Journal: Frontiers in Immunology

Article Title: Single cell transcriptomics of bone marrow derived macrophages reveals Ccl5 as a biomarker of direct IFNAR-independent responses to DNA sensing

doi: 10.3389/fimmu.2023.1199730

Figure Lengend Snippet: Integrated single cell transcriptomes reveal cell-cycling and non-cycling BMDM subsets. (A) Bone marrow derived macrophages (BMDMs) were isolated from adult male C57BL6 and IFNAR KO mice and differentiated in culture with m-CSF. BMDM were stimulated for 24 hours with one of the following i) vehicle control, ii) dsDNA complexed to LTX transfection reagent, or iii) dsDNA + IFNAR Ab, or iv) dsDNA-transfected IFNAR KO. Cells were then collected, stained with DAPI, FACS sorted to isolate live single cells, and processed for single cell RNA-Seq barcoding and sequencing using established 10X Genomics protocols. Condition 1 was designed to define the transcriptional heterogeneity and BMDM subsets in the unstimulated state. Condition 2 was designed to define the transcriptional response of BMDM subsets to dsDNA, an inducer of primary and secondary type I IFN responses. Condition 3 was designed to block IFN-dependent secondary responses and isolate the direct effects of dsDNA. (B) Data from all three experimental conditions, excluding the IFNAR KO due to strain differences, was integrated and clustered. Unsupervised clustering of integrated data (n= 19,343cells) revealed at least 6 distinct BMDM subsets. Data is dimensionally reduced using UMAP and displayed on a 2D plot to communicate relative similarities in transcriptional profiles between BMDM subsets. (C) Identification of replicating BMDMs from clusters based on cell cycle phase (color-coded legend) using cell cycle sorting. (D) Heatmap of biological replicates averaged, scaled expression defining differentially expressed genes for each BMDM subset. Cell cycling subsets (clusters 1-2, red & orange) and non-cycling subsets (clusters 3-6, yellow, green, and blue) are annotated.

Article Snippet: Single cell RNA-Seq was performed by microfluidic droplet-based encapsulation, barcoding, and library preparation (10X Genomics) as previously described ( ).

Techniques: Derivative Assay, Isolation, Control, Transfection, Staining, RNA Sequencing, Sequencing, Blocking Assay, Expressing

Journal: Science immunology

Article Title: The myeloid type I interferon response to myocardial infarction begins in bone marrow and is regulated by Nrf2-activated macrophages

doi: 10.1126/sciimmunol.aaz1974

Figure Lengend Snippet: (A) Infarcted hearts D1, D2, and D4 after MI were enzymatically digested, FACS sorted (singlets, DAPI−Ter119−) and barcoded for single cell RNA sequencing using custom inDrop barcoding platform (N = 3 biological replicates per day post-MI; 10,666 cells). (B) UMAP of bioinformatically isolated and integrated neutrophils (S100a8, S100a9, Cxcr2, Csf3r)HI. (C) Spearman’s rank correlation coefficient comparing heart and blood neutrophil subsets. (D) Integrated heatmap. (E,F) Distribution of neutrophil subsets by time (data are shown as mean +/− S.E.M). (G) Violin plots of subclustered ISG− and ISG+ cells (D1, D2 and D4 post-MI combined). Top marker gene for each neutrophil subset shown. (H, I) Pseudotime trajectory (Monocle) on ISG− and ISG+ (H) and heatmap of neutrophil maturation and ISG expression vs pseudotime (I) from RetnlgHISiglecfLOW to RetnlgLOWSiglecfHI.

Article Snippet: Single Cell RNA-seq Single cell RNA-seq was performed by microfluidic droplet-based encapsulation, barcoding, and library preparation, (inDrop and 10X Genomics) as previously described ( 30 ).

Techniques: RNA Sequencing, Isolation, Hi-C, Marker, Expressing

Journal: Science immunology

Article Title: The myeloid type I interferon response to myocardial infarction begins in bone marrow and is regulated by Nrf2-activated macrophages

doi: 10.1126/sciimmunol.aaz1974

Figure Lengend Snippet: (A) Bone marrow and blood leukocytes from non- and post-infarcted WT (D0, D1, D2, D4) and Irf3−/− (D0, D2) mice were FACS sorted for single cell barcoding (10X Genomics, N = 1). Lin−Sca1+/−c-Kit+ progenitor cells from WT (pre- and D1 post-infarct) mice were also sorted for single cell barcoding (N = 1). (B) Integrated UMAP plot of 87,822 cells color coded by major lineage type as indicated in plot. (C,H) UMAP plots of clustered (C) neutrophils (39,611 cells) and (H) monocytes (20,295 cells). (D,I) Cluster membership by sample (WT only, normalized by column) for (D) neutrophils and (I) monocytes. (E,J) Feature plots of ISG scores of (E) neutrophils and (J) monocytes. (F,K) Violin plots of ISG scores by cluster of (F) neutrophil and (K) monocyte populations. (G, L) Heatmaps showing select differential expressed genes of (G) neutrophil and (L) monocyte subsets. (M) ISG scores from bone marrow and blood leukocytes on D0, D1, D2 after MI. (N) ISG scores of bone marrow and blood leukocytes from WT and Irf3−/− mice on D0 and D2 post-MI. Data are shown as mean +/− S.D. with individual points (cells). **** P< .0001, Kolmogorov-Smirnov test.

Article Snippet: Single Cell RNA-seq Single cell RNA-seq was performed by microfluidic droplet-based encapsulation, barcoding, and library preparation, (inDrop and 10X Genomics) as previously described ( 30 ).

Techniques: