HO-1 upregulation promotes mitophagy-dependent ferroptosis in PM2.5-exposed hippocampal neurons.

Fine particulate matter (PM2.5) has been extensively implicated in the pathogenesis of neurodevelopmental disorders, but the underlying mechanism remains unclear. Recent studies have revealed that PM2.5 plays a role in regulating iron metabolism and redox homeostasis in the brain, which is closely associated with ferroptosis. In this study, the role and underlying mechanism of ferroptosis in PM2.5-induced neurotoxicity were investigated in mice, primary hippocampal neurons, and HT22 cells. Our findings demonstrated that exposure to PM2.5 could induce abnormal behaviors, neuroinflammation, and neuronal loss in the hippocampus of mice. These effects may be attributed to ferroptosis induced by PM2.5 exposure in hippocampal neurons. RNA-seq analysis revealed that the upregulation of iron metabolism-related protein Heme Oxygenase 1 (HO-1) and the activation of mitophagy might play key roles in PM2.5-induced ferroptosis in HT22 cells. Subsequent in vitro experiments showed that PM2.5 exposure significantly upregulated HO-1 in primary hippocampal neurons and HT22 cells. Moreover, PM2.5 exposure activated mitophagy in HT22 cells, leading to the loss of mitochondrial membrane potential, alterations in the expression of autophagy-related proteins LC3, P62, and mTOR, as well as an increase in mitophagy-related protein PINK1 and PARKIN. As a heme-degradation enzyme, the upregulation of HO-1 promotes the release of excess iron, genetically inhibiting the upregulation of HO-1 in HT22 cells could prevent both PM2.5-induced mitophagy and ferroptosis. Furthermore, pharmacological inhibition of mitophagy in HT22 cells reduced levels of ferrous ions and lipid peroxides, thereby preventing ferroptosis. Collectively, this study demonstrates that HO-1 mediates PM2.5-induced mitophagy-dependent ferroptosis in hippocampal neurons, and inhibiting mitophagy or ferroptosis may be a key therapeutic target to ameliorate neurotoxicity following PM2.5 exposure.

Fine particulate matter 2.5 induces susceptibility to Pseudomonas aeruginosa infection via expansion of PD-L1high neutrophils in mice.

BACKGROUND: Exposure to PM2.5 has been implicated in a range of detrimental health effects, particularly affecting the respiratory system. However, the precise underlying mechanisms remain elusive. METHODS: To address this objective, we collected ambient PM2.5 and administered intranasal challenges to mice, followed by single-cell RNA sequencing (scRNA-seq) to unravel the heterogeneity of neutrophils and unveil their gene expression profiles. Flow cytometry and immunofluorescence staining were subsequently conducted to validate the obtained results. Furthermore, we assessed the phagocytic potential of neutrophils upon PM2.5 exposure using gene analysis of phagocytosis signatures and bacterial uptake assays. Additionally, we utilized a mouse pneumonia model to evaluate the susceptibility of PM2.5-exposed mice to Pseudomonas aeruginosa infection. RESULTS: Our study revealed a significant increase in neutrophil recruitment within the lungs of PM2.5-exposed mice, with subclustering of neutrophils uncovering subsets with distinct gene expression profiles. Notably, exposure to PM2.5 was associated with an expansion of PD-L1high neutrophils, which exhibited impaired phagocytic function dependent upon PD-L1 expression. Furthermore, PM2.5 exposure was found to increase the susceptibility of mice to Pseudomonas aeruginosa, due in part to increased PD-L1 expression on neutrophils. Importantly, monoclonal antibody targeting of PD-L1 significantly reduced bacterial burden, dissemination, and lung inflammation in PM2.5-exposed mice upon Pseudomonas aeruginosa infection. CONCLUSIONS: Our study suggests that PM2.5 exposure promotes expansion of PD-L1high neutrophils with impaired phagocytic function in mouse lungs, contributing to increased vulnerability to bacterial infection, and therefore targeting PD-L1 may be a therapeutic strategy for reducing the harmful effects of PM2.5 exposure on the immune system.

