Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Biliary epithelial injury-induced regenerative response by IL-33 promotes cholangiocarcinogenesis from peribiliary glands

doi: 10.1073/pnas.1619416114

Figure Lengend Snippet: Blocking IL-33 suppresses ECC development. (A) Protocol of anti–IL-33 treatment of KTC-K19CreERT mice. (B and C) Representative macroscopic (B) and H&E-stained (C) images of EHBDs from KTC-K19CreERT mice treated with anti–IL-33 antibody or control IgG. (Scale bar: 500 μm.) Bar graph shows maximum diameter of the CBD (means ± SEM; n = 10 per group). *P < 0.05. (D) H&E-stained images of the IHBDs of anti–IL-33- or control IgG-administered KTC-K19CreERT mice. (Scale bar: 100 μm.) (E) Relative AREG and IL-13 mRNA levels in EHBDs from anti–IL-33 or control IgG-administered KTC-K19CreERT mice were determined by real-time PCR (means ± SD; n = 6 per group). *P < 0.05. (F) Numbers of hepatic ILC2s in anti–IL-33- or control IgG-administered KTC-K19CreERT mice on day 20 after TAM administration were analyzed by flow cytometry. Representative dot plots and numbers from three independent experiments (bar graph) are shown (means ± SD; n = 3 per group). *P < 0.05. (G) Representative Ki67-stained image of anti–IL-33 or control IgG-administered KTC-K19CreERT mice on day 10 after TAM administration. (Scale bar: 50 μm.) Bar graph shows the frequencies of Ki67-positive cells among the surface BECs and in PBGs (means ± SD; n = 5 per group). *P < 0.05. (H) Proposed mechanism of ECC development in KTC-K19CreERT mice.

Article Snippet: A neutralizing anti–IL-33 antibody or control IgG (3.6 μg/mouse) (R&D Systems) was administered intraperitoneally to mice every other day ( 25 ).

Techniques: Blocking Assay, Staining, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Biliary epithelial injury-induced regenerative response by IL-33 promotes cholangiocarcinogenesis from peribiliary glands

doi: 10.1073/pnas.1619416114

Figure Lengend Snippet: Anti–IL-33 treatment suppressed the growth of tumor xenografts. (A) Photograph and H&E-stained image of the KTC-BO cell-derived s.c. tumor that developed in C57BL/6 mice. (B and C) C57BL/6 mice were transplanted subcutaneously with KTC-BO cells and then treated with anti–IL-33 antibody or control IgG every other day from day 8 to day 20. Tumor weights were analyzed on day 21. Photograph of the tumors (B) and tumor weights (C) are shown (n = 12 per group). *P < 0.05.

Article Snippet: A neutralizing anti–IL-33 antibody or control IgG (3.6 μg/mouse) (R&D Systems) was administered intraperitoneally to mice every other day ( 25 ).

Techniques: Staining, Derivative Assay

Journal: JCI Insight

Article Title: Sex differences in IL-17 contribute to chronicity in male versus female urinary tract infection

doi: 10.1172/jci.insight.122998

Figure Lengend Snippet: (A–C) Female and male mice were infected with 1 × 107 CFU of UPEC UTI89-RFP-kanR and bladders analyzed at 24 hours PI. Graphs show (A) IL-33 protein levels in homogenized bladder, (B) IL-4Rα expression on bladder resident macrophages (Mϕ), and (C) the number of ILCs (CD90+CD3–CD4–NK1.1–MHC II–CD11b–) in bladders. (D–F) Female mice were implanted with empty tubing (Mock) or slow-release tubing containing testosterone (T tube) and allowed to recover 1 week before infection with 1 × 107 CFU UPEC strain UTI89-RFP-kanR. Graphs show (D) IL-33 protein levels in homogenized bladder tissue, (E) IL-4Rα expression on bladder resident macrophages, and (F) the number of ILCs in bladders. Data are pooled from 2–3 experiments, n = 4–5 mice/group in each experiment. Each dot is 1 mouse, red dots depict female mice and blue dots are male mice, and lines are medians. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Mann-Whitney test. Analyses in this figure were corrected for multiple testing by the Holm–Bonferroni method; all P < 0.05 had q < 0.05.

