Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet: CD163 + macrophages accumulate in the lung in severe COVID-19 (A) Overview of study design and analyses. CT, computed tomography; BAL, bronchoalveolar lavage; scRNA-seq, single-cell RNA sequencing; snRNA-seq, single-nucleus RNA sequencing; IHC, immunohistochemistry; IF, immunofluorescence microscopy; MELC, multi-epitope ligand cartography; EM, electron microscopy; VCin, inspiratory vital capacity; PBMC, peripheral blood mononuclear cells; IAV, Influenza A virus. (B) Postmortem analysis of consecutive histological sections of non-COVID-19 (left) and COVID-19 autopsy lung samples (right) by hematoxylin and eosin (H&E; top) and CD68 IHC (bottom). Scale bar, 100 μm. (C) IF of CD68 (green) and CD163 (red) in lung tissue autopsy samples of COVID-19 patients and non-COVID-19 controls. Arrows indicate CD68 + CD163 – macrophages, and arrowheads indicate CD68 + CD163 + macrophages. Scale bar, 20 μm. (D) Quantification of CD68 + macrophage density (left) and the proportion of CD163 + macrophages (right) in lung autopsy samples from fifteen donors (as in C). Mann-Whitney test; ∗ p < 0.05. (E) Representative images of consecutive histological sections of lung autopsy samples. H&E (left), CD68 IHC (middle), and SARS-CoV-2 RNA-FISH (right). Arrowheads indicate SARS-CoV-2 RNA-positive macrophages. Scale bars, 50 μm, 25 μm. RNA-FISH, RNA-fluorescence in situ hybridization. (F) Lung autopsy samples of 9 COVID-19 patients were analyzed by MELC with a panel of 22 markers on 19 fields of view (FOVs). Two-dimensional embedding computed by UMAP on 9,684 computationally identified CD45 positive cells (T cells, CD3 + ; B cells, CD20 + ; NK cells, CD56 + ; neutrophils, MRP14 + /CD66b + ; monocytes, MRP14 + /CCR2 + ; macrophages, MRP14 + /HLA-DR + ). (G) Relative proportion (of total CD45 + cells) of cell types in all 19 FOVs (left), and average cell numbers (summary, right).

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Computed Tomography, RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, Electron Microscopy, MANN-WHITNEY, Fluorescence, In Situ Hybridization

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet:

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Immunohistochemistry, Plasmid Preparation, Recombinant, Staining, Lysis, Protease Inhibitor, Mass Spectrometry, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Journal of Biological Chemistry

Article Title: B Cell Signaling Is Regulated by Induced Palmitoylation of CD81

doi: 10.1074/jbc.m404410200

Figure Lengend Snippet: FIG. 1. Coligation of the BCR and the CD19/CD21/CD81 complex on Daudi B cells induces the stable partitioning of the coligated complex into lipid rafts in a cholesterol-dependent fashion. Daudi B cells were incubated for 30 min at 4 °C either in medium alone (untreated) or media containing biotinylated Abs specific for Ig alone (BCR-cross-linked) or with Abs specific for Ig and CD19 (BCR-CD19 coligated), washed, and incubated with avidin for 30 min at 4 °C. The cells were lysed either immediately at 4 °C in 1% Triton X-100 detergent (0 m) or warmed to 37 °C for 60 min and then lysed (60 m). The soluble and insoluble membranes were separated on discontinuous sucrose density gradients, and the resulting gradient fractions were analyzed by SDS-PAGE and immunoblotting probing for CD19, CD81, Ig, Ig, Lyn, and CD45. To determine the cholesterol dependence of the partitioning of the CD19/CD21 complex into detergent insoluble membranes, Daudi cells were pretreated with MCD (10 mM) for 15 min at 37 °C to remove membrane cholesterol before coligating the BCR and CD19/CD21/CD81 complex. To determine the reversibility of the MCD treatment, MCD-treated cells were incubated in cholesterol-containing medium for 3 h at 37 °C prior to coligation.

Article Snippet: Wild type Daudi cells were cultured in IMDM (ATCC, Manassas, VA) supplemented with 10% fetal calf serum (Invitrogen) and penicillin (100 units/ml) and streptomycin (100 g/ml) (Sigma).

Techniques: Incubation, Avidin-Biotin Assay, SDS Page, Western Blot, Membrane

Journal: Journal of Biological Chemistry

Article Title: B Cell Signaling Is Regulated by Induced Palmitoylation of CD81

doi: 10.1074/jbc.m404410200

Figure Lengend Snippet: FIG. 4. The coligation-induced palmitoylation of CD81 is selective and transient and occurs in rafts. The Daudi cells were serum- starved for 1 h and incubated with 125IC16 for 2 h at 37 °C as described for Fig. 3. A, Daudi B cells were either untreated, treated to coligate the BCR and the CD19/CD21/CD81 complex (COL) as described for Fig. 3, or treated with biotinylated Abs specific for Ig and avidin to cross-link the BCR alone (BCR-XL). The cells were lysed in Triton X-100 at 4 °C and CD81, Lyn, and CD45 immunoprecipitated from the lysates. The 125IC16 associated with the immunoprecipitates was determined. Shown are average values for three separate experiments. B, Daudi B cells were either untreated or treated to coligate the BCR and CD19/CD21/CD81 complex as described for Fig. 3 and lysed either immediately following ligation (COL (0h)) or incubated at 37 °C for 1 h (COL (1h)). The cells were lysed in 1% Triton X-100 at 4 °C and the lysates subjected to discontinuous sucrose gradient centrifugation. CD81 was immunoprecipitated from pooled gradient fractions in RIPA buffer and the 125IC16 associated with the resulting CD81 immunoprecipitates determined. An aliquot of the pooled gradient fractions 3–6 in RIPA buffer was immunoprecipitated using an isotype-matched control Ab (IC). Shown are results from one representative experiment of two. C, Daudi B cells were either untreated or treated to coligate the BCR and the CD19/CD21/CD81 complex, washed, and lysed either immediately (0 h) or after 1- or 3-h incubation in complete medium at 37 °C. The cells were lysed in Triton X-100 buffer at 4 °C, and CD81 was immunoprecipitated from the lysates in RIPA buffer. The 125IC16 associated with the CD81 immunoprecipitates was determined. Shown are the average values for three separate experiments. D, Daudi B cells expressing both an endogenous CD19 and a transfected CD4/19 chimeric protein were serum-starved for 1 h and incubated with 125IC16 for 2 h at 37 °C as described for Fig. 3. The cells were either untreated, treated to coligate the endogenous CD19/CD21 complex and the BCR as described for Fig. 3 (CD19 COL), or treated to coligate the chimeric CD4/19 complex to the BCR by incubating cells with biotinylated antibodies specific for CD4 and the BCR followed by incubation with avidin (CD4 COL). The cells were lysed in 1% Triton X-100 and CD81, Lyn, and CD45 immunoprecipitated from aliquots of the lysates in RIPA buffer. The 125IC16 associated with the immunoprecipitates was determined and shown as an average fold increase over the cpm of the untreated controls in four separate experiments.

Article Snippet: Wild type Daudi cells were cultured in IMDM (ATCC, Manassas, VA) supplemented with 10% fetal calf serum (Invitrogen) and penicillin (100 units/ml) and streptomycin (100 g/ml) (Sigma).

Techniques: Incubation, Avidin-Biotin Assay, Immunoprecipitation, Ligation, Gradient Centrifugation, Control, Expressing, Transfection