Journal: bioRxiv

Article Title: IL-1-conferred gene expression pattern in ERα + BCa and AR + PCa cells is intrinsic to ERα − BCa and AR − PCa cells and promotes cell survival

doi: 10.1101/773978

Figure Lengend Snippet: RT-qPCR was performed for select genes for MCF7 and LNCaP cell lines treated with vehicle control or 25 ng/ml IL-1α or IL-1β for 5 days. MDA-MB-231, BT549, PC3 and DU145 cell lines were treated with vehicle control only. (A) RT-qPCR shows that CCL20 , CD68 , IL8 , p62 , SOX9 and Zeb1 are induced by IL-1 in MCF7 and/or LNCaP cell lines and are basally high in MDA-MB-231, BT549, PC3 and/or DU145 cell lines. (B) RT-qPCR shows that CXCR7 and MMP16 , but not CDK2 or PLK1 , are downregulated by IL-1 in MCF7 and/or LNCaP cell lines and are basally high in MDA-MB-231, BT549, PC3 and/or DU145 cell lines. N = 3 biological replicates; error bars, +/−STDEV; p-value, * ≤ 0.05, ** ≤ 0.005, ***≤ 0.0005. mRNA fold change is normalized to MCF7 vehicle control for the BCa cell lines and to LNCaP vehicle control for the PCa cell lines.

Article Snippet: Human recombinant IL-1α (GoldBio, St Louis MO; 1110-01A-100) and IL-1β (GoldBio, St Louis MO; 1110-01B-100) were resuspended in 0.1% bovine serum albumin (BSA, Thermo Fisher Scientific; BP 1600-1) in 1X phosphate buffered saline (PBS).

Techniques: Quantitative RT-PCR

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 2 PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- mice show reduced levels of circulating cytokines. (A) Volcano plot of proteins regulated in Olink mouse exploratory panel comparing serum protein levels of mice fed a NC vs. PCSK9-WT mice. Red data points indicate the difference of the NPX mean between PCSK9-WT and NC for each protein. The dashed line intersecting the y-axis indicates the significance of p<0.05. (B) Shiny GO (19) (Version 0.77) enrichment analysis annotating the significantly regulated proteins to the Gene Ontology (GO) Biological Process (false detection rate (FDR) cutoff: 0.1). The top 10 regulated pathways are presented as a barplot with the colors indicating the -log10 FDR, with red as the highest and blue as the lowest. (C) Heatmap depicting the dynamics of proteins significantly regulated in Il1a-/- compared to PCSK9-WT. Data is presented as fold change of PCSK9-WT, PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- to NC. (D) Venn Diagram representing serum proteins specifically regulated in only NC or PCSK9-Il1a-/- animals compared to PCSK9-WT. Shared regulated proteins are displayed at the intersection of both areas. (E) Bargraphs of serum IL-1a levels measured in the Olink 96 mouse exploratory panel. The dashed line indicates the limit of detection of the assay. Data is presented with the Olink NPX value as mean ± SD, *p<0.05. PCSK9-WT (n=5), PCSK9-Il1a-/- (n=6), PCSK9-Nlrp3-/- (n=5), and PCSK9-Il1b-/- (n=4).

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques:

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 3 Cell surface translocation of IL-1a is not influenced by NLRP3 and IL-1b in murine BMDC. (A) Schematic overview of the experimental setup. Bone marrow-derived dendritic cells (BMDCs) were replated after 7 days of differentiation and stimulated with 100 ng/ml ultrapure Lipopolysaccharide (upLPS) overnight. Afterward, cells were harvested, and fractions were isolated using a detergent-based method. (B) Representative immunoblot of the membrane and cytoplasmic fraction of BMDCs with or without upLPS stimulation. Protein levels of IL-1a, GAPDH (glycerinaldehyde-3- phosphate-dehydrogenase, cytoplasmatic marker), and NaK ATPase (sodium–potassium ATPase, membrane marker) are presented. (C) Densiometric quantification of IL-1a in cytoplasmic fraction normalized to GAPDH and IL-1a in membrane fraction normalized to NaK ATPase. (D) Representative flow cytometry scatter plot of staining for IL-1a on stimulated BMDCs after gating for viable, non-fixated (7AAD-) cells. Total IL-1a was measured on fixed cells. (E) Barplot depicting the percentage of total IL-1a positive monocytes (7AAD+) with or without upLPS stimulation. (F) Barplot depicting the percentage of csIL-1a positive monocytes (7AAD-) with or without upLPS stimulation. (G) Barplot representing the percentage of non-permeable monocytes with or without upLPS stimulation. (H) Representative immunoblot of membrane and cytosolic fraction from upLPS-stimulated BMDCs of PCSK9-WT, PCSK9-Il1a-/-, PCSK9-Nlrp3-/-, and PCSK9-Il1b-/- animals. Protein levels of IL-1a, GAPDH (cytoplasmatic marker), and NaK ATPase (membrane marker) are presented with corresponding densiometric quantification (I) of log-transformed IL-1a expression. All data are presented as mean ± SEM, *p<0.05.