Article Snippet: For IL-33 neutralization, 3.6 μg/mouse α–IL-33 (catalog number AF3626, BioTechne/R&D Systems) or 3.6 μg/mouse polyclonal goat IgG isotype control (catalog number AB-108-C, BioTechne/R&D Systems) was delivered intraperitoneally in 100 μL PBS ( 44 ).

Techniques: Infection, Expressing, MANN-WHITNEY

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A-H. Immunoblot analysis of intracellular IL-33 and cellular components from total cell lysates of cells expressing endogenous p IL-33 and exposed to various environmental allergens. Left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (cl, cleaved; fl, full length; m, mature; p, precursor; tot, total; ph, phosphorylated). EPC2 cells were incubated with control medium alone (Mock), 0.4 nM (A, D, E), 2 nM (A, D, E) or 10 nM (A-K) of Poly (I:C); or 10 nM Poly(I:C) or LPS (A-K); 1 μg/mL (A, D, E), 5 μg/mL (A, D, E), or 25 μg/mL (A-K) of A. alternata ( A.Alt ) , house dust mite (HDM), A. fumigatus ( A.Fum ) , cat dander, canary feathers, cockroach, birch pollen, Bermuda grass, peanut, whole wheat or cow milk extracts for 8 hours. B, C, F-H, K . Cells were treated with control medium or 20 μM pan-caspase inhibitor (Q-VD-OPH), selective calpain inhibitor (PD151746), cysteine protease inhibitor (E64D), or transcription inhibitor (actinomycin D) for 2 (G) or 8 (B, C, F, H, K) hours. I-K . Quantification of extracellular IL-33 released from cells overexpressing p IL-33. A-K . Data are representative or a summary of n = 3 independent experiments. Each data point is a mean of a technical duplicate ± SD from in vitro assays. Statistics were performed by 2-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0007 (***), p value ≤ 0.003 (**), and p value ≤ 0.03 (*), N/S, not significant.

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Western Blot, Expressing, Molecular Weight, Incubation, Protease Inhibitor, In Vitro

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A-E. Immunoblot analysis of intracellular IL-33 and cellular components from total cell lysates of cells expressing endogenous p IL-33 and exposed to various stimuli. EPC2 cells were treated for 8 hours with control medium alone (Mock), 10 nM of Poly (I:C) or LPS, or 25 μg/mL of A. alternata (A.Alt), house dust mite (HDM), or A. fumigatus (A.Fum) allergen extracts as indicated. EPC2 cells were treated in the presence of control medium (Mock) or in the presence of 20 μM pan-caspase inhibitor (Q-VD-OPH), caspase 8 inhibitor (Z-IETD-FMK), caspase 3 and 7 inhibitor (Z-DEVD-FMK), or caspase 1 inhibitor (Ac-YVAD-CHO); 50 μM inactive necrostatin 1 (Inact Ctr); and/or 20 μM or 50 μM of necrostatin 1 (NEC-1), necrostatin 5 (NEC-5), necrostatin 7 (NEC-7), or necrostatin 1s (NEC-1s) as indicated. E. Immunoblot analysis of EPC2 cells treated for 8 hours with control medium (Mock), 10 nM of Poly (I:C) or 25 μg/mL of A. alternata (A.Alt), house dustmite (HDM), or A. fumigatus (A.Fum) allergen extracts in medium alone or pre-mixed with complete protease inhibitor cocktail (Prot Inhib;Roche see ). F-G. LDH (F) and IL-33 (G) release analysis in cell supernatants of EPC2 cells expressing endogenous p IL-33 and exposed to various stimuli. Cells were treated as above (A-E) for 2, 4 and 8 hours as indicated. Lysis control are cells treated with Triton 100 lysis buffer for 45 minutes to determine maximum LDH and IL-33 release (CyQUANT LDH cytotoxicity assay - see ). Each data point is a mean of a technical duplicate ±s.d. of in vitro assays. Statistics were performed by two-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0003 (***). H-I. Immunoblot analysis of intracellular IL-33 and cellular components from total cell lysates of cells expressing endogenous p IL-33. H. Immunoblot analysis of control (TLR3 +) and CRISPR/Cas9 TLR3 knockout (TLR3 -) EPC2 cells treated as above (A-E). I. Immunoblot analysis of total cell lysates of human esophageal epithelial cells (EPC2), skin epithelial cells (HaCaT), bronchial epithelial cells (HBEC3-KT) expressing endogenous IL-33, and fibroblasts (FEF3) cells; IL-33 expression in FEF3 cells was induced with 100 pg/mL TNF-α for 16 hours. Then all the cells were incubated with either control medium or 10 nM Poly (I:C)

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Western Blot, Expressing, Protease Inhibitor, Inhibition, Lysis, CyQUANT Assay, LDH Cytotoxicity Assay, In Vitro, CRISPR, Knock-Out, Incubation

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A . Immunoblot analysis of recombinant IL-33 (100 ng) incubated for 2 hours with control medium or 10, 30, or 100 units of recombinant human active caspases 3, 7 or 8 (100 units). B . Immunoblot analysis of necrotic supernatants of TE-7 cells overexpressing p IL-33 (1–270). Cells were pre-incubated with control medium or Poly (I:C) (10 nM) for 8 hours. Control supernatants were incubated with medium or 100 units of recombinant human active caspases 3, 7, or 8 for 2 hours. C . Immunoblot analysis of TE-7 cells overexpressing wildtype (1–270), single-point mutated (D175N; D178N), and double-point mutated (D175N/D178N) p IL-33. Necrotic supernatants were incubated with control medium or 100 units of recombinant human active caspases 3 or 7 for 2 hours. D . X-ray complex structure of ST2 receptor (S117-S268; PDB ID: 4KC3) juxtaposed with the NMR-based structure of IL-33 (S111-T270; PDB ID: 2KLL) showing IL-33 minimal binding domain interacting with ST2 and the non-interacting IL-33 flexible loop with a.a. D175 and D178 (yellow); D179-T270 is posterior to the IL-33 and is not interacting with ST2 receptor binding interface (grey). E . Schematic representation of (D) inclusive of the M1-H109 and caspase cleavage site (D175, D178) as determined by MS/tandem LC-MS. F . Immunoblot analysis of coimmunoprecipitation of IL-33 and ST2: TE-7 cells overexpressing p IL-33 (1–270) were incubated with Poly (I:C) (10 nM) for 8 hours and lysed. Lysate input control (left) and co-immunoprecipitation of IL-33 with ST2-Fc (middle) or normal goat IgG (right) are shown. A-C, F. Data are representative of n = 3 independent experiments. Left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (cl, cleaved; fl, full length; m, mature; p, precursor).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Western Blot, Recombinant, Incubation, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Molecular Weight

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A. Precursor IL-33 reference sequence (UniProtKB O95760). Peptides covered by analysis are underlined. Bold letter Ds indicate residues 175 (left) and 178 (right). B-F. Summary table and peptide profiles of recombinant human IL-33 peptides identified by MALDI-TOF Mass Spectrometry and Tandem Liquid Chromatography MALDI-TOF Mass Spectrometry (LCMS) before and after cleavage by recombinant human caspases. C-F. Exact profiles corresponding to identified peptides sequences of precursor (full-length control; C, E) and cleaved (D, F) GST–IL-33 are labeled with colors in the inserts, and the first letter of each color in the histogram peptide profiles as indicated: green (G), blue (B), and purple (P). Bold red indicates extra sequence identified by nano-LCMS. G-H. The 159-VLLSYYESQHPSNESGD-175 peptide profiles were generated by caspase 3 and caspase 7 cleavage. I-J. 159-VLLSYYESQHPSNESGDGVD-178 peptide profiles generated by caspase 3 and caspase 7 cleavage, respectively. A-J. Data are a summary of n = 4 independent experiments. m/z, mass-to-charge ratio

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Sequencing, Recombinant, Mass Spectrometry, Liquid Chromatography, Labeling, Generated

