anti il33 neutralizing antibody af3626  (R&D Systems)

Journal: Diabetes & Metabolism Journal

Article Title: Interleukin 33 Promotes Liver Sinusoidal Endothelial Cell Dysfunction and Hepatic Fibrosis in Diabetic Mice

doi: 10.4093/dmj.2024.0532

Figure Lengend Snippet: Diabetic liver fibrosis is alleviated in interleukin 33 (IL33)-deficient mice. (A) Schematic diagram of the experimental procedure, mice were fed with normal diet (ND) or high-fat diet (HFD) for a total of 26 weeks. After 12 weeks, HFD-fed mice were consecutively injected with streptozotocin (STZ, 50 mg/kg) for 5 days. Diabetic phenotype was validated by fasting blood glucose ≥11.1 mmol/L at 7 days later (14 weeks). HFD-fed mice were consecutively injected with αIL33, immunoglobulin G (IgG), recombinant IL33 (rIL33), or vehicle for 12 weeks. (B) Serum IL33 level in control (Ctrl) mice and diabetic (DM) mice assessed by enzyme-linked immunosorbent assay (ELISA) ( n =12). (C) Western blot and measurement for IL33 expression of whole liver ( n =6). (D) Representative images of H&E and Sirius Red staining of liver sections from DM mice and IL33 knockout (KO) diabetic mice. Scale bar, 100 μm. (E) A positive area of Sirius Red staining is used to quantify liver fibrosis (right) ( n =4). (F) Hepatic hydroxyproline levels in mice ( n =4). (G, H) Representative images and quantify of immunohistochemical (IHC) staining of alpha smooth muscle actin (αSMA) in liver sections ( n =4). Scale bar, 100 μm. Data was shown as mean±standard error of the mean. ST2, suppression of tumorigenicity 2; i.p., intraperitoneal; PBS, phosphate buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a P <0.01.

Article Snippet: Wild-type diabetic mice were given an intraperitoneal injection of anti-IL33 neutralizing antibody (αIL33, 37 μg/kg body weight, R&D Systems, Minneapolis, MN, USA) or recombinant IL33 (rIL33, 12.5 μg/kg body weight, R&D Systems) twice a week for last 12 weeks.

Techniques: Injection, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Staining, Knock-Out, Immunohistochemical staining, Immunohistochemistry, Saline

Journal: Diabetes & Metabolism Journal

Article Title: Interleukin 33 Promotes Liver Sinusoidal Endothelial Cell Dysfunction and Hepatic Fibrosis in Diabetic Mice

doi: 10.4093/dmj.2024.0532

Figure Lengend Snippet: Interleukin 33 (IL33) promotes liver sinusoidal endothelial cell (LSEC) dysfunction in diabetic mice. (A) Representative scanning electron micrographs of livers of mice. Scale bar, 5 μm. (B) Hepatic nitric oxide (NO) levels ( n =8). (C, D) Representative immunofluorescent images and analysis of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and CD31 of liver sections ( n =4) (scale bar, 100 μm). (E) Relative mRNA abundance of vascular cell adhesion molecule 1 (Vcam1), intercellular adhesion molecule 1 (Icam1), nitric oxide synthase 2 (Nos2), endothelin 1 (Edn-1), nitric oxide synthase 3 (Nos3), and KLF transcription factor 2 (Klf2) of liver tissue ( n =8). Data was shown as mean±standard error of the mean. Ctrl, control; DM, diabetes mellitus; IgG, immunoglobulin G; PBS, phosphate buffered saline; rIL33, recombinant IL33; NS, no significant. a P <0.05, b P <0.01.

Article Snippet: Wild-type diabetic mice were given an intraperitoneal injection of anti-IL33 neutralizing antibody (αIL33, 37 μg/kg body weight, R&D Systems, Minneapolis, MN, USA) or recombinant IL33 (rIL33, 12.5 μg/kg body weight, R&D Systems) twice a week for last 12 weeks.

Techniques: Control, Saline, Recombinant

Journal: Diabetes & Metabolism Journal

Article Title: Interleukin 33 Promotes Liver Sinusoidal Endothelial Cell Dysfunction and Hepatic Fibrosis in Diabetic Mice

doi: 10.4093/dmj.2024.0532

Figure Lengend Snippet: Interleukin 33 (IL33) amplifies palmitic acid and high glucose-induced liver sinusoidal endothelial cell (LSEC) dysfunction in vitro. (A) Representative fluorescent images of adhered monocytes (upper), and microscopy images of reactive oxygen species (ROS) in LSEC (lower). (B) Analysis of adherent monocytes (upper) and average fluorescent intensity of ROS (lower) ( n =4) (scale bar, 25 μm). (C) Nitric oxide (NO) levels of culture medium from LSEC administrated with recombinant IL33 in vitro (0, 1, 5, 10, 50, or 100 ng/mL) for 24 and 48 hours in the presence of palmitic acid (PA) plus high glucose (PAHG; 0.2 mM PA and 30 mM glucose) ( n =4). (D) Schematic representation of Transwell coculture system. (E, F) Representative fluorescent images and analysis of alpha smooth muscle actin (αSMA) in LX-2 co-cultured with LSEC ( n =4) (scale bar, 25 μm). (G) Western blot images and quantification of suppression of tumorigenicity 2 (ST2) from L02 cells transfected with small interfering RNA targeting ST2 (siST2) or scramble RNA. (H) Representative fluorescent images of adhered monocytes (upper), and microscopy images of ROS in LSEC (lower) after transfected with siST2 or scramble RNA ( n =3) (scale bar, 25 μm). (I) Representative fluorescent images and analysis of αSMA in LSEC transfected with siST2 or scramble RNA ( n =3) (scale bar, 25 μm). (J) Representative fluorescent images and analysis of αSMA in LX-2 cultured with culture medium collected from LSECs transfected with siST2 or scramble RNA ( n =3) (scale bar, 25 μm). Data was shown as mean±standard error of the mean. NG, normal glucose; NS, no significant; HSC, hepatic stellate cell; DAPI, 4’,6-diamidino-2-phenylindole; wt, wild-type; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. a P <0.05, b P <0.01.

Article Snippet: Wild-type diabetic mice were given an intraperitoneal injection of anti-IL33 neutralizing antibody (αIL33, 37 μg/kg body weight, R&D Systems, Minneapolis, MN, USA) or recombinant IL33 (rIL33, 12.5 μg/kg body weight, R&D Systems) twice a week for last 12 weeks.

Techniques: In Vitro, Microscopy, Recombinant, Cell Culture, Western Blot, Transfection, Small Interfering RNA

Journal: Diabetes & Metabolism Journal

Article Title: Interleukin 33 Promotes Liver Sinusoidal Endothelial Cell Dysfunction and Hepatic Fibrosis in Diabetic Mice

doi: 10.4093/dmj.2024.0532

Figure Lengend Snippet: Interleukin 33 (IL33) drives liver sinusoidal endothelial cell (LSEC) dysfunction by blocking autophagy in vitro. (A) Western blot and measurement for light chain 3 (LC3) and p62 of LSEC ( n =4). (B) Nitric oxide (NO) levels of culture medium from LSEC cultured with different administration ( n =4). (C) Representative fluorescent images of adhered monocytes (upper), and microscopy images of reactive oxygen species (ROS) in LSEC (lower) (scale bar, 25 μm). (D) Analysis of adherent monocytes number (left) ( n =4) and average fluorescent intensity of ROS in LSEC (right) ( n =4). (E, F) Representative fluorescent images and analysis of alpha smooth muscle actin (αSMA) in LX-2 (scale bar, 25 μm). Data was shown as mean±standard error of the mean. PAHG, palmitic acid plus high glucose; Rapa, rapamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NG, normal glucose; NS, no significant; DAPI, 4’,6-diamidino-2-phenylindole. a P <0.05, b P <0.01.

Article Snippet: Wild-type diabetic mice were given an intraperitoneal injection of anti-IL33 neutralizing antibody (αIL33, 37 μg/kg body weight, R&D Systems, Minneapolis, MN, USA) or recombinant IL33 (rIL33, 12.5 μg/kg body weight, R&D Systems) twice a week for last 12 weeks.

