Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: In mice, treatment with angiotensin II (Ang II) and β-aminopropionitrile (BAPN) causes acute inflammation before aortic dissection occurs. ( A ) Gross morphology of aortic dissection (AD) in male C57BL/6J mice; aortas in control mice without Ang II or BAPN treatment (Control), aortas without AD after exposure to ANGII (1 µg/kg/µl) and BAPN (5 g/drinking water) (No-AD), and aortas with AD after exposure to ANGII and BAPN (AD). ( B , C ) Flow cytometric analysis showing percentages with plots after gating of CD45 + immune cells excluding doublets ( B ) and cell numbers of total CD45 + cells, neutrophils, Ly6C high monocytes, and macrophages ( C ). (n = 5 controls, n = 4 Non-AD, n = 5 AD). One-way analysis of variance with Tukey’s multiple comparison test; * P < 0.05. ( D ) Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis identifying unique immune populations in male mice exposed to Ang II (1 µg/kg/µl) and BAPN (5 g/l in drinking water) for 1 week. ( E ) Dot plots displaying the signature cell gene expression markers. ( F , G ) CellChat showing the activated interaction between each cell population in the interleukin-1 (IL-1) signaling pathway. ( H ) Feature plots of IL-1β and Il1r1 expression on UMAP. CellChat; a tool that can quantitatively infer and analyze intercellular communication networks from scRNA-seq data. Macs macrophages, Monos monocytes, DCs dendritic cells, NK cells natural killer cells, ILCs Innate lymphoid cells, VSMCs vascular smooth muscle cells, ECs endothelial cells.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 µg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Comparison, Expressing

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: IL-1β + inflammatory macrophages are accumulated before the onset of macroscopic aortic dissection (AD) in mice. ( A ) Re-clustering of monocytes and macrophages into three groups: mouse aortas without Ang II or BAPN (control), mouse aortas without AD after exposure to Ang II and BAPN (non-AD), and mouse aortas with AD after exposure to Ang II and BAPN (AD). ( B ) Violin plots depicting single-cell gene expression of each canonical monocyte or macrophage marker for clustering. ( C ) Proportion of each monocyte-macrophage cluster. ( D ) Heatmap of the top-5 differentially expressed genes in each monocyte and macrophage cluster. ( E ) Trajectory pseudo-time analysis in Monocle3 with Seurat cluster annotations (left) and change in the expression of IL-1β across pseudo-time for the monocyte and monocyte-derived macrophage partitions (clusters 0, 1, 2, 3, 4) in the non-AD and AD groups (right). Macs macrophages, Monos monocytes, Ang II Angiotensin II, BAPN β-aminopropionitrile.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 µg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Expressing, Marker, Derivative Assay

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Anti-IL-1β neutralizing antibody improves survival rate in mice. ( A ) Kaplan–Meier survival curve tracking death due to ruptured aortic dissection (AD) in male C57BL/6J mice exposed to Ang II (1 µg/kg/µl) and BAPN (1 g/l in drinking water) for 2 weeks after treatment with isotype control or anti-IL-1β neutralizing antibody (isotype IgG or anti-IL-1β neutralizing antibody; 200 β g i.p./mouse/every 3 days, n = 14 sham; n = 14 anti-IL-1β neutralizing antibody). Log-rank (Mantel–Cox) test, *P < 0.05. ( B ) Incidence of AD in AD model mice treated with the isotype control or anti-IL-1β antibody (n = 11/14 sham; n = 7/14 anti-IL-1β neutralizing antibody). ( C ) Blood pressure of AD model mice treated with the isotype control or anti-IL-1β neutralizing antibody (n = 8, sham; n = 9, anti-IL-1β neutralizing antibody). ( D ) Representative images of Elastica van Gieson (EVG) staining in the ascending (upper) and thoracoabdominal aorta (bottom). ( E ) Percentage of EVG-stained area per total tunica media without AD in the ascending aorta (upper) and thoracoabdominal aorta (bottom). *P < 0.05. Ang II Angiotensin II, BAPN β-aminopropionitrile.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 µg every 3 days for 2 weeks (Fig. ).

