Journal: Blood Advances

Article Title: The alarmin interleukin-33 modulates platelet proteome, function, and biogenesis

doi: 10.1182/bloodadvances.2025018363

Figure Lengend Snippet: IL-33 is not expressed by circulating platelets or MKs. (A-B) Representative immunofluorescence images showing nonspecific IL-33 staining in bone marrow (BM)–derived MKs enriched on BSA gradient (A) and platelet-rich plasma (PRP) (B) from WT and IL-33KO mice. (C) Flow-cytometric detection of IL-33 nonspecific signal in blood platelets from WT and IL-33KO mice (PRP), using an isotype control as a negative reference. (D) Western blot analysis of IL-33 expression in platelets and MK-enriched BM cells, with total lung lysates from WT and IL-33 KO mice used as positive and negative controls, respectively. Following quantification, an equal amount of protein (20 μg) was loaded in each lane. (E) IL-33 promoter activity in total BM cells, blood platelets, and BM MK from IL-33–Citrine reporter mice assessed by flow cytometry. (F) Identification of Citrine + cells as stromal cells (CD41 − CD45 − ) in Citrine reporter mice (Citrine +/+ ) by flow cytometry. Panels A-D used a polyclonal goat anti-mouse IL-33 antibody. FL, full length; IF, immunofluorescence; KO, knockout; mIL, mouse interleukin; SSC-A, side scatter.

Article Snippet: However, IL-33 mRNA and protein have not been detected in previous platelet transcriptomic or proteomic studies., To resolve these discrepancies, we performed analyses with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems, catalog no. AF3626) that has been validated for immunofluorescence, flow cytometry, and western blot analyses in previous studies , including several studies by our team., , , IL-33 is a nuclear cytokine and the Ab AF3626 stains the nucleus of IL-33 producing cells in WT mice but not in IL-33KO mice.

Techniques: Immunofluorescence, Staining, Derivative Assay, Clinical Proteomics, Control, Western Blot, Expressing, Activity Assay, Flow Cytometry, Knock-Out

Journal: Blood Advances

Article Title: The alarmin interleukin-33 modulates platelet proteome, function, and biogenesis

doi: 10.1182/bloodadvances.2025018363

Figure Lengend Snippet: IL-33 deficiency alters the platelet proteome. (A) Proteomic profiling of sorted platelets from WT and IL-33KO mice. (B) Volcano plot comparing IL-33KO and WT platelet proteomes. The x-axis shows log fold change (IL-33KO/WT), and the y-axis shows −log( P -value). Proteins upregulated in IL-33KO platelets are highlighted in blue; downregulated proteins in gray. (C) Number of proteins detected and significantly altered ( P < .03, fold change > 0.2). (D,F) Gene Ontology (GO) analysis of enriched biological processes among upregulated (D) and downregulated (F) proteins ( https://geneontology.org ). (E,G) Heat maps of the top 20 most abundant upregulated (E) and downregulated (G) proteins. Data were obtained from n = 5 mice per group. KO, knockout. Figure created with biorender.com . Blanchard L. (2026) https://BioRender.com/b78qj95 .

Article Snippet: However, IL-33 mRNA and protein have not been detected in previous platelet transcriptomic or proteomic studies., To resolve these discrepancies, we performed analyses with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems, catalog no. AF3626) that has been validated for immunofluorescence, flow cytometry, and western blot analyses in previous studies , including several studies by our team., , , IL-33 is a nuclear cytokine and the Ab AF3626 stains the nucleus of IL-33 producing cells in WT mice but not in IL-33KO mice.

