Alveolar Epithelial Cells Research Articles

Saharan dust induces the lung disease-related cytokines granulocyte–macrophage colony-stimulating factor and granulocyte colony-stimulating factor

Desert dust exposure is associated with adverse respiratory health effects. Desert dust is a complex pollutant mixtures that includes respirable crystalline and amorphous particles, metals, and microbial constituents. Given the health effects of desert dust and its heterogeneity, as yet unidentified harmful biological pathways may be triggered. Therefore, we exposed human in vitro air–liquid interface co-cultures of alveolar epithelial A549 cells and THP-1 macrophages to Saharan dust (SD). For comparison, we used the known pulmonary toxicant DQ12 quartz dust. Via RNA sequencing, we identified that SD but not DQ12 increased the gene expression of granulocyte–macrophage colony-stimulating factor (GMCSF) and granulocyte colony-stimulating factor (GCSF). These findings were confirmed by quantitative reverse transcriptase PCR. SD dose-dependently upregulated GMCSF and GCSF expression with significant 7 and 9-fold changes, respectively, at the highest tested concentration of 31 µg/cm2. Furthermore, we observed that SD significantly enhanced the secretion of GM-CSF and G-CSF by 2-fold. Both cytokines have previously been associated with lung diseases such as asthma and fibrosis. Hence, we present two molecular messengers that may contribute to the adverse health effects of desert dust and might serve as drug targets for this globally relevant non-anthropogenic air pollutant.

Down-regulating lncRNA KCNQ1OT1 relieves type II alveolar epithelial cell apoptosis during one-lung ventilation via modulating miR-129-5p/HMGB1 axis induced pulmonary endothelial glycocalyx.

Endothelial glycocalyx (EG) maintains vascular homeostasis and is destroyed after one-lung ventilation (OLV)-induced lung injury. Long noncoding RNAs (lncRNAs) are critically involved in various lung injuries. This study aimed to investigate the role and regulatory mechanism of KCNQ1 overlapping transcript 1 (KCNQ1OT1) in OLV-induced lung injury and LPS-induced type II alveolar epithelial cell (AECII) apoptosis. The rat OLV model was established, and the effects of KCNQ1OT1 on OLV-induced ALI in vivo were explored. Bax and Caspase-3 expression in rat lung tissues was measured by immunochemistry (IHC). AECIIs were isolated from rat lungs and treated with LPS or normal saline (control) for in vitro analysis. The expression of KCNQ1OT1, miR-129-5p, and HMGB1 was measured by quantitative real-time PCR (qRT-PCR) or Western blot (WB). Cell proliferation and apoptosis were examined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and flow cytometry. The downstream targets of KCNQ1OT1 were predicted by bioinformatics, and the binding relationship between KCNQ1OT1 and miR-129-3p was verified by dual-luciferase reporter assays. The potential target of miR-129-5p was further explored on the Targetscan website and revealed to target HMGB1. Enzyme-linked immunosorbent assay (ELISA) or WB was adopted to determine the levels of IL-1β, TNF-α, MDA, SOD, heparanase (HPA), matrix metalloproteinase 9 (MMP9), heparan sulfate (HS) and syndecan-1 (SDC-1). KCNQ1OT1 and HMGB1 were up-regulated during OLV-induced lung injury, and their expression was positively correlated. KCNQ1OT1 knockdown reduced OLV-induced pulmonary edema and lung epithelial cell apoptosis, increased vascular permeability, reduced IL-1β, TNF-α, MDA, and SOD levels and glycocalyx markers by targeting miR-129-5p or upregulating HMGB1. Overexpressing KCNQ1OT1 promoted cell apoptosis, reduced cell proliferation, aggravated inflammation and oxidative stress, and up-regulated HMGB1, HPA and MMP9 in LPS-treated AECIIs, while the HMGB1 silencing showed the opposite effects. MiR-129-5p mimics partially eliminated the KCNQ1OT1-induced effects, while recombinant HMGB1 restored the effects of miR-129-5p overexpression on AECIIs. Additionally, KCNQ1OT1 was demonstrated to promote the activation of the p38 MAPK/Akt/ERK signaling pathways in AECIIs via HMGB1. KCNQ1OT1 knockdown alleviated AECII apoptosis and EG damage during OLV by targeting miR-129-5p/HMGB1 to inactivate the p38 MAPK/Akt/ERK signaling. The findings of our study might deepen our understanding of the molecular basis in OLV-induced lung injury and provide clues for the targeted disease management.

