Single-cell communication patterns and their intracellular information flow in synovial fibroblastic osteoarthritis and rheumatoid arthritis.

BACKGROUND: Synovial fibroblasts are critical for maintaining homeostasis in major autoimmune diseases involving joint inflammation, including osteoarthritis and rheumatoid arthritis. However, little is known about the interactions among different cell subtypes and the specific sets of signaling pathways and activities that they trigger. METHODS: Using social network analysis, pattern recognition, and manifold learning approaches, we identified patterns of single-cell communication in OA (osteoarthritis) and RA (rheumatoid arthritis). RESULTS: Our results suggest that OA and RA have distinct cellular communication patterns and signaling pathways. The LAMININ (Laminin) and COLLAGEN (Collagen) pathways predominate in osteoarthritis, while the EGF (Epidermal growth factor), NT (Neurotrophin) and CDH5 (Cadherin 5) pathways predominate in rheumatoid arthritis, with a central role for THY1 (Thy-1 cell surface antigen) +CDH11 (Cadherin 11) + cells. The OA opens the PDGF (Platelet-derived growth factors) pathway (driver of bone angiogenesis), the RA opens the EGF pathway (bone formation) and the SEMA3 (Semaphorin 3A) pathway (involved in immune regulation). Interestingly, we found that OA no longer has cell types involved in the MHC complex (Major histocompatibility complex) and their activity, whereas the MHC complex functions primarily in RA in the presentation of inflammatory antigens, and that the complement system in OA has the potential to displace the function of the MHC complex. The specific signaling patterns of THY1+CDH11+ cells and their secreted ligand receptors are more conducive to cell migration and lay the foundation for promoting osteoclastogenesis. This subpopulation may also be involved in the accumulation of lymphocytes, affecting the recruitment of immune cells. Members of the collagen family (COL1A1 (Collagen Type I Alpha 1 Chain), COL6A2 (Collagen Type VI Alpha 2 Chain) and COL6A1 (Collagen Type VI Alpha 1 Chain)) and transforming growth factor (TGFB3) maintain the extracellular matrix in osteoarthritis and mediate cell migration and adhesion in rheumatoid arthritis, including the PTN (Pleiotrophin) / THBS1 (Thrombospondin 1) interaction. CONCLUSION: Increased understanding of the interaction networks between synovial fibroblast subtypes, particularly the shared and unique cellular communication features between osteoarthritis and rheumatoid arthritis and their hub cells, should help inform the design of therapeutic agents for inflammatory joint disease.

Single-cell RNA sequencing deciphers the mechanism of sepsis-induced liver injury and the therapeutic effects of artesunate.

Liver, as an immune and detoxification organ, represents an important line of defense against bacteria and infection and a vulnerable organ that is easily injured during sepsis. Artesunate (ART) is an anti-malaria agent, that also exhibits broad pharmacological activities including anti-inflammatory, immune-regulation and liver protection. In this study, we investigated the cellular responses in liver to sepsis infection and ART hepatic-protective mechanisms against sepsis. Cecal ligation and puncture (CLP)-induced sepsis model was established in mice. The mice were administered ART (10 mg/kg, i.p.) at 4 h, and sacrificed at 12 h after the surgery. Liver samples were collected for preparing single-cell RNA transcriptome sequencing (scRNA-seq). The scRNA-seq analysis revealed that sepsis-induced a dramatic reduction of hepatic endothelial cells, especially the subtypes characterized with proliferation and differentiation. Macrophages were recruited during sepsis and released inflammatory cytokines (Tnf, Il1b, Il6), chemokines (Ccl6, Cd14), and transcription factor (Nfkb1), resulting in liver inflammatory responses. Massive apoptosis of lymphocytes and abnormal recruitment of neutrophils caused immune dysfunction. ART treatment significantly improved the survival of CLP mice within 96 h, and partially relieved or reversed the above-mentioned pathological features, mitigating the impact of sepsis on liver injury, inflammation, and dysfunction. This study provides comprehensive fundamental proof for the liver protective efficacy of ART against sepsis infection, which would potentially contribute to its clinical translation for sepsis therapy. Single cell transcriptome reveals the changes of various hepatocyte subtypes of CLP-induced liver injury and the potential pharmacological effects of artesunate on sepsis.

Single-nucleus RNA sequencing unveils critical regulators in various hippocampal neurons for anti-N-methyl-D-aspartate receptor encephalitis.

Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a neuropsychiatric disease with variable clinical manifestations caused by NMDAR autoantibody. The underlying molecular underpinnings of this disease are rarely characterized on a genomic scale. Anti-NMDAR encephalitis mainly affects the hippocampus, however, its effect on gene expression in hippocampal neurons is unclear at present. Here, we construct the active and passive immunization mouse models of anti-NMDAR encephalitis, and use single-nucleus RNA sequencing to investigate the diverse expression profile of neuronal populations isolated from different hippocampal regions. Dramatic changes in cell proportions and differentially expressed genes were observed in excitatory neurons of the dentate gyrus (DG) subregion. In addition, we found that ATP metabolism and biosynthetic regulators related genes in excitatory neurons of DG subregion were significantly affected. Kcnq1ot1 in inhibitory neurons and Meg3 in interneurons also changed. Notably, the latter two molecules exhibited opposite changes in different models. Therefore, the above genes were used as potential targets for further research on the pathological process of anti-NMDAR encephalitis. These data involve various hippocampal neurons, which delineate a framework for understanding the hippocampal neuronal circuit and the potential molecular mechanisms of anti-NMDAR encephalitis.

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice.

BACKGROUND: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. METHODS: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson's trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. RESULTS: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. CONCLUSIONS: TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Dissection of cellular and molecular mechanisms of aristolochic acid-induced hepatotoxicity via single-cell transcriptomics.

Background: Aristolochic acids (AAs), a class of carcinogenic and mutagenic natural products from Aristolochia and Asarum plants, are well-known to be responsible for inducing nephrotoxicity and urothelial carcinoma. Recently, accumulating evidence suggests that exposure to AAs could also induce hepatotoxicity and even hepatocellular carcinoma, though the mechanisms are poorly defined. Methods: Here, we aimed to dissect the underlying cellular and molecular mechanisms of aristolochic acid I (AAI)-induced hepatotoxicity by using advanced single-cell RNA sequencing (scRNA-seq) and proteomics techniques. We established the first single-cell atlas of mouse livers in response to AAI. Results: In hepatocytes, our results indicated that AAI activated NF-κB and STAT3 signaling pathways, which may contribute to the inflammatory response and apoptosis. In liver sinusoidal endothelial cells (LSECs), AAI activated multiple oxidative stress and inflammatory associated signaling pathways and induced apoptosis. Importantly, AAI induced infiltration of cytotoxic T cells and activation of proinflammatory macrophage and neutrophil cells in the liver to produce inflammatory cytokines to aggravate inflammation. Conclusions: Collectively, our study provides novel knowledge of AAs-induced molecular characteristics of hepatotoxicity at a single-cell level and suggests future treatment options for AAs associated hepatotoxicity.

Integrated single-cell transcriptomics and proteomics reveal cellular-specific responses and microenvironment remodeling in aristolochic acid nephropathy.