Techniques: Dissection, Control, Staining

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Single-cell RNA sequencing reveals a characteristic immune cell landscape in the ascending aorta in patients with Stanford type A acute aortic dissection (AAD). ( A ) Representative contrast-enhanced computed tomography (CT) image of Stanford type A AAD. ( B ) Histological images stained with hematoxylin and eosin (HE) and Elastica van Gieson (EVG). ( C , D ) Uniform manifold approximation and projection (UMAP) dimensionality reduction analysis identifying a unique single-cell immune landscape in the aortas of combined ( C ) and each individual Control or AAD group ( D ) (n = 3 Controls from Li et al. ; n = 2 AAD). ( E ) Dot plots displaying signature cell gene expression markers for each immune cell cluster. ( F ) Comparison of the proportions of each cluster between controls and patients with AADs.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 µg every 3 days for 2 weeks (Fig. ).

Techniques: RNA Sequencing Assay, Dissection, Computed Tomography, Staining, Control, Expressing, Comparison

Journal: Scientific Reports

Article Title: Intense impact of IL-1β expressing inflammatory macrophages in acute aortic dissection

doi: 10.1038/s41598-024-65931-3

Figure Lengend Snippet: Monocytes and inflammatory macrophages expressing IL-1β are accumulated in ascending aorta in patients with Stanford type A aortic dissection (AAD). ( A ) Sub-clustering of myeloid cells by uniform manifold approximation and projection (UMAP) in Controls and AADs (n = 3 Controls from LiY et al ; n = 2 AAD). ( B ) Dot plots displaying signature cell gene expression markers for each subcluster of myeloid cells. ( C ) Featured plots displaying characteristic gene expression of IL1B, NLRP3, and CCL2 in myeloid cells. ( D ) Pie charts showing the proportion of each myeloid cell cluster in the Control and AAD groups. Percentage of partitioned monocytes and monocyte-derived macrophages (clusters 0, 1, 2, and 3). ( E ) Gene ontology (GO) terms showing enriched biological processes (BP) (left) and molecular functions (MF) of clusters 0, 1, 2, 3, and 4. ( F ) Trajectory pseudo-time analysis in Monocle3 with Seurat cluster annotations (left) and change in the expression of IL-1β across pseudo-time for monocytes and monocyte-derived macrophage partitions (clusters 0, 1, 2, and 3) in AAD samples (right). ( G ) Histological staining with EVG, CD68 and, IL-1β. The scale bar represents 100 μm. Macs macrophages, Monos monocytes, cDC conventional dendritic cells, pDCs plasmacytoid dendritic cells.

Article Snippet: A blocking antibody against IL-1β (BE0246, BioXCell Therapeutics, New Haven, CT, USA) or an isotype control antibody (BE0091, BioXCell) were injected intraperitoneally at a dose of 200 µg every 3 days for 2 weeks (Fig. ).

Techniques: Expressing, Dissection, Control, Derivative Assay, Staining

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: MyD88 deficiency in Foxp3 + cells reduces oral mucosa T reg accumulation in vivo and during heat killed Candida albicans germ tube (HKGT) activation in vitro. Cells were isolated from spleen (SPLN) and cervical lymph nodes (CLN) and mouse oral intra-epithelial lamina propria leukocytes (MOIL) derived from FYcre and MFYcre mice. (A) Flow cytometric plots of CD25 and Foxp3 ex vivo . (B) Statistics of T reg frequency (above) and numbers (below) from individual mouse from FYcre and MFYcre groups ex vivo . (C) 3 x 10 5 cells from the indicated tissue were stimulated with α-CD3(1μg/ml, α-CD28 (2μg/ml), TGF-β1 (5 ng/ml) and heat killed Candida albicans ( CA ) germ tube (HKGT) (10 7 /ml) for 5 days before assessing CD25 and Foxp3 by flow cytometry. (D) Statistics of CD25 + Foxp3 + cell frequency (above) and numbers (below) in cultures stimulated as in (C) , from indicated groups (Each data point corresponds to an individual mouse). (E, F) MyD88 signaling expands T regs . CLN CD4 + CD25 + Foxp3-YFP + T regs from FYcre and MFYcre mice, and CD4 + CD25 + T regs from TLR-2 -/- were FACS sorted and labelled with CPD-670. 5 x 10 4 T regs were stimulated with APC as in C . Flow cytometric plots showing CPD-670 dilution and Foxp3 (E) , and statistics showing T reg expansion (F) are depicted. Mean values ± SEM are plotted. (*P < 0.05; Mann Whitney test). Data represent at least triplicate experiments. **P < 0.005.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: In Vivo, Activation Assay, In Vitro, Isolation, Derivative Assay, Ex Vivo, Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: T reg specific deletion of MyD88 reduces results in impaired resistance to oropharyngeal candidiasis (OPC) in mice, and T reg reconstitution reduces fungal burden and immunopathology. FYcre and MFYcre mice were sublingually infected with CA or PBS (Sham) in vivo (n= 4-5/group). (MFYcre +CA + T regs ) group received 1 x 10 6 CD4 + CD25 + GFP + T regs from Foxp3-GFP reporter mice. (A) Periodic Acid Schiff’s (PAS) staining was done on tongue sections isolated from mice on day 7 after infection. (B) Foxp3 (left) and PAS (right) immunohistochemistry evaluation was performed on tongues derived from mice on day 7 and 18 after infection, respectively. Microscopic images of the slides viewed at 20X magnification (Epi, epithelium; E.D, epithelial damage; F, fungus; IF, immune cell infiltration; Red arrows indicate some of the Foxp3 + cells). (C) Statistical analyses of # Foxp3 + (top) cells, inflammation score (middle) from 20X images of the tongues, and fungal burden (CFU/gm of tongue) (bottom panel) assessed in tongue lysates from mice on day 6 or 7 after infection (* P<0.05; Mann Whitney test). (D) MOILs were isolated on day-6 after infection and processed for flow cytometric staining of F4/80 and Ly6C (left, Figure S5B ) and Gr-1 (right, Figure S5C ). Data represent two experiments. (E) Mouse body weight was monitored every other day after infection until the day of sacrifice. The percent weight change in mice in all groups. Mean values ± SEM are plotted. (2 way ANOVA, multiple comparison; alpha= 0.05* significant). At least 3-5 independent experiments showed similar results. **P < 0.005, ***P< 0.0005, ****P < 0.00005.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: Infection, In Vivo, Staining, Isolation, Immunohistochemistry, Derivative Assay, MANN-WHITNEY, Comparison