IRF4-mediated Treg phenotype switching can aggravate hyperoxia-induced alveolar epithelial cell injury

Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.

The expression of the surfactant proteins SP-A and SP-B during postnatal alveolarization of the rat lung.

Surfactant-specific proteins (SP) are responsible for the functional and structural integrity as well as for the stabilization of the intra-alveolar surfactant. Morphological lung maturation starts in rat lungs after birth. The aim of this study was to investigate whether the expression of the hydrophilic SP-A and the hydrophobic SP-B is associated with characteristic postnatal changes characterizing morphological lung maturation. Stereological methods were performed on the light microscope. Using immunohistochemical and molecular biological methods (Western Blot, RT-qPCR), the SP-A and SP-B of adult rat lungs and of those with different postnatal developmental stages (3, 7, 14 and 21 days after birth) were characterized. As signs of alveolarization the total septal surface and volume increased and the septal thickness decreased. The significantly highest relative surface fraction of SP-A labeled alveolar epithelial cells type II (AEII) was found together with the highest relative SP-A gene expression before the alveolarization (3th postnatal day). With the downregulation of SP-A gene expression during and after alveolarization (between postnatal days 7 and 14), the surface fraction of the SP-A labeled AEII also decreased, so they are lowest in adult animals. The surface fraction of SP-B labeled AEII and the SP-B gene expression showed the significantly highest levels in adults, the protein expression increased also significantly at the end of morphological lung maturation. There were no alterations in the SP-B expression before and during alveolarization until postnatal day 14. The protein expression as well as the gene expression of SP-A and SP-B correlated very well with the total surface of alveolar septa independent of the postnatal age. The expression of SP-A and SP-B is differentially associated with morphological lung maturation and correlates with increased septation of alveoli as indirect clue for alveolarization.

The Role of Cytokines and Molecular Pathways in Lung Fibrosis Following SARS-CoV-2 Infection: A Physiopathologic (Re)view.

SARS-CoV-2 infection is a significant health concern that needs to be addressed not only during the initial phase of infection but also after hospitalization. This is the consequence of the various pathologies associated with long COVID-19, which are still being studied and researched. Lung fibrosis is an important complication after COVID-19, found in up to 71% of patients after discharge. Our research is based on scientific articles indexed in PubMed; in the selection process, we used the following keywords: "lung fibrosis", "fibrosis mediators", "fibrosis predictors", "COVID-19", "SARS-CoV-2 infection", and "long COVID-19". In this narrative review, we aimed to discuss the current understanding of the mechanisms of initiation and progression of post-COVID-19 lung fibrosis (PC-19-LF) and the risk factors for its occurrence. The pathogenesis of pulmonary fibrosis involves various mediators such as TGF-β, legumain, osteopontin, IL-4, IL-6, IL-13, IL-17, TNF-α, Gal-1, Gal-3, PDGF, and FGFR-1. The key cellular effectors involved in COVID-19 lung fibrosis are macrophages, epithelial alveolar cells, neutrophils, and fibroblasts. The main fibrosis pathways in SARS-CoV-2 infection include hypoxemia-induced fibrosis, macrophage-induced fibrosis, and viral-fibroblast interaction-induced fibrosis.

Characteristics of Polycyclic Aromatic Hydrocarbons in Size-Resolved Particles in the Roadside Environment of Beijing: Seasonality, Source, and Toxicological Effects

