to detect pathological changes in lung tissue. Immunofluorescence staining was used to detect the polarization of macrophages in lung tissue. Immunohistochemistry was used to detect changes in cytokine expression in lung tissues. Western Blot was used to detect the expression of proteins related to the NF-κB signalling pathway. ResultsLung function test results showed that lung function decreased in the model group and improved in the treatment group.HE staining showed that inflammatory response was evident in the model group and decreased in the treatment group. Immunohistochemistry results showed that the expression of pro-inflammatory factors was significantly higher in the model group, and the expression of pro-inflammatory factors was significantly higher in the treatment group. The expression of pro-inflammatory factors in the treatment group was significantly lower than that in the model group, and the expression of anti-inflammatory factors in the treatment group was higher than that in the model group. Immunofluorescence showed that the expression of M1 subtype macrophages was up-regulated in the model group and down-regulated in the treatment group. The expression of M2 subtype macrophages was up-regulated in the treatment group relative to the model group. Western Blot showed that P–NF-κB p65/NF-κB p65 was significantly increased in the model group, and P–NF-κB p65/NF-κB p65 was decreased in the treatment group. ConclusionHydrogen therapy promotes macrophage polarization from M1 to M2 subtypes by inhibiting the NF-κB signalling pathway, thereby attenuating the inflammatory response to radiation lung injury.

Effects of environmental exposure to iron powder on healthy and elastase-exposed mice

Prolonged exposure to iron powder and other mineral dusts can threaten the health of individuals, especially those with COPD. The goal of this study was to determine how environmental exposure to metal dust from two different mining centers in Brazil affects lung mechanics, inflammation, remodeling and oxidative stress responses in healthy and elastase-exposed mice. This study divided 72 male C57Bl/6 mice into two groups, the summer group and the winter group. These groups were further divided into six groups: control, nonexposed (SAL); nonexposed, given elastase (ELA); exposed to metal powder at a mining company (SAL-L1 and ELA-L1); and exposed to a location three miles away from the mining company (SAL-L2 and ELA-L2) for four weeks. On the 29th day of the protocol, the researchers assessed lung mechanics, bronchoalveolar lavage fluid (BALF), inflammation, remodeling, oxidative stress, macrophage iron and alveolar wall alterations (mean linear intercept-Lm). The Lm was increased in the ELA, ELA-L1 and ELA-L2 groups compared to the SAL group (p < 0.05). There was an increase in the total number of cells and macrophages in the ELA-L1 and ELA-L2 groups compared to the other groups (p < 0.05). Compared to the ELA and SAL groups, the exposed groups (ELA-L1, ELA-L2, SAL-L1, and SAL-L2) exhibited increased expression of IL-1β, IL-6, IL-10, IL-17, TNF-α, neutrophil elastase, TIMP-1, MMP-9, MMP-12, TGF-β, collagen fibers, MUC5AC, iNOS, Gp91phox, NFkB and iron positive macrophages (p < 0.05). Although we did not find differences in lung mechanics across all groups, there were low to moderate correlations between inflammation remodeling, oxidative stress and NFkB with elastance, resistance of lung tissue and iron positive macrophages (p < 0.05). Environmental exposure to iron, confirmed by evaluation of iron in alveolar macrophages and in air, exacerbated inflammation, initiated remodeling, and induced oxidative stress responses in exposed mice with and without emphysema. Activation of the iNOS, Gp91phox and NFkB pathways play a role in these changes.

TSPO deficiency exacerbates acute lung injury via NLRP3 inflammasome-mediated pyroptosis.