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: IL-1β promotes T reg 17 cells and constrains induction of T regDys cells in Candida activated oral mucosal cells in vitro . (A) HKGT mediated T reg 17 induction is slightly reduced in TLR-2 -/- T regs , but significantly lower in MFYcre T regs in vitro . Pooled cervical lymph nodes (CLN) and mouse oral intra-epithelial and lamina propria leukocytes (MOIL) cells from FYcre and MFYcre were stimulated with heat killed Candida albicans germ tube (HKGT) as in Figure 1C for 5 days and were restimulated with PMA-Ionomycin for flow cytometry assessment. For TLR-2 -/- cultures, T regs were purified from TLR-2 -/- mice and stimulated with WT APC from T cell depleted CLN and MOIL cells. Flow cytometry plots showing Foxp3 and IL-17A expression (gated on CD3 + CD4 + Foxp3 + cells). (B, C) WT CLN and MOIL cells were examined ex vivo or stimulated with HKGT as in Figure 1C , in the presence of IL-1β (10 ng/ml) or α- IL-β antibody (10 μg/ml) for 5 days. Flow cytometry plots showing Foxp3 and IL-17A expression (gated on CD3 + CD4 + cells) ( B , top) and statistical data points from experimental replicates ( B , bottom). Flow plots showing Foxp3 and IFN-γ expression (gated on CD3 + CD4 + Foxp3 + cells) (C) and statistical data points from experimental replicates ( C , bottom). NS, non-significant. *P < 0.005, **P < 0.0005, ****P < 0.00005.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: In Vitro, Flow Cytometry, Purification, Expressing, Ex Vivo

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: Loss of endogenous IL-1R signaling diminishes T reg 17 but increases T regDys cells in CD4 + T cells in vitro . IL-6 expands T regDys cells in the absence of IL-1β. Naïve CD4 + T cells from WT C57BL/6 and IL-1R (IL-1R1 -/- ) knockout mice were stimulated with heat killed Candida albicans germ tube (HKGT) and TGF-β1 for 5 days as in Figure 1C with WT APC and stained for Foxp3, IL-17A ( A , top and bottom), and Foxp3 and IFN-γ ( B , top and bottom) (gated on CD3 + CD4 + cells). (C) Naïve CD4 + T cells from WT C57BL/6 and IL-1R (IL-1R1 -/- ) knockout mice were stimulated with HKGT and TGF-β1 for 3 days. CD4 T cells were purified from these cultures for qPCR assessment of indicated transcripts. (D, E) WT and IL-1R1 -/- mice were orally infected with CA, and cervical lymph nodes (CLN) and mouse oral intra-epithelial and lamina propria leukocytes (MOIL) were collected 3 days after infection (n=5/group) for HKGT restimulation for 2 days and flow cytometry. Statistical analyses of Foxp3 + ROR-γt + IL-17A + (D) , and Foxp3 + IFN-γ + (E) , expressing cells after PMA/Iono restimulation for 4 h in vitro (gated on CD3 + CD4 + cells). α- IL-6 antibody (10 μg/ml) was added in some cultures. Data are representative of at least 3 independent experiments. NS, non-significant. *P < 0.05, **P < 0.005, ***P < 0.0005.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: In Vitro, Knock-Out, Staining, Purification, Infection, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: IL-1β induces IRAK, Akt, TOR, and p-70-S6K activation in T regs (A) Naïve CD4 + T (T n ) and CD4 + CD25 + T reg cells were CFSE labeled and stimulated as in Figure 1C for 3 days without or with IL-1β. Cells were restimulated with 10 ng/ml of IL-1β for 4 h before fixation and staining for p-IRAK (A) , p-Akt (B) , p-mTOR (C) , and p-70-S6K (D) for flow cytometry analyses, or cytospun on slides for confocal analyses. (E) Mouse oral intra-epithelial and lamina propria leukocytes (MOILs) were collected from oral CA infected WT and IL-1R1 -/- mice 3 days after infection (n=5/group) and stained for p-mTOR as in (C) . ( F , left and right) Pooled cervical lymph nodes (CLN) and MOIL cells were stimulated with HKGT as in Figure 1C for 5 days with or without Rapamycin (1 ng/ml Rapa). Cells were restimulated with PMA/Ionomycin before intracellular staining and flow cytometry analyses (gated on Foxp3 + cells). Geometric mean fluorescence intensities (GM) are shown with the histogram plots. Data represent triplicate experiments with similar results. NS, non-significant. *P < 0.05.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: Activation Assay, Labeling, Staining, Flow Cytometry, Infection, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: Aged mice show diminished IL-1β but excessive IL-6 expression and immunopathology during OPC. Young (6 weeks) and Aged (age 12–18 months) mice were sublingually infected and re-infected with CA or Sham (n= 4–5/group) as in Figures 2 and 3 . Histological analyses of PAS staining (A) , body weight ( B above), tongue inflammation score ( B , below) and fungal burden in tongue lysates (C) on day 7 of infection are shown. (D) Flow cytometric analyses of oral mucosal CD3 + CD4 + gated cells for CD25 and Foxp3; Geometric means of CD25 in Foxp3+ cells: Young + Sham= 776; Young +CA= 851; Aged + Sham= 334; Aged + CA=618. (E) Supernatants were collected from MOIL cells restimulated with PMA/Ionomycin, and IL-1β (on day 2 after infection), and IFN-γ, IL-6, and TNF-α levels (day 5 after infection) were quantified by ELISA. NS, non-significant. *P < 0.005, **P < 0.0005, ****P < 0.00005.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: Expressing, Infection, Staining, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: IL-1β-MyD88-mTOR Axis Promotes Immune-Protective IL-17A + Foxp3 + Cells During Mucosal Infection and Is Dysregulated With Aging

doi: 10.3389/fimmu.2020.595936

Figure Lengend Snippet: Aged individuals have decreased T reg 17 cells and increased T regDys , correlating with CD4 T cell hyperactivation in oral mucosa. 32 human participants were recruited to the study (Male n=13, Female n=19; Aged < 60 n = 25; Aged > 60 n=7). Gingival biopsies and peripheral blood mononuclear cells (PBMC) were collected. Human oral intra-epithelial and lamina propria leukocytes (HOIL) and PBMC were obtained by processing gingival biopsy tissues and blood, respectively. Flow cytometric plots showing HOIL Foxp3 and CD25 expression; Geometric Mean of CD25; Young=1387 Aged= 744 (A) and statistical analysis of T reg proportions in CD3 + CD4 + of HOIL (above) and PBMC (below) (n=32). (B) Flow cytometric plots showing ROR-γt and IL-17A (left), ROR-γt, p-mTOR(middle), CD25, and IFN-γ (right) in HOIL Foxp3+ cells. (C) Flow cytometric analyses of non-T reg CD4 + HOIL (above) and PBMC (below) cells showing IFN-γ expression ex vivo (n=12) (CD3 + CD4 + Foxp3 neg gated). ELISA quantification of IL-6 (D) , and IL-1β (E) , in saliva (above) and serum (below) collected from the participants. Mean values ± SEM are plotted. (*P < 0.05; Mann Whitney test). **P < 0.005. NS, non-significant.

Article Snippet: Anti-mouse blocking IL-1β blocking antibody was bought from Novus Biologicals.

Techniques: Expressing, Ex Vivo, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY