Analysis Of Intercellular Communication Research Articles

Deciphering the immune-metabolic nexus in sepsis: a single-cell sequencing analysis of neutrophil heterogeneity and risk stratification.

Metabolic dysregulation following sepsis can significantly compromise patient prognosis by altering immune-inflammatory responses. Despite its clinical relevance, the exact mechanisms of this perturbation are not yet fully understood. Single-cell RNA sequencing (scRNA-seq) was utilized to map the immune cell landscape and its association with metabolic pathways during sepsis. This study employed cell-cell interaction and phenotype profiling from scRNA-seq data, along with pseudotime trajectory analysis, to investigate neutrophil differentiation and heterogeneity. By integrating scRNA-seq with Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning techniques, key genes were identified. These genes were used to develop and validate a risk score model and nomogram, with their efficacy confirmed through Receiver Operating Characteristic (ROC) curve analysis. The model's practicality was further reinforced through enrichment and immune characteristic studies based on the risk score and in vivo validation of a critical gene associated with sepsis. The complex immune landscape and neutrophil roles in metabolic disturbances during sepsis were elucidated by our in-depth scRNA-seq analysis. Pronounced neutrophil interactions with diverse cell types were revealed in the analysis of intercellular communication, highlighting pathways that differentiate between proximal and core regions within atherosclerotic plaques. Insight into the evolution of neutrophil subpopulations and their differentiation within the plaque milieu was provided by pseudotime trajectory mappings. Diagnostic markers were identified with the assistance of machine learning, resulting in the discovery of PIM1, HIST1H1C, and IGSF6. The identification of these markers culminated in the development of the risk score model, which demonstrated remarkable precision in sepsis prognosis. The model's capability to categorize patient profiles based on immune characteristics was confirmed, particularly in identifying individuals at high risk with suppressed immune cell activity and inflammatory responses. The role of PIM1 in modulating the immune-inflammatory response during sepsis was further confirmed through experimental validation, suggesting its potential as a therapeutic target. The understanding of sepsis immunopathology is improved by this research, and new avenues are opened for novel prognostic and therapeutic approaches.

A specific inflammatory suppression fibroblast subpopulation characterized by MHCII expression in human dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a significant cause of heart failure that requires heart transplantation. Fibroblasts play a central role in the fibro-inflammatory microenvironment of DCM. However, their cellular heterogeneity and interaction with immune cells have not been well identified. An integrative analysis was conducted on single-cell RNA sequencing (ScRNA-Seq) data from human left ventricle tissues, which comprised 4 hearts from healthy donors and 6 hearts with DCM. The specific antigen-presenting fibroblast (apFB) was explored as a subtype of fibroblasts characterized by expressing MHCII genes, the existence of which was confirmed by immunofluorescence staining of 3 cardiac tissues from DCM patients with severe heart failure. apFB highly expressed the genes that response to IFN-γ, and it also have a high activity of the JAK-STAT pathway and the transcription factor RFX5. In addition, the analysis of intercellular communication between apFBs and CD4+T cells revealed that the anti-inflammatory ligand-receptor pairs TGFB-TGFR, CLEC2B-KLRB1, and CD46-JAG1 were upregulated in DCM. The apFB signature exhibited a positive correlation with immunosuppression and demonstrated diagnostic and prognostic value when evaluated using a bulk RNA dataset comprising 166 donors and 166 DCM samples. In conclusion, the present study identified a novel subpopulation of fibroblasts that specifically expresses MHCII-encoding genes. This specific apFBs can suppress the inflammation occurring in DCM. Our findings further elucidate the composition of the fibro-inflammatory microenvironment in DCM, and provide a novel therapeutic target.

Revealing the role of necroptosis microenvironment: FCGBP + tumor-associated macrophages drive primary liver cancer differentiation towards cHCC-CCA or iCCA.

Previous research has demonstrated that the conversion of hepatocellular carcinoma (HCC) to intrahepatic cholangiocarcinoma (iCCA) can be stimulated by manipulating the tumor microenvironment linked with necroptosis. However, the specific cells regulating the necroptosis microenvironment have not yet been identified. Additionally, further inquiry into the mechanism of how the tumor microenvironment regulates necroptosis and its impact on primary liver cancer(PLC) progression may be beneficial for precision therapy. We recruited a single-cell RNA sequencing dataset (scRNA-seq) with 34 samples from 4 HCC patients and 3 iCCA patients, and a Spatial Transcriptomic (ST) dataset including one each of HCC, iCCA, and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). Quality control, dimensionality reduction and clustering were based on Seurat software (v4.2.2) process and batch effects were removed by harmony (v0.1.1) software. The pseudotime analysis (also known as cell trajectory) in the single cell dataset was performed by monocle2 software (v2.24.0). Calculation of necroptosis fraction was performed by AUCell (v1.16.0) software. Switch gene analysis was performed by geneSwitches(v0.1.0) software. Dimensionality reduction, clustering, and spatial image in ST dataset were performed by Seurat (v4.0.2). Tumor cell identification, tumor subtype characterization, and cell type deconvolution in spot were performed by SpaCET (v1.0.0) software. Immunofluorescence and immunohistochemistry experiments were used to prove our conclusions. Analysis of intercellular communication was performed using CellChat software (v1.4.0). ScRNA-seq analysis of HCC and iCCA revealed that necroptosis predominantly occurred in the myeloid cell subset, particularly in FCGBP + SPP1 + tumor-associated macrophages (TAMs), which had the highest likelihood of undergoing necroptosis. The existence of macrophages undergoing necroptosis cell death was further confirmed by immunofluorescence.Regions of HCC with poor differentiation, cHCC-CCA with more cholangiocarcinoma features, and the tumor region of iCCA shared spatial colocalization with FCGBP + macrophages, as confirmed by spatial transcriptomics, immunohistochemistry and immunofluorescence. Pseudotime analysis showed that premalignant cells could progress into two directions, one towards HCC and the other towards iCCA and cHCC-CCA. Immunofluorescence and immunohistochemistry experiments demonstrated that the number of macrophages undergoing necroptosis in cHCC-CCA was higher than in iCCA and HCC, the number of macrophages undergoing necroptosis in cHCC-CCA with cholangiocarcinoma features was more than in cHCC-CCA with hepatocellular carcinoma features. Further investigation showed that myeloid cells with the highest necroptosis score were derived from the HCC_4 case, which had a severe inflammatory background on pathological histology and was likely to progress towards iCCA and cHCC-CCA. Switchgene analysis indicated that S100A6 may play a significant role in the progression of premalignant cells towards iCCA and cHCC-CCA. Immunohistochemistry confirmed the expression of S100A6 in PLC, the more severe inflammatory background of the tumor area, the more cholangiocellular carcinoma features of the tumor area, S100A6 expression was higher. The emergence of necroptosis microenvironment was found to be significantly associated with FCGBP + SPP1 + TAMs in PLC. In the presence of necroptosis microenvironment, premalignant cells appeared to transform into iCCA or cHCC-CCA. In contrast, without a necroptosis microenvironment, premalignant cells tended to develop into HCC, exhibiting amplified stemness-related genes (SRGs) and heightened malignancy.

Exploring key genes associated with neutrophil function and neutrophil extracellular traps in heart failure: a comprehensive analysis of single-cell and bulk sequencing data