Acute respiratory distress syndrome (ARDS) is a common cause of respiratory failure in many critically ill patients. Although inflammasome activation plays an important role in the induction of acute lung injury (ALI) and ARDS, the regulatory mechanism of this process is still unclear. When cells are stimulated by inflammation, the integrity and physiological function of mitochondria play a crucial part in pyroptosis. However, the underlying mechanisms and function of mitochondrial proteins in the process of pyroptosis are largely not yet known. Here, we identified the 18-kDa translocator protein (TSPO), a mitochondrial outer membrane protein, as an important mediator regulating nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in macrophages during ALI. TSPO gene knockout (KO) and lipopolysaccharide (LPS)-induced ALI/ARDS mouse models were employed to investigate the biological role of TSPO in the pathogenesis of ARDS. Murine macrophages were used to further characterize the effect of TSPO on the NLRP3 inflammasome pathway. Activation of NLRP3 inflammasome was preformed through LPS+adenosine triphosphate (ATP) co-stimulation, followed by detection of mitochondrial membrane potential, reactive oxygen species (ROS) production, and cell death to evaluate the potential biological function of TSPO. Comparisons between two groups were performed with a two-sided unpaired t -test. TSPO- KO mice exhibited more severe pulmonary inflammation in response to LPS-induced ALI. TSPO deficiency resulted in enhanced activation of the NLRP3 inflammasome pathway, promoting more proinflammatory cytokine production of macrophages in LPS-injured lung tissue, including interleukin (IL)-1β, IL-18, and macrophage inflammatory protein (MIP)-2. Mitochondria in TSPO -KO macrophages tended to depolarize in response to cellular stress. The increased production of mitochondrial damage-associated molecular pattern led to enhanced mitochondrial membrane depolarization and pyroptosis in TSPO -KO cells. TSPO may be the key regulator of cellular pyroptosis, and it plays a vital protective role in ARDS occurrence and development.

Pyroptosis and polarization of macrophages in septic acute lung injury induced by lipopolysaccharide in mice.

Pyroptosis and polarization are significant contributors to the onset and development of many diseases. At present, the relationship between pyroptosis and polarization in acute lung injury (ALI) caused by sepsis remains unclear. The ALI model for sepsis was created in mice and categorized into the blank control, lipopolysaccharide(LPS) group, LPS + low-dose Belnacasan group, LPS + high-dose Belnacasan group, LPS + low-dose Wedelolactone group, LPS + high-dose Wedelolactone group, and positive control group. The wet-dry specific gravity was evaluated to compare pulmonary edema. Hematoxylin-eosin, Masson, and terminal deoxynucleotidyl transferase dUTP nick end labelingstaining techniques were conducted to observe and contrast the pathological changes in lung tissue. ELISA was utilized to identify M1 and M2 macrophages and correlated inflammatory factors. Immunohistochemical staining and flow cytometry were employed to identify markers of M1 and M2 macrophages in lung tissue. Propidium iodidestaining, together with flow cytometry, was utilized to observe the degree and positive rate of pyroptosis of alveolar macrophages. Western blot analysis was conducted to detect the expression levels of Caspase 1, Caspase 11, GSDMD, and IL-18 in the lung tissues of each group. The real-time quantitative polymerase chain reactionmethod was used to ascertain relative expression levels of NLRP3, Caspase 1, Caspase 11, GSDMD, IL-18, iNOS, and Arg-1 in lung tissues of all groups. In mice with sepsis-induced ALI, both classical and nonclassical pathways of pyroptosis are observed. Inhibiting pyroptosis has been found to ameliorate lung injury, pulmonary edema, and inflammation induced by LPS. Notably, the expression of NLRP3, Caspase 1, Caspase 11, GSDMD, IL-1β, IL-18, TGF-β, CD86, CD206, iNOS, and Arg-1 were all altered in this process. Additionally, alveolar macrophages were polarized along with pyroptosis in mice with ALI caused by sepsis. Pyroptosis of alveolar macrophages in the context of ALI in mice infected with sepsis has been linked to the polarization of alveolar macrophages towardtype M1.

CD274 (PD-L1) negatively regulates M1 macrophage polarization in ALI/ARDS.