Techniques: Blocking Assay, In Vitro, Western Blot, Cell Culture, Microscopy

Journal: Diabetes & Metabolism Journal

Article Title: Interleukin 33 Promotes Liver Sinusoidal Endothelial Cell Dysfunction and Hepatic Fibrosis in Diabetic Mice

doi: 10.4093/dmj.2024.0532

Figure Lengend Snippet: Interleukin 33 (IL33) elicits liver sinusoidal endothelial cell (LSEC) dysfunction by activating the extracellular signal-regulated kinase 1 (ERK1)/mitogen-activated protein kinase (MAPK) pathway. (A, B) Western blot and measurement for p-ERK, ERK, light chain 3 (LC3), and p62 of LSEC ( n =3). (C) Nitric oxide (NO) levels of culture medium from LSEC cultured with different administration ( n =4). (D) Representative fluorescent images and analysis of adhered monocytes (upper), and microscopy images of reactive oxygen species (ROS) in LSEC (lower) (scale bar, 25 μm). (E) Representative fluorescent images and analysis of alpha smooth muscle actin (αSMA) in LX-2 cultured with culture medium collected from different groups of LSECs ( n =4) (scale bar, 25 μm). (F) Representative fluorescent images and analysis of αSMA in LSEC cultured with different administration ( n =4) (scale bar, 25 μm). Data was shown as mean±standard error of the mean. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NG, normal glucose; NS, no significant; PAHG, palmitic acid plus high glucose; DAPI, 4’,6-diamidino-2-phenylindole. a P <0.05, b P <0.01.

Article Snippet: Wild-type diabetic mice were given an intraperitoneal injection of anti-IL33 neutralizing antibody (αIL33, 37 μg/kg body weight, R&D Systems, Minneapolis, MN, USA) or recombinant IL33 (rIL33, 12.5 μg/kg body weight, R&D Systems) twice a week for last 12 weeks.

Techniques: Western Blot, Cell Culture, Microscopy

Journal: Nature

Article Title: Active eosinophils regulate host defence and immune responses in colitis

doi: 10.1038/s41586-022-05628-7

Figure Lengend Snippet: a , Activity of A-Eos-specific regulons across clusters. g, number of genes in regulon. b , Expression of NF-κB signalling components. c , Quantification of pNF-κB p65 + cells in colonic A-Eos and B-Eos ( n = 3, B6J). Data are mean ± s.e.m. Two-tailed unpaired Student’s t -test. d , e , A-Eos and B-Eos frequencies in antibiotic-treated ( d ) ( n = 16, B6J) and germ-free (GF) ( e ) ( n = 9, B6J) mice relative to controls. SPF, specific pathogen free. d , Data are pooled from two independent experiments. Medians are shown. Two-tailed unpaired Student’s t -test. f , Depleted gene sets in PD-L1 + CD80 + A-Eos (red) and PD-L1 − CD80 − eosinophils (grey), relative to BM stem cells. Kolmogorov–Smirnov test. Dot size indicates gene-set size. Dashed line indicates P = 0.05. g , A-Eos frequencies after conditioning of BM-Eos with IL-33, colon CM and anti-IL-33 ( n = 2, pooled B6J). Technical replicates and mean ± s.e.m. are shown. One-way ANOVA. h , Colonic A-Eos and B-Eos frequencies in B6J ( n = 21) and Myd88 −/− ( n = 15) mice treated with IL-33, relative to untreated controls. Medians are shown. Two-tailed unpaired Student’s t -test. WT, wild type.

Article Snippet: The NF-κB inhibitor BAY11-7082 (B5556, Sigma) was added at a concentration of 5 μM and anti-IL-33 neutralizing antibody (AF3626, Biotechne) at 30 ng ml −1 .

Techniques: Activity Assay, Expressing, Two Tailed Test

Journal: Nature

Article Title: Active eosinophils regulate host defence and immune responses in colitis