Background: Heart failure (HF) is a complex and heterogeneous manifestation of multiple cardiovascular diseases that usually occurs in the advanced stages of disease progression. The role of neutrophil extracellular traps (NETs) in the pathogenesis of HF remains to be explored.Methods: Bioinformatics analysis was employed to investigate general and single-cell transcriptome sequencing data downloaded from the GEO datasets. Differentially expressed genes (DEGs) associated with NETs in HF patients and healthy controls were identified using transcriptome sequencing datasets and were subsequently subjected to functional enrichment analysis. To identify potential diagnostic biomarkers, the random forest algorithm (RF) and the least absolute shrinkage and selection operator (LASSO) were applied, followed by the construction of receiver operating characteristic (ROC) curves to assess accuracy. Additionally, single-cell transcriptome sequencing data analysis identified key immune cell subpopulations in TAC (transverse aortic constriction) mice potentially involved in NETs regulation. Cell-cell communication analysis and trajectory analysis was then performed on these key cell subpopulations.Results: We identified thirteen differentially expressed genes (DEGs) associated with NET through differential analysis of transcriptome sequencing data from HF (heart failure) samples. Utilizing the Random Forest and Lasso algorithms, along with experimental validation, we successfully pinpointed four diagnostic markers (CXCR2, FCGR3B, VNN3, and FPR2) capable of predicting HF risk. Furthermore, our analysis of intercellular communication, leveraging single-cell sequencing data, highlighted macrophages and T cells as the immune cell subpopulations with the closest interactions with neutrophils. Pseudo-trajectory analysis sheds light on the differentiation states of distinct neutrophil subpopulations.Conclusion: In this study, we conducted an in-depth investigation into the functions of neutrophil subpopulations that infiltrate cardiac tissue in TAC mice. Additionally, we identified four biomarkers (CXCR2, FCGR3B, VNN3, and FPR2) associated with NETs in HF. Our findings enhance the understanding of immunology in HF.

Identification of Cell-Cell Communications by Single-Cell RNA Sequencing in End Stage Renal Disease Provides New Insights into Immune Cell Heterogeneity.

Impaired immune system characterized by low-grade inflammation is closely associated with kidney chronic kidney disease (CKD) progression. To reveal the alterations of the function, component, and intercellular communication of immune cells during the progression of CKD. We conducted a case-control study enrolling regular hemodialysis patients and healthy controls. Clinical data, serum and peripheral blood mononuclear cell (PBMC) samples were collected. Flow cytometry and single-cell RNA sequencing were performed to quantitatively analyze the immune cell subsets and T-cell subsets of PBMCs. scRNA data of GSE140023 containing mouse unilateral ureteral obstruction (UUO) models were analyzed the heterogeneity of immune cells. Overall reduction in peripheral blood lymphocyte subsets in patients with end-stage renal disease (ESRD) was observed. A higher ratio of Th17/Treg, Th1/Treg, and b-cell/Treg in the ESRD group was associated with a decrease in eGFR, PTH, and ferritin. Among T cell subsets identified by scRNA analysis, Th17 cells were significantly increased in the ESRD and UU0 group. TFH, Th1, and Th2 cells are located at the final stage in the developmental tree, while Treg and memory CD8+ T cells are at the beginning site. Early developmental differentiation of Th17, Th1, and Tfh cells was observed in the ESRD and UUO group. Analysis of intercellular communication between t-cell subpopulations identified two major input and output signaling pathways: the CD40 and macrophage inhibitory factor (MIF) pathways. The MIF signaling pathway primarily mediates intercellular communication among th17 effects, CD8+ t-cell, and Th17-Treg in the ESRD group, the serum level of MIF showed significant upregulation, which was closely related to Th17/Treg cells. A global immune imbalance was closely associated with the deterioration in renal function and complication development. The MIF signaling pathway mediates Th17/Treg communication and promotes the trans-differentiation of Treg cells to Th17 cells in CKD progression.

Abstract EC.09: Single Nuclei RNA Sequencing Analysis Of Mybpc-3- Associated HCM To Identify Common Pathological Mechanisms In Human, Feline, And Murine Hearts

Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease characterized by unexplained hypertrophy, often associated with left ventricular outflow tract (LVOT) obstruction, ventricular tachyarrhythmias, diastolic heart failure, and sudden cardiac death. Mutations in mybpc3 are the most common genetic mutations associated with human and feline HCM. To understand how mutations in mybc3 causing sarcomere dysfunction result in the development of non-myocyte phenotypes such as fibrosis, mitral valve anomalies, and microvascular occlusion across species, we generated single nuclei RNA-sequencing datasets from feline, human and murine heart tissue carrying mybpc3 variants. We analyzed these datasets for differential gene expression by genotype for each cell type and assessed changes in cell-cell communication by ligand-receptor analysis. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in feline cardiomyocytes revealed alteration in regulation of calcium ion transmembrane transporter activity, regulation of cation channel activity, and extracellular matrix structure and organization, consistent with known phenotypic aspects of HCM such as calcium dyshomeostasis and fibrosis. DEGs in cardiomyocytes of human tissues revealed novel obstruction of cGMP-mediated signaling, protein O-linked glycosylation and negative regulation of cAMP-mediated signaling. DEGs in murine cardiomyocytes affected cardiac muscle thin filament assembly, CDP-diacylglycerol biosynthesis and cell adhesion molecule production. Analysis of intercellular communication in feline and murine hearts revealed an overall loss of ligands broadcast from all cell types, with notable losses from fibroblast cells and cardiomyocytes. These findings are consistent with recent studies implicating altered intercellular communication between cardiomyocytes and non-myocyte cell types as a potential disease mechanism in human HCM. We will further identify common and species-specific disease-promoting pathways in HCM tissue that will allow identification of conserved disease mechanisms that may be targeted in both human and feline disease and will facilitate an understanding of the relevance of mouse models.

Analysis of intercellular communication in the osteosarcoma microenvironment based on single cell sequencing data

Osteosarcoma (OS) is the most common primary bone cancer in children and young adults, patient survival rates have not improved in recent decades. To further understand the interrelationship between different cell types in the tumor microenvironment of osteosarcoma, we comprehensively analyzed single-cell sequencing data from six patients with untreated osteosarcoma. Nine major cell types were identified from a total of 46,046 cells based on unbiased clustering of gene expression profiles and canonical markers. Osteosarcoma from different patients display heterogeneity in cellular composition. Myeloid cells were the most commonly represented cell type, followed by osteoblastic and TILs. Copy number variation (CNV) results identified amplifications and deletions in malignant osteoblastic cells and fibroblasts. Trajectory analysis based on RNA velocity showed that osteoclasts in the OS microenvironment could be differentiated from myeloid cells. Furthermore, we explored the intercellular communications in OS microenvironment and identified multiple ligand-receptor pairs between myeloid cells, osteoblastic cells and their cells, including 21 ligand-receptor pair genes that significantly associated with survival outcomes. Importantly, we found chemotherapy may have an effect on cellular communication in the OS microenvironment by analyzing single-cell sequencing data from seven primary osteosarcoma patients who received chemotherapy. We believe these observations will improve our understanding of potential mechanisms of microenvironment contributions to OS progression and help identify potential targets for new treatment development in the future.

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

BackgroundCancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.MethodsIn this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.ResultsWe discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.ConclusionsOur study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

Interaction of vitamin D with membrane-based signaling pathways

Many studies in different biological systems have revealed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) modulates signaling pathways triggered at the plasma membrane by agents such as Wnt, transforming growth factor (TGF)-β, epidermal growth factor (EGF), and others. In addition, 1α,25(OH)2D3 may affect gene expression by paracrine mechanisms that involve the regulation of cytokine or growth factor secretion by neighboring cells. Moreover, post-transcriptional and post-translational effects of 1α,25(OH)2D3 add to or overlap with its classical modulation of gene transcription rate. Together, these findings show that vitamin D receptor (VDR) cannot be considered only as a nuclear-acting, ligand-modulated transcription factor that binds to and controls the transcription of target genes. Instead, available data support the view that much of the complex biological activity of 1α,25(OH)2D3 resides in its capacity to interact with membrane-based signaling pathways and to modulate the expression and secretion of paracrine factors. Therefore, we propose that future research in the vitamin D field should focus on the interplay between 1α,25(OH)2D3 and agents that act at the plasma membrane, and on the analysis of intercellular communication. Global analyses such as RNA-Seq, transcriptomic arrays, and genome-wide ChIP are expected to dissect the interactions at the gene and molecular levels.

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

Intercellular Ca(2+) waves are propagation of Ca(2+) transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca(2+) waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca(2+) signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5'-triphosphate successfully induced the propagation of intercellular Ca(2+) waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.