Acute lung injury (ALI)/severe acute respiratory distress syndrome (ARDS) is a serious clinical syndrome characterized by a high mortality rate. The pathophysiological mechanisms underlying ALI/ARDS remain incompletely understood. Considering the crucial role of immune infiltration and macrophage polarization in the pathogenesis of ALI/ARDS, this study aims to identify key genes associated with both ALI/ARDS and M1 macrophage polarization, employing a combination of bioinformatics and experimental approaches. The findings could potentially reveal novel biomarkers for the diagnosis and management of ALI/ARDS. Gene expression profiles relevant to ALI were retrieved from the GEO database to identify co-upregulated differentially expressed genes (DEGs). GO and KEGG analyses facilitated functional annotation and pathway elucidation. PPI networks were constructed to identify hub genes, and differences in immune cell infiltration were subsequently examined. The expression of hub genes in M1 versus M2 macrophages was evaluated using macrophage polarization datasets. The diagnostic utility of CD274 (PD-L1) for ARDS was assessed by receiver operating characteristic (ROC) analysis in a validation dataset. Experimental confirmation was conducted using two LPS-induced M1 macrophage models and an ALI mouse model. The role of CD274 (PD-L1) in M1 macrophage polarization and associated proinflammatory cytokine production was further investigated by siRNA-mediated silencing. A total of 99 co-upregulated DEGs were identified in two ALI-linked datasets. Enrichment analysis revealed that these DEGs were mainly involved in immune-inflammatory pathways. The following top 10 hub genes were identified from the PPI network: IL-6, IL-1β, CXCL10, CD274, CCL2, TLR2, CXCL1, CCL3, IFIT1, and IFIT3. Immune infiltration analysis revealed a significantly increased abundance of M1 and M2 macrophages in lung tissue from the ALI group compared to the control group. Subsequent analysis confirmed that CD274 (PD-L1), a key immunological checkpoint molecule, was highly expressed within M1 macrophages. ROC analysis validated CD274 (PD-L1) as a promising biomarker for the diagnosis of ARDS. Both in vitro and in vivo experiments supported the bioinformatics analysis and confirmed that the JAK-STAT3 pathway promotes CD274 (PD-L1) expression on M1 macrophages. Importantly, knockdown of CD274 (PD-L1) expression potentiated M1 macrophage polarization and enhanced proinflammatory cytokines production. This study demonstrates a significant correlation between CD274 (PD-L1) and M1 macrophages in ALI/ARDS. CD274 (PD-L1) functions as a negative regulator of M1 polarization and the secretion of proinflammatory cytokines in macrophages. These findings suggest potential new targets for the diagnosis and treatment of ALI/ARDS.

Pla2g5 contributes to viral-like-induced lung inflammation through macrophage proliferation and LA/Ffar1 lung cell recruitment.

Macrophages expressing group V phospholipase A2 (Pla2g5) release the free fatty acid (FFA) linoleic acid (LA), potentiating lung type 2 inflammation. Although Pla2g5 and LA increase in viral infections, their role remains obscure. We generated Pla2g5flox/flox mice, deleted Pla2g5 by using the Cx3cr1cre transgene, and activated bone marrow-derived macrophages (BM-Macs) with poly:IC, a synthetic double-stranded RNA that triggers a viral-like immune response, known Pla2g5-dependent stimuli (IL-4, LPS + IFNγ, IL-33 + IL-4 + GM-CSF) and poly:IC + LA followed by lipidomic and transcriptomic analysis. Poly:IC-activated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs had downregulation of major bioactive lipids and critical enzymes producing those bioactive lipids. In addition, AKT phosphorylation was lower in poly:IC-stimulated Pla2g5flox/flox;Cx3cr1cre/+ BM-Macs, which was not restored by adding LA to poly:IC-stimulated BM-Macs. Consistently, Pla2g5flox/flox;Cx3cr1cre/+ mice had diminished poly:IC-induced lung inflammation, including inflammatory macrophage proliferation, while challenging Pla2g5flox/flox;Cx3cr1cre/+ mice with poly:IC + LA partially restored lung inflammation and inflammatory macrophage proliferation. Finally, mice lacking FFA receptor-1 (Ffar1)-null mice had reduced poly:IC-induced lung cell recruitment and tissue macrophage proliferation, not corrected by LA. Thus, Pla2g5 contributes to poly:IC-induced lung inflammation by regulating inflammatory macrophage proliferation and LA/Ffar1-mediated lung cell recruitment and tissue macrophage proliferation.