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A . Immunoblot analysis of TE-7 cells overexpressing WT p IL-33 (1–270) and m IL-33 forms (1–175; 1–178) or single-point mutated (D175N; D178N) or double-point mutated (D175N/D178N) p IL-33 (1–270). Cells were treated with control medium or TLR3 agonist (Poly (I:C); 10 nM) for 8 hours and lysed. B-C . IL-33 bioactivity assay as a function of IL-8 secretion by HMC-I human mast cells with and without ST2 neutralization (anti-ST2 antibody and control IgG). TE-7 cells overexpressing IL-33 (same as A) were incubated with control medium or Poly (I:C) (10 nM) for 8 hours. Then HMC-I cells were co-incubated with medium alone (Mock), normalized necrotic supernatants from the TE-7 cells (A): 1–270 expressing cells treated with Poly(I:C) (1–270/Poly(I:C); containing both p IL-33 and secreted m IL-33 forms), untreated 1–270, 1–175, 1–178, D175N, D178N and D175/D178N expressing cells for 8 hours. The IL-8 concentration in the medium was measured by ELISA. D . Immunoblot analysis of recombinant IL-33 as used in (E) E . IL-33 bioactivity assay as a function of IL-8 secretion by HMC-I human mast cells with ST2 neutralization (anti-ST2 antibody) and control IgG. Cells were treated for 8 hours with equimolar quantities (10 nM) of recombinant IL-33 forms (same as D) in medium or medium alone (Mock). F-H . Primary murine eosinophils were stimulated with ST2 neutralization (anti-ST2 antibody) and control IgG. Cells were treated for 18 hours with equimolar quantities (10 nM) of recombinant IL-33 forms (same as D) in medium or medium alone (Mock). The cytokines concentrations were measured by ELISA. A-E. Data are representative of n = 3 independent experiments. A-D. Left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (m, mature; p, precursor). F-H. Data are summary of n = 3 independent experiments. B, C, E-H. Each data point is a mean of a technical duplicate ± SD from the in vitro assays. Statistics were performed by 2-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0001 (****), p value ≤ 0.0007 (***), and p value ≤ 0.0013 (**).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Western Blot, Neutralization, Incubation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Molecular Weight, In Vitro

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A-D. IL-33 knock out (KO) mice were intraperitoneally injected with equimolar quantities (10 nM) of recombinant p IL-33 and m IL-33 forms in PBS or PBS alone (Mock). Single-cell dot plot data and gating strategy for live, mouse, intraperitoneal cells (A) where P0 are neutrophils (Neut; GR1/Ly6ChighCD11b+c-KIT-) and inflammatory macrophages (iMɸ; GR1/Ly6CmediumCD11b+c-KIT-). A-B. Flow cytometry analysis of single-cell dot plot data with corresponding gates (B) for neutrophils (P1; GR1high CD11b+) and inflammatory macrophages (P2; GR1medium CD11b+). Summary plots show neutrophil (C) and inflammatory macrophages (D) influx in peritoneal cavity. Data are representative of n = 3 independent experiments. C-D. Data are summary of n = 3 independent experiments. Each data point is a mean of a technical duplicate ± SD of in vivo (individual mouse) assays. Statistics were performed by one-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0001 (****), p value ≤ 0.0002 (***), and p value ≤ 0.008 (**).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Knock-Out, Injection, Recombinant, Flow Cytometry, In Vivo

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A. Quantification analysis of released (cell medium) IL-33 from TE-7 cells overexpressing p IL-33 (1–270). Cells were treated with control medium or Poly (I:C) (10 nM) for 0–24 hours, and medium was collected for each time point. B. Immunoblot analysis of released (concentrated cell medium) and intracellular (cellular; total cell lysates) IL-33 from the corresponding TE-7 cells. GAPDH was used as a loading control. C. UV absorption plot of size-exclusion column fractions 1–95. D. Immunoblot analysis of TE-7 cells overexpressing p IL-33 (1–270). Cells were treated with TLR3 agonist (Poly (I:C)) for 8 hours in serum-free medium (Opti-MEM). Medium containing secreted IL-33 was supplemented with complete protease inhibitors, filtered through 45 μM pores, DNase treated, and concentrated 10-fold using a 10-kDa cutoff membrane filter. Samples were run on the size-exclusion column. Fractions 1–95 were collected, concentrated 20-fold using a 10-kDa cutoff membrane filter, and analyzed via immunoblotting in the following order: protein standard ladder (L), secreted IL-33 medium loading control (LC), and fractions 1–95. E. Size-exclusion column standard curve by fraction number as a function of molecular weight (MW). F, G. IL-33 bioactivity assay as function of IL-8 secretion by HMC-I human mast cells. Cells were treated for 8 h with 2.5–10 nM of wheat germ extract–produced IL-33 forms in medium alone (Mock) or medium supplemented with 500 ng of acetone-purified histones in the presence of IgG control (IgG) or anti-ST2 blocking antibody (aST2). A-G. Data are representative of n = 3 independent experiments. Immunoblot left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (m, mature; p, precursor); # are non-specific bands. A, F, G. Each data point is a mean of a technical duplicate ± SD. Statistics were performed by 2-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0001 (****) and p value ≤ 0.015 (*).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Western Blot, Molecular Weight, Produced, Purification, Blocking Assay

