Journal: PLOS One

Article Title: Glucocorticoids suppress early lung inflammation and impair control of SARS-CoV-2 in non-human primates

doi: 10.1371/journal.pone.0342849

Figure Lengend Snippet: A) Experimental design and sample collection. Created in part with BioRender under CC BY license, Nelson, C., (2025). B) Quantification of total lung lesions detected with 18 FDG-PET/CT imaging at the peak of the response, per animal and per group with mean. Animal #ID represented by color and shape in legend. C) Example 18 FDG-PET/CT images from control animal #DGT7 and GC treated animal #DHXD. D) Quantification of the lung lesion volume (dot size) and the metabolic intensity (normalized FDG uptake = SUV of lesion/SUV muscle). Significance calculated with 2way Anova multiple comparison test at the indicated timepoints between GC treatment and control. E) Quantification of subgenomic RNA of the SARS-CoV-2 N1 protein in copies per mL of BAL fluid. Individual animals and the mean of each group with standard error mean represented. Significance calculated with 2way Anova. Fold change between GC treatment and control indicated. Limit of detection (LOD) is 2,000 copies/mL fluid. F) Subgenomic N1 in copies per gram of tissue in the pulmonary lymph nodes, PET+ involved lung, and PET- uninvolved lung. Significance calculated with 2way Anova multiple comparison test. LOD is 1,000 copies per gram of tissue. p > 0.05 not shown, *p < 0.05, ***p < 0.001.

Article Snippet: Peptide pool consisted of Peptivator SARS-CoV-2 Prot_S Complete (Miltenyi Cat#130-127-953), Peptivator SARS-CoV-2 Prot_S B.1.617.2 Mutation (Miltenyi Cat#130-128-763), and Peptivator SARS-CoV-2 Prot_N (Miltenyi Cat# 130-126-699).

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Control, Comparison

Journal: PLOS One

Article Title: Glucocorticoids suppress early lung inflammation and impair control of SARS-CoV-2 in non-human primates

doi: 10.1371/journal.pone.0342849

Figure Lengend Snippet: A) Representative flow cytometry of SARS-CoV-2 specific CD4 and CD8 T cell responses (IFNγ + /TNF+) in blood at day 7 post-infection after ex vivo peptide stimulation with Spike and Nucleocapsid peptide pools, gated on live/CD45 + /CD3 + /CD95 + . B) Quantification of the frequency of SARS-CoV-2 specific CD4 and CD8 T cell responses in the blood overtime after subtracting background staining in unstimulated samples. Significance calculated with 2-way Anova and multiple comparison test. C) Representative flow cytometry of SARS-CoV-2 specific CD4 and CD8 T cell responses (IFNγ + /TNF+) in BAL at day 13 post-infection after ex vivo peptide stimulation with Spike and Nucleocapsid peptide pools, gated on live/CD45 + /CD3 + /CD95 + . D) Quantification of the frequency of SARS-CoV-2 specific CD4 and CD8 T cell responses in the BAL overtime after subtracting background staining in unstimulated samples. Significance calculated with 2-way Anova and multiple comparison test. E) Geometric mean fluorescence intensity (GMFI) of IFNγ production by IFNγ + SARS-CoV-2 specific CD4 and CD8 T cells. F) GMFI of TNF production by TNF+ SARS-CoV-2 specific CD4 and CD8 T cells. G) Frequency of IL-2+ of SARS-CoV-2 specific (IFNγ + /TNF+) CD4 and CD8 T cells in the BAL at day 13 post-infection. Significance calculated with unpaired t-test. H) Frequency of Granzyme B+ of SARS-CoV-2 specific (IFNγ + /TNF+) CD4 and CD8 T cells in the BAL at day 13 post-infection. I) Frequency of IL-17A+ of SARS-CoV-2 specific (IFNγ + /TNF+) CD4 and CD8 T cells in the BAL at day 13 post-infection. p > 0.05 ns or not shown, *p < 0.05, ***p < 0.001. ****p < 0.000.

Article Snippet: Peptide pool consisted of Peptivator SARS-CoV-2 Prot_S Complete (Miltenyi Cat#130-127-953), Peptivator SARS-CoV-2 Prot_S B.1.617.2 Mutation (Miltenyi Cat#130-128-763), and Peptivator SARS-CoV-2 Prot_N (Miltenyi Cat# 130-126-699).