Techniques: Knock-Out

Journal: Blood Advances

Article Title: The alarmin interleukin-33 modulates platelet proteome, function, and biogenesis

doi: 10.1182/bloodadvances.2025018363

Figure Lengend Snippet: IL-33 deficiency impairs platelets adhesion to ECM components. (A) Schematic and (B) analysis of platelet adhesion assays to soluble fibrinogen, with or without ADP (1μM), assessed by flow cytometry. PRP from WT and IL-33 KO mice was analyzed, and results are expressed as a ratio relative to the WT mean. Data were obtained from n = 11 mice per group across 3 independent experiments. (C) Schematic of platelet spreading assays on fixed substrates, analyzed by immunofluorescence in WT and IL-33 KO platelets. Representative images and quantitative analyses of adherent and spreading PRP from WT or IL-33 KO mice on (D) fibrinogen, (E) podoplanin, and (F) laminin at 5 and 15 minutes following ADP (1μM) stimulation. Platelets were stained with CD41-APC. Data were obtained from n = 11 mice per group across 3 independent experiments, except for the laminin spreading assay, which was performed on n = 5 mice in a single experiment. (G-H) Platelet spreading assays without ADP stimulation on fibrinogen (G) and podoplanin (H) at 5 and 15 minutes (n = 7 mice per group per experiment). Statistical analyses were performed using t tests (D-F) and 2-way analysis of variance (ANOVA) with Sidak multiple comparisons test (B). Scale bar, 20 μm. Statistical analysis was conducted using t tests; # P < .05; ## P < .01; ### P < .001; #### P < .0001. IF, immunofluorescence. Figure created with biorender.com . Blanchard L. (2026) https://BioRender.com/z523pur .

Article Snippet: However, IL-33 mRNA and protein have not been detected in previous platelet transcriptomic or proteomic studies., To resolve these discrepancies, we performed analyses with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems, catalog no. AF3626) that has been validated for immunofluorescence, flow cytometry, and western blot analyses in previous studies , including several studies by our team., , , IL-33 is a nuclear cytokine and the Ab AF3626 stains the nucleus of IL-33 producing cells in WT mice but not in IL-33KO mice.

Techniques: Flow Cytometry, Immunofluorescence, Staining

Journal: Blood Advances

Article Title: The alarmin interleukin-33 modulates platelet proteome, function, and biogenesis

doi: 10.1182/bloodadvances.2025018363

Figure Lengend Snippet: IL-33 deficiency reduces thrombus formation under high shear stress. (A) Schematic representation of the thrombus formation assay on collagen during arterial flow of whole blood from WT or IL-33 KO mice. (B) Representative images and quantitative analysis of thrombus volume, expressed as a percentage relative to the WT mean, formed on collagen under physiological arterial shear rate (1500 s ˗1 ) using whole blood from WT or IL-33 KO mice. (C) Representative images and quantitative analysis of thrombus volume, expressed as a percentage relative to the WT mean, formed on collagen under pathological arterial shear rate (3000 s ˗1 ) using whole blood from WT or IL-33 KO mice. Data are representative of 3 independent experiments (n = 4-5 mice/group). Statistical analysis was performed using 2-way ANOVA with Sidak multiple comparisons test, # P < .05. Investigators were blinded to genotype during the thrombus formation assay. Figure created with biorender.com . Blanchard L. (2026) https://BioRender.com/tt7i113 .

Article Snippet: However, IL-33 mRNA and protein have not been detected in previous platelet transcriptomic or proteomic studies., To resolve these discrepancies, we performed analyses with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems, catalog no. AF3626) that has been validated for immunofluorescence, flow cytometry, and western blot analyses in previous studies , including several studies by our team., , , IL-33 is a nuclear cytokine and the Ab AF3626 stains the nucleus of IL-33 producing cells in WT mice but not in IL-33KO mice.

Techniques: Shear, Tube Formation Assay

Journal: Blood Advances

Article Title: The alarmin interleukin-33 modulates platelet proteome, function, and biogenesis

doi: 10.1182/bloodadvances.2025018363

Figure Lengend Snippet: IL-33 treatment in vivo modifies platelet morphology and activation, expands the MK-biased LT-HSC pool, and enhances platelet release within lung capillaries. (A) Schematic overview of the IL-33 treatment protocol: WT mice received 3 intranasal doses of recombinant human IL-33 or PBS. (B) Flow cytometry analysis of platelet morphology in WT mice following IL-33 or PBS treatment. (C) Quantification of platelet counts by flow cytometry in IL-33- or PBS-treated WT mice. Data are from n = 14 mice per group, collected across 3 independent experiments. (D) Flow cytometry assessment of platelet degranulation, measuring alpha granule (P-selectin) and dense granule (CD63) expression after IL-33 or PBS administration. (E) Plasma levels of sCD40L, measured by ELISA in IL-33–treated or PBS-treated WT mice. (D-E) Data are from n = 8 to 9 mice per group, collected across 2 independent experiments. (F) Quantification of BM LT-HSC, separated into CD41 ˗ (canonical) and CD41 + (MK-biased) populations, shown as a percentage of LSK cells. Data are obtained from n = 22 mice per group, collected across 5 independent experiments. (G) Lung intravital microscopy was performed in PF4-mTmG mice treated with IL-33 or PBS, 24 hours after the first (IL-33 1×) or third (IL-33 3×) administration to assess MKs/proplatelets generating platelets. (H) Representative time-lapse intravital lung microscopy images showing PF4 + small proplatelets and large proplatelet/MK structures releasing platelets within pulmonary capillaries. Scale bar, 50 μm. (I-J) Quantification of proplatelet structures producing platelets per hour per ROI in the lung, categorized as small (<200 platelet volume equivalent), medium (200-1000 platelets), and large (>1000 platelets). (K) Total platelet release per hour across the entire lung. Statistical analysis was performed using t test; # P < .05, ## P < .01. A total of n = 5 to 21 videos were analyzed, collected from 3 to 11 mice per group across 5 independent experiments. ns, not significant. Figure created with biorender.com . Blanchard L. (2026) https://BioRender.com/qdgtyov .

Article Snippet: However, IL-33 mRNA and protein have not been detected in previous platelet transcriptomic or proteomic studies., To resolve these discrepancies, we performed analyses with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems, catalog no. AF3626) that has been validated for immunofluorescence, flow cytometry, and western blot analyses in previous studies , including several studies by our team., , , IL-33 is a nuclear cytokine and the Ab AF3626 stains the nucleus of IL-33 producing cells in WT mice but not in IL-33KO mice.

Techniques: In Vivo, Activation Assay, Recombinant, Flow Cytometry, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Intravital Microscopy, Microscopy

Journal: Blood Advances

Article Title: The alarmin interleukin-33 modulates platelet proteome, function, and biogenesis

doi: 10.1182/bloodadvances.2025018363

Figure Lengend Snippet: IL-33 in vivo treatment alters platelet proteome, modulating inflammatory and coagulation-related pathways. (A) Proteomic profiling of sorted platelets from WT mice treated with PBS or recombinant (rIL-33). (B) Volcano plot comparing human rIL-33 and PBS-treated mice platelet proteomes. The x-axis shows log fold change (IL-33KO/WT), and the y-axis shows ˗log( P -value). Proteins upregulated in IL-33 treated mice platelets are highlighted in orange; downregulated proteins in gray. (C) Number of proteins detected and significantly altered ( P < .03, fold change > 0.2). (D,F) Gene Ontology (GO) analysis of enriched biological processes among upregulated (D) and downregulated (F) proteins ( https://geneontology.org ). (E,G) Heat maps of the top 20 most abundant upregulated (E) and downregulated (G) proteins. Data represent n = 10 mice per group, collected from 2 independent experiments. Figure created with biorender.com . Blanchard L. (2026) https://BioRender.com/og48c3y .

Article Snippet: However, IL-33 mRNA and protein have not been detected in previous platelet transcriptomic or proteomic studies., To resolve these discrepancies, we performed analyses with a polyclonal goat anti-mouse IL-33 antibody (R&D Systems, catalog no. AF3626) that has been validated for immunofluorescence, flow cytometry, and western blot analyses in previous studies , including several studies by our team., , , IL-33 is a nuclear cytokine and the Ab AF3626 stains the nucleus of IL-33 producing cells in WT mice but not in IL-33KO mice.