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A-I . Wildtype (WT; A, B, D-F) and IL-33 knock out (KO; A-C, G-I) mice were treated intratracheally with A. alternata ( A.Alt ) extract in PBS or PBS alone (Mock). Mice were challenged three consecutive times 24 hours apart and sacrificed 4 hours after the last challenge. Mice were IV injected with vehicle (PBS, DMSO 0.1%) alone (Mock) or with a caspase 8–specific inhibitor preparation (Z-IETD-FMK; PBS, DMSO 0.1%) 1 hour before and after each A. alternata challenge. A-C . ELISA quantification (A, C) and immunoblot (B) analysis of secreted m IL-33 in bronchoalveolar lavage fluid (BALF) from WT (A, B) and IL-33 KO (B, C) mice. p IL-33 was not detected (ND). D-I . Total BALF cells counts (D, G) and flow cytometry analysis of BALF ST2-positive neutrophils (E, H) and eosinophils (F, I). From WT (D-F) and IL-33 KO (G-I) mice. Data are representative (B) or a summary (A, C-I) of n = 3 independent experiments. Immunoblot (B) left margin (throughout): protein molecular weight (kDa). Right margin (throughout): protein names (m, mature; p, precursor). Each data point is a mean of a technical duplicate ± SD of in vivo (individual mouse) assays. Statistics were performed by one-way ANOVA with Tukey’s multiple comparisons test: p value ≤ 0.0001 (****), p value ≤ 0.008 (**), and p value ≤ 0.03 (*), N/S, not significant. Arrowheads are comparison of A. alternata challenges alone between WT and IL-33 KO mice. Statistics were performed by unpaired one-sided t-test: p value ≤ 0.0001 (****), p value ≤ 0.0067 (**).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Knock-Out, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Molecular Weight, In Vivo

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A. Single-cell dot plot data and gating strategy for the live, mouse bronchoalveolar fluid (BALF) cells: the P1 CD45+CD11c- population is derived from total single cells; the P2 population was identified on the basis of total single cells and applied on the P1 population. Finally, the P2-derived ST2+ cells are neutrophils (Neut; SiglecF-GR1/Ly6Chigh) and eosinophils (Eos; SiglecF+GR1/Ly6Cmedium/low). Data are representative of n = 3 independent experiments. B-C. BALF cytokines in WT (B) and IL-33 KO (C) mice were measured by ELISA with and without treatment with a specific inhibitor of caspase 8 (Z-IETD-FMK). Data are summary of n = 3 independent experiments. Each data point is a mean ± SD of in vivo (individual mouse) assays. Statistics were performed by unpaired t-test: p value ≤ 0.0001 (****), p value ≤ 0.0067 (***), p value ≤ 0.0099 (**), p value ≤ 0.0431 (*). N/S is not significant. Arrowheads are comparison of A. alternata challenges alone between WT and IL-33 KO mice. Statistics were performed by unpaired one-sided t-test: p value ≤ 0.0001 (****), p value ≤ 0.0006 (***), p value ≤ 0.0034 (**), p value ≤ 0.028 (*).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A-F . Wildtype (WT; +) and caspase 8 knock out (KO; -) mice were treated intratracheally with A. alternata ( A.Alt ) extract in PBS or PBS alone (Mock). Caspase 8 KO mice had targeted deletion of caspase 8 in bronchial epithelial cells by generating CC10-CreER +/− /Casp8 fl/fl mice (see ). Mice were challenged three consecutive times 24 hours apart and sacrificed 4 hours after the last challenge. A-B . ELISA quantification (A) and immunoblot (B) analysis of secreted m IL-33 in bronchoalveolar lavage fluid (BALF). p IL-33 was not detected (ND). C-E . Total BALF cells counts (C) and flow cytometry analysis of BALF ST2-positive neutrophils (D) and eosinophils (E). Data are representative (B) or a summary (A, C-E) of n = 3 independent experiments. Immunoblot (B) left margin: protein molecular weight (kDa). Right margin: protein names (m, mature; p, precursor). Each data point is a mean of a technical duplicate ± SD of in-vivo (individual mouse) assays. Statistics were performed by one-way ANOVA with Tukey’s multiple comparisons test: p-value ≤ 0.0001 (****).

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Western Blot, Flow Cytometry, Molecular Weight, In Vivo

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A-E. Wildtype (WT; +) and caspase 8 knock out (KO; -) mice were treated intratracheally with A. alternata (A.Alt) extract in PBS or PBS alone (Mock) - see . A. Representative images of active caspase 8 and IL-33 staining in murine lungs bronchi epithelial cells in mice treated with PBS (Mock) and A. alternata (A.Alt). Positive (grey) and negative (white) staining is indicated with arrowheads. The 100μm scale bars included in all images. B. Active caspase 8 quantification. C. IL-33 quantification. D-E. Correlation of IL-33 with active caspase 8 in WT (D) and caspase 8 KO (E) mice. Statistics are by Pearson correlation (D-E): R2 (r) and p values are as indicated. Data are representative (A) or a summary (B-E) of n = 3 independent experiments. Each data point is a mean ± SD of multiple sections measurement in an individual mouse. Statistics were performed by one-way ANOVA with Tukey’s multiple comparisons test: p-value ≤ 0.0001 (****), p value ≤ 0.0025 (**), p value ≤ 0.0139 (*). N/S is not significant.

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Knock-Out, Staining

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: A . Representative images of hematoxylin and eosin (H&E, I-III), active caspase 8 (IV-VI), and active caspase 3 (VII-IX) staining of esophageal biopsies from control individuals (ctr; I, IV, VII) and patients with EoE in remission (II, V, VIII) or active EoE (III, VI, IX). Scale bars, 100 μm in all images and 10 μm in all enlarged regions of interest. The dashed line represents the basement membrane. The enlarged region of interest with scale bars shows eosinophils per high power field (HPF) (III) and active caspase 8 (VI)- or active caspase 3 (IX)–positive cells. B . Active caspase 8 quantification. C . active caspase 3 quantification. D . Correlation of active caspase 8 with active caspase 3. E . Eosinophil quantification per high power field (HPF) from H&E images. F . Correlation of active caspase 8 with eosinophil counts. G . Correlation of active caspase 3 with eosinophil counts. H . Representative immunoblot analysis of esophageal biopsy protein lysates from control individuals and patients with EoE in remission or active EoE. Left margin: protein molecular weight (kDa). Right margin: protein names (m, mature; p, precursor). I . m IL-33 quantification of immunoblots (H). HSP90 is used as a loading control for m IL-33. J . Correlation of m IL-33 with active caspase 8. K . Correlation of m IL-33 with active caspase 3. L . Correlation of m IL-33 with eosinophil counts. A-L . Data are from n = 6 control individuals, n = 3 patients with EoE in remission, and n = 7 patients with active EoE. Statistics are by Pearson (E) and Spearman correlation (F, G, J-L): R 2 (r) and p values are as indicated. Each data point is a single data point for an individual biopsy measurement mean ± SD. Statistics were performed by 2-way ANOVA with Tukey’s multiple comparisons test (B, C, E, I): ****P ≤ 0.0001 and ***P ≤ 0.0005.

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: Staining, Western Blot, Molecular Weight

Journal: Nature immunology

Article Title: Environmental Allergens Trigger Type 2 Inflammation Through Ripoptosome Activation

doi: 10.1038/s41590-021-01011-2

Figure Lengend Snippet: Allergen exposure triggers RIP phosphorylation and ripoptosome assembly: RIP (RIP) in complex with cFLIPL, FADD, TRADD, and pro-caspase 8. Following RIP phosphorylation ( p RIP), FADD-bound pro-caspase 8 is self-cleaved and activated. Active caspase 8 cleaves and deactivates p RIP and activates effector pro-caspases 3 and 7. Active effector caspases in turn target and cleave histone-bound p IL-33 at amino acids D175 and D178. m IL-33 is released to initiate type 2 innate immune responses.

Article Snippet: Next IL-33 concentrations were determined by ELISA (DY3626: R&D) or by immunoblotting as indicated.

Techniques: