Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Schematic of the primary mouse astrocyte cultures. (B) Representative immunofluorescence images of cultured astrocytes stained for GFAP, showing characteristic astrocyte morphology. (C) Overview of RNA-seq and capped small (cs)RNA-seq data generation from astrocytes. The schematic shows the typical distribution of csRNA-seq and RNA-seq at various genomic locations, which allows the identification of Transcriptional Start Site (TSSs) using HOMER2. (D) Volcano plot of RNA-seq differential expression in astrocytes treated with vehicle (Veh) or IL-1B (10 ng/mL, 1 h). (E) Pathway enrichment analysis of IL-1B-induced differentially expressed genes. (F) RT-qPCR validation of selected IL-1B-responsive genes in astrocytes

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: Immunofluorescence, Cell Culture, Staining, RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Biomarker Discovery

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: Genome browser views showing RNA-seq signal at the Cxcl1, Ccl20, Csf2 , and Il6 loci in astrocytes treated with vehicle or IL-1B. These representative examples illustrate the robust transcriptional induction of canonical inflammatory genes following IL-1B stimulation. Gene models and genomic coordinates are shown in mm10 .

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: RNA Sequencing

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Pairwise correlation analysis of csRNA-seq replicates from untreated and IL-1B-treated astrocytes. (B) Distribution of strand-specific csRNA-seq reads around GENCODE-annotated TSSs, showing strong enrichment at annotated transcriptional start sites. (C) Genomic annotation of astrocytes TSRs, partitioned across promoter, intronic, intergenic, and other genomic features. (D) Average chromatin profiles at promoter-distal TSRs, showing ATAC-seq and H3K27ac enrichment around transcribed regulatory elements. (E) Average csRNA-seq signal centered on promoter-distal ATAC-seq peaks, comparing transcribed and non-transcribed accessible regions (No Tx: 225,150 peaks, Tx: 14,836 peaks). (F) Average mCH profiles at promoter-proximal TSRs, promoter-distal transcribed TSRs, and non-transcribed distal accessible regions in astrocytes (data from ). (G) Distribution of NFIA and TEAD4 ChIP-seq peaks found overlapping TSRs and ATAC-seq peaks in astrocytes. (H)ChIP-seq read density for NFIA and TEAD4 centered on csRNA-seq defined TSRs, showing transcription factor binding immediately upstream of the primary TSS.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A)Volcano plot of differentially regulated csRNA-seq signal at astrocyte TSRs following IL-1B stimulation, identifying induced and repressed regulatory elements. (B) Genome browser tracks depicting induction of promoter and distal regulatory enhancer transcription by csRNA-seq at the Ccl2 locus. (C) GREAT functional enrichment analysis of IL-1B induced TSRs. (D) Spatial clustering of IL-1B induced TSRs, depicting the density of TSRs in each category adjacent to Induced TSRs. (E) De novo motif enrichment analysis of IL-1B induced TSRs by HOMER. (F) Average csRNA-seq signal centered on promoter-distal NF-κB/p65 peaks, showing increased bidirectional transcription at p65-bound distal elements after IL-1B stimulation, consistent with eRNA induction. (G) Spatial density of NF-κB, AP1, and IRF motifs relative to TSRs (TF binding sites per bp per TSS), showing upstream enrichment of these motifs at IL-1B-induced TSRs compared with unchanged or repressed TSRs.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: Functional Assay, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Genome browser example of an IL-1B-induced locus (Cxcl10) in astrocytes, showing increased transcription initiation after stimulation. (B) Scatter plot comparing IL-1B-induced Log2 csRNA-seq changes at the promoter vs. RNA-seq changes across genes, highlighting genes regulated primarily at initiation (along x-axis) versus those showing stronger changes at the mRNA level (along y-axis). (C) Genome browser tracks at the Junb locus showing increased gene expression and RNAPII elongation in the gene body with limited change in promoter initiation and RNAPII promoter levels, consistent with regulation being mediated primarily at the level of transcription elongation rather than increased initiation. (D) RNAPII ChIP-seq levels at the promoters of IL-1B induced genes stratified by genes with minimal versus strong increases in csRNA-seq initiation activity. (E) Scatter plot comparing changes in overall TSR levels (Log2 Fold change, NT vs. IL-1B) versus their WIP score significance (the −Log10 p-value), identifying TSRs with altered initiation patterns independent of changes in total transcriptional output. (F) Representative examples of TSRs exhibiting a strong change in initiation pattern (top, WIP score −1.61, Lg10 p-value = 9.54e-05) versus a strong change in overall initiation levels with minimal change in initiation shape(WIP score −0.13, Log10 p-value = 0.84). (G) Scatter plot of TF motif enrichment in TSRs with significant changes in overall activity versus changes in TSS positions, highlighting differential associations of NF-κB, TEAD, and NFI motifs with these TSRs classes. (H) Venn diagram showing the overlap of ChIP-seq peaks for NFIA and TEAD4 before and after IL-1B stimulation in astrocytes. (I) Motif enrichment analysis of condition-specific (either Veh or IL-1B) TEAD4- and NFIA-bound regions, showing IL-1B-specific enrichment for inflammatory TF motifs, including NF-κB, IRF (IRF8), and AP1 (i.e. Fra1).

