Transcriptional effect of GSK3 inhibition on monocyte-derived GM-MØ.

A. MAFB, total GSK3α, total GSK3β, Ser9-phosphorylated GSK3β and Ser21-phosphorylated GSK3α levels in M-MØ and GM-MØ, as determined by Western blot (left panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of the MAFB/GAPDH, Ser21-phosphorylated GSK3α/total GSK3α and Ser9-phosphorylated GSK3β/total GSK3β protein ratios from three independent experiments are shown (right panel) (paired Student’s t test: ***, p<0.005; ****, p<0.001). A representative Western blot experiment is shown in each case in the upper panel. B. p-Tyr279– GSK3α and p-Tyr216–GSK3β levels in four independent samples of M-MØ and GM-MØ, as determined by Western blot (left panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of the p-Tyr279–GSK3α/GAPDH and p-Tyr216–GSK3β/GAPDH protein ratios from four independent experiments are shown (right panel) (paired Student’s t test: n.s., not significant). C. Schematic representation of the exposure of GM-MØ to CHIR-99021 for 48 hours. This panel was created with BioRender.com. D. Tyr279–phosphorylated GSK3α and Tyr216–phosphorylated GSK3β levels in three independent preparations of DMSO- or CHIR-99021-treated GM-MØ, as determined by Western blot (left panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of the p-Tyr216–GSK3β/GAPDH and p-Tyr279–GSK3α/GAPDH protein ratios from the three independent experiments are shown (right panel) (paired Student’s t test: ****, p<0.001). E. MAFB protein levels in DMSO-GM-MØ and CHIR-GM-MØ, as determined by Western blot. A representative experiment is shown. F. Number of differentially expressed genes ([log2FC] >1; adjp<0.05) between DMSO-GM-MØ and CHIR-GM-MØ. Differential gene expression was assessed using DESeq2. G. GSEA of the indicated gene sets (from GSE68061, left panel; from GSE188278, right panel) on the ranked comparison of the CHIR-GM-MØ vs. DMSO-GM-MØ transcriptomes. Normalized Enrichment Score (NES) and FDRq values are indicated in each case. H. Summary of GSEA of the gene sets that define the tissue-resident monocyte and macrophage states (MoMac-VERSE) 2 on the ranked comparison of the CHIR-GM-MØ vs. DMSO-GM-MØ transcriptomes. FDRq values are indicated only if FDRq>0.0; empty dots, Not Significant. I. Summary of GSEA of the gene sets that characterize the macrophage subsets identified in severe COVID-19 23–25 on the ranked comparison of the CHIR-GM-MØ vs. DMSO-GM-MØ transcriptomes. FDRq values are indicated only if FDRq>0.0; empty dots, Not Significant.

Phenotypic effects of GSK3 inhibition on monocyte-derived GM-MØ: Enhanced expression of MAFB-dependent genes and proteins.

A. DoRoThEA 51 analysis on the ranked comparison of the DMSO-GM-MØ and CHIR-GM-MØ transcriptomes. B. GSEA of MAFB-regulated gene sets (from GSE155719) on the ranked comparison of the DMSO-GM-MØ and CHIR-GM-MØ transcriptomes. NES and FDRq values are indicated in each case C. Overlap between the genes upregulated (|log2FC|>1; adjp<0.05) in CHIR-GM-MØ (relative to DMSO M-MØ) and MAFB-dependent genes (from GSE155719, left panel) or the 75-geneset of MAFB-regulated genes (right panel) 52, with indication of some of the overlapping genes. D. Relative mRNA levels of the indicated genes in DMSO-GM-MØ and CHIR-GM-MØ, as determined by RNA-Seq on three independent samples (GSE256208). Adjp of the comparison, shown in each case, was assessed using DESeq2. E. CD163, MAF and IL7R protein levels in DMSO-GM-MØ and CHIR-GM-MØ, as determined by Western blot (left panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of the MAF/GAPDH, IL7R/GAPDH and CD163/GAPDH protein ratios from seven independent experiments are shown (right panels) (paired Student’s t test: ***, p<0.005; ****, p<0.001). A representative Western blot experiment is shown in each case. F. FOLR2 protein levels in DMSO-GM-MØ and CHIR-GM-MØ, as determined by Western blot (left panel). Vinculin protein levels were determined as protein loading control. Mean ± SEM of the FOLR2/Vinculin protein ratio from three independent experiments are shown (lower panel) (paired Student’s t test: **, p<0.01). A representative Western blot experiment is shown. G. Production of the indicated soluble factors by DMSO-GM-MØ and CHIR-GM-MØ, as determined by ELISA. Mean ± SEM of seven independent samples are shown (paired Student’s t test: *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001).

Functional consequences of GSK3 inhibition on monocyte-derived GM-MØ.

A. Production of IL-10 by untreated (−) or stimulated (LPS, TNF, IFNγ or CL264) DMSO-GM-MØ and CHIR-GM-MØ, as determined by ELISA. Mean ± SEM of four independent samples are shown (paired Student’s t test: *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001). B. Phagocytosis of pHRodo-labeled bacterial particles by DMSO-GM-MØ and CHIR-GM-MØ, as determined by flow cytometry. Mean ± SEM of three independent samples are shown (paired Student’s t test: *, p<0.05). A representative flow cytometry analysis is shown in the left panel. C. Efferocytosis capacity of DMSO-GM-MØ, CHIR-GM-MØ and DMSO-M-MØ, as determined by flow cytometry using staurosporine-induced CellTrace Violet-labeled apoptotic Jurkat cells. The percentage of positive cells and mean fluorescence intensity are shown. Mean⍰±⍰SEM of 3 independent samples are shown (one-way ANOVA with Fisher LSD test: *, p<0.05; **, p⍰<⍰0.01; ***, p⍰<⍰0.005). Representative flow cytometry histograms for CellTrace Violet emission of DMSO-GM-MØ, CHIR-GM-MØ and DMSO-M-MØ are shown.

Transcriptional effects of GSK3 knock-down in monocyte-derived GM-MØ.

A. Schematic representation of the siRNA-mediated GSK3 knock-down procedure in monocyte-derived GM-MØ. This panel was created with BioRender.com. B. Total GSK3α, GSK3β and MAFB protein levels in monocyte-derived GM-MØ after siRNA-mediated GSK3A and/or GSK3B silencing, as determined by Western blot (upper panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of GSK3β/GAPDH and GSK3α/GAPDH protein ratios from the three independent experiments are shown (lower panel) (one-way ANOVA with Dunnet test:*, p<0.05). C. Heatmap of the relative expression of the genes significantly altered after GSK3A and GSK3B knock-down in siCNT-GM-MØ (lanes A-C), siGSK3A-GM-MØ (lanes D-F), siGSK3B-GM-MØ (lanes G-I), siGSK3A/B-GM-MØ (lanes J-L). Representative genes are indicated. D. Number of differentially expressed genes (adjp<0.05) between siGSK3A-GM-MØ, siGSK3B-GM-MØ or siGSK3A/B-GM-MØ and siCNT-GM-MØ. Differential gene expression was assessed using DESeq2. E. GSEA of the genes whose expression is significantly modulated by CHIR-99021 in either GM-MØ (from Figure 1, this manuscript) or in ex vivo isolated Alveolar Macrophages (AMØ, see below in Figure 6) on the ranked comparison of the siGSK3A/B-GM-MØ vs. siCNT-GM-MØ transcriptomes. F. GSEA of the indicated gene sets (from GSE68061) on the ranked comparison of the siGSK3A/B-GM-MØ vs. siCNT-GM-MØ transcriptomes. G. Summary of GSEA of the indicated gene sets (from GSE188278) on the ranked comparison of siGSK3A/B-GM-MØ vs. siCNT-GM-MØ. H. Summary of GSEA of the gene sets that define the tissue-resident monocyte and macrophage states (MoMac-VERSE) 2 on the ranked comparison of the siGSK3A/B-GM-MØ vs. siCNT-GM-MØ transcriptomes. FDRq values are indicated only if FDRq>0.0; empty dots, Not Significant. I. Summary of GSEA of the gene sets that characterize the macrophage subsets identified in severe COVID-19 23–25 on the ranked comparison of siGSK3A/B-GM-MØ vs. siCNT-GM-MØ. FDRq values are indicated only if FDRq>0.0; empty dots, Not Significant.

Transcriptional effects of GSK3 inhibition on human peripheral blood monocytes.