Oxidative stress-mediated activation of FTO exacerbates impairment of the epithelial barrier by up-regulating IKBKB via N6-methyladenosine-dependent mRNA stability in asthmatic mice exposed to PM2.5.

In order to comprehend the underlying mechanisms contributing to the development and exacerbation of asthma resulting from exposure to fine particulate matter (PM2.5), we established an asthmatic model in fat mass and obesity-associated gene knockdown mice subjected to PM2.5 exposure. Histological analyses using hematoxylin-eosin (HE) and Periodic Acid-Schiff (PAS) staining revealed that the down-regulation of the fat mass and obesity-associated gene (Fto) expression significantly ameliorated the pathophysiological alterations observed in asthmatic mice exposed to PM2.5. Furthermore, the down-regulation of Fto gene expression effectively attenuated damage to the airway epithelial barrier. Additionally, employing in vivo and in vitro models, we elucidated that PM2.5 modulated FTO expression by inducing oxidative stress. Asthmatic mice exposed to PM2.5 exhibited elevated Fto expression, which correlated with increased levels of reactive oxygen species. Similarly, when cells were exposed to PM2.5, FTO expression was up-regulated in a ROS-dependent manner. Notably, the administration of N-acetyl cysteine successfully reversed the PM2.5-induced elevation in FTO expression. Concurrently, we performed transcriptome-wide Methylated RNA immunoprecipitation Sequencing (MeRIP-seq) analysis subsequent to PM2.5 exposure. Through the implementation of Gene Set Enrichment Analysis and m6A-IP-qPCR, we successfully identified inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB) as a target gene regulated by FTO. Interestingly, exposure to PM2.5 led to increased expression of IKBKB, while m6A modification on IKBKB mRNA was reduced. Furthermore, our investigation revealed that PM2.5 also regulated IKBKB through oxidative stress. Significantly, the down-regulation of IKBKB effectively mitigated epithelial barrier damage in cells exposed to PM2.5 by modulating nuclear factor-kappa B (NF-κB) signaling. Importantly, we discovered that decreased m6A modification on IKBKB mRNA facilitated by FTO enhanced its stability, consequently resulting in up-regulation of IKBKB expression. Collectively, our findings propose a novel role for FTO in the regulation of IKBKB through m6A-dependent mRNA stability in the context of PM2.5-induced oxidative stress. Therefore, it is conceivable that the utilization of antioxidants or inhibition of FTO could represent potential therapeutic strategies for the management of asthma exacerbated by PM2.5 exposure.

UPP1 promotes lung adenocarcinoma progression through the induction of an immunosuppressive microenvironment.

The complexity of the tumor microenvironment (TME) is a crucial factor in lung adenocarcinoma (LUAD) progression. To gain deeper insights into molecular mechanisms of LUAD, we perform an integrative single-cell RNA sequencing (scRNA-seq) data analysis of 377,574 cells from 117 LUAD patient samples. By linking scRNA-seq data with bulk gene expression data, we identify a cluster of prognostic-related UPP1high tumor cells. These cells, primarily situated at the invasive front of tumors, display a stronger association with the immunosuppressive components in the TME. Our cytokine array analysis reveals that the upregulation of UPP1 in tumor cells leads to the increased release of various immunosuppressive cytokines, with TGF-ß1 being particularly prominent. Furthermore, this UPP1 upregulation also elevates the expression of PD-L1 through the PI3K/AKT/mTOR pathway, which contributes to the suppression of CD8 + T cells. Cytometry by time-of-flight (CyTOF) analysis provides additional evidence of the role of UPP1 in shaping the immunosuppressive nature of the TME. Using patient-derived organoids (PDOs), we discover that UPP1high tumors exhibit relatively increased sensitivity to Bosutinib and Dasatinib. Collectively, our study highlights the immunosuppressive role of UPP1 in LUAD, and these findings may provide insights into the molecular features of LUAD and facilitate the development of personalized treatment strategies.

Integrative Analyses of Pyrimidine Salvage Pathway-Related Genes Revealing the Associations Between UPP1 and Tumor Microenvironment.

