Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: Control = blue CoV-1 = teal, CoV-2 = green, CoV-2 D614G = yellow. (A) A PCA analysis of the proteomics data, depicting variation of each time point and replicate on PC1 and PC2. (B) Correlation plots of each proteomic replicate compared to all proteomics replicates at each time point, separated by treatment type (indicated by the box color) with highly positive correlations shown in orange. (C) A PCA analysis of the metabolic data, depicting variation of each time point and replicate on PC1 and PC2. (D) Correlation plots of each metabolic replicate compared to all metabolic replicates at each time point, separated by treatment type (indicated by the box color) with highly positive correlations shown in orange. (E) A PCA analysis of the lipidomic data, depicting variation of each time point and replicate on PC1 and PC2. (F) Correlation plots of each lipidomic replicate compared to all lipidomics replicates at each time point, separated by treatment type (indicated by the box color) with highly positive correlations shown in orange. (G) Lines reflect ECHO fitted models for core circadian clock proteins detected in the proteomics data set. Shading depicts ±1 standard deviation of the data. (H-K) Abundance values (points) and ECHO-fitted models (line) with statistical significance for all detected central metabolism proteins in (H) glycolysis, (I) pentose phosphate pathway, (J) TCA cycle, and (K) OXPHOS. (L-M) ECHO fit of circadian OXPHOS proteins in Complex I (L) , Complex III (M) , Complex IV (N) , and Complex V (O) . Statistical significance was determined using the Friedman test and post-hoc Dunn’s Test, * = p<0.05, ** = p<0.01, ***= p<0.001, ****= p<0.0001

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques: Control, Standard Deviation

Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: (A) ECHO fitted models of the log2FC of all proteins within the four central metabolism pathways in response to CoV-1, CoV-2, and CoV-2 D614G spike proteins at different times of the day (HPS/CT). Significant circadian fits are represented with an asterisk. (B) Bubble plot showing log2FC values of all detected central metabolite responses to spike proteins (left panel CoV-1, middle panel CoV-2, right panel CoV-2 D614G , delineated by order in the pathway. Bubble size represents - log10(FDR), bubble color represents spike treatment, with lighter/darker shades representing significant up/downregulation, and outlined bubbles have a significant FDR < 0.05, as in the key.

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques:

Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: Cytokine levels in response to vehicle control, CoV-1, CoV-2, or CoV-2 D614G spike protein for (A) IL-6, (B) TNFa, (C) TGFβ-1, and (D) IL-10. (E) TGF- β 1 cytokine levels normalized to vehicle control levels at each time point of exposure to CoV-1 spike protein (ratio of CoV-1:vehicle control represented as bars), with significant ECHO fitted model (line). Cytokine levels in response to treatment with LPS and IL-4 for (F) TGF- β 1 and (G) IL-10. HPS = hours post serum shock, CT = circadian time. (H-K) Violin plots showing all protein log2FC values (each point represents one protein log2FC value) and significant circadian ECHO models (lines) for response to spike proteins in (H) glycolysis, (I) pentose phosphate pathway (PPP), (J) TCA, and (K) OXPHOS pathways. Statistical significance for cytokines determined using RM 2-way ANOVA with Geisser-Greenhouse correction and post-hoc Dunnett’s multiple comparison test. Statistical significance for central metabolic pathway violin plots determined using Kruskall-Wallis and post-hoc Dunn’s Test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.000.

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: (A) A chart of significantly up- or down-regulated log2FC values of mitochondrial proteins separated by functional category in response to CoV-2 spike protein (MitoCarta3.0 ). Internal numbers represent significant log2FC upregulation (≥1.0, light green) or downregulation (≤1.0, dark green). Timepoints shown are in HPS and CT. (B) Volcano plot showing differential level analysis of lipids responding to CoV-1, CoV-2, and CoV-2 D614G spike proteins at HPS20/CT8, colored by treatment if significant (ns = not significant, in gray). (C) Confocal maximum projections of representative fixed mBMDMs stained with MitoTracker TM CMXRos Red following treatment with either PBS control or CoV-2 spike protein at HPS20. The original image (left) contains an ROI labeled in a red box, zoomed and represented to the right. Scale bar represents 2μM; images pseudo-colored using the inverted look-up table (LUT) in ImageJ/FIJI. (D) SuperPlots quantifying significant mitochondrial parameters at each time point. Biological replicates are represented by large, circled points; smaller points represent technical replicates; the line represents group mean; and error bars are ±1SD. Matching technical and biological replicates are represented by the same color saturation as indicated in the key. Statistics: linear mixed model, Holm-Šídák adjusted.

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques: Functional Assay, Staining, Control, Labeling

Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: Significant GO ontologies (PantherGO) of mitochondrial-related significant DEPs in response to CoV-2 D614G spike protein. (B) A chart of all significant mitochondrial DEPs (MitoCarta 3.0) in response to treatment by CoV-1, CoV-2, and CoV-2 D614G spike proteins (colors in key) at HPS16, 20, 24, 28, 32, 36, and 40. Internal numbers represent the fold increase (light) or decrease (dark) relative to PBS control.

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques: Control

Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: (A) Measurements of JC-10 590/525 ratios under non-stimulatory PBS control conditions across 24 hrs. Line represents ECHO model filtered for circadian parameters (period = 20-28hrs, BH-adj p-value = <0.05). (B) Mitochondrial membrane potential (MMP) at each treatment time point, as a percentage of the vehicle control, for CoV-1, CoV-2, and CoV-2 D614G spike protein treatments. (C) JC-10 590/525 ratios of mBMDMs treated at HPS16, 20, 24, 28, 32, 36, or 40 with LPS (left) or IL-4 (right), as a percentage of the PBS-treated control. Overlaid line on all graphs indicates significant ECHO fit if response was determined to be circadian (e.g. period = 20-28h, BH-adj p-val = <0.05). (A-C) Bar colors indicate treatment type, and individual point colors represent each biological replicate as indicated in the key. Error bars represent ±1 SD. (D) Confocal maximum projections of representative fixed mBMDMs stained with MitoTracker TM CMXRos Red following treatment with either PBS control, CoV-2 spike protein, LPS, or IL-4 at HPS20, 24, 32, and 36. Images pseudo-colored using the inverted look-up table (LUT) in ImageJ/FIJI. Omitted images indicated within the panels are reported in . Scale bar represents 2μM.

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques: Control, Membrane, Staining

Journal: bioRxiv

Article Title: Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

doi: 10.64898/2026.02.24.707668

Figure Lengend Snippet: (A) Diagram showing the experimental setup for exposing circadianly-synchronized human macrophages to SARS-CoV-2 spike protein and harvesting cell pellets for mass spectrometric analysis using the MPLEx method. (B) Volcano plot showing differential levels of circadian core clock proteins at each treatment time point. Shape indicates clock protein; color denotes the time of spike protein treatment. Dotted lines indicate significant log2FC and -log10(FDR) cutoff values. (C) ECHO model fit lines to the log2FC values of all central metabolic proteins, separated by pathway, in response to CoV-2 spike. Significant fits are denoted by (*). (D) Bubble plot of all detected metabolites within central metabolic pathways glycolysis and TCA cycle. Color indicates significant up (light blue) or down (dark blue) differential levels, while bubble size denotes –log10 FDR value. Metabolites are listed in the order they follow in each pathway. (E) ECHO fit (line) and corresponding log2FC (bar) of circadian response proteins DLST at each treatment time point.

Article Snippet: Starting at 16hrs post-50% FBS shock (HPS), mouse BMDMs or human macrophages were exposed to 11.2nM (1μg/mL) of SARS spike protein in PBS as indicated every 4 hours for 24 hours, for a total of 7 time points (at HPS16, 20, 24, 28, 32, 36, and 40). mBMDMs were incubated for 6 hours with either PBS (vehicle control), SARS-1 spike protein, SARS-COV-2 S1 spike protein, or SARS-COV-2 D614G S1 spike protein (SinoBiological Cat#40150-V08B1, 40591-V08B1, 40591-V08H3, respectively) as indicated.

Techniques:

Journal: medRxiv

Article Title: Early Fc-effector antibody signatures impact COVID-19 disease trajectory

doi: 10.64898/2026.02.18.26346542

Figure Lengend Snippet: Box-and-whisker plots showing (A) the binding antibodies titers against SARS-CoV-2 S1, RBD and S2 spike antigens (B) the ratios between S1/S2 and RBD/S2 binding titers and (C) the Fc-receptor antibody-mediated ADCC and ADCP activities against SARS-CoV-2 full-length spike in mild and severe COVID-19 patients. Box indicates interquartile range (IQR, Q1-Q3), with horizontal lines showing the median and vertical lines indicating minimum and maximum. Mann-Whitney U test was performed to compare differences between mild vs. severe patients at each time point. Statistical significance was considered when p ≤ 0.05. D) Heatmap of Spearman correlation matrices between SARS-CoV-2 antibody titers (ADCC, ADCP, nAb and IgG binding titers against S, S1, S2 and RBD) at days 0, 3, 7 and 46 post-recruitment. Statistically significant correlations in the underlined intersections are indicated with asterisk (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). E) Spearman correlations between binding IgG antibodies against S1, RBD or S2 domains and functional ADCC (blue) or ADCP (purple) antibodies at each timepoint. Each dot represents a sample. Shown are the Spearman r coefficient, 95% confidence interval (CI) and p value (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant). Data is also shown in Supplementary Table 2.

Article Snippet: All recombinant proteins used in this study were purchased from Sino Biological: SARS-CoV-2 full-length spike (S) protein (Cat. #40589-V08H4), seasonal HCoV-OC43 full-length S (Cat. #40607-V08B), seasonal HCoV-229E full-length S (Cat. #40605-V08B), SARS-CoV-2 S1 domain (Cat. #40591-V08H), SARS-CoV-2 S2 domain (Cat. #40590-V08H1), SARS-CoV-2 RBD subdomain (Cat. #40592-V08H80), HCoV-OC43 S1 domain (Cat. #40607-V08H1) and HCoV-OC43 S2 domain (Cat. #40607-V08B1).

Techniques: Whisker Assay, Binding Assay, MANN-WHITNEY, Functional Assay

Journal: medRxiv

Article Title: Early Fc-effector antibody signatures impact COVID-19 disease trajectory

doi: 10.64898/2026.02.18.26346542

Figure Lengend Snippet: A) Heatmap of FcγR-binding antibody responses to SARS-CoV-2 S antigens in mild and severe COVID-19 patients. Binding activity of antibodies to FcγRs was measured for SARS-CoV-2 full-length S, S1 and S2 antigens at days 0, 3, 7, and 46 post-recruitment. Box-and-whisker diagrams showing (B) FcγR2AH and FcγR2AR-binding antibody responses (C) FcγR3AV and FcγR3AF-binding titers, (D) functional monocyte antibody-dependent cellular phagocytosis (ADCP) responses and (E) functional antibody-dependent neutrophil phagocytosis (ADNP) responses against SARS-CoV-2 full-length S, S1 and S2 antigens in mild and severe COVID-19 patients. Box indicates interquartile range (IQR, Q1-Q3), with horizontal line showing the median and vertical lines indicating minimum and maximum. Mann-Whitney U test was performed to compare differences between mild vs. severe patients at each time point. Statistical significance was considered when p ≤ 0.05. F) PCA of antibody responses according to day of recruitment (day 0: blue; day 3: red; day 7: green and day 46: purple). Empty circles represent mild cases, and filled circles represent severe cases. Ellipses show 95% confidence regions. G) Spider plot of the antibody landscape (IgG antibody titers, FcγR-binding activity and FcγR-effector functions) in mild and severe COVID-19 patients. Median percentile for each antibody feature is shown at each timepoint and for each SARS-CoV-2 S antigen.

Article Snippet: All recombinant proteins used in this study were purchased from Sino Biological: SARS-CoV-2 full-length spike (S) protein (Cat. #40589-V08H4), seasonal HCoV-OC43 full-length S (Cat. #40607-V08B), seasonal HCoV-229E full-length S (Cat. #40605-V08B), SARS-CoV-2 S1 domain (Cat. #40591-V08H), SARS-CoV-2 S2 domain (Cat. #40590-V08H1), SARS-CoV-2 RBD subdomain (Cat. #40592-V08H80), HCoV-OC43 S1 domain (Cat. #40607-V08H1) and HCoV-OC43 S2 domain (Cat. #40607-V08B1).

Techniques: Binding Assay, Activity Assay, Whisker Assay, Functional Assay, MANN-WHITNEY