A. Schematic representation of the exposure of human monocytes to CHIR-99021 or DMSO for 16h. This panel was created with BioRender.com. B. MAFB protein levels in monocytes exposed for 16h to DMSO, CHIR-99021 or M-CSF, as determined by Western blot (left panel). Vinculin protein levels were determined as protein loading control. Mean ± SEM of the MAFB/Vinculin protein ratio from the three independent experiments shown (right panel) (one-way ANOVA with Fisher LSD test: ***, p<0.005). C. Number of differentially expressed genes ([log2FC] >1; adjp<0.05) between DMSO-Mon and CHIR-Mon. Differential gene expression was assessed using DESeq2. D. GSEA of the indicated gene sets (from GSE68061, upper panel; from GSE188278, lower panel) on the ranked comparison of the DMSO-Mon and CHIR-Mon transcriptomes. NES and FDRq values are indicated in each case. E. Relative mRNA levels of the indicated genes in monocytes exposed for 16h to DMSO, CHIR-99021 or M-CSF, as determined by RNA-Seq on three independent samples (GSE256538). Adjp of the comparison, shown in each case, was assessed using DESeq2. F. GSEA of MAFB-regulated gene sets (from GSE155719) on the ranked comparison of the DMSO-Mon and CHIR-Mon transcriptomes. NES and FDRq values are indicated in each case. G. Overlap between the genes upregulated (|log2FC|>1; adjp<0.05) in CHIR-Mon (relative to DMSO-Mon) and M-MØ-specific marker genes (from GSE68061) (left panel) and genes downregulated (|log2FC|>1; adjp<0.05) in CHIR-Mon (relative to DMSO-Mon) and GM-MØ-specific marker genes (from GSE68061) (right panel), with indication of some of the overlapping genes. H. Schematic representation of the exposure of human monocytes to synovial fluid from rheumatoid arthritis patients (RASF) for 48h in the presence or absence of CHIR-99021. This panel was created with BioRender.com. I. MAFB protein levels in monocytes exposed to three independent samples of RASF in the presence of DMSO or CHIR-99021, as determined by Western blot (upper panel). Vinculin protein levels were determined as protein loading control. Mean ± SEM of the MAFB/Vinculin protein ratio from seven independent experiments using three monocyte preparations and three unrelated RASF (lower panel) (paired Student’s t test: **, p<0.01). J. Relative expression of IL10 and LGMN in monocytes exposed to RASF in the presence of DMSO or CHIR-99021, as determined by RT-PCR. Mean ± SEM of the results from the three independent samples are shown (paired Student’s t test: **, p<0.01).

Transcriptional and phenotypic effects of GSK3 inhibition on ex vivo isolated human alveolar macrophages.

A. Schematic representation of the exposure of ex vivo isolated human alveolar macrophages to CHIR-99021 or DMSO for 24h. This panel was created with BioRender.com. B. Volcano plot depicting the differentially expressed genes between CHIR-AMØ and DMSO-AMØ. C. Heatmap of the relative expression of the genes within the CD163+/LGMN+ pro-fibrotic monocyte-derived macrophage cluster (from EGAS00001005634) 23 in DMSO-AMØ (lanes A-C) and CHIR-AMØ (lanes D-F). Differentially expressed between CHIR-AMØ and DMSO-AMØ are shown in greeen, and MAFB-dependent genes (GSE155719) are shown in blue. Representative genes are indicated. D. (Upper panel) GSEA of the gene sets that characterize the monocyte-derived profibrotic macrophage subsets identified in severe COVID-19, namely CD163+/LGMN+ (EGAS00001005634) 23, MoAM3 (GSE155249) 25 and SPP1+ (GSE145926) 24, on the ranked comparison of the transcriptomes of CHIR-AMØ vs. DMSO-AMØ. NES and FDRq values are indicated in each case. (Lower panel) GSEA of the gene sets that characterize tissue-resident alveolar macrophages in severe COVID-19, namely AMØ1 (EGAS00001005634) 23, TRAM1+ (GSE155249) 25 and FABP4+ (GSE145926) 24, on the ranked comparison of the transcriptomes of CHIR-AMØ vs. DMSO-AMØ. E. Heatmap of the relative expression of the genes within the Alveolar Macrophage signature (including all the genes overexpressed in the AMØ1 and AMØ2 macrophages clusters from EGAS00001005634) 23 in DMSO-AMØ (lanes A-C) and CHIR-AMØ (lanes D-F). Differentially expressed between CHIR-AMØ and DMSO-AMØ are indicated in greeen, and selected genes are shown. F. MAFB protein levels in three independent samples of ex vivo isolated AMØ exposed to DMSO or CHIR-99021 for 24h, as determined by Western blot. GAPDH protein levels were determined as protein loading control. G. Production of the indicated soluble factors by three independent preparations of DMSO-AMØ and CHIR-AMØ, as determined by ELISA. H. Expression of inactive GSK3 (S9, pSer9-GSK3β; S21, pSer21-GSK3α), MAFB and CD163 in human lung macrophages. Representative human lung tissues from COVID (n= 2) and control (n= 3) patients co-stained for CD68 (macrophage marker, red), Ser21-phosphorylated GSK3α, Ser9-phosphorylated GSK3β, MAFB or CD163 (green), and DAPI (nuclei, blue), as indicated (Scale bar, 50 μm) (left panel). Plots show MFI (in arbitrary units, a.u.) of single-cell CD68+ macrophages stained for each antibody (right panel) (paired Student’s t test: ****, p<0.001).

Differential expression of GSK3-regulated genes in human lung interstitial and alveolar macrophages.

A. Embedding of 21310 single-cell transcriptomes from human lungs in UMAP (Uniform Manifold Approximation and Projection) with the scVI reduction. Cell-type annotation is based on expression of canonical marker genes, as reported in 47. B. Two-dimensional embedding computed by UMAP with the scVI reduction on computationally identified macrophages after filtering according to number of genes per cell (nFeature, > 200 and < 6000), Unique Molecular Identifiers (nCount, > 1000) and % of mitochondrial genes (< 15 %). C. Relative expression of the indicated genes in the four macrophage subsets defined upon re-clustering of the macrophages identified in the single cell RNA sequencing reported in GSE128033 47. D. Volcano plot illustrating the differentially expressed genes between the Interstitial Macrophage (IMØ) and Alveolar Macrophage (AMØ) subsets defined upon re-analysis of the single cell RNA sequencing reported in GSE128033 47. E-G. GSEA of the gene sets that define human lung AMØ or IMØ macrophage subsets (GSE128033 and GSE193782) 47,55 on the ranked comparison of CHIR-GM-MØ vs. DMSO-GM-MØ transcriptomes (E), CHIR-AMØ vs. DMSO-AMØ transcriptomes (F) or siGSK3A/B-GM-MØ vs. siCNT-GM-MØ (G).

A. MAFB protein levels in GM-MØ exposed to DMSO or distinct CHIR-99021 concentrations (0.1μM, 1μM, 10μM) for 6-48 hours, as determined by Western blot (left panel). GAPDH protein levels were determined as protein loading control. Mean ± SEM of the MAFB/GAPDH protein ratio from three independent experiments are shown (right panel) (one-way ANOVA with Fisher LSD test: ***, p⍰<⍰0.005; ****, p<0.001). B. p-Ser9-GSK3β and p-Tyr216– GSK3β levels in GM-MØ treated with DMSO, CHIR-99021 (10 μM), SB-216763 (10 μM) or LiCl (10 mM) for 48 hours, as determined by Western blot. A representative experiment of three independent experiments is shown in the left panel. GAPDH protein levels were determined as protein loading control. C. Gene ontology analysis of the genes upregulated (|log2FC|>1; adjp<0.05) in CHIR-GM-MØ (relative to DMSO GM-MØ) using Enrichr and the indicated databases. D. Oxygen Consumption rate (OCR) profile of DMSO-GM-MØ and CHIR-GM-MØ monitored using the Seahorse Biosciences extracellular flux analyzer at the indicated time points. Cells were treated sequentially, as indicated, with 1 mM oligomycin (Oligo), 0.6 plus 0.4 mM FCCP, and 1 mM rotenone plus 1 mM antimycin A (Rot/AA). E. Extracellular acidification rate (ECAR), a proxy for the rate of lactate production, measured in DMSO-GM-MØ and CHIR-GM-MØ under basal conditions and after the stimulation with 1 mM oligomycin and at the indicated time points. F. Metabolic parameters obtained from the OCR and ECAR profiling after subtraction of the rotenone/antimycin-insensitive respiration. Basal OCR is the oxygen consumption rate in the absence of effectors, ATP turnover is considered as the oligomycin-sensitive respiration, maximal respiration is the OCR value in the presence of the uncoupler FCCP, glycolytic capacity is the ECAR value after the inhibition with oligomycin of the mitochondrial ATP synthesis (paired Student’s t test: ***, p<0.005; n.s., not significant). In D-F, results are normalized according to protein concentrations and presented as mean ± SEM of six independent samples.

A. Summary of GSEA of the gene sets that characterize the macrophage subsets identified in severe COVID-19 23–25 on the ranked comparison of siGSK3A-GM-MØ vs. siCNT-GM-MØ (square symbols), siGSK3B-GM-MØ vs. siCNT-GM-MØ (star symbols) and siGSK3A/B-GM-MØ vs. siCNT-GM-MØ. FDRq values are indicated only if FDRq>0.0; empty dots, Not Significant. B. GSEA of bona fide MAFB-bound genes (“75-genelist”) 52 on the ranked comparison of the transcriptomes of CHIR-Mon vs DMSO-Mon. NES and FDRq value is indicated. C. Summary of GSEA of the gene sets that characterize the macrophage subsets identified in severe COVID-19 23–25 on the ranked comparison of the transcriptomes of CHIR-Mon vs. DMSO-Mon. FDRq values are indicated only if FDRq>0.0; empty dots, Not Significant. D. Overlap between the genes upregulated (|log2FC|>1; adjp<0.05) in CHIR-Mon (relative to M-CSF-Mon) and M-MØ-specific marker genes (left panel) and genes downregulated (|log2FC|>1; adjp<0.05) in CHIR-Mon (relative to M-CSF-Mon) and GM-MØ-specific marker genes (right panel), with indication of some shared genes. E. Overlap between the genes upregulated or downregulated in CHIR-AMØ (|log2FC|>1 and adjp<0.05) and the CD163+/LGMN+ pro-fibrotic macrophage cluster defined in EGAS00001005634 23 (left panel), or the gene sets of MAFB-dependent (middle panel) and MAFB-inhibited (right panel) genes defined in GSE155719 52. F. Overlap between the genes upregulated or downregulated in CHIR-AMØ (|log2FC|>1 and adjp<0.05, or just adjp<0.05) and the Alveolar Macrophage signature as defined in EGAS00001005634 (AMØ1 and AMØ2 clusters) 23 (left panel), GSE155249 (TRAM1 and TRAM2 clusters) 25 (middle panel) or in 54 (right panel). G. Summary of GSEA of the indicated gene sets (from GSE68061 and GSE188278) on the ranked comparison of the DMSO-AMØ and CHIR-AMØ transcriptomes. FDRq values are indicated only if FDRq>0.0.

A. Number of cells from single cell-RNA sequencing on human lungs from eight independent donors (from GSE128033) 47, after filtering according to number of genes per cell (nFeature, > 200 and < 6000), Unique Molecular Identifiers (nCount, > 1000) and % of mitochondrial genes (< 15 %). B. UMAP (Uniform Manifold Approximation and Projection), with the scVI reduction, embedding of 21310 single-cell transcriptomes from human lungs as reported in GSE128033 47, with color-coding indicating distinct cell clusters. C. UMAP (Uniform Manifold Approximation and Projection), with the scVI reduction, embedding of 21310 single-cell transcriptomes from human lungs as reported in GSE128033 47, with color-coding indicating donor origin. D. Color-coded expression of the indicated genes after projection onto the UMAP embedding shown in B-C.

A. UMAP (Uniform Manifold Approximation and Projection), with the scVI reduction, embedding of single-cell transcriptomes from human lung macrophages as reported in GSE128033 47, with color-coding indicating distinct cell clusters. B. UMAP (Uniform Manifold Approximation and Projection), with the scVI reduction, embedding of single-cell transcriptomes from human lungs as reported in GSE128033 47, with color-coding indicating donor origin. C. Color-coded expression of the genes used for macrophage definition (CD163, FABP4, LYVE1, FCN1), as well as proliferation-associated genes (TYMS, MKI67, TOP2A, NUSAP1) and other bona fide macrophage marker genes (SPI1, FOLR2), after projection onto the UMAP embedding shown in B-C.

Transcriptional datasets used in the present study.