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Spatial transcriptomics Visium data for mouse bleomycin model of lung fibrosis

ABSTRACT: Visium (10x Genomics) spatially resolved transcriptomics data generated from the mouse model of bleomycin (BLM)-induced lung fibrosis, with lung tissue collected at 7 or 21 days post single-dose BLM (40 µg/mouse, o.p) or saline vehicle challenge. Data from a total of 24 tissue sections, from 18 unique mouse left lung lobes. The mice were all female C57BL/6NCrl, purchased from Charles River (Germany), and were 8 weeks + 5 days old at the time of study initiation. Animal handling conformed to standards established by the Council of Europe ETS123 AppA, the Helsinki Convention for the Use and Care of Animals, Swedish legislation, and AstraZeneca global internal standards. All mouse experiments were approved by the Gothenburg Ethics Committee for Experimental Animals in Sweden and conformed to Directive 2010/63/EU. The present study was approved by the local Ethical committee in Gothenburg (EA000680-2017) and the approved site number is 31-5373/11. Data included in this repository: - Visium data in the format of selected Space Ranger output files ("filtered_feature_bc_matrix.h5", "raw_feature_bc_matrix.h5", "metrics_summary.csv", and the "spatial/" folder) for each individual section analysed. Zipped into one folder: "mm_visium_spaceranger_output.zip" - Sample metadata containing information for each sample with linked subject information: "mm_visium_metadata.tsv" - R objects produced using STUtility and contains the processed data used for downstream analyses, most importantly all spot metadata with assigned data and deconvolution results (NMF, cell2location): "mm_visium_all_stutility_obj.rds" (all), "mm_visium_d7_stutility_obj.rds " (day 7 data), "mm_visium_d21_stutility_obj.rds" (day 21 data) - Cell2location output files ("*_spot_cell_abundances_5pc.csv"), zipped into one folder: "cell2location_strunz2020.zip" - Full resolution H&E images ("*.jpg") of each tissue section that was used as input for spaceranger together with alignment json and sequencing fastq files. Zipped into one folder: "he_fullres_jpgs.zip" - Spot alignment files ("*.json") created in Loupe Browser using the corresponding full resolution H&E image in which spots under the tissue was identified. Zipped into one folder: "loupe_alignment_jsons.zip" Space Ranger output found within the zipped files in folders named "V*****-***-*1". To generate these files, raw FastQ files from the NovaSeq sequencing were processed with the Space Ranger pipeline (v. 1.2.2, 10x Genomics), where the reads were mapped to the mm10 reference genome. Manual spot alignment was performed in the Loupe Browser (v. 6, 10x Genomics) software. Cell type mapping results were obtained using the cell2location (v. 0.1) method, integrating the Space Ranger output data with annotated single cell RNA-seq data produced from mouse BLM injured lungs collected at multiple time points, published by Strunz et al., 2020 (DOI: 10.1038/s41467-020-17358-3, GEO accession: GSE141259). Seurat/STUtility objects were generated from the Space Ranger output files, using the R packages STUtility (v. 1.1.1) and Seurat (v. 4.1.1) in R (v. 4.0.5). The d7 and d21 subset were derived from the object with all samples processed jointly, and thereafter NMF deconvolution and subsequent clustering (d21) was performed. All R scripts used for the data analyses can be found at https://github.com/lfranzen/spatial-lung-fibrosis. The deposited data is presented in the article "Mapping spatially resolved transcriptomes in human and mouse pulmonary fibrosis" by Franzén L & Olsson Lindvall M, et al. (preprint: "Translational mapping of spatially resolved transcriptomes in human and mouse pulmonary fibrosis", bioRxiv, https://doi.org/10.1101/2023.12.21.572330).

ORGANISM(S): Mus musculus (mouse)

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PROVIDER: S-BSST1409 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:IntroductionIn lung fibrosis, alveolar epithelium degenerates progressively. The goal of regenerative medicine is to aid repair and regeneration of the lost tissues in parenchyma and airways for which mobilization of tissue-resident endogenous or bone marrow-derived exogenous stem cells niches is a critical step. We used a lung injury model in mice to identify and characterize functional lung stem cells to clarify how stem cell niches counteract this degenerative process.MethodsShort term assay (STA) - Bleomycin-induced lung inflammation and fibrosis were assessed in a model of idiopathic pulmonary fibrosis in wild-type (WT), gp91phox-/- (NOX-/-), and gp91phoxMMP-12 double knockout (DKO) mice on C57Bl/6 background and Hoechst 33322 dye effluxing side population (SP) cells characterized. Long term assay (LTA) - In a bleomycin induced lung fibrosis model in C57Bl6 mice, the number of mature cells were quantified over 7, 14, and 21 days in bone marrow (BM), peripheral blood (PB), lung parenchyma (LP) and bronchoalveolar lavage (BAL) fluid by FACS. BrdU pulse chase experiment (10 weeks) was used to identify label retaining cells (LRC). BrdU+ and BrdU- cells were characterized by hematopoietic (CD45+), pluripotency (TTF1+, Oct3/4+, SSEA-3+, SSEA-4+, Sca1+, Lin-, CD34+, CD31+), and lung lineage-specific (SPC+, AQP-5+, CC-10+) markers. Clonogenic potential of LRCs were measured by CFU-c assays.ResultsSTA- In lung, cellularity increased by 5-fold in WT and 6-fold in NOX-/- by d7. Lung epithelial markers were very low in expression in all SP flow sorted from lung of all three genotypes cultured ex vivo. (p < 0.01). Post-bleomycin, the SP in NOX-/- lung increased by 3.6-fold over WT where it increased by 20-fold over controls. Type I and II alveolar epithelial cells progressively diminished in all three genotypes by d21 post-bleomycin. D7 post-bleomycin, CD45+ cells in BALf in NOX-/- was 1.7-fold > WT, 57% of which were Mf that decreased by 67% in WT and 83% in NOX-/- by d21.LTA- Cellularity as a factor of time remained unchanged in BM, PB, LP and BAL fluid. BrdU+ (LRC) were the putative stem cells. BrdU+CD45+ cells increased by 0.7-fold and SPC+CC10+ bronchoalveolar stem cells (BASC), decreased by ~40-fold post-bleomycin. BrdU+VEGF+ cells decreased by 1.8-fold while BrdU-VEGF+ cells increased 4.6-fold. Most BrdU- cells were CD45-. BrdU- BASCs remained unchanged post-bleomycin. CFU-c of the flow-sorted BrdU+ cells remained similar in control and bleomycin-treated lungs.ConclusionSTA- Inflammation is a pre-requisite for fibrosis; SP cells, being the putative stem cells in the lungs, were increased (either by self renewal or by recruitment from the exogenous bone marrow pool) post-bleomycin in NOX-/- but not in DKO indicating the necessity of cross-talk between gp91phox and MMP-12 in this process; ex vivo cultured SP progressively lose pluripotent markers, notably BASC (SPC+CC10+) - significance is unknown. LTA- The increase in the hematopoietic progenitor pool in lung indicated that exogenous progenitors from circulation contribute to lung regeneration. Most non-stem cells were non-hematopoietic in origin indicating that despite tissue turnover, BASCs are drastically depleted possibly necessitating recruitment of progenitors from the hematopoietic pool. Loss of VEGF+ LRC may indicate a signal for progenitor mobilization from niches. BrdU- BASC population may be a small quiescent population that remains as a reserve for more severe lung injury. Increase in VEGF+ non-LRC may indicate a checkpoint to counterbalance the mobilization of VEGF+ cells from the stem cell niche.

Project description:Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the aberrant accumulation of extracellular matrix in the lungs. nintedanib is one of the two FDA-approved drugs for IPF treatment; however, the exact pathophysiological mechanisms of fibrosis progression and response to therapy are still poorly understood. In this work, the molecular fingerprint of fibrosis progression and response to nintedanib treatment have been investigated by mass spectrometry-based bottom-up proteomics in paraffin-embedded lung tissues from bleomycin-induced (BLM) pulmonary fibrosis mice. Our proteomics results unveiled that (i) samples clustered depending on the tissue fibrotic grade (mild, moderate, and severe) and not on the time course after BLM treatment; (ii) the dysregulation of different pathways involved in fibrosis progression such as the complement coagulation cascades, advanced glycation end products (AGEs) and their receptors (RAGEs) signaling, the extracellular matrix-receptor interaction, the regulation of actin cytoskeleton, and ribosomes; (iii) Coronin 1A (Coro1a) as the protein with the highest correlation when evaluating the progression of fibrosis, with an increased expression from mild to severe fibrosis; and (iv) a total of 10 differentially expressed proteins (padj-value ≤ 0.05 and Fold change ≤-1.5 or ≥1.5), whose abundance varied in the base of the severity of fibrosis (mild and moderate), were modulated by the antifibrotic treatment with nintedanib, reverting their trend. Notably, nintedanib significantly restored lactate dehydrogenase B (Ldhb) expression but not lactate dehydrogenase A (Ldha). Notwithstanding the need for further investigations to validate the roles of both Coro1a and Ldhb, our findings provide an extensive proteomic characterization with a strong relationship with histomorphometric measurements. These results unveil some biological processes in pulmonary fibrosis and drug-mediated fibrosis therapy.

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Spatial transcriptomics Visium data for mouse bleomycin model of lung fibrosis

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