Blockage of mechanosensitive Piezo1 channel alleviates the severity of experimental malaria-associated acute lung injury

BackgroundMalaria-associated acute lung injury (MA-ALI) is a well-recognized clinical complication of severe, complicated malaria that is partly driven by sequestrations of infected red blood cells (iRBCs) on lung postcapillary induced impaired blood flow. In earlier studies the mechanosensitive Piezo1 channel emerged as a regulator of mechanical stimuli, but the function and underlying mechanism of Piezo1 impacting MA-ALI severity via sensing the impaired pulmonary blood flow are still not fully elucidated. Thus, the present study aimed to explore the role of Piezo1 in the severity of murine MA-ALI.MethodsHere, we utilized a widely accepted murine model of MA-ALI using C57BL/6 mice with Plasmodium berghei ANKA infection and then added a Piezo1 inhibitor (GsMTx4) to the model. The iRBC-stimulated Raw264.7 macrophages in vitro were also targeted with GsMTx4 to further explore the potential mechanism.ResultsOur data showed an elevation in the expression of Piezo1 and number of Piezo1+-CD68+ macrophages in lung tissues of the experimental MA-ALI mice. Compared to the infected control mice, the blockage of Piezo1 with GsMTx4 dramatically improved the survival rate but decreased body weight loss, peripheral blood parasitemia/lung parasite burden, experimental cerebral malaria incidence, total protein concentrations in bronchoalveolar lavage fluid, lung wet/dry weight ratio, vascular leakage, pathological damage, apoptosis and number of CD68+ and CD86+ macrophages in lung tissues. This was accompanied by a dramatic increase in the number of CD206+ macrophages (M2-like subtype), upregulation of anti-inflammatory cytokines (e.g. IL-4 and IL-10) and downregulation of pro-inflammatory cytokines (e.g. TNF-α and IL-1β). In addition, GsMTx4 treatment remarkably decreased pulmonary intracellular iron accumulation, protein level of 4-HNE (an activator of ferroptosis) and the number of CD68+-Piezo1+ and CD68+-4-HNE+ macrophages but significantly increased protein levels of GPX4 (an inhibitor of ferroptosis) in experimental MA-ALI mice. Similarly, in vitro study showed that the administration of GsMTx4 led to a remarkable elevation in the mRNA levels of CD206, IL-4, IL-10 and GPX-4 but to a substantial decline in CD86, TNF-α, IL-1β and 4-HNE in the iRBC-stimulated Raw264.7 cells.ConclusionsOur findings indicated that blockage of Piezo1 with GsMTx4 alleviated the severity of experimental MA-ALI in mice partly by triggering pulmonary macrophage M2 polarization and subsequent anti-inflammatory responses but inhibited apoptosis and ferroptosis in lung tissue. Our data suggested that targeting Piezo1 in macrophages could be a promising therapeutic strategy for treating MA-ALI.Graphical

MiR-146a alleviates acute lung injury via inhibiting Notch 1 signaling pathway targeting macrophage.

Acute lung injury (ALI) is associated with the leukocyte infiltration and inflammation. Previous studies have shown that miR-146a is a valid regulator of the macrophage polarization in vitro inflammatory model. However, it is unclear whether miR-146a plays a protective role in ALI via modulating macrophage inflammation. To explore the potential therapeutic effect mechanism of miR-146a on ALI. We analyzed the expression of miR-146a in acute injured lung tissues and differentiated macrophage. Lipopolysaccharide (LPS) and interleukin-4 (IL-4) were employed in provoking the macrophage to polarization. We used miR-146a mimics to improve the overexpression of miR-146a and investigated the effect of increased miR-146a on LPS-induced ALI mice via the target of macrophage polarization. We showed that the expression of miR-146a markedly decreased in injured lung tissue and type M1 macrophage, while increased miR-146a expression exhibited in type M2 macrophage. Moreover, overexpression of miR-146a in LPS-induced macrophage reversed inflammatory M1 phenotype to anti-inflammatory M2 phenotype and mitigated inflammatory level via inhibiting Notch 1 signaling pathway. Hence, inflammation, infiltration, integrity of capillary barrier, and histology in ALI model were corrected after miR-146a overexpression treatment. These results suggested that miR-146a promotes type M2 macrophage polarization via restraining Notch 1 signaling pathway. Overexpression of miR-146a prevents inflammation damage and ameliorates lung damage after LPS induction. Therefore, miR-146a may serve as a promising target for the therapy of ALI in the future.

Exogenous IL-25 ameliorates airway neutrophilia via suppressing macrophage M1 polarization and the expression of IL-12 and IL-23 in asthma

BackgroundSevere asthma is associated with substantial mortality and has unmet therapeutic need. A subset of severe asthma is characterized by neutrophilic airway inflammation. Classically activated (or M1) macrophages which express IL-12 and IL-23 are associated with airway neutrophilia in asthma. Exogenous IL-25 was reported to suppress intestinal inflammation in animal models of inflammatory bowel diseases via suppressing IL-12 and IL-23 production. We hypothesize that IL-25 ameliorates airway neutrophilia via inhibiting macrophage M1 polarization and the expression of IL-12 and IL-23 in asthma.MethodsIn a mouse model of neutrophil-dominant allergic airway inflammation, the effect of mouse recombinant IL-25 on airway inflammation were assessed by H&E staining and bronchoalveolar lavage (BAL) cell counting. The percentage of M1 macrophages in lung tissue and BAL cells were analyzed by flow cytometry. Quantitative PCR and immunostaining were performed to measure the expression of Il12, Il23, and inflammatory cytokines. Mechanistic experiments were performed in primary culture of macrophages from mouse lungs. The expression of IL-12, IL-23 and IL-25 in sputum was analyzed in a cohort of severe asthma and subjects with eosinophilic or non-eosinophilic asthma.ResultsIntranasal administration of IL-25 markedly decreased the number of neutrophils in BAL cells in a murine model of neutrophil-dominant allergic airway inflammation. Moreover, exogenous IL-25 decreased the number of M1 macrophages, and reduced the expression of IL-12, IL-23 in the lungs of the mouse model. Exogenous IL-25 also inhibited the expression of inflammatory cytokines IL-1β, IFN-γ, TNF-α and IL-17 A. In vitro, IL-25 suppressed IL-12 and IL-23 expression in lipopolysaccharide (LPS)-stimulated primary culture of mouse pulmonary macrophages. Mechanistically, IL-25 inhibited LPS-induced c-Rel translocation to nucleus via STAT3-dependent signaling. In a cohort of severe asthma, IL-25 protein levels in sputum were significantly lower than control subjects. The transcript levels of IL-12 and IL-23 were increased whereas IL-25 transcripts were decreased in sputum cells from subjects with non-eosinophilic asthma compared to eosinophilic asthma.ConclusionsIL-25 expression is downregulated in subjects with severe or non-eosinophilic asthma. Exogenous IL-25 ameliorates airway neutrophilia, at least in part, via inhibiting macrophage M1 polarization and the expression of IL-12 and IL-23.

Bletilla striata polysaccharides protect against ARDS by modulating the NLRP3/caspase1/GSDMD and HMGB1/TLR4 signaling pathways to improve pulmonary alveolar macrophage pyroptosis

Ethnopharmacological relevanceBletilla striata polysaccharides (BSP) extracted from the B. striata tuber, have been demonstrated to possess anti-inflammatory properties. However, their potential protective effect against ARDS and their role in regulating cell pyroptosis remained unexplored. Aim of the studyThe aim of this study was to investigate the therapeutic effect of BSP in the alleviation of lipopolysaccharide (LPS)-induced ARDS, and to explore its mechanism of action. MethodsThe effect of BSP was assessed by LPS injection into the intraperitoneal cavity in vivo; pathological changes of ARDS mice were gauged by immunohistochemical, hematoxylin and eosin staining, and immunofluorescence assays. MH-S cells were used to model the pyroptosis in vitro. Finally, the pyroptosis of alveolar macrophage was detected by western blots, qPCR, and flow cytometry for NLRP3/caspase1/GSDMD and HMGB1/TLR4 pathway-associated proteins and mRNA. ResultsBSP could significantly increase the weight and survival rate of mice with ARDS, alleviate the cytokine storm in the lungs, and reduce lung damage in vivo. BSP inhibited the inflammation caused by LPS/Nigericin significantly in vitro. Compared with the control group, there was a remarkable surge in the incidence of pyroptosis observed in ARDS lung tissue and alveolar macrophages, whereas BSP significantly diminished the pyroptosis ratio. Besides, BSP reduced NLRP3/caspase1/GSDMD and HMGB1/TLR4 levels in ARDS lung tissue and MH-S cells. ConclusionsThese findings proved that BSP could improve LPS-induced ARDS via inhibiting pyroptosis, and this effect was mediated by NLRP3/caspase1/GSDMD and HMGB1/TLR4, suggesting a therapeutic potential of BSP as an anti-inflammatory agent for ARDS treatment.