The polycyclic aromatic hydrocarbons (PAHs) in size-resolved particles emitted from diverse sources are required for quantification to reduce the emissions in order to protect public health. Twenty-four PAHs in size-segregated particles in the roadside environment of Beijing were observed from 1 October 2021 to 30 September 2022. The size distributions of PAHs were bimodal, with peak concentrations ranging from size fractions of 0.43 to 0.65 μm and 4.7 to 5.8 μm in all four seasons, respectively. The highest concentration of PAHs in fine particles (PM2.1) was 35.3 ng m−3 in winter, followed by 16.0 ng m−3 in autumn, 15.3 ng m−3 in spring, and 6.5 ng m−3 in summer. Conversely, the concentration of PAHs in coarse particles (PM2.1–9) ranged from 6.8 ng m−3 (summer) to 20.5 ng m−3 (winter) from low to high. The size fractions of 0.43–2.1 μm PAHs increased most from clear to polluted days, which could be ascribed to the heterogeneous reactions. Source apportionment using positive matrix factorization showed that four sources, namely biomass combustion, coal combustion, diesel vehicles, and gasoline vehicles accounted for PAHs with the estimation of 17.4%, 22.1%, 26.4%, and 23.2% to PAHs in PM2.1; and 19.6%, 24.3%, 23.6%, and 20.1% in PM2.1–9, respectively. Furthermore, we used the human alveolar epithelial cell (BEAS-2B) to assess the toxicological effects of size-resolved atmospheric PAHs. The results showed that the cell survival rate caused by fine particles was lower than that of coarse particles with the same concentrations of PAHs, which is mainly related to the higher content of highly toxic PAHs in fine particles.

Epithelial MAPK signaling directs endothelial NRF2 signaling and IL-8 secretion in a tri-culture model of the alveolar-microvascular interface following diesel exhaust particulate (DEP) exposure

BackgroundParticulate matter 2.5 (PM2.5) deposition in the lung’s alveolar capillary region (ACR) is significantly associated with respiratory disease development, yet the molecular mechanisms are not completely understood. Adverse responses that promote respiratory disease development involve orchestrated, intercellular signaling between multiple cell types within the ACR. We investigated the molecular mechanisms elicited in response to PM2.5 deposition in the ACR, in an in vitro model that enables intercellular communication between multiple resident cell types of the ACR.MethodsAn in vitro, tri-culture model of the ACR, incorporating alveolar-like epithelial cells (NCI-H441), pulmonary fibroblasts (IMR90), and pulmonary microvascular endothelial cells (HULEC) was developed to investigate cell type-specific molecular responses to a PM2.5 exposure in an in-vivo-like model. This tri-culture in vitro model was termed the alveolar capillary region exposure (ACRE) model. Alveolar epithelial cells in the ACRE model were exposed to a suspension of diesel exhaust particulates (DEP) (20 µg/cm2) with an average diameter of 2.5 µm. Alveolar epithelial barrier formation, and transcriptional and protein expression alterations in the directly exposed alveolar epithelial and the underlying endothelial cells were investigated over a 24 h DEP exposure.ResultsAlveolar epithelial barrier formation was not perturbed by the 24 h DEP exposure. Despite no alteration in barrier formation, we demonstrate that alveolar epithelial DEP exposure induces transcriptional and protein changes in both the alveolar epithelial cells and the underlying microvascular endothelial cells. Specifically, we show that the underlying microvascular endothelial cells develop redox dysfunction and increase proinflammatory cytokine secretion. Furthermore, we demonstrate that alveolar epithelial MAPK signaling modulates the activation of NRF2 and IL-8 secretion in the underlying microvascular endothelial cells.ConclusionsEndothelial redox dysfunction and increased proinflammatory cytokine secretion are two common events in respiratory disease development. These findings highlight new, cell-type specific roles of the alveolar epithelium and microvascular endothelium in the ACR in respiratory disease development following PM2.5 exposure. Ultimately, these data expand our current understanding of respiratory disease development following particle exposures and illustrate the utility of multicellular in vitro systems for investigating respiratory tract health.

Identification of HK3 as a promising immunomodulatory and prognostic target in sepsis-induced acute lung injury

BackgroundSepsis is a life-threatening global disease with a significant impact on human health. Acute lung injury (ALI) has been identified as one of the primary causes of mortality in septic patients. This study aimed to identify candidate genes involved in sepsis-induced ALI through a comprehensive approach combining bioinformatics analysis and experimental validation. MethodsThe datasets GSE65682 and GSE32707 obtained from the Gene Expression Omnibus database were merged to screen for sepsis-induced ALI related differentially expressed genes (DEGs). Functional enrichment and immune infiltration analyses were conducted on DGEs, with the construction of protein-protein interaction (PPI) networks to identify hub genes. In vitro and in vivo models of sepsis-induced ALI were used to study the expression and function of hexokinase 3 (HK3) using various techniques including Western blot, real-time PCR, immunohistochemistry, immunofluorescence, Cell Counting Kit-8, Enzyme-linked immunosorbent assay, and flow cytometry. ResultsThe results of bioinformatics analysis have identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic significance for sepsis-induced ALI. The HK3 has profound effects on sepsis-induced ALI and exhibits a correlation with immune regulation. Experimental results showed increased HK3 expression in lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro studies demonstrated upregulation of HK3 in lipopolysaccharide (LPS)-stimulated lung epithelial cells, with cytoplasmic localization around the nucleus. Interestingly, following the knockdown of HK3 expression, lung epithelial cells exhibited a significant decrease in proliferation activity and glycolytic flux, accompanied by an increase in cellular inflammatory response, oxidative stress, and cell apoptosis. ConclusionsIt was observed for the first time that HK3 plays a crucial role in the progression of sepsis-induced ALI and may be a valuable target for immunomodulation and therapy.Bioinformatics analysis identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic relevance in sepsis-induced ALI. Experimental findings showed increased HK3 expression in the lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro experiments demonstrated increased HK3 levels in lung epithelial cells stimulated with LPS, with cytoplasmic localization near the nucleus. Knockdown of HK3 expression resulted in decreased proliferation activity and glycolytic flux, increased inflammatory response, oxidative stress, and cell apoptosis in lung epithelial cells.

Circular RNA MKLN1 promotes epithelial-mesenchymal transition in pulmonary fibrosis by regulating the miR-26a/b-5p/CDK8 axis in human alveolar epithelial cells and mice models.

Pulmonary fibrosis involves destruction of the lung parenchyma and extracellular matrix deposition. Effective treatments for pulmonary fibrosis are lacking and its pathogenesis is still unclear. Studies have found that epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) plays an important role in progression of pulmonary fibrosis. Thus, an in-depth exploration of its mechanism might identify new therapeutic targets. In this study, we revealed that a novel circular RNA, MKLN1 (circMKLN1), was significantly elevated in two pulmonary fibrosis models (intraperitoneally with PQ, 50mg/kg for 7days, and intratracheally with BLM, 5mg/kg for 28days). Additionally, circMKLN1 was positively correlated with the severity of pulmonary fibrosis. Inhibition of circMKLN1 expression significantly reduced collagen deposition and inhibited EMT in AECs. EMT was aggravated after circMKLN1 overexpression in AECs. MiR-26a-5p/miR-26b-5p (miR-26a/b), the targets of circMKLN1, were confirmed by luciferase reporter assays. CircMKLN1 inhibition elevated miR-26a/b expression. Significantly decreased expression of CDK8 (one of the miR-26a/b targets) was observed after inhibition of circMKLN1. EMT was exacerbated again, and CDK8 expression was significantly increased after circMKLN1 inhibition and cotransfection of miR-26a/b inhibitors in AECs. Our research indicated that circMKLN1 promoted CDK8 expression through sponge adsorption of miR-26a/b, which regulates EMT and pulmonary fibrosis. This study provides a theoretical basis for finding new targets or biomarkers in pulmonary fibrosis.

Deciphering the molecular regulatory of RAB32/GPRC5A axis in chronic obstructive pulmonary disease

BackgroundChronic obstructive pulmonary disease (COPD) is a significant public health problem characterized by persistent airflow limitation. Despite previous research into the pathogenesis of COPD, a comprehensive understanding of the cell-type-specific mechanisms in COPD remains lacking. Recent studies have implicated Rab GTPases in regulating chronic immune response and inflammation via multiple pathways. In this study, the molecular regulating mechanism of RAB32 in COPD was investigated by multiple bioinformatics mining and experimental verification.MethodsWe collected lung tissue surgical specimens from Zhongshan Hospital, Fudan University, and RT-qPCR and western blotting were used to detect the expression of Rabs in COPD lung tissues. Four COPD microarray datasets from the Gene Expression Omnibus (GEO) were analyzed. COPD-related epithelial cell scRNA-seq data was obtained from the GSE173896 dataset. Weighted gene co-expression network analysis (WGCNA), mfuzz cluster, and Spearman correlation analysis were combined to obtain the regulatory network of RAB32 in COPD. The slingshot algorithm was used to identify the regulatory molecule, and the co-localization of RAB32 and GPRC5A was observed with immunofluorescence.ResultsWGCNA identified 771 key module genes significantly associated with the occurrence of COPD, including five Rab genes. RAB32 was up-regulated in lung tissues from subjects with COPD as contrast to those without COPD on both mRNA and protein levels. Integrating the results of WGCNA, Mfuzz clusters, and Spearman analysis, nine potential interacting genes with RAB32 were identified. Among these genes, GPRC5A exhibited a similar molecular expression pattern to RAB32. Co-expression density analysis at the cell level demonstrated that the co-expression density of RAB32 and GPRC5A was higher in type I alveolar epithelial cells (AT1s) than in type II alveolar epithelial cells (AT2s). The immunofluorescence also confirmed the co-localization of RAB32 and GPRC5A, and the Pearson correlation analysis found the relationship between RAB32 and GPRC5A was significantly stronger in the COPD lungs (r = 0.65) compared to the non-COPD lungs (r = 0.33).ConclusionsOur study marked endeavor to delineate the molecular regulatory axis of RAB32 in COPD by employing diverse methods and identifying GPRC5A as a potential interacting molecule with RAB32. These findings offered novel perspectives on the mechanism of COPD.

Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed “organoids.” Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Tetrandrine alleviates pulmonary fibrosis by inhibiting alveolar epithelial cell senescence through PINK1/Parkin-mediated mitophagy

Idiopathic pulmonary fibrosis (IPF) is a fatal and insidious interstitial lung disease. So far, there are no effective drugs for preventing the disease process. Cellular senescence plays a critical role in the development of IPF, with the senescence and insufficient mitophagy of alveolar epithelial cells being implicated in its pathogenesis. Tetrandrine is a natural alkaloid which is now produced synthetically. It was known that the tetrandrine has anti-fibrotic effects, but the efficacy and mechanisms are still not well evaluated. Here, we reveal the roles of tetrandrine on AECs senescence and the antifibrotic effects by using a bleomycin challenged mouse model of pulmonary fibrosis and a bleomycin-stimulated mouse alveolar epithelial cell line (MLE-12). We performed the β-galactosidase staining, immunohistochemistry and fluorescence to assess senescence in MLE-12 cells. The mitophagy levels were detected by co-localization of LC3 and COVIX. Our findings indicate that tetrandrine suppressed bleomycin-induced fibroblast activation and ultimately blocked the increase of collagen deposition in mouse model lung tissue. It has significantly inhibited the bleomycin-induced senescence and senescence-associated secretory phenotype (SASP) in alveolar epithelial cells (AECs). Mechanistically, tetrandrine suppressed the decrease of mitochondrial autophagy-related protein expression to rescue the bleomycin-stimulated impaired mitophagy in MLE-12 cells. We revealed that knockdown the putative kinase 1 (PINK1) gene by a short interfering RNA (siRNA) could abolish the ability of tetrandrine and reverse the MLE-12 cells senescence, which indicated the mitophagy of MLE-12 cells is PINK1 dependent. Our data suggest the tetrandrine could be a novel and effective drug candidate for lung fibrosis and senescence-related fibrotic diseases.

Cell-free DNA methylation reveals cell-specific tissue injury and correlates with disease severity and patient outcomes in COVID-19

BackgroundThe recently identified methylation patterns specific to cell type allows the tracing of cell death dynamics at the cellular level in health and diseases. This study used COVID-19 as a disease model to investigate the efficacy of cell-specific cell-free DNA (cfDNA) methylation markers in reflecting or predicting disease severity or outcome.MethodsWhole genome methylation sequencing of cfDNA was performed for 20 healthy individuals, 20 cases with non-hospitalized COVID-19 and 12 cases with severe COVID-19 admitted to intensive care unit (ICU). Differentially methylated regions (DMRs) and gene ontology pathway enrichment analyses were performed to explore the locus-specific methylation difference between cohorts. The proportion of cfDNA derived from lung and immune cells to a given sample (i.e. tissue fraction) at cell-type resolution was estimated using a novel algorithm, which reflects lung injuries and immune response in COVID-19 patients and was further used to evaluate clinical severity and patient outcome.ResultsCOVID‑19 patients had globally reduced cfDNA methylation level compared with healthy controls. Compared with non-hospitalized COVID-19 patients, the cfDNA methylation pattern was significantly altered in severe patients with the identification of 11,156 DMRs, which were mainly enriched in pathways related to immune response. Markedly elevated levels of cfDNA derived from lung and more specifically alveolar epithelial cells, bronchial epithelial cells, and lung endothelial cells were observed in COVID-19 patients compared with healthy controls. Compared with non-hospitalized patients or healthy controls, severe COVID-19 had significantly higher cfDNA derived from B cells, T cells and granulocytes and lower cfDNA from natural killer cells. Moreover, cfDNA derived from alveolar epithelial cells had the optimal performance to differentiate COVID-19 with different severities, lung injury levels, SOFA scores and in-hospital deaths, with the area under the receiver operating characteristic curve of 0.958, 0.941, 0.919 and 0.955, respectively.ConclusionSevere COVID-19 has a distinct cfDNA methylation signature compared with non-hospitalized COVID-19 and healthy controls. Cell type-specific cfDNA methylation signature enables the tracing of COVID-19 related cell deaths in lung and immune cells at cell-type resolution, which is correlated with clinical severities and outcomes, and has extensive application prospects to evaluate tissue injuries in diseases with multi-organ dysfunction.

Mast cell degranulation-triggered by SARS-CoV-2 induces tracheal-bronchial epithelial inflammation and injury

SARS-CoV-2 infection-induced hyper-inflammation is a key pathogenic factor of COVID-19. Our research, along with others', has demonstrated that mast cells (MCs) play a vital role in the initiation of hyper-inflammation caused by SARS-CoV-2. In previous study, we observed that SARS-CoV-2 infection induced the accumulation of MCs in the peri-bronchus and bronchioalveolar-duct junction in humanized mice. Additionally, we found that MC degranulation triggered by the spike protein resulted in inflammation in alveolar epithelial cells and capillary endothelial cells, leading to subsequent lung injury. The trachea and bronchus are the routes for SARS-CoV-2 transmission after virus inhalation, and inflammation in these regions could promote viral spread. MCs are widely distributed throughout the respiratory tract. Thus, in this study, we investigated the role of MCs and their degranulation in the development of inflammation in tracheal-bronchial epithelium. Histological analyses showed the accumulation and degranulation of MCs in the peri-trachea of humanized mice infected with SARS-CoV-2. MC degranulation caused lesions in trachea, and the formation of papillary hyperplasia was observed. Through transcriptome analysis in bronchial epithelial cells, we found that MC degranulation significantly altered multiple cellular signaling, particularly, leading to upregulated immune responses and inflammation. The administration of ebastine or loratadine effectively suppressed the induction of inflammatory factors in bronchial epithelial cells and alleviated tracheal injury in mice. Taken together, our findings confirm the essential role of MC degranulation in SARS-CoV-2-induced hyper-inflammation and the subsequent tissue lesions. Furthermore, our results support the use of ebastine or loratadine to inhibit SARS-CoV-2-triggered degranulation, thereby preventing tissue damage caused by hyper-inflammation.

Effects of chitosan and TiO2 nanoparticles on the antibacterial property and ability to self-healing of cracks and retrieve mechanical characteristics of dental composites

The aim of this study was to improve the self-healing properties of dental nanocomposite using nanoparticles of TiO2 and chitosan. We evaluated flexural and compressive strength, crack-healing, and self-healing lifespan after 3 months of water aging. The effect of the developed composite on cell viability and toxicity was assessed by an MTT assay on human alveolar basal epithelial cells (A549 cell line). The nanocomposite included 7.5 wt% polyurea-formaldehyde (PUF) and 0, 0.5, and 1 wt% n-TiO2 and chitosan. After the fracture, the samples were put in a mold for 1–90 days to enable healing. Then, the fracture toughness of the healed nanocomposites and the healing yield were measured. The flexural strength of the nanocomposite improved by adding 0.5 wt% n-TiO2, while the compressive strength increased after adding 0.5 wt% chitosan (p > 0.1). When these two materials were used simultaneously, the flexural strength was improved by around 2%; however, the compressive strength was unaffected. Compared to the other sample, the nanocomposite with 0.5 wt% n-TiO2 and chitosan had higher KIC-healing and self-healing efficiency. Self-healing efficacy had no significant effect of water aging over 90 days compared to one day (p > 0.1), demonstrating that the PUF nanocapsules were not damaged.

Role of microbiota-derived corisin in coagulation activation during SARS-CoV-2 infection

BackgroundCoagulopathy is a major cause of morbidity and mortality in COVID-19 patients. Hypercoagulability in COVID-19 results in deep vein thrombosis, thromboembolic complications, and diffuse intravascular coagulation. Microbiome dysbiosis influences the clinical course of COVID-19. However, the role of dysbiosis in COVID-19-associated coagulopathy is not fully understood. ObjectivesThe present study tested the hypothesis that the microbiota-derived proapoptotic corisin is involved in the coagulation system activation during SARS-CoV-2 infection. MethodsThis cross-sectional study included 47 consecutive patients who consulted for symptoms of COVID-19. A mouse acute lung injury model was used to recapitulate the clinical findings. A549 alveolar epithelial, THP-1, and human umbilical vein endothelial cells were used to evaluate procoagulant and anticoagulant activity of corisin. ResultsCOVID-19 patients showed significantly high circulating levels of corisin, thrombin-antithrombin complex, D-dimer, tumor necrosis factor-α, and monocyte-chemoattractant protein-1 with reduced levels of free protein S compared with healthy subjects. The levels of thrombin-antithrombin complex, D-dimer, and corisin were significantly correlated. A monoclonal anticorisin-neutralizing antibody significantly inhibited the inflammatory response and coagulation system activation in a SARS-CoV-2 spike protein-associated acute lung injury mouse model, and the levels of corisin and thrombin-antithrombin complex were significantly correlated. In an in vitro experiment, corisin increased the tissue factor activity and decreased the anticoagulant activity of thrombomodulin in epithelial, endothelial, and monocytic cells. ConclusionThe microbiota-derived corisin is significantly increased and correlated with activation of the coagulation system during SARS-CoV-2 infection, and corisin may directly increase the procoagulant activity in epithelial, endothelial, and monocytic cells.

Astragaloside IV inhibits idiopathic pulmonary fibrosis through activation of autophagy by miR-21-mediated PTEN/PI3K/AKT/mTOR pathway.

As the main active ingredient of Astragalus, Astragaloside IV (AS-IV) can ameliorate pulmonary fibrosis. In this experiment, we studied how AS-IV reduces idiopathic pulmonary fibrosis (IPF). Bleomycin (BLM) or TGF-β1 was treated in mice or alveolar epithelial cells to mimic IPF in vivo as well as in vitro. ASV-IV alleviated levels of inflammatory cytokines and fibrosis markers in IPF model. Through detection of autophagy-related genes, ASV-IV was observed to induce autophagy in IPF. Besides, ASV-IV inhibited miR-21 expression in IPF models, and overexpression of miR-21 could reverse the protective potential of ASV-IV on IPF. PTEN was targeted by miR-21 and was up-regulated by ASV-IV in IPF models. In addition, levels of inflammatory cytokines and fibrosis markers, autophagy, as well as the PI3K/AKT/mTOR pathway regulated by ASV-IV could be neutralized after treatment with autophagy inhibitors, miR-21 mimics, or si-PTEN. Our study demonstrates that ASV-IV inhibits IPF through activation of autophagy by miR-21-mediated PTEN/PI3K/AKT/mTOR pathway, suggesting that ASV-IV could be acted to be a promising therapeutic method for IPF.

Alterations in the molecular control of mitochondrial turnover in COPD lung and airway epithelial cells

Abnormal mitochondria have been observed in bronchial- and alveolar epithelial cells of patients with chronic obstructive pulmonary disease (COPD). However, it is unknown if alterations in the molecular pathways regulating mitochondrial turnover (mitochondrial biogenesis vs mitophagy) are involved. Therefore, in this study, the abundance of key molecules controlling mitochondrial turnover were assessed in peripheral lung tissue from non-COPD patients (n = 6) and COPD patients (n = 11; GOLDII n = 4/11; GOLDIV n = 7/11) and in both undifferentiated and differentiated human primary bronchial epithelial cells (PBEC) from non-COPD patients and COPD patients (n = 4–7 patients/group). We observed significantly decreased transcript levels of key molecules controlling mitochondrial biogenesis (PPARGC1B, PPRC1, PPARD) in peripheral lung tissue from severe COPD patients. Interestingly, mRNA levels of the transcription factor TFAM (mitochondrial biogenesis) and BNIP3L (mitophagy) were increased in these patients. In general, these alterations were not recapitulated in undifferentiated and differentiated PBECs with the exception of decreased PPARGC1B expression in both PBEC models. Although these findings provide valuable insight in these pathways in bronchial epithelial cells and peripheral lung tissue of COPD patients, whether or not these alterations contribute to COPD pathogenesis, underlie changes in mitochondrial function or may represent compensatory mechanisms remains to be established.

Microfluidics produced ATRA-loaded PLGA NPs reduced tuberculosis burden in alveolar epithelial cells and enabled high delivered dose under simulated human breathing pattern in 3D printed head models

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is second only to COVID-19 as the top infectious disease killer worldwide. Multi-drug resistant TB (MDR-TB) may arise because of poor patient adherence to medications due to lengthy treatment duration and side effects. Delivering novel host directed therapies (HDT), like all trans retinoic acid (ATRA) may help to improve drug regimens and reduce the incidence of MDR-TB. Local delivery of ATRA to the site of infection leads to higher bioavailability and reduced systemic side effects. ATRA is poorly soluble in water and has a short half-life in plasma. Therefore, it requires a formulation step before it can be administered in vivo. ATRA loaded PLGA nanoparticles suitable for nebulization were manufactured and optimized using a scalable nanomanufacturing microfluidics (MF) mixing approach (MF-ATRA-PLGA NPs). MF-ATRA-PLGA NPs demonstrated a dose dependent inhibition of Mtb growth in TB-infected A549 alveolar epithelial cell model while preserving cell viability. The MF-ATRA-PLGA NPs were nebulized with the Aerogen Solo vibrating mesh nebulizer, with aerosol droplet size characterized using laser diffraction and the estimated delivered dose was determined. The volume median diameter (VMD) of the MF-ATRA-PLGA NPs was 3.00 ± 0.18 μm. The inhaled dose delivered in adult and paediatric 3D printed head models under a simulated normal adult and paediatric breathing pattern was found to be 47.05 ± 3 % and 20.15 ± 3.46 % respectively. These aerosol characteristics of MF-ATRA-PLGA NPs supports its suitability for delivery to the lungs via inhalation. The data generated on the efficacy of an inhalable, scalable and regulatory friendly ATRA-PLGA NPs formulation provides a foundation on which further pre-clinical testing can be built. Overall, the results of this project are promising for future research into ATRA loaded NPs formulations as inhaled host directed therapies for TB.

Curcumin as a promising treatment for pulmonary fibrosis: Mechanism and therapeutic potential

IntroductionPulmonary fibrosis (PF) is characterized by excessive proliferation of alveolar epithelial cells and fibroblasts, abnormal deposition of fibrous connective tissue, altered alveolar structure, impaired lung function, and increased airway resistance. Curcumin has been studied as an inhibitor of pulmonary fibrosis. MethodsThis study investigated curcumin's inhibitory effect and mechanism on bleomycin (BLM) -induced pulmonary fibrosis in mice. Curcumin was administered to the mice to assess its therapeutic potential. The mice were weighed every two days, and the respiratory function of the mice was measured on day 7, 14, and 21. After 21 days, the mice's lung tissue was examined for hydroxyproline (HYP), malondialdehyde (MDA), superoxide dismutase (SOD) levels, as well as relevant protein expression. ResultsThe administration of curcumin significantly alleviated respiratory dysfunction and improved the quality of life of mice with pulmonary fibrosis. It led to an increase in the expression of oxidative stress-related proteins Nuclear factor erythroid 2-related factor 2 (Nrf2) and Heme Oxygenase-1 (HO-1), while reducing apoptosis-related factors. Pathological examination revealed a notable reduction in lung tissue fibrosis. ConclusionsCurcumin demonstrated promising therapeutic effects in mice with BLM-induced pulmonary fibrosis. The activation of the Akt pathway, upregulating the expression of Nrf2 and HO-1, contributed to curcumin's antioxidant effects and its potential for treating pulmonary fibrosis in mice.