Aristolochic acid nephropathy (AAN) is characterized by acute proximal tubule necrosis and immune cell infiltration, contributing to the global burden of chronic kidney disease and urothelial cancer. Although the proximal tubule has been defined as the primary target of aristolochic acids I (AAI), the mechanistic underpinning of gross renal deterioration caused by AAI has not been explicitly explained, prohibiting effective therapeutic intervention. To this point, we employed integrated single-cell RNA-Seq, bulk RNA-Seq, and mass spectrometry-based proteomics to analyze the mouse kidney after acute AAI exposure. Our results reveal a dramatic reduction of proximal tubule epithelial cells, associated with apoptotic and inflammatory pathways, indicating permanent damage beyond repair. We found the enriched development pathways in other nephron segments, suggesting activation of reparative programs triggered by AAI. The divergent response may be attributed to the segment-specific distribution of organic anion channels along the nephron, including OAT1 and OAT3. Moreover, we observed dramatic activation and recruitment of cytotoxic T and macrophage M1 cells, highlighting inflammation as a principal contributor to permanent renal injury. Ligand-receptor pairing revealed that critical intercellular crosstalk underpins damage-induced activation of immune cells. These results provide potentially novel insight into the AAI-induced kidney injury and point out possible pathways for future therapeutic intervention.

Identification and Validation of Serum CST1 as a Diagnostic Marker for Differentiating Early-Stage Non-Small Cell Lung Cancer from Pulmonary Benign Nodules.

BACKGROUND: Effective means for early diagnosis are imperative to reduce death rate of non-small cell lung cancer (NSCLC) patients. We aimed to find out high-performance serologic markers to distinguish early-stage NSCLC patients from benign pulmonary nodule patients and healthy controls (HC). Cystatin-SN (CST1) is an active cysteine protease inhibitor of the CST superfamily, involving in the processes of inflammation and tumorigenesis. This is the first exploration of the diagnostic and prognostic values of serum CST1 in NSCLC. METHODS: We analyzed the transcriptome data from The Cancer Genome Atlas and the Gene Expression Omnibus database, screened biomarkers for NSCLC, and verified the candidate markers via the ONCOMINE database. Then, we performed ELISA, western blotting, and immunohistochemistry analysis to detect the expression levels of CST1 in NSCLC cell lines, tumor tissues, and serum samples of clinical cohorts. RESULTS: We identified 3 up-regulated secreted protein-encoding genes, validated the expression levels of CST1 in NSCLC tumor tissues and cell lines, and found that serum CST1 levels of NSCLC (4289 ± 2405 pg/mL) were significantly higher than those of PBN patients (1558 ± 441 pg/mL, P < .0001) and healthy controls (1529 ± 416 pg/mL, P < .0001). The AUC of the combination of CST1, Cytokeratin 19 fragment (Cyfra21-1), and Carcinoembryonic antigen (CEA) for distinguishing early-stage NSCLC from PBN/HC was as high as .914/0.925. Furthermore, our results suggested that the NSCLC patient with low serum CST1 level had a better survival rate. CONCLUSIONS: Serum CST1 may serve as a novel diagnostic marker for differentiating early-stage NSCLC from PBN and HC, and could be used as a prognosis predictor in NSCLC patients.

Single-Cell Transcriptome Analysis Reveals RGS1 as a New Marker and Promoting Factor for T-Cell Exhaustion in Multiple Cancers.

T-cell exhaustion is one of the main reasons of tumor immune escape. Using single-cell transcriptome data of CD8+ T cells in multiple cancers, we identified different cell types, in which Pre_exhaust and exhausted T cells participated in negative regulation of immune system process. By analyzing the coexpression network patterns and differentially expressed genes of Pre_exhaust, exhausted, and effector T cells, we identified 35 genes related to T-cell exhaustion, whose high GSVA scores were associated with significantly poor prognosis in various cancers. In the differentially expressed genes, RGS1 showed the greatest fold change in Pre_exhaust and exhausted cells of three cancers compared with effector T cells, and high expression of RGS1 was also associated with poor prognosis in various cancers. Additionally, RGS1 protein was upregulated significantly in tumor tissues in the immunohistochemistry verification. Furthermore, RGS1 displayed positive correlation with the 35 genes, especially highly correlated with PDCD1, CTLA4, HAVCR2, and TNFRSF9 in CD8+ T cells and cancer tissues, indicating the important roles of RGS1 in CD8+ T-cell exhaustion. Considering the GTP-hydrolysis activity of RGS1 and significantly high mRNA and protein expression in cancer tissues, we speculated that RGS1 potentially mediate the T-cell retention to lead to the persistent antigen stimulation, resulting in T-cell exhaustion. In conclusion, our findings suggest that RGS1 is a new marker and promoting factor for CD8+ T-cell exhaustion and provide theoretical basis for research and immunotherapy of exhausted cells.