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: RNA Sequencing, Gene Expression, ChIP-sequencing, Activity Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Venn diagram showing overlap between IL-1B-induced genes in astrocytes and KLA-induced genes in bone marrow-derived macrophages (BMDMs). (B) Venn diagram showing overlap between induced TSRs in astrocytes and BMDMs, revealing largely distinct stimulus-responsive enhancer landscapes despite partial overlap in induced genes. (C) Genome browser view of Tnfaip3 locus illustrating that astrocytes and BMDMs induce the same gene but use different enhancers upstream a shared promoter. (D) Top: Venn diagram showing overlap of NF-κB binding sites between activated astrocytes and BMDMs. Bottom: Fraction of astrocyte-specific, shared, and BMDM-specific p65 peaks associated with TSRs induced in astrocytes only, in both cell types, and in BMDM only. (E) Heatmap of de novo motif enrichment at cell type-specific induced TSRs, showing enrichment of lineage-associated motifs for each cell type. (F) Fraction of induced TSR classes bound by lineage-associated TFs, showing preferential association of astrocyte-induced TSRs with NFI and TEAD4 and macrophage-induced TSRs with PU.1 and CEBPα.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: Derivative Assay, Binding Assay

Journal: bioRxiv

Article Title: Transcription initiation profiling defines the regulatory logic of astrocyte gene regulation

doi: 10.64898/2026.05.03.722406

Figure Lengend Snippet: (A) Average eRNA signal centered on astrocyte-specific, shared, and BMDM-specific NF-κBp65 peaks, showing cell type-matched induction of regulatory transcription at p65 bound sites. (B) Fraction of astrocyte-specific, shared, and BMDM-specific p65 peaks overlapping accessible chromatin regions in astrocytes or BMDMs, indicating that NF-κB recruitment occurs preferentially at cell-type-specific open chromatin regions. (C) Violin plots showing increase in ChIP-seq signal for p65, NFIA, TEAD4 at astrocyte IL-1B-induced TSRs and for p65, PU.1, and CEBPβ in macrophage KLA-induced TSRs.

Article Snippet: Two days before collection, the astrocytes were plated at a density of 500,000 cells per well on 4-well chamber slides to perform immunocytochemistry or a density of 5.5 million cells per 15 cm dish to be treated with 10ng/mL of recombinant human interleukin 1 beta (IL-1B) (Invivogen, Cat#rcyec-h) for 1hr before sample collection.

Techniques: ChIP-sequencing

Journal: Stem Cell Research & Therapy

Article Title: IL-1β modulates inflammatory response of human bone marrow-derived MSCs and neutrophil recruitment in vitro via NF-kB-associated signaling

doi: 10.1186/s13287-026-05029-x

Figure Lengend Snippet: IL-1β stimulation changes the transcriptomic profile of BM-hMSCs. Schematic of experimental design A . BM-hMSCs were exposed to IL-1 β (3 replicates, 1 donor) or left unstimulated (3 replicates, 1 donor) for one hour, followed by bulk RNA sequencing. PCA plot was used to show the variance between the unstimulated control (green) and IL-1 β exposed (purple) samples B . Heatmap displaying the Z-score of the top 100 varying genes across all samples. Red color indicates higher expression of the genes and blue color indicates decreased expression C . Volcano plot of differentially expressed genes, showing their log2 fold change (X-axis) and -log10 adjusted p-values (Y-axis) D . IL-1 β Interleukin-1β, BM-hMSCs, Bone marrow derived human mesenchymal cells, PCA Principal component analysis, FC Fold change, NS not significant. Figure 1A was created using Biorender.com

Article Snippet: Following synchronization, medium was removed, and cells were stimulated with IL-1β (20 ng/ml in DPBS with 0.1% Bovine Serum Albumin, cat# 208-IL-010, R&D Systems) for 1 h (RNA-sequencing) or 24 h. Unstimulated control BM-hMSCs were exposed to serum free DMEM only.

Techniques: RNA Sequencing, Control, Expressing, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: IL-1β modulates inflammatory response of human bone marrow-derived MSCs and neutrophil recruitment in vitro via NF-kB-associated signaling

doi: 10.1186/s13287-026-05029-x

Figure Lengend Snippet: IL-1β stimulation increases expression of neutrophil recruitment and NF-kB signaling genes by BM-hMSCs. Heatmap clustering of the Z-score of the top 20 differentially expressed genes between unstimulated control BM-hMSCs (3 replicates, 1 donor, green) and IL-1 β stimulated BM-hMSCs (3 replicates, 1 donor, purple). Red color indicates higher expression of the genes and blue color indicates decreased expression A . Protein expression of CXCL1 (10 replicates, 4 donors), CCL2 (4 replicates, 2 donors), CXCL5 (4 replicates, 2 donors), and CXCL8/IL-8 (7 replicates, 3 donors) in BM-hMSCs secretome measured by ELLA or ELISA. Data are presented as median, and statistical analysis was performed using unpaired t-test with Welch’s correction B . Bar plot of the top 20 significant Gene Ontology (GO) biological processes representing IL- 1β induced upregulated differentially expressed genes C IL-1 β, Interleukin-1β; BM-hMSCs, Bone marrow derived human mesenchymal cells; CCL2, chemokine (C–C motif) ligand 2; CXCL1, chemokine (C-X-C motif) ligand 1; CXCL2, chemokine (C-X-C motif) ligand 1; CXCL8, chemokine (C-X-C motif) ligand 8; ****, p < 0,0001

Article Snippet: Following synchronization, medium was removed, and cells were stimulated with IL-1β (20 ng/ml in DPBS with 0.1% Bovine Serum Albumin, cat# 208-IL-010, R&D Systems) for 1 h (RNA-sequencing) or 24 h. Unstimulated control BM-hMSCs were exposed to serum free DMEM only.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: IL-1β modulates inflammatory response of human bone marrow-derived MSCs and neutrophil recruitment in vitro via NF-kB-associated signaling

doi: 10.1186/s13287-026-05029-x

Figure Lengend Snippet: IL-1β stimulated BM-hMSCs increased neutrophil recruitment partly via the NF-kB signaling pathway. Schematic of experimental design of the neutrophil migration assay. Illustration was created with BioRender.com A . Number of neutrophils that migrated through the transwell membrane from the top well to the bottom towards control conditioned medium, conditioned medium from IL-1β stimulated BM-hMSCs, and conditioned medium from IL-1β with the NF-kB inhibitor stimulated BM-hMSCs. The experiment was performed using neutrophils from four different donors (each donor represent one data point) and the experiments were performed on three different days. B . Protein expression of CXCL1 was measured in conditioned medium from unstimulated BM-hMSCs (4 replicates, 1 donor, green), IL-1β stimulated BM-hMSCs (4 replicates, 1 donor, purple), and ILβ stimulated BM-hMSCs with 10 μM BAY 11–7082 (4 replicates, 1 donor, grey) C . Phospho-p65 levels were quantified and normalized to total p65. Data are expressed relative to control, which was set to 1 D . IL-1β Interleukin-1β BM-hMSCs Bone marrow-derived human mesenchymal cells, Ctrl control, CXCL1 chemokine (C-X-C motif) ligand 1, ns not significant; * p < 0,05; ****, p < 0,0001

Article Snippet: Following synchronization, medium was removed, and cells were stimulated with IL-1β (20 ng/ml in DPBS with 0.1% Bovine Serum Albumin, cat# 208-IL-010, R&D Systems) for 1 h (RNA-sequencing) or 24 h. Unstimulated control BM-hMSCs were exposed to serum free DMEM only.

Techniques: Migration, Membrane, Control, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: (A-F) NHBE/HMVEC-L co-cultures were infected with SARS-CoV-2 (MOI 1) or mock-infected and treated with 10 mg/mL Anakinra immediately post-infection. Cells were fixed at 72h post-infection. A) Immunofluorescence staining for ICAM-1 (green), F-actin (Phalloidin; grey), and nuclei (DAPI; blue). Images are representative of n = 5 independent experiments. B) ICAM-1 intensity between conditions was analysed by one-way ANOVA, with Sidak’s multiple comparison test. C) Immunofluorescence staining for VE-cadherin (magenta), F-actin (Phalloidin; grey), and nuclei (DAPI; blue). Images are representative of n = 3 independent experiments. D) Quantification of gaps in the endothelial monolayer under different conditions was determined by calculating the percentage of the image area covered by gaps, and analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. E) Zombie Red-stained cells (magenta) indicate cells (containing F-actin and nuclei) undergoing cell death. Images are representative of n = 3 independent experiments. F) The percentage of Zombie Red positive cells was analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. G) IL-1β levels in the apical supernatant of NHBE monocultures infected with SARS-CoV-2 (MOI 1) or mock-infected, at 24, 48, and 72h post-infection (n = 3 independent experiments). H) TNF levels in the apical (left panel) and basal (right panel) supernatant of SARS-CoV-2-infected NHBE/HMVEC-L co-cultures, untreated or treated with Anakinra, at 24, 48, and 72h post-infection (n = 2 independent experiments). Scale bar for all images = 50 µm. 5 ROIs per experiment were quantified (small dots) and are colour-coded per experiment. The average of the 5 ROIs is represented with the large dot (colour-coded per experiment), and the data shows mean ± SEM. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HMVEC-L monocultures were stimulated with indicated concentrations of recombinant human TNF (Thermo Fisher Scientific; PHC3011), recombinant human IL-1β (RnD systems; 201-LB ) , recombinant IL-6 (Peprotech; 200-06-20UG) or recombinant human soluble IL-6R (Peprotech; 200-06RC-20UG) diluted in hydrocortisone-free 50:50 ALI/EGM-2MV medium for 24h at 37 °C, 5% CO 2 .

Techniques: Infection, Immunofluorescence, Staining, Comparison

Journal: bioRxiv

Article Title: IL-1β and TNF drive endothelial dysfunction and coagulopathy in acute COVID-19

doi: 10.64898/2026.03.21.713333

Figure Lengend Snippet: A) Representative immunohistochemistry (IHC) images of lungs from SARS-CoV-2-infected (10 4 TCID50) or mock-infected wild-type (WT), Tnf -/- , Il1b -/- , and Tnf -/- IL1b -/- mice, harvested at 3 days post-infection (dpi). Tissues were stained with CD31 (magenta), ICAM-1 (green), and DAPI (blue). Scale bar = 50 µm. Images are representative of 5 images per mouse, 3 mice per group. B) Quantification of ICAM-1 intensity in areas of CD31 staining, analysed by 2-way ANOVA with Sidak’s multiple testing correction. 5 ROIs per mouse were quantified (small dots) and are colour-coded per mouse. The average of the 5 ROIs is represented with the large dot (colour-coded per mouse), and the data shows mean ± SEM. C-F) Aged K18-hACE c57BL/6□J mice infected with SARS-CoV-2 (10 3 PFU) and treated with an isotype control or anti-IL-1β antibody at 1h or 3 days post-infection. Lungs were harvested at day 6 post-infection. C) Lung tissues were stained with CD31 (magenta), ICAM-1 (green), and DAPI (blue). Scale bar = 100 µm. Images are representative of 5 images per mouse, 4-6 mice per group. D) Quantification of ICAM-1 intensity in areas of CD31 staining, analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. 5 ROIs per mouse were quantified (small dots) and are colour-coded per mouse. The average of the 5 ROIs is represented by the large dot (colour-coded per mouse), and the data show the mean ± SEM. E) Lung tissues were stained for fibrinogen. Scale bar = 50 µm. Images are representative of 4-6 mice per group. F) Quantification of fibrinogen staining intensity analysed by one-way ANOVA, with Sidak’s multiple comparison’s test. Asterisks indicate statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HMVEC-L monocultures were stimulated with indicated concentrations of recombinant human TNF (Thermo Fisher Scientific; PHC3011), recombinant human IL-1β (RnD systems; 201-LB ) , recombinant IL-6 (Peprotech; 200-06-20UG) or recombinant human soluble IL-6R (Peprotech; 200-06RC-20UG) diluted in hydrocortisone-free 50:50 ALI/EGM-2MV medium for 24h at 37 °C, 5% CO 2 .

Techniques: Immunohistochemistry, Infection, Staining, Control