il 33 concentrations  (R&D Systems)

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:

Journal: Shock (Augusta, Ga.)

Article Title: THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS

doi: 10.1097/SHK.0000000000002114

Figure Lengend Snippet: IL-33 increased in the lung and BALF 1 h after LPS-induced lung injury. WT mice were administered an intratracheal injection of LPS, and then the lung tissue and BALF were collected for 1, 3, and 24 h after ARDS induction. A, The mRNA expression level of IL-33 was detected by quantitative polymerase chain reaction (n = 5). In the BALF (B) and lung (C), IL-33 protein concentrations were determined via ELISA (n = 5–8) and western blotting (E–D) (n = 5–8). Total protein in the lung (C) was measured by BCA. F, An anti-IL-33 antibody was used for the immunohistochemistry analysis of lung tissue (n = 5). IL-33 − / − mice were used as the negative controls. Arrowheads point to the nuclear translocation of IL-33. Scale bar = 50 μm. G, Lung protein concentrations in lung tissue were determined via western blotting (n = 3), and (H) was the typical picture. The results are shown as the mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n. s., not significant. ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; and KO, knockout; WT, wild-type.

Article Snippet: According to the manufacturer’s instructions, IL-33 concentrations in mouse serum and lung tissue were tested using ELISA kits (R&D Systems, Abingdon, United Kingdom).

Techniques: Injection, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Immunohistochemistry, Translocation Assay, Knock-Out

Journal: Shock (Augusta, Ga.)

Article Title: THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS

doi: 10.1097/SHK.0000000000002114

Figure Lengend Snippet: The IL-33/ST2 axis promotes lung damage in LPS-induced ARDS. BALF and lung tissue were harvested after LPS administration for 24 h. A, The lung D/W ratio was assessed to evaluate lung edema (n = 5). B, Protein concentration and neutrophil count (C) in the BALF were analyzed with a BCA kit and flow cytometry to detect epithelial permeability (n = 5). D and E, Histopathological images of the lung tissues are based on one representative image among five (HE staining), and the scale bar = 50 μm. The results are shown as the mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n. s., not significant. ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; ctr, control; D/W, dry/wet; HE, hematoxylin-eosin; KO, knockout; WT, wild-type.

Article Snippet: According to the manufacturer’s instructions, IL-33 concentrations in mouse serum and lung tissue were tested using ELISA kits (R&D Systems, Abingdon, United Kingdom).

Techniques: Protein Concentration, Flow Cytometry, Permeability, Staining, Knock-Out

Journal: Shock (Augusta, Ga.)

Article Title: THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS

doi: 10.1097/SHK.0000000000002114

Figure Lengend Snippet: IL-33 and ST2 deletion leads to reduced levels of circulating and BALF proinflammatory cytokines. The concentrations of cytokines in the serum and BALF were measured 24 h after ARDS induction and by CBA. The IL-6, MCP-1, and TNF levels in the plasma (A) and BALF (B) of WT mice, IL-33 − / − mice, and ST2 − / − mice were measured to evaluate the systemic inflammatory response (n = 5). The data are shown as the mean + SD * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n. s., not significant. ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; CBA, bead-based multiplex immunoassay; ctr, control; KO, knockout; WT, wild-type.

Article Snippet: According to the manufacturer’s instructions, IL-33 concentrations in mouse serum and lung tissue were tested using ELISA kits (R&D Systems, Abingdon, United Kingdom).

Techniques: Multiplex Assay, Knock-Out

Journal: Shock (Augusta, Ga.)

Article Title: THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS

doi: 10.1097/SHK.0000000000002114

Figure Lengend Snippet: Mice lacking IL-33 and ST showed reduced ability to recruit and activate iNKT cells after ARDS induction. WT, IL-33 − / − , and ST2 − / − mice were subjected to a sham treatment or intratracheal LPS injection. After 24 h, the lung and spleen were obtained to prepare single-cell suspensions, and the cells were stained with anti-CD45, anti-CD3, anti-CD1d-tetramer, anti-NK1.1, anti-CD4, anti-CD8, and anti-CD69 antibodies. A and B, Representative dot plots. iNKT cells were identified as CD45 + CD3 + CD1d + tetramers (n = 5). C, The data are presented as the ratio of iNKT to CD3 + T cells and the CD69 geometric mean expression on iNKT cells in the lung and spleen (n = 3–5). D and E, Representative dot plots. NK cells were identified as CD45 + CD3 − NK1.1 + cells. F, The data are presented as the ratio of NK cells to CD3 − T cells and CD69 geometric mean expression on NK cells in the lung and spleen (n = 3–5). Three independent experiments were conducted to obtain the results shown. The data are shown as the mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n. s., not significant. ARDS, acute respiratory distress syndrome; Ctr, control; KO, knockout; WT, wild-type.

Article Snippet: According to the manufacturer’s instructions, IL-33 concentrations in mouse serum and lung tissue were tested using ELISA kits (R&D Systems, Abingdon, United Kingdom).

Techniques: Injection, Staining, Expressing, Knock-Out

Journal: Shock (Augusta, Ga.)

Article Title: THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS

doi: 10.1097/SHK.0000000000002114

Figure Lengend Snippet: The ratio of CD4 + T cells and CD8 + T cells to CD3 + T cells was not different after ARDS induction in IL-33 − / − and ST − / − mice. The lung and spleen were obtained to prepare single-cell suspensions, and the cells stained with anti-CD45, anti-CD3, anti-CD1d-tetramer, anti-NK1.1, anti-CD4, anti-CD8, and anti-CD69 antibodies 24 h after LPS-induced ARDS. A, The data are presented as the ratio of CD4 + T cells to CD3 + T cells and the CD69 geometric mean in CD4 + T cells in the lung and spleen (n = 5). B, The data are presented as the ratio of CD8+ T cells to CD3 + T cells and the CD69 geometric mean expression on CD8 + T cells in the lung and spleen (n = 5). The results shown are based on one of three independent experiments. The data are shown as the mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and n. s., not significant. ARDS, acute respiratory distress syndrome; ctr, control; KO, knockout; WT, wild-type.

Article Snippet: According to the manufacturer’s instructions, IL-33 concentrations in mouse serum and lung tissue were tested using ELISA kits (R&D Systems, Abingdon, United Kingdom).

Techniques: Staining, Expressing, Knock-Out

Journal: Shock (Augusta, Ga.)

Article Title: THE IL-33/ST2 AXIS PROMOTES ACUTE RESPIRATORY DISTRESS SYNDROME BY NATURAL KILLER T CELLS

doi: 10.1097/SHK.0000000000002114

Figure Lengend Snippet: IL-33 contributes to an uncontrolled inflammatory response in ARDS via activation of NKT cells. WT and Vα14Τg mice were pretreated with an anti-ST2 antibody 1 h before LPS administration and sacrificed 24 h after ARDS induction. BALF and lung tissue were harvested. A, The D/W ratio was calculated to evaluate lung edema (n = 5). B, Protein concentration and neutrophil count (C) in the BALF were measured with a BCA kit and by flow cytometry to evaluate epithelial permeability (n = 5). D and E, One of five representative histopathological images showing the lung tissues (HE staining). The lung injury score was determined by two blinded pathologists. Scale bar = 50 μm. F, The levels of IL-6, MCP-1, and TNF in the plasma and BALF were tested by CBA. (n = 5, * P < 0.05). The data are shown as the mean ± SD and are representative of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001. ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; ctr, control; D/W, dry/wet; KO, knockout; WT, wild-type.

Article Snippet: According to the manufacturer’s instructions, IL-33 concentrations in mouse serum and lung tissue were tested using ELISA kits (R&D Systems, Abingdon, United Kingdom).

Techniques: Activation Assay, Protein Concentration, Flow Cytometry, Permeability, Staining, Knock-Out

Journal: Journal of Indian Society of Periodontology

Article Title: Higher interleukin-33 levels in aggressive periodontitis cases

doi: 10.4103/jisp.jisp_217_19

Figure Lengend Snippet: Descriptive statistics of the study population and gingival crevicular fluid and plasma interleukin-33 concentration

Article Snippet: IL-33 concentration in GCF and plasma, obtained from the study subjects, were measured using Human IL-33 DuoSet ELISA kit (R and D Systems, USA, Imported by Biotech-India, India) and estimated using the standard curve, in accordance with the instructions provided in the kit insert.

Techniques: Concentration Assay

Journal: Journal of Indian Society of Periodontology

Article Title: Higher interleukin-33 levels in aggressive periodontitis cases

doi: 10.4103/jisp.jisp_217_19

Figure Lengend Snippet: Distribution of interleukin-33 allelic variants of single-nucleotide polymorphism rs1157505 in the study population

Article Snippet: IL-33 concentration in GCF and plasma, obtained from the study subjects, were measured using Human IL-33 DuoSet ELISA kit (R and D Systems, USA, Imported by Biotech-India, India) and estimated using the standard curve, in accordance with the instructions provided in the kit insert.

Techniques:

Journal: Journal of Indian Society of Periodontology

Article Title: Higher interleukin-33 levels in aggressive periodontitis cases

doi: 10.4103/jisp.jisp_217_19

Figure Lengend Snippet: Correlation of genotype rs1157505 (C/C, C/G, G/G) with respect to gingival crevicular fluid and plasma interleukin-33

Article Snippet: IL-33 concentration in GCF and plasma, obtained from the study subjects, were measured using Human IL-33 DuoSet ELISA kit (R and D Systems, USA, Imported by Biotech-India, India) and estimated using the standard curve, in accordance with the instructions provided in the kit insert.

Techniques: Concentration Assay

Journal: Journal of Indian Society of Periodontology

Article Title: Higher interleukin-33 levels in aggressive periodontitis cases

doi: 10.4103/jisp.jisp_217_19

Figure Lengend Snippet: Correlation of genotype rs7044343 (C/C, T/C, T/T) with respect to gingival crevicular fluid and plasma interleukin -33 levels irrespective of groups

Article Snippet: IL-33 concentration in GCF and plasma, obtained from the study subjects, were measured using Human IL-33 DuoSet ELISA kit (R and D Systems, USA, Imported by Biotech-India, India) and estimated using the standard curve, in accordance with the instructions provided in the kit insert.

Techniques:

Journal: Journal of Indian Society of Periodontology

Article Title: Higher interleukin-33 levels in aggressive periodontitis cases

doi: 10.4103/jisp.jisp_217_19

Figure Lengend Snippet: Correlation of genotype rs7044343 (C/C, T/C, T/T) with respect to gingival crevicular fluid and plasma interleukin-33

Article Snippet: IL-33 concentration in GCF and plasma, obtained from the study subjects, were measured using Human IL-33 DuoSet ELISA kit (R and D Systems, USA, Imported by Biotech-India, India) and estimated using the standard curve, in accordance with the instructions provided in the kit insert.

Techniques: Concentration Assay