AutoVEM2: A flexible automated tool to analyze candidate key mutations and epidemic trends for virus.

In our previous work, we developed an automated tool, AutoVEM, for real-time monitoring the candidate key mutations and epidemic trends of SARS-CoV-2. In this research, we further developed AutoVEM into AutoVEM2. AutoVEM2 is composed of three modules, including call module, analysis module, and plot module, which can be used modularly or as a whole for any virus, as long as the corresponding reference genome is provided. Therefore, it's much more flexible than AutoVEM. Here, we analyzed three existing viruses by AutoVEM2, including SARS-CoV-2, HBV and HPV-16, to show the functions, effectiveness and flexibility of AutoVEM2. We found that the N501Y locus was almost completely linked to the other 16 loci in SARS-CoV-2 genomes from the UK and Europe. Among the 17 loci, 5 loci were on the S protein and all of the five mutations cause amino acid changes, which may influence the epidemic traits of SARS-CoV-2. And some candidate key mutations of HBV and HPV-16, including T350G of HPV-16 and C659T of HBV, were detected. In brief, we developed a flexible automated tool to analyze candidate key mutations and epidemic trends for any virus, which would become a standard process for virus analysis based on genome sequences in the future.

Prediction and evolution of B cell epitopes of surface protein in SARS-CoV-2.

BACKGROUND: In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. METHODS: The analyses were related to epitopes on 3 proteins, including spike (S), envelope (E) and membrane (M) proteins, which are located on the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. RESULTS: 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all 6 identified epitopes were conserved in nearly 3500 SARS-CoV-2 genomes, showing that it is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, which deserved further study at the experiment level. CONCLUSION: The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.

Characterization of Glycolysis-Associated Molecules in the Tumor Microenvironment Revealed by Pan-Cancer Tissues and Lung Cancer Single Cell Data.

Altered metabolism is a hallmark of cancer and glycolysis is one of the important factors promoting tumor development. There is however still a lack of molecular characterization glycolysis and comprehensive studies related to tumor glycolysis in the pan-cancer landscape. Here, we applied a gene expression signature to quantify glycolysis in 9229 tumors across 25 cancer types and 7875 human lung cancer single cells and verified the robustness of signature using defined glycolysis samples from previous studies. We classified tumors and cells into glycolysis score-high and -low groups, demonstrated their prognostic associations, and identified genome and transcriptome molecular features associated with glycolysis activity. We observed that glycolysis score-high tumors were associated with worse prognosis across cancer types. High glycolysis tumors exhibited specific driver genes altered by copy number aberrations (CNAs) in most cancer types. Tricarboxylic acid (TCA) cycle, DNA replication, tumor proliferation and other cancer hallmarks were more active in glycolysis-high tumors. Glycolysis signature was strongly correlated with hypoxia signature in all 25 cancer tissues (r > 0.7) and cancer single cells (r > 0.8). In addition, HSPA8 and P4HA1 were screened out as the potential modulating factors to glycolysis as their expression were highly correlated with glycolysis score and glycolysis genes, which enables future efforts for therapeutic options to block the glycolysis and control tumor progression. Our study provides a comprehensive molecular-level understanding of glycolysis with a large sample data and demonstrates the hypoxia pressure, growth signals, oncogene mutation and other potential signals could activate glycolysis, thereby to regulate cell cycle, energy material synthesis, cell proliferation and cancer progression.