doi: 10.1038/s41586-022-05628-7

Figure Lengend Snippet: a , Experimental workflow of the CRISPR inhibition screen. b , Log10 negative score per gene, as calculated by MAGeCK. Cd80 and Cd274 evidenced in orange. Genes involved in TNF signalling pathway via NF-κB in red, and MAPK signalling pathway in darkred. c , IL-33 concentrations measured by ELISA in colon of DSS-treated mice (n = 12, B6J) and colon and blood of C. rodentium -infected mice (n = 7, B6J), compared to untreated controls (n = 7, B6J). Medians are shown. Two-tailed unpaired Student’s t -test. d , IFNγ, TNF and IL-22 concentrations measured by LEGENDplex in colon (left) and blood (right) of C. rod entium-infected mice (n = 7, B6J), compared to untreated controls (n = 7, B6J). Medians are shown. Two-tailed unpaired Student’s t -test. e , A-Eos (PD-L1 + CD80 + ) frequencies upon conditioning of BM-Eos with colon CM, IL-22, IL-25, TNF or IL-33 (n = 4, B6J). Data are pooled from two independent experiments. Medians are shown. One-way ANOVA. f , A-Eos frequencies after conditioning with increasing doses of IL-33. Input: BM-derived (n = 5, B6J), blood (n = 5, Il5 -tg) and splenic (n = 5, Il5 -tg) eosinophils. Medians are shown. One-way ANOVA. g , Western blot of phospho-p38 and phospho-p65 upon conditioning of BM-Eos with colon CM or IL-33 (n = 3, B6J). h , Gene expression normalized to Hprt measured by qRT–PCR of BM-Eos upon conditioning with IL-33 (n = 4, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t -test. i , ST2 expression in BM-Eos upon IL-33 treatment (n = 4, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t -test. j , ST2 expression in colonic A-Eos and B-Eos (n = 5, B6J). Data represents mean ± SEM. Two-tailed unpaired Student’s t -test. k , ST2 expression across organs (n = 5, B6J). Data represents mean ± SEM. One-way ANOVA. l , A-Eos frequencies upon conditioning of WT (n = 2, pooled B6J) or ST2 −/− (n = 2, pooled) BM-Eos with colon CM or IL-33. Technical replicates and mean ± SEM are shown. Two-tailed unpaired Student’s t -test. m , Left: Representative FACS plots of A-Eos (PD-L1 + CD80 + ) and PD-L1 − CD80 − eosinophils in the blood (top) and spleen (bottom). Numbers indicate % of eosinophils. Right: A-Eos frequencies in mice treated with IL-33 (n = 6-7, B6J). Medians are shown. Two-tailed unpaired Student’s t -test. n , A-Eos and B-Eos frequencies in the indicated organs of B6J (n = 7) and Il33 −/− (n = 5) mice at steady state. Medians are shown. Two-tailed unpaired Student’s t -test.

Article Snippet: The NF-κB inhibitor BAY11-7082 (B5556, Sigma) was added at a concentration of 5 μM and anti-IL-33 neutralizing antibody (AF3626, Biotechne) at 30 ng ml −1 .

Techniques: CRISPR, Inhibition, Enzyme-linked Immunosorbent Assay, Infection, Two Tailed Test, Derivative Assay, Western Blot, Expressing, Quantitative RT-PCR

Journal: Nature

Article Title: Active eosinophils regulate host defence and immune responses in colitis

doi: 10.1038/s41586-022-05628-7

Figure Lengend Snippet: a , Regulon activity in A-Eos across conditions (n = 4, Il5 -tg). b , Multidimensional scaling (MDS) plot of bulk RNA-seq samples shown in Fig. . c , Heat map of signature gene expression across conditions of samples shown in Fig. . d , A-Eos (PD-L1 + CD80 + ) frequencies upon treatment of BM-Eos with IL-33 and/or IFNγ. (n = 4, B6J). Data represents mean ± SEM. One-way ANOVA. e , EPX immunofluorescence of A-Eos upon exposure to IFNγ for 90 min. Splenic eosinophils were magnetically enriched (n = 2, Il5 -tg), treated overnight with colon CM and A-Eos sorted by flow cytometry. Scale bar, 10 µm. f , Frequencies of PD-L1 + and CD80 + in colonic eosinophils of WT (n = 6, B6J) and Eo- Cre;Ifngr fl/fl mice (n = 4) upon C. rodentium infection, relative to uninfected controls (n = 2, B6J). Medians are shown. Two-tailed unpaired Student’s t -test. g , Left: UMAP of single-cell eosinophil transcriptomes isolated from the colon of anti-IFNγR-treated, C. rodentium -infected or control Il5 -tg mice (n = 3). Middle: expression of IFNγ target genes. Right: Expression of granule and antimicrobial signatures. Data represents mean ± SD. Two-sided Wilcoxon test. h , Observed vs. expected number of contacts between clusters of SIGLEC8 and CD4 molecules shown per slide. P Values are computed based on a two-sided permutation test (see ). i , Proportions of segmented cells expressing SIGLEC8 only (blue) or co-expressing both SIGLEC8 and CD4 (red) across slides. Dotted horizontal line shows mean. j , Mean count per slide of molecules of a given transcript in the proximity (<10 µm) of SIGLEC8 RNA molecules spatially associated with CD4 molecules vs SIGLEC8 molecules not associated with CD4 molecules. The central line in the box plot represents the median count per slide, the lower and upper hinge corresponds to the first quartiles and the whisker extends from the hinge to the smallest or largest value no further than 1.5 x IQR from the hinge. Two-sided paired Wilcoxon test (17 ROIs, n = 4 patients).

Article Snippet: The NF-κB inhibitor BAY11-7082 (B5556, Sigma) was added at a concentration of 5 μM and anti-IL-33 neutralizing antibody (AF3626, Biotechne) at 30 ng ml −1 .

Techniques: Activity Assay, RNA Sequencing Assay, Expressing, Immunofluorescence, Flow Cytometry, Infection, Two Tailed Test, Isolation, Whisker Assay

Journal: Nature

Article Title: Active eosinophils regulate host defence and immune responses in colitis

doi: 10.1038/s41586-022-05628-7

Figure Lengend Snippet: a , Top, Venn diagram of significant differentially expressed genes (DEGs) (false discovery rate (FDR) < 0.05, logFC > |2|) in BM-Eos treated with IL-33 and/or IFNγ ( n = 4, B6J). All DEGs are listed in Supplementary Table . Bottom, expression of subset markers across conditions. Columns are clustered, rows are scaled. CPM, counts per million. b , Proliferation of anti-CD3/CD28-activated, CFSE-labelled naive CD4 + T cells co-cultured with BM-Eos conditioned with IL-33 and/or IFNγ ( n = 4, B6J). Data are pooled from two independent experiments. Medians are shown. One-way ANOVA. c , A-Eos frequencies in mice treated with IL-33 and/or IFNγ ( n = 5, B6J). Medians are shown. One-way ANOVA. d , A-Eos and B-Eos frequencies in DSS-treated B6J ( n = 5) and Il33 −/ − (n = 4) mice. Medians are shown. Two-tailed unpaired Student’s t -test. e , Frequencies of IFNγ-, IL-17- and TNF-expressing colonic CD4 + T cells from mice in d . Medians are shown. Two-tailed unpaired Student’s t -test. f , Left, representative haematoxylin and eosin (H&E)-stained colonic sections of mice in d . Scale bar, 100 µm. Right, colitis score assessed by histopathological examination. Medians are shown. Two-tailed unpaired Student’s t -test. g , Representative molecular cartography images of human ulcerative colitis samples. Nuclei are stained with DAPI; CD4 , SIGLEC8 and CD80 RNA molecules are shown in blue, red and yellow, respectively. Scale bar, 200 µm. h , Pairwise proximity score of transcripts across slides. The score indicates the fraction of slides in which the proximity of a pair of transcripts is significantly higher than expected by chance. P values are computed using a permutation test . T reg cells, regulatory T cells. i , Mean counts per slide of CD80 and NFKB1 transcripts in the proximity (<10 µm) of SIGLEC8 transcripts spatially associated with CD4 molecules versus SIGLEC8 molecules not associated with CD4 molecules. The central line in the box plot represents the median count per slide, the lower and upper hinge correspond to the first quartiles and the whisker extends from the hinge to the smallest or largest value no further than 1.5 times the interquartile range (IQR) from the hinge. Two-sided paired Wilcoxon test (17 regions of interest (ROIs), n = 4 patients).

Article Snippet: The NF-κB inhibitor BAY11-7082 (B5556, Sigma) was added at a concentration of 5 μM and anti-IL-33 neutralizing antibody (AF3626, Biotechne) at 30 ng ml −1 .

Techniques: Expressing, Cell Culture, Two Tailed Test, Staining, Whisker Assay