Background: The pyrimidine salvage pathway plays a critical role in tumor progression and patient outcomes. The roles of pyrimidine salvage pathway-related genes (PSPGs) in cancer, however, are not fully understood. This study aims to depict the characteristics of PSPGs across various cancers. Methods: An integrative pan-cancer analysis of six PSPGs (CDA, UCK1, UCK2, UCKL1, UPP1, and UPP2) was conducted using TCGA data, single-cell RNA sequencing datasets, and patient samples. Single-cell transcriptome analysis and RT-qPCR were used to validate the relation between UPP1 and cytokines. Flow cytometry was performed to validate the role of UPP1 in immune checkpoint regulation. The correlation between UPP1 and tumor associated neutrophils (TAN) were investigated and validated by single-cell transcriptome analysis and tissue microarrays (TMAs). Results: PSPGs showed low mutation rates but significant copy number variations, particularly amplifications in UCKL1, UPP1, and UCK2 across various cancers. DNA methylation patterns varied, with notable negative correlations between methylation and gene expression in UPP1. PSPGs were broadly up-regulated in multiple cancers, with correlations to clinical staging and prognosis. Proteomic data further confirmed these findings. Functional analysis revealed PSPGs' associations with tumor proliferation, metastasis, and various signaling pathways. UPP1 showed strong correlations with the tumor microenvironment (TME), particularly with cytokines, immune checkpoints, and various immune cells. Single-cell transcriptome analysis confirmed these associations, highlighting UPP1's influence on cytokine expression and immune checkpoint regulation. In esophageal squamous cell carcinoma (ESCC), UPP1-high tumor cells were significantly associated with immunosuppressive cells in the TME. Spatial analysis using TMAs revealed that UPP1+ tumor cells were predominantly located at the invasive margin and closely associated with neutrophils, correlating with poorer patient prognosis. Conclusion: Our study depicted the multi-dimensional view of PSPGs in cancer, with a particular focus on UPP1's role in the TME. Targeting UPP1 holds promise as a potential strategy for cancer therapy.

MMP14high macrophages orchestrate progressive pulmonary fibrosis in SR-Ag-induced hypersensitivity pneumonitis.

Fibrotic hypersensitivity pneumonitis (FHP) is a fatal interstitial pulmonary disease with limited treatment options. Lung macrophages are a heterogeneous cell population that exhibit distinct subsets with divergent functions, playing pivotal roles in the progression of pulmonary fibrosis. However, the specific macrophage subpopulations and underlying mechanisms involved in the disease remain largely unexplored. In this study, a decision tree model showed that matrix metalloproteinase-14 (MMP14) had higher scores for important features in the up-regulated genes in macrophages from mice exposed to the Saccharopolyspora rectivirgula antigen (SR-Ag). Using single-cell RNA sequencing (scRNA-seq) analysis of hypersensitivity pneumonitis (HP) mice profiles, we identified MMP14high macrophage subcluster with a predominant M2 phenotype that exhibited higher activity in promoting fibroblast-to myofibroblast transition (FMT). We demonstrated that suppressing toll-like receptor 2 (TLR2) and nuclear factor kappa-B (NF-κB) could attenuate MMP14 expression and exosome secretion in macrophages stimulation with SR-Ag. The exosomes derived from MMP14-overexpressing macrophages were found to be more effective in regulating the transition of fibroblasts through exosomal MMP14. Importantly, it was observed that the transfer of MMP14-overexpressing macrophages into mice promoted lung inflammation and fibrosis induced by SR-Ag. NSC-405020 binding to the hemopexin domain (PEX) of MMP-14 ameliorated lung inflammation and fibrosis induced by SR-Ag in mice. Thus, MMP14-overexpressing macrophages may be an important mechanism contributing to the exacerbation of allergic reactions. Our results indicated that MMP14 in macrophages has the potential to be a therapeutic target for HP.

Integrated analysis of ATAC-seq and RNA-seq unveils the role of ferroptosis in PM2.5-induced asthma exacerbation.