Techniques: Flow Cytometry, Infection, Ex Vivo, Staining, Comparison, Fluorescence

Journal: PLOS One

Article Title: Glucocorticoids suppress early lung inflammation and impair control of SARS-CoV-2 in non-human primates

doi: 10.1371/journal.pone.0342849

Figure Lengend Snippet: A) Quantification of total B cells (CD3 - /CD20 + ) by flow cytometry in the blood over time. Mean of each group with standard error mean (SEM) represented. Significance calculated with 2way Anova. B) Representative flow cytometry plots and gating strategy for identifying total B cells (CD3 - /CD20 + ) from the BAL at day 4 after infection. C) Quantification of total B cells by flow cytometry in the BAL over time. Mean, SEM, and 2way Anova. D) Quantification of total B cells by scRNAseq (cluster 9) in the BAL over time. Mean, SEM, and 2way Anova. E) Differentially expressed genes in B cells (cluster 9) at day 4 post-infection between GC treatment vs. control. Red is upregulated with GC treatment, log 2 FC > 1 and adjusted p-value <0.05. Blue is downregulated with GC treatment, log 2 FC < −1 and adjusted p-value <0.05. Grey with large dot is adjusted p-value <0.05 but absolute |log 2 FC| < 1. Grey with small dot is adjusted p-value >0.05. F) Representative flow cytometry plots and gating strategy for identifying naïve B cells (CD3 - /CD20 + /IgD + ) and activated B cell (CD3 - /CD20 + /IgD - /CD95 + ) from the BAL at day 4 after infection. G) Quantification of naïve B cells by flow cytometry in the BAL over time. Mean, SEM, and 2way Anova. H) Quantification of activated B cells by flow cytometry in the BAL over time. Mean, SEM, and 2way Anova. I) Quantification of total B cells, J) naïve B cells, K) and activated B cells in the spleen, bone marrow, axillary lymph nodes, and pulmonary lymph nodes at necropsy day 13 post-infection. Significance calculated with 2way Anova. L) Representative flow cytometry plots of Spike-specific B cells (CD3 - /CD20 + /CD95 + /Spike-tetramer + ) isolated from the axillary or pulmonary lymph nodes at necropsy, day 13 post-infection. M) Quantification of Spike-specific B cells (CD3 - /CD20 + /CD95 + /Spike (B.1.617.2) tetramer + ) as a percentage of non-naïve (IgD - ) B cells in the spleen, bone marrow, axillary lymph nodes, and pulmonary lymph nodes at necropsy day 13 post-infection. N) Quantification of the frequency of IgG + expression by Spike-specific (tetramer + ) B cells in the pulmonary lymph node. Significance calculated with 2way Anova. p > 0.05 ns or not shown, *p < 0.05, ***p < 0.001. ****p < 0.0001.

Article Snippet: Peptide pool consisted of Peptivator SARS-CoV-2 Prot_S Complete (Miltenyi Cat#130-127-953), Peptivator SARS-CoV-2 Prot_S B.1.617.2 Mutation (Miltenyi Cat#130-128-763), and Peptivator SARS-CoV-2 Prot_N (Miltenyi Cat# 130-126-699).

Techniques: Flow Cytometry, Infection, Control, Isolation, Expressing

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD expression in vivo and in vitro after SARS-CoV-2 infection (A) Volcano plot relative to the reanalysis of transcriptomic data from ten studies of healthy lungs and four studies of patients with COVID-19 (Delorey et al.). (B and C) Calu3 cells are infected with 2020B.1 (MOI 0.01), Delta (MOI 0.01), or Omicron (MOI 0.01) SARS-CoV-2 variants, and PAD4 (B) and PAD2 (C) mRNA are assessed by RT-qPCR. Data are normalized to the housekeeping gene PGK1 and expressed as mean fold change ±SEM over mock-infected cells of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test. (D) Protein lysates from Calu3 infected with SARS-CoV-2 (2020B.1 variant, MOI 0.1 PFU/cell) or uninfected cells (mock) at different hours post-infection (hpi) are analyzed for citrullinated proteins using an Rh-PG citrulline-specific probe (left panel) or an anti-CCP antibody (right panel). Samples are subjected to gel electrophoresis, and SARS-CoV-2 infection is confirmed using an anti-SARS-CoV-2 Spike (S SARS-CoV-2) antibody. β-actin is used as a loading control. One representative blot of three independent experiments is shown. (E) Volcano plot depicts host (gray dots) and viral (red dots) citrullinated proteins of SARS-infected vs. mock-infected cells at 48 hpi (2020B.1 variant, MOI 0.1 PFU/cell). The x axis represents the ratio of citrullination between mock and infected cells at the indicated time points, while the y axis indicates the statistical significance. Both variables are plotted on a logarithmic scale ( n = 3). (F) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Expressing, In Vivo, In Vitro, Infection, Quantitative RT-PCR, Variant Assay, Nucleic Acid Electrophoresis, Control

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: SARS-CoV-2 infection affects protein citrullination by modulating PAD expression in vivo (A and B) PAD4 and PAD2 mRNA expression in mouse lungs (A) and brains (B) assessed by RT-qPCR. Each dot represents an individual mouse sample ( n = 6). Data are normalized to the housekeeping gene β-actin and presented relative to one randomly selected non-infected sample. Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (C) Western blot analysis of protein lysates from pooled uninfected (mock) and SARS-CoV-2-infected (6 dpi, Delta variant) mouse lungs and brains. The analysis is performed using antibodies against PAD2, PAD4, and β-actin, the latter to ensure equal loading. A representative blot from three independent experiments is shown. (D) Multiplex immunofluorescence staining for the detection of SARS-CoV-2-targeted cells in mouse brains and lung sections at 6 dpi and mock control. S SARS-CoV-2 protein, PAD2, and PAD4 positive signals are represented in green, pink, and yellow, respectively. Cell nuclei are visualized by DAPI (blue). Original magnification 20×. (E) Quantification of PAD2-and PAD4-positive cells is performed using the inForm Image Analysis software (Akoya Biosciences). For each mouse ( n = 6), one representative section is analyzed to determine cell density (cells/mm 2 ). Data represent mean ± SEM. Statistical significance is determined using non parametric t test, ∗ p < 0.05. (F) Volcano plot shows citrullinated proteins in pooled protein lysates from SARS-CoV-2-infected vs. mock-infected mouse lungs at 6 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples, and the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (G) PANTHER classification of over-citrullinated cellular proteins based on protein classes.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Infection, Expressing, In Vivo, Quantitative RT-PCR, Western Blot, Variant Assay, Multiplex Assay, Immunofluorescence, Staining, Control, Software, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: Antiviral effects of PAD inhibitors against SARS-CoV-2 in vitro (A and B) Quantification of SARS-CoV-2 2020B.1 (MOI 0.1) viral particle production in Calu-3 and Huh7.5 cells (at 24 hpi and 48 hpi, respectively) treated with the increasing concentrations of BB-Cl (A) or GSK199 (B), determined by plaque assay. Results are expressed as a percentage relative to vehicle-treated cells (DMSO, marked as 0 on the graph). Data are presented as means ± SEM from four independent experiments and are analyzed by one-way ANOVA followed by Bonferroni’s post-test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (C and D) Cell viability of uninfected Calu3 and Huh7.5 cells treated with the indicated concentrations of BB-Cl (C) and GSK199 (D) for 72 h, determined for each concentration by MTT assay. Values are expressed as means ± SEM of three independent experiments. (E) Viral titers of different SARS-CoV-2 variants are measured in Calu-3 cells at 24 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (F) Viral titers of different SARS-CoV-2 variants (as indicated in the figure) are measured in Huh7.5 cells at 48 hpi (MOI = 0.1) following treatment with BB-Cl (5 μM) or GSK199 (20 μM). Viral particle production is determined by plaque assay. Data are expressed as mean ± SEM from four independent experiments and analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: In Vitro, Plaque Assay, Concentration Assay, MTT Assay