Techniques: In Vivo, Coagulation, Recombinant

Journal: Nature Communications

Article Title: Single-cell multiomics uncovers an endothelial mechanosensitive PIEZO1-IL-33 axis driving pulmonary fibrosis

doi: 10.1038/s41467-026-70193-w

Figure Lengend Snippet: A CellChat analysis of human lung snRNA-seq data. B Venn of 47 shared DEGs from four datasets ( GSE159354 , GSE132771 , GSE128033 , GSE122960 ). C Heatmap of 47-gene expression across cell types. D Correlation analysis (Pearson Correlation) of expression of IGFBP5, IL33, MGP, SPRY1, ACKR1 and mechanical stress scores in ECs. E Correlation analysis (Pearson Correlation) of expression of PIEZO1 and IGFBP5, IL33, MGP, SPRY1, ACKR1 in ECs. F Human snRNA-seq UMAP and violin of IL33 distribution and subpopulations. G Bar chart showed expression of IL33 (mean ± SEM) in IPF (n = 7966 cells) vs control (n = 4174 cells). P value was calculated using the two-tailed unpaired t-test. H Violin of IL33 in PIEZO1 - vs PIEZO1 + ECs. I Bulk-RNA-seq of saline and BLM (7 days and 14 days) induced mice (n = 3 per group). Gene expression levels were Z-score normalized. P value was calculated using the two-tailed unpaired t-test. J qPCR of Il33 in BLM mouse lung ECs. K IL-33 (green) and CD31 (red) co-staining in human IPF vs normal, quantification is shown at right (n = 10). L IL-33 (green) and CD31 (red) co-staining in Yoda1- or GsMTx4-treated fibrotic mouse lungs, quantification is shown at right (n = 3). M IL-33 (green) and CD31 (red)co-staining in Piezo1 WT and Piezo1 ΔEC BLM lungs, quantification is shown at right (n = 3). Error bars represent mean ± SEM, H, K, J, M: two-tailed unpaired t-test; L: one-way ANOVA followed Tukey’s multiple comparwasons test, All n values indicate biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: 30–60 μg of protein was loaded per well on 4–20% SDS-PAGE gels (ACE, #ET15420LGel), transferred to ethanol-activated PVDF membranes (Thermo Fisher Scientific, #88518), blocked with 5% skim milk in TBST, and incubated overnight at 4 °C with primary antibodies diluted in TBST: IL-33 (R&D Systems, #AF3626, 1:1000), PIEZO1 (Proteintech, #15939-1-AP, 1:1 000), CALPAIN2 (Labome, #A03492, 1:1000), P-STAT3 (CST, #9134 s, 1:1000), STAT3 (Abcam, #ab68153, 1:2 000), P-STAT1 (CST, #9171, 1:1000), STAT1 (Proteintech, #10144-2-AP, 1:1000), GAPDH (Servicebio, #GB12002, 1:5000), β-actin (Proteintech, #66009-1, 1:5000), β-tubulin (CST, #2146, 1:5000).

Techniques: Gene Expression, Expressing, Control, Two Tailed Test, RNA Sequencing, Saline, Staining

Journal: Nature Communications

Article Title: Single-cell multiomics uncovers an endothelial mechanosensitive PIEZO1-IL-33 axis driving pulmonary fibrosis

doi: 10.1038/s41467-026-70193-w

Figure Lengend Snippet: A Schematic of BLM-induced fibrosis in Il33 WT vs Il33 ΔEC mice. B Il33 mRNA (n = 4) and IL-33 protein in lung ECs (n = 3). C Representative Masson/PSR images and quantification (n = 5). D αSMA immunofluorescence and quantification (n = 4). E Lung hydroxyproline content (n = 5). F Schematic: EC-specific Il33 overexpression via AAV-Tie1 in Piezo1 WT and Piezo1 ΔEC mice; 4-week pretreatment, BLM at week 4, sacrifice at week 7. G IL-33 expression in ECs, immune and epithelial cells after AAV-OE- Il33 (n = 3). H Masson/PSR images and quantification (n = 5). I αSMA immunofluorescence and quantification (n = 4). J Hydroxyproline content (n = 5). Error bars: mean ± SEM; B,G: two-tailed unpaired t-test; C,D-E,I: two-way ANOVA followed Tukey’s multiple comparisons test; H,J: three-way ANOVA followed Tukey’s multiple comparisons test. All n values indicate biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: 30–60 μg of protein was loaded per well on 4–20% SDS-PAGE gels (ACE, #ET15420LGel), transferred to ethanol-activated PVDF membranes (Thermo Fisher Scientific, #88518), blocked with 5% skim milk in TBST, and incubated overnight at 4 °C with primary antibodies diluted in TBST: IL-33 (R&D Systems, #AF3626, 1:1000), PIEZO1 (Proteintech, #15939-1-AP, 1:1 000), CALPAIN2 (Labome, #A03492, 1:1000), P-STAT3 (CST, #9134 s, 1:1000), STAT3 (Abcam, #ab68153, 1:2 000), P-STAT1 (CST, #9171, 1:1000), STAT1 (Proteintech, #10144-2-AP, 1:1000), GAPDH (Servicebio, #GB12002, 1:5000), β-actin (Proteintech, #66009-1, 1:5000), β-tubulin (CST, #2146, 1:5000).

Techniques: Immunofluorescence, Over Expression, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Single-cell multiomics uncovers an endothelial mechanosensitive PIEZO1-IL-33 axis driving pulmonary fibrosis

doi: 10.1038/s41467-026-70193-w

Figure Lengend Snippet: HUVECs under 20% stretch for 0,6,24 h or 2,12,25 kPa substrates; IL-33 secretion ( n = 3) A and mRNA (n = 3) B . C PIEZO1 knock-down with sh PIEZO1 ( n = 3). HUVECs were transduced with sh PIEZO1 for 48 h, cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch before measuring IL-33 secretion ( n = 3) D and mRNA ( n = 3) E . HUVECs were cultured on 2, 12, 25 kPa substrates for 24 h or stretched (20%) for 0, 6, 24 h, followed by measurement of calpain activity F and CAPN2 protein G ( n = 3). HUVECs (sh PIEZO1 ) were cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch, followed by measurement of calpain activity ( n = 3) H and CAPN2 protein levels ( n = 3) I . J CAPN2 knock-down with shCAPN2 ( n = 3). K HUVECs (sh CAPN2 ) were cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch, IL-33 secretion(left) and mRNA(right) ( n = 3). L Model for paracrine IL-33 regulation. M Cistrome: STAT3 predicted to drive IL-33 regulation. N intersection of top 100 motifs in BLM-treated mouse ECs with top 10 TFs. O The protein levels of P-STAT3 and STAT3 in HUVECs treated with 20% stretch for 0 and 6 hours ( n = 3). P The protein levels of P-STAT3 and STAT3 in HUVECs (sh PIEZO1 or sh CAPN2 ) treated with 20% stretch for 6 h ( n = 3). Q STAT3 knock-down with sh STAT3 ( n = 3). R HUVECs (sh STAT3 ) were cultured on 25 kPa substrates for 24 h or subjected to 6 h mechanical stretch; IL-33 secretion(left) and mRNA(right) ( n = 3). Error bars: mean ± SEM; C , G , J , O – Q : two-tailed unpaired t-test; A , B , F : one-way ANOVA followed Šídák’s multiple comparisons test, D – E , H – I , K , R : two-way ANOVA Tukey’s multiple comparisons test. All n denote biologically independent samples. Source data are provided as a Source Data file.

Article Snippet: 30–60 μg of protein was loaded per well on 4–20% SDS-PAGE gels (ACE, #ET15420LGel), transferred to ethanol-activated PVDF membranes (Thermo Fisher Scientific, #88518), blocked with 5% skim milk in TBST, and incubated overnight at 4 °C with primary antibodies diluted in TBST: IL-33 (R&D Systems, #AF3626, 1:1000), PIEZO1 (Proteintech, #15939-1-AP, 1:1 000), CALPAIN2 (Labome, #A03492, 1:1000), P-STAT3 (CST, #9134 s, 1:1000), STAT3 (Abcam, #ab68153, 1:2 000), P-STAT1 (CST, #9171, 1:1000), STAT1 (Proteintech, #10144-2-AP, 1:1000), GAPDH (Servicebio, #GB12002, 1:5000), β-actin (Proteintech, #66009-1, 1:5000), β-tubulin (CST, #2146, 1:5000).

Techniques: Knockdown, Transduction, Cell Culture, Activity Assay, Two Tailed Test