Anti-Ro52 positivity is associated with progressive interstitial lung disease in systemic sclerosis—an exploratory study

BackgroundInterstitial lung disease (ILD) is the most common cause of death in patients with systemic sclerosis (SSc). Prognostic biomarkers are needed to identify SSc-ILD patients at risk for progressive pulmonary fibrosis. This study investigates autoantibodies measured in bronchoalveolar lavage (BAL) fluid and in serum in reference to the clinical disease course of SSc-ILD.MethodsFifteen patients with new onset SSc-ILD underwent bronchoscopy. Autoantibody levels were analyzed using addressable laser bead immunoassay from BAL fluid and the serum. In a separate longitudinal cohort of 43 patients with early SSc-ILD, autoantibodies in serum were measured at baseline and pulmonary function tests were performed at least 2 times over the course of at least 2 or more years. Linear mixed effect models were created to investigate the relationship between specific autoantibodies and progression of SSc-ILD. Finally, lung tissue from healthy controls and from subjects with SSc was analyzed for the presence of the Ro52 antigen using immunohistochemistry.ResultsAmong SSc-ILD patients who were positive for anti-Ro52 (N = 5), 3 (60%) had enrichment of anti-Ro52 in BAL fluid at a ratio exceeding 50x. In the longitudinal cohort, 10/43 patients (23%) were anti-Ro52 positive and 16/43 (37%) were anti-scl-70 positive. Presence of anti-Scl-70 was associated with a lower vital capacity (VC) at baseline (-12.6% predicted VC [%pVC]; 95%CI: -25.0, -0.29; p = 0.045), but was not significantly associated with loss of lung function over time (-1.07%pVC/year; 95%CI: -2.86, 0.71; p = 0.230). The presence of anti-Ro52 was significantly associated with the loss of lung function over time (-2.41%pVC/year; 95% CI: -4.28, -0.54; p = 0.013). Rate of loss of lung function increased linearly with increasing anti-Ro52 antibody levels (-0.03%pVC per arbitrary units/mL and year; 95%CI: -0.05, -0.02; p < 0.001). Immunohistochemical staining localized the Ro52 antigen to alveolar M2 macrophages in peripheral lung tissue both in subjects with and without SSc.ConclusionsThis study suggests that antibodies targeting Ro52 are enriched in the lungs of patients with new-onset SSc-ILD, linking Ro52 autoimmunity to the pulmonary pathology of SSc. Clinical and immunohistochemical data corroborates these findings and suggest that anti-Ro52 may serve as a potential biomarker of progressive SSc-ILD.

Protective effect of β-glucan on Poly(I:C)-induced acute lung injury/inflammation: Therapeutic implications of viral infections in the respiratory system

AimsAcute lung inflammation, particularly acute respiratory distress syndrome (ARDS), is caused by a variety of pathogens including bacteria and viruses. β-Glucans have been reported to possess both anti-inflammatory and immunomodulatory properties. The current study evaluated the therapeutic effect of β-glucans on polyinosinic:polycytidylic acid (Poly(I:C)) induced lung inflammation in both hamster and mice models. Main methodsPoly(I:C)-induced ALI/inflammation models were developed in hamsters (2.5 mg/kg) and mice (2 mg/kg) by delivering the Poly(I:C) intratracheally, and followed with and without β-glucan administration. After treatment, lung mechanics were assessed and lung tissues were isolated and analyzed for mRNA/protein expression, and histopathological examinations. Key findingsPoly(I:C) administration, caused a significant elevation of inflammatory marker's expression in lung tissues and showed abnormal lung mechanics in mice and hamsters. Interestingly, treatment with β-glucan significantly (p < 0.001) reversed the Poly(I:C)-induced inflammatory events and inflammatory markers expression in both mRNA (IL-6, IL-1β, TNF-α, CCL2 and CCL7) and protein levels (TNF-α, CD68, myeloperoxidase, neutrophil elastase, MUC-5Ac and iNOS). Lung functional assays revealed that β-glucan treatment significantly improved lung mechanics. Histopathological analysis showed that β-glucan treatment significantly attenuated the Poly(I:C) induced inflammatory cell infiltration, injury and goblet cell population in lung tissues. Consistent with these findings, β-glucan treatment markedly reduced the number of neutrophils and macrophages in lung tissues. Our findings further demonstrated that β-glucan could reduce inflammation by suppressing the MAPK pathway. SignificanceThese results suggested that β-glucan may attenuate the pathogenic effects of Poly(I:C)-induced ALI/ARDS via modulating the MAPK pathway, indicating β-glucan as a possible therapeutic agent for the treatment of viral-pulmonary inflammation/injury.

Early ART reduces viral seeding and innate immunity in liver and lungs of SIV-infected macaques.

Identifying immune cells and anatomical tissues that contribute to the establishment of viral reservoirs is of central importance in HIV-1 cure research. Herein, we used rhesus macaques (RMs) infected with SIVmac251 to analyze viral seeding in the liver and lungs of either untreated or early antiretroviral therapy-treated (ART-treated) RMs. Consistent with viral replication and sensing, transcriptomic analyses showed higher levels of inflammation, pyroptosis, and chemokine genes as well as of interferon-stimulating gene (ISG) transcripts, in the absence of ART. Our results highlighted the infiltration of monocyte-derived macrophages (HLA-DR+CD11b+CD14+CD16+) in inflamed liver and lung tissues associated with the expression of CD183 and CX3CR1 but also with markers of tissue-resident macrophages (CD206+ and LYVE+). Sorting of myeloid cell subsets demonstrated that CD14+CD206-, CD14+CD206+, and CD14-CD206+ cell populations were infected, in the liver and lungs, in SIVmac251-infected RMs. Of importance, early ART drastically reduced viral seeding consistent with the absence of ISG detection but also of genes related to inflammation and tissue damage. Viral DNA was only detected in CD206+HLA-DR+CD11b+ cells in ART-treated RMs. The observation of pulmonary and hepatic viral rebound after ART interruption reinforces the importance of early ART implementation to limit viral seeding and inflammatory reactions.

Integrated multi-omics analyses reveal the altered transcriptomic characteristics of pulmonary macrophages in immunocompromised hosts with Pneumocystis pneumonia.

With the extensive use of immunosuppressants, immunosuppression-associated pneumonitis including Pneumocystis jirovecii pneumonia (PCP) has received increasing attention. Though aberrant adaptive immunity has been considered as a key reason for opportunistic infections, the characteristics of innate immunity in these immunocompromised hosts remain unclear. In this study, wild type C57BL/6 mice or dexamethasone-treated mice were injected with or without Pneumocystis. Bronchoalveolar lavage fluids (BALFs) were harvested for the multiplex cytokine and metabolomics analysis. The single-cell RNA sequencing (scRNA-seq) of indicated lung tissues or BALFs was performed to decipher the macrophages heterogeneity. Mice lung tissues were further analyzed via quantitative polymerase chain reaction (qPCR) or immunohistochemical staining. We found that the secretion of both pro-inflammatory cytokines and metabolites in the Pneumocystis-infected mice are impaired by glucocorticoids. By scRNA-seq, we identified seven subpopulations of macrophages in mice lung tissues. Among them, a group of Mmp12+ macrophages is enriched in the immunocompetent mice with Pneumocystis infection. Pseudotime trajectory showed that these Mmp12+ macrophages are differentiated from Ly6c+ classical monocytes, and highly express pro-inflammatory cytokines elevated in BALFs of Pneumocystis-infected mice. In vitro, we confirmed that dexamethasone impairs the expression of Lif, Il1b, Il6 and Tnf, as well as the fungal killing capacity of alveolar macrophage (AM)-like cells. Moreover, in patients with PCP, we found a group of macrophages resembled the aforementioned Mmp12+ macrophages, and these macrophages are inhibited in the patient receiving glucocorticoid treatment. Additionally, dexamethasone simultaneously impaired the functional integrity of resident AMs and downregulated the level of lysophosphatidylcholine, leading to the suppressed antifungal capacities. We reported a group of Mmp12+ macrophages conferring protection during Pneumocystis infection, which can be dampened by glucocorticoids. This study provides multiple resources for understanding the heterogeneity and metabolic changes of innate immunity in immunocompromised hosts, and also suggests that the loss of Mmp12+ macrophages population contributes to the pathogenesis of immunosuppression-associated pneumonitis.