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD4 inhibition blocks SARS-CoV-2 protein and genome synthesis (A) Relative viral RNA quantification in cell extracts from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO, determined by comparative RT-qPCR. Values are expressed relative to vehicle-treated samples and normalized to the housekeeping gene PGK1. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (B) Western blot analysis of viral protein expression in Huh7.5 and Calu3 cells, either mock-infected or infected with SARS-CoV-2 (MOI 0.1) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO. One representative blot from three independent experiments is shown. (C) Schematic representation of infection and treatment protocols used for the viral entry inhibition assay. (D) Viral entry assay on VERO-E6 (left) and TMPRSS2-rexpressing VERO-E6 (right) infected with VSV-Spike GFP (MOI 1). GFP-positive infected cells are microscopically counted, and the results are expressed as a percentage relative to vehicle-treated cells, and are analyzed by t test. Data represent mean ± SEM. (E) Absolute quantification of viral RNA released from SARS-CoV-2-infected Huh7.5 (left) or Calu3 (right) cells (MOI 0.1) treated with DMSO (vehicle) or GSK199 (20 μM), either using standard treatment (total) or added at 2 hpi (Post), determined by qRT-PCR. Supernatants are collected at 24 hpi for Calu3 and 48 hpi for Huh7.5. Data represent mean ± SEM from three independent experiments and are analyzed by t test. ∗ p < 0.05 and ∗∗ p < 0.01.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Inhibition, Infection, Quantitative RT-PCR, Western Blot, Expressing, Quantitative Proteomics

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: In vivo antiviral activity of PAD inhibitors against SARS-CoV-2 (A) Schematic representation of the treatment and infection protocols in K18-hACE2 transgenic mice. (B) Quantification of viral genome copies in mouse lungs—treated and infected as described in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (C) Plaque assay to determine the number of infectious viral particles in mouse nasal swabs. (D) Graphical representation of cumulative scores from for immunohistochemical staining of mouse lungs using an anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (E) Graphical representation of cumulative scores from for H&E staining of mouse lungs ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. (F) Quantification of viral genome copies in mouse brains—treated and infected, as represented in A— by ddPCR. Each dot represents an individual mouse sample ( n = 6). (G) Distribution of brains from vehicle- or PAD-inhibitor-treated infected mice based on viral load (low: < below 10 3 ; high: > below 10 3 ). (H) Graphical representation of cumulative scores from for immunohistochemical staining of mouse brains using anti-SARS-CoV-2 nucleocapsid antibody ( n = 4). Y axis represents the cumulative score calculated according to the criteria described in . (I) Graphical representation of cumulative scores from for H&E staining of mouse brains ( n = 4), showing histopathological alterations classified by anatomical site and summarized as a percentage of tissue involvement. Data from all experiments, which represent mean ± SEM, are analyzed using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: In Vivo, Activity Assay, Infection, Transgenic Assay, Plaque Assay, Immunohistochemical staining, Staining, Two Tailed Test

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD inhibition modulates SARS-CoV-2-induced inflammation (A–D) Relative mRNA levels of the indicated genes are quantified by comparative RT-qPCR from total RNA of Calu-3 cells infected with the SARS-CoV-2 Delta strain (gray bars; MOI 0.1, 24 hpi) and treated with GSK199 (20 μM), BB-Cl (5 μM), or DMSO (vehicle). Data from three independent experiments were normalized to the housekeeping gene PGK1 and plotted as mean fold change ±SEM over mock-infected cells (white bars). (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; one-way ANOVA followed by Bonferroni’s post-test). Red asterisks indicate comparisons with mock control, while black asterisks refer to comparisons among treatments. (E–J) Relative mRNA levels of the indicated cytokines are quantified by RT-qPCR from lung tissues of mice infected with SARS-CoV-2 Delta variant (10 5 PFU, i.n.) for 4 days and treated with BB-Cl (1 mg/kg), GSK199 (30 mg/kg), or DMSO (vehicle). Each dot represents one individual mouse. Data are normalized to the housekeeping gene β-actin and are presented relative to the mean ΔCt value of vehicle-treated mice. Statistical significance is assessed using an unpaired two-tailed t test (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Inhibition, Quantitative RT-PCR, Infection, Control, Variant Assay, Two Tailed Test

Journal: iScience

Article Title: Targeting peptidyl-arginine deiminase 4 suppresses SARS-CoV-2 replication and modulates the inflammatory response

doi: 10.1016/j.isci.2026.115038

Figure Lengend Snippet: PAD inhibition protects against SARS-CoV-2-induced pathological changes (A–C) Volcano plots show citrullinated proteins in pooled protein lysates from SARS-CoV-2- vs. mock-infected mouse lungs at 4 dpi. Lysates are incubated with biotin-PG to isolate citrullinated proteins on streptavidin agarose, followed by on-bead tryptic digestion and LC-MS/MS analysis. Each dot represents an identified citrullinated protein. The x axis shows the citrullination ratio between mock and infected samples (A) and between infected mice treated with vehicle (DMSO) or with PAD inhibitors BB-Cl-amidine (B) or GSK199 (C), while the y axis indicates statistical significance, both on a logarithmic scale ( n = 3). (D) Heatmap shows the fold changes of differentially citrullinated proteins identified in lung tissues across the indicated pairwise comparisons: uninfected vs. infected, and vehicle-treated infected vs. BB-Cl–treated or GSK199–treated infected mice. The color scale represents relative citrullination levels expressed as log 2 fold change.

Article Snippet: The following primary antibodies were sequentially applied to the slides: SARS-CoV-1/2 Spike Protein (clone 2B3E5, Cell Signaling), mouse anti-mouse β-tubulin IV (clone T7941, Merck), rat anti-mouse F4/80 (clone CI:A3-1, Bio-Rad Laboratories), rabbit anti-mouse PAD2 (polyclonal, Proteintech), rabbit anti-mouse PAD4 (polyclonal, Proteintech), rabbit anti-mouse Ly6g (clone EPR22909-135, Abcam), rat anti-mouse CD3 (clone CD3-12, Abcam), and rabbit anti-mouse cytokeratin 8 (clone EP1628Y, Abcam).

Techniques: Inhibition, Infection, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: PLOS One

Article Title: Glucocorticoids suppress early lung inflammation and impair control of SARS-CoV-2 in non-human primates

doi: 10.1371/journal.pone.0342849

Figure Lengend Snippet: A) Experimental design and sample collection. Created in part with BioRender under CC BY license, Nelson, C., (2025). B) Quantification of total lung lesions detected with 18 FDG-PET/CT imaging at the peak of the response, per animal and per group with mean. Animal #ID represented by color and shape in legend. C) Example 18 FDG-PET/CT images from control animal #DGT7 and GC treated animal #DHXD. D) Quantification of the lung lesion volume (dot size) and the metabolic intensity (normalized FDG uptake = SUV of lesion/SUV muscle). Significance calculated with 2way Anova multiple comparison test at the indicated timepoints between GC treatment and control. E) Quantification of subgenomic RNA of the SARS-CoV-2 N1 protein in copies per mL of BAL fluid. Individual animals and the mean of each group with standard error mean represented. Significance calculated with 2way Anova. Fold change between GC treatment and control indicated. Limit of detection (LOD) is 2,000 copies/mL fluid. F) Subgenomic N1 in copies per gram of tissue in the pulmonary lymph nodes, PET+ involved lung, and PET- uninvolved lung. Significance calculated with 2way Anova multiple comparison test. LOD is 1,000 copies per gram of tissue. p > 0.05 not shown, *p < 0.05, ***p < 0.001.

Article Snippet: B cell tetramers were prepared by combining Recombinant SARS-CoV-2 Spike-Prot B.1.617.2 (HEK) Biotin (Miltenyi cat#130-129-565) at 0.1 mg/mL with streptavidin BV605 at 50ug/mL and PEB buffer (PBS + 0.45% BSA, 2mM EDTA) for 15 minutes prior to staining.

Techniques: Positron Emission Tomography-Computed Tomography, Imaging, Control, Comparison

Journal: PLOS One

Article Title: Glucocorticoids suppress early lung inflammation and impair control of SARS-CoV-2 in non-human primates

doi: 10.1371/journal.pone.0342849

Figure Lengend Snippet: A) Representative flow cytometry of SARS-CoV-2 specific CD4 and CD8 T cell responses (IFNγ + /TNF+) in blood at day 7 post-infection after ex vivo peptide stimulation with Spike and Nucleocapsid peptide pools, gated on live/CD45 + /CD3 + /CD95 + . B) Quantification of the frequency of SARS-CoV-2 specific CD4 and CD8 T cell responses in the blood overtime after subtracting background staining in unstimulated samples. Significance calculated with 2-way Anova and multiple comparison test. C) Representative flow cytometry of SARS-CoV-2 specific CD4 and CD8 T cell responses (IFNγ + /TNF+) in BAL at day 13 post-infection after ex vivo peptide stimulation with Spike and Nucleocapsid peptide pools, gated on live/CD45 + /CD3 + /CD95 + . D) Quantification of the frequency of SARS-CoV-2 specific CD4 and CD8 T cell responses in the BAL overtime after subtracting background staining in unstimulated samples. Significance calculated with 2-way Anova and multiple comparison test. E) Geometric mean fluorescence intensity (GMFI) of IFNγ production by IFNγ + SARS-CoV-2 specific CD4 and CD8 T cells. F) GMFI of TNF production by TNF+ SARS-CoV-2 specific CD4 and CD8 T cells. G) Frequency of IL-2+ of SARS-CoV-2 specific (IFNγ + /TNF+) CD4 and CD8 T cells in the BAL at day 13 post-infection. Significance calculated with unpaired t-test. H) Frequency of Granzyme B+ of SARS-CoV-2 specific (IFNγ + /TNF+) CD4 and CD8 T cells in the BAL at day 13 post-infection. I) Frequency of IL-17A+ of SARS-CoV-2 specific (IFNγ + /TNF+) CD4 and CD8 T cells in the BAL at day 13 post-infection. p > 0.05 ns or not shown, *p < 0.05, ***p < 0.001. ****p < 0.000.

Article Snippet: B cell tetramers were prepared by combining Recombinant SARS-CoV-2 Spike-Prot B.1.617.2 (HEK) Biotin (Miltenyi cat#130-129-565) at 0.1 mg/mL with streptavidin BV605 at 50ug/mL and PEB buffer (PBS + 0.45% BSA, 2mM EDTA) for 15 minutes prior to staining.

Techniques: Flow Cytometry, Infection, Ex Vivo, Staining, Comparison, Fluorescence

Journal: PLOS One

Article Title: Glucocorticoids suppress early lung inflammation and impair control of SARS-CoV-2 in non-human primates

doi: 10.1371/journal.pone.0342849

Figure Lengend Snippet: A) Quantification of total B cells (CD3 - /CD20 + ) by flow cytometry in the blood over time. Mean of each group with standard error mean (SEM) represented. Significance calculated with 2way Anova. B) Representative flow cytometry plots and gating strategy for identifying total B cells (CD3 - /CD20 + ) from the BAL at day 4 after infection. C) Quantification of total B cells by flow cytometry in the BAL over time. Mean, SEM, and 2way Anova. D) Quantification of total B cells by scRNAseq (cluster 9) in the BAL over time. Mean, SEM, and 2way Anova. E) Differentially expressed genes in B cells (cluster 9) at day 4 post-infection between GC treatment vs. control. Red is upregulated with GC treatment, log 2 FC > 1 and adjusted p-value <0.05. Blue is downregulated with GC treatment, log 2 FC < −1 and adjusted p-value <0.05. Grey with large dot is adjusted p-value <0.05 but absolute |log 2 FC| < 1. Grey with small dot is adjusted p-value >0.05. F) Representative flow cytometry plots and gating strategy for identifying naïve B cells (CD3 - /CD20 + /IgD + ) and activated B cell (CD3 - /CD20 + /IgD - /CD95 + ) from the BAL at day 4 after infection. G) Quantification of naïve B cells by flow cytometry in the BAL over time. Mean, SEM, and 2way Anova. H) Quantification of activated B cells by flow cytometry in the BAL over time. Mean, SEM, and 2way Anova. I) Quantification of total B cells, J) naïve B cells, K) and activated B cells in the spleen, bone marrow, axillary lymph nodes, and pulmonary lymph nodes at necropsy day 13 post-infection. Significance calculated with 2way Anova. L) Representative flow cytometry plots of Spike-specific B cells (CD3 - /CD20 + /CD95 + /Spike-tetramer + ) isolated from the axillary or pulmonary lymph nodes at necropsy, day 13 post-infection. M) Quantification of Spike-specific B cells (CD3 - /CD20 + /CD95 + /Spike (B.1.617.2) tetramer + ) as a percentage of non-naïve (IgD - ) B cells in the spleen, bone marrow, axillary lymph nodes, and pulmonary lymph nodes at necropsy day 13 post-infection. N) Quantification of the frequency of IgG + expression by Spike-specific (tetramer + ) B cells in the pulmonary lymph node. Significance calculated with 2way Anova. p > 0.05 ns or not shown, *p < 0.05, ***p < 0.001. ****p < 0.0001.

Article Snippet: B cell tetramers were prepared by combining Recombinant SARS-CoV-2 Spike-Prot B.1.617.2 (HEK) Biotin (Miltenyi cat#130-129-565) at 0.1 mg/mL with streptavidin BV605 at 50ug/mL and PEB buffer (PBS + 0.45% BSA, 2mM EDTA) for 15 minutes prior to staining.

Techniques: Flow Cytometry, Infection, Control, Isolation, Expressing