BACKGROUND: PM2.5 exposure increases asthma exacerbation risk and worsens airway inflammation and mucus secretion, but the underlying mechanisms, especially the epigenetic modification changes, are not fully understood. METHODS: ATAC-seq was conducted in Beas-2B cells to explore the differential chromatin accessibilities before and after exposure to PM2.5. RNA-seq was applied to screen the differentially expressed genes (DEGs) as well. The integrated analysis of ATAC-seq and RNA-seq was performed. The key up-regulated genes in the ferroptosis signaling pathway were identified by combined analysis with the FerrDb database and then verified. Meanwhile, to access the role of PM2.5-induced ferroptosis in asthma mice, house dust mites (HDM) were employed to conduct an allergic asthma mice model, and the ferroptosis-specific inhibitor (Ferrostatin-1, Fer-1) was used. The H&E staining, PAS staining, airway hyperresponsiveness, and bronchoalveolar lavage fluid (BALF) cell counting were used to investigate the impact of PM2.5-induced ferroptosis in asthma mice. RESULTS: A total of 4,921 regions with differential accessibility were identified, encompassing 4,031 unique genes. Among these, 250 regions exhibited increased accessibility while 4,671 regions displayed reduced accessibility. Through the integrated analysis of ATAC-seq and RNA-seq, ferroptosis was determined as the key enriched pathway based on up-regulated DEGs and increased chromatin accessibilities. Furthermore, the decreased cell viability, accelerated lipid peroxide and morphological changes in mitochondria observed upon PM2.5 exposure were rescued by Fer-1, which are indicative of ferroptosis. By overlapping with ferroptosis-related genes from the FerrDb database, FTH1 and FTL were identified as the prominent up-regulated genes with increased chromatin accessibility in ferroptosis pathway. In addition, ChIP-qPCR analysis indicated that histone modification like H3K4me3 and H3K27ac positively regulated FTH1 and FTL expression. Subsequently, in PM2.5-exposed asthmatic mice, inhibition of ferroptosis effectively attenuated airway inflammation and mucus secretion. CONCLUSION: These findings shed light on the molecular mechanisms underlying PM2.5-induced asthma exacerbation, with epigenetic modifications playing a pivotal role. Furthermore, it suggests the therapeutic potential of targeting ferroptosis as an intervention strategy.

Single cell characteristics of patients with vaccine-related adverse reactions following inactivated COVID-19 vaccination.

A good safety and immunogenicity profile was reported in Phase I and II clinical trials of inactivated SARS-CoV-2 vaccines. Here, we report two cases associated with vaccine-associated adverse events, including one patient with fever and another with anaphylactic shock resulting from inactivated SARS-CoV-2 vaccination. Cell sub-types and the importance of genetic characteristics were assessed using single-cell mRNA sequencing and machine learning. Overall, the patient with fever showed a significant increase in the numbers of cytotoxic CD8 T cells and MKI67high CD8 T cells. A potential concurrent infection with the Epstein-Barr virus enhanced interferon type I responses to vaccination against the virus. STAT1, E2F1, YBX1, and E2F7 played a key role in the transcription regulation of MKI67high CD8 T cells. In contrast, the patient with allergic shock displayed predominant increases in the numbers of S100A9high monocytes, activated CD4 T cells, and PPBPhigh megakaryocytes. The decision tree showed that LYZ and S100A8 in S100A9high monocytes contributed to the degranulation of neutrophils and activation of neutrophils involved in allergic shock. PPBP and PF4 were major contributors to platelet degranulation. These findings highlight the diversity of adverse reactions following inactivated SARS-CoV-2 vaccination and show the emerging role of cellular subtypes and central genes in vaccine-associated adverse reactions.

The identification of cell sub-types may help in the diagnosis of COVID-19 vaccine-related adverse events.COVID-19 vaccination-related acute pulmonary edema may induce a higher risk of thrombosis.The long-term fever after vaccination may attribute to the excessive type I interferon responses.

Transcriptome analysis identifies IL24 as an autophagy modulator in PM2.5 caused lung dysfunction.

BACKGROUND: Evidence suggests that exposure to PM2.5 increased hospitalization and mortality rates of respiratory diseases. However, the potential biomarkers and targets associated with PM2.5-induced lung dysfunction are not fully discovered. METHODS: Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and HALLMARK enrichment analysis of the RNA-seq data (Beas-2B cells treated with PM2.5) were applied. Gene set enrichment analysis (GSEA) was performed to identify the biological processes correlated with autophagy. Three gene expression profile datasets (GSE158954, GSE155616 and GSE182199) were downloaded from the Gene Expression Omnibus (GEO) database to identify the potential targets. PM2.5-exposed mice were constructed. Real-time qPCR, siRNA transfection, western blot, immunofluorescence, and pathological staining were applied for validation both in vitro and in vivo studies. RESULTS: GO, KEGG and HALLMARK enrichment based on RNA-seq data showed that the differentially expressed genes (DEGs) were associated with autophagy like lysosome and macroautophagy. GSEA analysis revealed that PM2.5 was positively correlated with autophagy-related biological processes compared with control group. Venn diagrams identified IL24 was upregulated in our data as well as in these three datasets (GSE158954, GSE155616 and GSE182199) after PM2.5 exposure. Consistent with the analysis, activation of autophagy by PM2.5 was validated in vivo and in vitro. In PM2.5-exposed mice, lung pathological changes were observed, including airway inflammation and mucus secretion. The mRNA and protein levels of the key gene, IL24, were significantly increased. Moreover, Bafilomycin A1, the inhibitor of autophagy, inhibited the autophagy and ameliorated lung injury induced by PM2.5. Furthermore, downregulation of IL24 decreased autophagy activity. Meanwhile, IL24 was regulated by mTOR signaling. CONCLUSIONS: In summary, we discovered a potential relationship between IL24 and autophagy during PM2.5 exposure. IL24 might be a novel potential biomarker or therapeutic target in PM2.5 caused lung dysfunction through regulation of autophagy.

Integrated analysis of single-cell and bulk RNA sequencing reveals pro-fibrotic PLA2G7high macrophages in pulmonary fibrosis.

Pulmonary fibrosis (PF) is the pathological change of end-stage interstitial lung diseases with high mortality and limited therapeutic options. Lung macrophages have distinct subsets with divergent functions, and play critical roles in the pathogenesis of PF. In this study, integrative analysis of lung single-cell and bulk RNA-seq data from patients with fibrotic hypersensitivity pneumonitis and idiopathic pulmonary fibrosis was utilized to identify particular macrophage subsets during the development of PF. We find a specific macrophage subpopulation highly expressing PLA2G7 in fibrotic lungs. We performed additional single-cell RNA-seq analysis to identify analogous macrophage population in bleomycin (BLM)-induced mouse pulmonary fibrosis models. By in vitro and in vivo experiments, we further reveal the pro-fibrotic role for this PLA2G7high macrophage subset in fibroblast-to-myofibroblast transition (FMT) during pulmonary fibrosis. PLA2G7 promotes FMT via LPC/ATX/LPA/LPA2 axis in macrophages. Moreover, PLA2G7 is regulated by STAT1, and pharmacological inhibition of PLA2G7 by Darapladib ameliorates pulmonary fibrosis in BLM-induced mice. The results of this study support the view that PLA2G7high macrophage subpopulation contributes importantly to the pathogenesis of PF, which provides a potential way for targeted therapy.

Methyltransferase-like 3 leads to lung injury by up-regulation of interleukin 24 through N6-methyladenosine-dependent mRNA stability and translation efficiency in mice exposed to fine particulate matter 2.5.

Fine particulate matter 2.5 (PM2.5) exposure leads to the progress of pulmonary disease. It has been reported that N6-methyladenosine (m6A) modification was involved in various biological processes and diseases. However, the critical role of m6A modification in pulmonary disease during PM2.5 exposure remains elusive. Here, we revealed that lung inflammation and mucus production caused by PM2.5 were associated with m6A modification. Both in vivo and in vitro assays demonstrated that PM2.5 exposure elevated the total level of m6A modification as well as the methyltransferase like 3 (METTL3) expression. Integration analysis of m6A RNA immunoprecipitation-seq (meRIP-seq) and RNA-seq discovered that METTL3 up-regulated the expression level and the m6A modification of Interleukin 24 (IL24). Importantly, we explored that the stability of IL24 mRNA was enhanced due to the increased m6A modification. Moreover, the data from qRT-PCR showed that PM2.5 also increased YTH N6-Methyladenosine RNA Binding Protein 1 (YTHDF1) expression, and the up-regulated YTHDF1 augmented IL24 mRNA translation efficiency. Down-regulation of Mettl3 reduced Il24 expression and ameliorated the pulmonary inflammation and mucus secretion in mice exposed to PM2.5. Taken together, our finding provided a comprehensive insight for revealing the significant role of m6A regulators in the lung injury via METTL3/YTHDF1-coupled epitranscriptomal regulation of IL24.

Characterizing cellular heterogeneity in fibrotic hypersensitivity pneumonitis by single-cell transcriptional analysis.

Fibrotic hypersensitivity pneumonitis (FHP) remains one of fatal interstitial pulmonary disease. Comprehensively dissecting the cellular heterogeneity of FHP paves the way for developing general gene therapeutic solutions for FHP. Here, utilizing an integrated strategy based on scRNA-seq, scTCR-seq, and bulk RNA-seq analysis of FHP profiles, we identified ten major cell types and 19 unique subtypes. FHP exhibited higher features of EMT and inflammation-promoting than normal control. In distinct subsets of lung macrophages in FHP, FN1high, PLA2G7high, and MS4A6Ahigh macrophages with predominant M2 phenotype exhibited higher activity of inflammatory responses and para-inflammation than other macrophages. KRT17high basal-like epithelial cells were significantly increased in FHP, and showed higher ability to induce EMT. We identified roles for ACTA2high, COL1A1high, and PLA2G2Ahigh fibroblasts in FHP, which were significantly related to interstitial fibrosis. NK cells and KLRG1+ effector CD8+ T cells had greater activity in inflammation-promoting. Our results provide a comprehensive portrait of cellular heterogeneity in FHP, and highlight the indispensable role of cell subpopulations in shaping the complexity and heterogeneity of FHP. These subpopulations are potentially key players for FHP pathogenesis.

Transcriptome and pan-cancer system analysis identify PM2.5-induced stanniocalcin 2 as a potential prognostic and immunological biomarker for cancers.

Epidemiological studies have shown that air pollution and particulate matter (PM) are closely related to the occurrence of cancer. However, the potential prognostic and immunological biomarkers for air pollution related cancers are lacking. In this study, we proved PM2.5 exposure was correlated with lung cancer through transcriptome analysis. Importantly, we identified STC2 as a key gene regulated by PM2.5, whose expression in epithelial cells was significantly increased after PM2.5 treatment and validated by using RT-qPCR and immunofluorescence. Kaplan-Meier OS curves suggested that high STC2 expression positively correlated with a poor prognosis in lung cancer. Furthermore, we discovered that STC2 was associated with multiple cancers and pathways in cancer. Next, Pan-Cancer Expression Landscape of STC2 showed that STC2 exhibited inconsistent expression across 26 types of human cancer, lower in KIRP in cancer versus adjacent normal tissues, and significantly higher in another cancers. Cox regression results suggested that STC2 expression was positively or negatively associated with prognosis in different cancers. Moreover, STC2 expression was associated with clinical phenotypes including age, gender, stage and grade. Mutation features of STC2 were also analyzed, in which the highest alteration frequency of STC2 was presented in KIRC with amplification. Meanwhile, the effects of copy number variation (CNV) on STC2 expression were investigated across various tumor types, suggesting that STC2 expression was significantly correlated with CNV in tumors. Additionally, STC2 was closely related to tumor heterogeneity, tumor stemness and tumor immune microenvironment like immune cell infiltration. In the meantime, we analyzed methylation modifications and immunological correlation of STC2. The results demonstrated that STC2 expression positively correlated with most RNA methylation genes and immunomodulators across tumors. Taken together, the findings revealed that PM2.5-induced STC2 might be a potential prognostic and immunological biomarker for cancers related to air pollution.