{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Siddharth Jhunjhunwala"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16194"],"description":["This study investigates the mechanism underlying increased morbidity in individuals with chronic inflammatory conditions upon a secondary inflammatory challenge. Using mouse models, we identified a pronounced expansion of circulating immature neutrophils during chronic inflammation via single-cell RNA sequencing. Functional assays revealed these neutrophils are dysregulated. We established their causal role in exacerbating immunopathology and demonstrated that therapeutic intervention with antibodies or small molecules to block their migration or function significantly reduces tissue damage. This work defines a pathological cell population and proposes a novel therapeutic strategy for patients with chronic inflammatory diseases."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Treatment - 2) Acute Lung Injury: Mice were anesthetized with ketamine (120 mg/kg) and xylazine (40 mg/kg) and placed on a board at an angle of 50 degrees in a supine position. The tongue was carefully pulled using sterile forceps, and the chest was illuminated using a cold source light to visualize the tracheal opening. IV cannula (22G) was inserted into the trachea. LPS (0.4 mg/kg, Escherichia coli O127:B8; Sigma-Aldrich, Merck) was administered through the cannula in 25 μL of sterile saline using a 200 μL pipette tip. The mice were gently massaged and allowed to recover on a heating pad.","Sample Collection - Mice were first anesthetized using high dose ketamine-xylazine solution. After anesthesia, about 500-1000 μL blood was collected through the retro-orbital vein. Following euthanasia, bronchoalveolar lavage fluid (BALF) was collected using a 20-G cannula was inserted into the trachea and fixed with a 93 ligature. Lungs were then lavaged with 1 mL sterile Ice-cold 1X phosphate buffered saline (PBS) two times (total volume 2 mL) to collect BALF. Fluid recovery was >85% each time. Blood and BALF samples were then centrifuged at 400 RCF for 5 mins at 4°C. The supernatant was stored at -80°C and the cell pellet was subjected to RBC lysis in ACK Lysis buffer and resuspended in 1mL PBS for counting using a hemacytometer.","Sequencing - Sequencing was performed by Eurofin Advinus (Bengaluru, India). Sequencing was performed using Illumina NovaSeq 6000","Nucleic Acid Extraction - Immune cells from blood and BALF were isolated as described in earlier sections. Sample tagging was performed by labeling cells with BD® Mouse Single-Cell Multiplexing Kit. Neutrophils in the samples were tagged with Ly6G antibody-derived tags (Ly6G-ADTs, BD® AbSeq Ab-Oligos). These cells were subjected to single cell capture and cDNA Synthesis, library construction using a Whole Transcriptome Analysis (WTA, BD) kit with the BD Rhapsody™ single-cell analysis system following standard procedures provided by the manufacturer","Sample Treatment - 1) Biomaterial implantation: Chitosan microspheres (337± 47 micrometers) were prepared using the Spraybase® Electrospray system inside a sterile biosafety cabinet and chemically sterilized as described22. Microspheres were surgically implanted into the peritoneal cavity of mice to induce chronic inflammation. In brief, microspheres (450 µL) were suspended in 450 µL sterile saline for implantation. The entire volume (900 µL) was then implanted into the peritoneal space22,27 following a laparotomy procedure. In controls (also called mock-surgery), the animals went through the surgical procedure and were injected with 900 µL sterile saline.","Library Construction - Sample tagging was performed by labeling cells with BD® Mouse Single-Cell Multiplexing Kit. Neutrophils in the samples were tagged with Ly6G antibody-derived tags (Ly6G-ADTs, BD® AbSeq Ab-Oligos). These cells were subjected to single cell capture and cDNA Synthesis, library construction using a Whole Transcriptome Analysis (WTA, BD) kit with the BD Rhapsody™ single-cell analysis system following standard procedures provided by the manufacturer."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Pre-processing of the raw FASTQ files obtained from the BD Rhapsody™ single-cell analysis system, including read quality filtering, alignment, and quantification, was performed on the Seven Bridges platform using the BD Rhapsody™ sequence analysis pipeline with default references including RhapRef_Mouse_WTA_2023-02 for mRNA and Mouseneutrophil_Ly6G for AbSeq. Further, downstream analyses were performed using R 4.4.1 and Seurat 5.1.0. Cells marked \\\"Undetermined\\\" or \\\"Multiplet\\\" by BD Rhapsody™ Sequence Analysis Pipeline, cells failing to have a minimum of 300 genes (min.features = 300), and genes that were not present in at least three cells (min.cells = 3) were filtered out. To retain only high-quality cells, cells with nFeature_RNA in the range 500-4500 and 500-8000, nCount_RNA in the range 1000-25000 and 1000-50000 for blood and BALF, respectively, and mitochondrial percentage (percent.mt) < 15 in all groups were used for downstream analyses.  This filtering led to a total of 9843 cells from the single inflammation model of blood (3506 mock-treated with an average of 2560 genes/cell, 6337 chitosan implanted with an average of 2601 genes/cell), and 6843 (2577 mock-treated with an average of 2132 genes/cell, 2075 chitosan implanted with an average of 2075 genes/cell) and 9772 (5548 mock-treated with an average of 3302 genes/cell, 4224 chitosan implated with an average of 3695 genes/cell) cells from the double inflammation model of blood and BALF, respectively."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Mus musculus"],"pubmed_authors":["Siddharth Jhunjhunwala","Vinod Kumar Dorai"],"additional_accession":[]},"is_claimable":false,"name":"ScRNA-seq of Neutrophils upon acute lung injury in a biomaterial-induced chronic inflammatory mouse model","description":"This study investigates the mechanism underlying increased morbidity in individuals with chronic inflammatory conditions upon a secondary inflammatory challenge. Using mouse models, we identified a pronounced expansion of circulating immature neutrophils during chronic inflammation via single-cell RNA sequencing. Functional assays revealed these neutrophils are dysregulated. We established their causal role in exacerbating immunopathology and demonstrated that therapeutic intervention with antibodies or small molecules to block their migration or function significantly reduces tissue damage. This work defines a pathological cell population and proposes a novel therapeutic strategy for patients with chronic inflammatory diseases.","dates":{"release":"2025-12-14T00:00:00Z","modification":"2026-05-27T14:40:24.049Z","creation":"2025-11-19T11:31:21.222Z"},"accession":"E-MTAB-16194","cross_references":{"ENA":["ERP185322"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184","EFO_0003969"]}}