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: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Mechanism underlying tumor necrosis factor-like ligand 1A (TL1A)–C-C motif chemokine ligand 8 (CCL8) axis activation in the context of allergic inflammation. (A and B) Expression levels of TL1A induced by different stimuli in human airway epithelial cells or macrophages. (C) TL1A expression in uninduced macrophages, macrophages induced by interleukin 33 (IL-33), and macrophages induced by IL-33 plus interleukin 2 (IL-2). (D and E) Comparison of the expression levels of CCL8 induced by different concentrations of TL1A, quantified using reverse transcription polymerase chain reaction (RT-PCR) or transcriptomic sequencing. The messenger RNA (mRNA) levels of CCL8 after the transfection of TL1A-M plasmids, quantified using RT-PCR (F) or transcriptomic sequencing (G). (H) Death receptor 3 (DR3) knockdown abolished the elevation of CCL8 induced by TL1A stimulation. (I) CCL8 level in CD14 + macrophages treated or not with TL1A. (J) RT-PCR analysis of CCL8 transcript following DR3 small interfering RNA (siRNA) treatment. Representative data from CD14 + cells from a single donor. (K) A schematic for the administration of different signaling pathway inhibitors to investigate the pathways involved in TL1A–CCL8 axis activation (BAY11-7082, a nuclear factor kappa B [NF-κB] inhibitor; LY294002, a phosphoinositide 3-kinase [PI3K inhibitor]; PD98059, an extracellular signal-regulated kinase [ERK] inhibitor; SB203580, a p38 inhibitor; and SP600125, a c-Jun N-terminal kinase [JNK] inhibitor). Data are presented as the mean ± SD of triplicate measurements from at least 3 independent cell culture experiments. For primary human macrophage experiments (I and J), cells were derived from n = 4 healthy donors. * P < 0.05 and ** P < 0.01 compared with the respective groups. All in vitro stimulation and inhibitor treatments were analyzed in a blinded manner.

Article Snippet: The recombinant proteins, small-molecular compounds, or inhibitors were purchased as indicated: recombinant mouse IL-13, IL-33, and TSLP protein (MCE); recombinant human IL-2, IL-4, TNF-α, and IFN-γ protein (Abbkine); poly(I:C) (Selleck); lipopolysaccharide (Sigma-Aldrich); IL-1β (Thermo Fisher); and small-molecule inhibitors BAY11-7082, LY294002, PD98059, SB203580, and SP600125 (Selleck).

Techniques: Activation Assay, Expressing, Comparison, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Knockdown, Small Interfering RNA, Cell Culture, Derivative Assay, In Vitro