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Translocation Assay, Derivative Assay, Isolation, Western Blot, Membrane, Marker, Cytometry, Staining, Transformation Assay, Expressing

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 5 Myristoylation regulates csIL-1a in murine bone marrow cells and human monocytes. (A) Barplot depicting the percentage of murine bone marrow cells with myristoylated proteins. Bone marrow cells were cultured and myristoylated proteins were labeled overnight. Data are presented as mean ± SEM of NC (n=3) and PCSK9-WT (n=3), *p<0.05. (B) Mean fluorescence intensity of human primary monocytes under culture conditions (con) or with overnight incubation of N-myristoyltransferase inhibitor IMP-1088 [1 µM]. Data are presented as mean ± SEM of three independent experiments. One-sided paired t-test was performed, *p<0.05. (C) Percentage of csIL-1a presenting monocytes stimulated with 100 ng/ml upLPS and 1 µM IMP-1088 as indicated. Data are presented as mean ± SEM of four independent experiments, *p<0.05.

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Cell Culture, Labeling, Incubation

Journal: Frontiers in immunology

Article Title: Membrane-bound Interleukin-1α mediates leukocyte adhesion during atherogenesis.

doi: 10.3389/fimmu.2023.1252384

Figure Lengend Snippet: FIGURE 4 IL-1a surface expression on human monocytes induces VCAM1 expression and leads to increased adhesion on endothelial cells. (A) Schematic principle of proximity ligation assay (PLA) and representative picture of PLA. Direct binding of csIL-1a to the Interleukin-1 receptor 1 (IL1R1) leads to a fluorescence signal detectable at 594 nm. Human umbilical vein endothelial cells (HUVECs) were treated with monocytes for 6h, stimulated with and without upLPS (100 ng/ml). Cells were imaged at a 40× magnification (scale bar 50µm). HUVECs and monocytes are presented in blue, IL-1a/IL1R1 PLA signal is visible as red dots. (B) Representative flow cytometry histogram of vascular cell adhesion molecule–1 (VCAM1) stained HUVECs after 4h treatment with upLPS-stimulated monocytes. 10 µg/ml neutralizing IL1R1 (nIL1R1) antibody was added 1h before HUVEC-monocyte co-incubation. (C) Bar graph depicting the percentage of VCAM1- positive HUVECs after treatment with unstimulated and upLPS-stimulated monocytes. HUVECs were incubated with and without nIL1R1 antibody (10 µg/ml) for 1h before co-incubation. Data are presented as mean ± SEM of seven independent experiments; *p< 0.05. (D): Schematic experimental setup of monocyte adhesion assay. Primary monocytes were treated as indicated, labeled with Calcein and 4x washings. HUVECs were treated with and without 1 µg/ml neutralizing IL-1a antibody (nIL-1a) 1h before co-incubation. Then, HUVECs were treated with labeled monocytes for 4h. The initial fluorescence of adhering monocytes was measured as well as after two washes. Cells were imaged (4× magnification), scalebar 100 µM. (E) Quantification of adhering monocytes to HUVECs presented as mean ± SEM of four independent experiments. Repeated measure ANOVA was performed, followed by Sidak’s multiple comparison test (*p< 0.05).

Article Snippet: The primary antibodies hIL-1a (1:50, sc-271618, clone G10, Santa Cruz, USA) and IL1R1 Polyclonal Antibody (1:100, PA5-117479, Invitrogen, USA) were diluted in Duolink antibody dilution and stained overnight.

Techniques: Expressing, Proximity Ligation Assay, Binding Assay, Cytometry, Staining, Incubation, Cell Adhesion Assay, Labeling, Comparison

Journal: The Journal of Infectious Diseases

Article Title: Interleukin 1α (IL-1α) Promotes Pathogenic Immature Myeloid Cells and IL-1β Favors Protective Mature Myeloid Cells During Acute Lung Infection

doi: 10.1093/infdis/jiy049

Figure Lengend Snippet: Interleukin 1α (IL-1α) elicits an immature myeloid cell response, but IL-1β is required for mature myeloid cells. Wild-type (Wt) mice were treated with anti–IL-1α or anti–IL-1β and infected with Francisella tularensis. A, Tissue bacterial burden and total numbers of mature neutrophils (mPMNs), immature neutrophils (iPMNs), mature macrophages (mMΦ), and immature macrophages (iMΦ) in the lungs of IL-1α–neutralized mice at day 6. Data are median values from 2 independent experiments. *P < .05 and **P < .01, by the unpaired Student t test. B, Tissue bacterial burden and total numbers of mPMNs, iPMNs, mMΦ, and iMΦ in lungs of IL-1β–neutralized mice at day 5. Data are median values from 2 independent experiments. *P < .05 and **P < .01, by the unpaired Student t test. C, Levels of cytokines and chemokines measured by the Luminex assay in lung homogenates of IL-1α–neutralized mice at day 6. Data are median values from 2 independent experiments. *P < .05, by the nonparametric Mann-Whitney test. D, Levels of cytokines and chemokines measured by the Luminex assay in lung homogenates of IL-1β–neutralized mice at day 5. Data are median values from 2 independent experiments. *P < .05 and **P < .01, by the nonparametric Mann-Whitney test. Ab, antibody; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-17, interleukin 17; IsoAb, isotype control antibody.

Article Snippet: Cytokine Neutralization F. tularensis –infected mice were injected intraperitoneally with 200 µg/mouse of anti–IL-1β (clone B122), anti–IL-1α (clone ALF-161), or isotype control antibody (BioXcell, West Lebanon, NH) at −1, 1, 3, 4, and 5 days after infection.

Techniques: Infection, Luminex, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Interleukin 1α (IL-1α) Promotes Pathogenic Immature Myeloid Cells and IL-1β Favors Protective Mature Myeloid Cells During Acute Lung Infection

doi: 10.1093/infdis/jiy049

Figure Lengend Snippet: Interleukin 1β (IL-1β) favors myeloid cell maturation, differentiation, and phagocytosis. A and B, Ly6C+ cells from bone marrow (A) or blood (B) were or were not treated with recombinant IL-1α (rIL-1α) or rIL-1β. Left, Percentage of cells that phagocytosed the bioparticles. Data are median values with ranges, from 2 experiments. *P < .05, by the unpaired Student t test. Right, Intracellular bacterial burden. Data are median values from 2 experiments. *P < .05, by the nonparametric Mann-Whitney test. C and D, Ly6G+ cells from bone marrow (C) or blood (D) show the percentage of cells that phagocytosed the bioparticles. Data are median values with ranges, from 2 experiments. *P < .05, by the unpaired Student t test.

Article Snippet: Cytokine Neutralization F. tularensis –infected mice were injected intraperitoneally with 200 µg/mouse of anti–IL-1β (clone B122), anti–IL-1α (clone ALF-161), or isotype control antibody (BioXcell, West Lebanon, NH) at −1, 1, 3, 4, and 5 days after infection.

Techniques: Recombinant, MANN-WHITNEY

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A to D) Representative gross findings (A), histology (hematoxylin and eosin staining) (B), epidermal thickness (C), and numbers of total infiltrating cells and neutrophils in the dorsal skin (D) of wild-type (n =6 or 8) and Elovl6 -/- (n =6 or 8) mice before and on day 9 after the start of tape stripping. (E, F) Epidermal thickness (E) and numbers of total infiltrating cells and neutrophils in the skin (F) of Elovl6 fl/fl (n = 5) and Elovl6 fl/fl K14 -Cre (n = 4) on day 9 after the start of tape stripping. Black bars indicate scale (50 μm) (B). Error bars indicate 1 SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of three (A-D) and two (E, F) independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Staining, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: ( A ) The fetuses at E13.5 and E17.5 from WT and Elovl6 -/- mice were stained with 0.1% toluidine blue for 24 h and photographed. ( B ) The transepidermal water loss of 6-8 weeks old WT and Elovl6 -/- mice was measured before and after tape stripping (n = 14). Error bars indicate SD. NS, not significant. Data are representative of three independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Staining, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Quantitative RT-PCR analysis of epidermis of wild-type and Elovl6 -/- mice isolated 6 h after tape stripping (n = 10 in each group). (B) Epidermis was isolated before, and 12 h after, tape stripping from wild-type and Elovl6 -/- mice and cultured for 24 h. The concentrations of IL-1β and CXCL-1 in the supernatants were measured by using cytometric bead array (n = 11 per group). (C) Quantitative RT-PCR analysis of ¡lift and Cxcl1 in the epidermis of wild-type, Myd88 -/- , and Ticami -/- mice before, and 6 h after, tape stripping (n = 7 to 11 per group). Error bars indicate 1 SD; *, P < 0.05; ***, P < 0.001. Data are representative of three independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Quantitative RT-PCR, Isolation, Stripping Membranes, Cell Culture

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Representative histopathology of wild-type and Elovl6 -/- mice 4 h after tape stripping. Black arrows indicate degenerated keartinocytes with irregularly shaped nuclei. Scale bar, 50 pm. (B, C) Flow cytometry of epidermal cells isolated from the skin of wild-type and Elovl6 -/- mice at the indicated time points after tape stripping. Cells were stained with anti-CD45.2, anti-CD49f and propidium iodide (PI) and the proportion of PI+ cells in CD45.2-CD49f+ cells were shown (n = 6 to 10 per group). Error bars indicate 1 SD; *, P < 0.05. Data are representative of two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Histopathology, Stripping Membranes, Flow Cytometry, Isolation, Staining

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Live cell number of primary peritoneal macrophages 16 h after stimulation with oleic acid (OA) or CVA (300 µM) (n = 3 in each group). (B, E) Primary mouse keratinocytes were cultured for 6 h in the presence or absence of 10 μM of triacsin C (B), or 1 mM necrostatin (Nec-1), 1 mM necrosulfonamide (NSA), 2 mM IM-54, or 1 mM cyclosporine (CyA) (E), followed by stimulation by adding 300 μM CVA; live cells were counted 16 h afterward (n = 3). (C) A representative dead primary keratinocyte induced by stimulation with 300 μM CVA for 10 h under a transmission electron microscope. (D) Immunofluorescence microscopic study of primary keratinocyte 10 h after stimulation with CVA or 6 h after ultraviolet irradiation. Cells were stained with anti-cleaved caspase 9, followed by Alexa Fluor 594-conjugated secondary antibody and DAPI. White bars indicate a scale (20 μm). Percentage of cleaved caspase 9-positive cells was calculated (n = 3). Error bars indicate SD. NS, not significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of more than two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Cell Culture, Transmission Assay, Microscopy, Immunofluorescence, Irradiation, Staining

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: (A) Enzyme-linked immunosorbent assay of HMGB-1 (n = 4 per group) and cytokine bead array of IL-1α (n = 3 per group) in the supernatant of cultured primary keratinocytes 10 h after initiation of stimulation with 300 pM OA or CVA. (B) Quantitative RT-PCR analysis of Il1β and Cxcl1 in the epidermis of wild-type mice 6 h after topical application of ethanol (control) (n = 10) or 15mM of OA (n = 13) or CVA (n = 14) (B). (C, D) Wild-type and Elovl6 -/- mice were treated with PBS (n = 10 and 12, respectively), an IL-1 receptor antagonist (n = 9 and 8, respectively), or a CXCR-2 antagonist (n = 5 and 4, respectively) daily for 9 days, from the beginning on the day of tape stripping. Epidermal thickness (C) and the number of infiltrating neutrophils (D) were analyzed on day 9. (E) A proposed signal pathway from mechanical damage onto the skin to skin inflammation. Error bars indicate SD; *, P < 0.05; **, P < 0.01, ***, P < 0.001; NS, not significant. Data are representative of at least two independent experiments.

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Stripping Membranes

Journal: bioRxiv

Article Title: A long-chain fatty acid elongase Elovl6 regulates mechanical damage–induced keratinocyte death and skin inflammation

doi: 10.1101/264838

Figure Lengend Snippet: Quantitative RT-PCR analysis of Il1β and Cxcl1 in keratinocytes of wild-type and Elovl6 -/- mice after stimulation or not with HMGB-1 or IL-1α in vitro (n=10 per group) (A) and in the epidermis isolated 4 h after injection intradermally with PBS, HMGB-1, or IL-1α (n = 8 per each group) (B).

Article Snippet: Primary mouse keratinocytes were stimulated with bovine HMGB-1 (Chondrex, Redmond, Washington, USA) or mouse IL-1α (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Quantitative RT-PCR, In Vitro, Isolation, Injection