Journal: JCI Insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: (A) Human PBMC death induced by compounds at 3 concentrations after incubation for 20 hours (duplicate per compound at each concentration). IC50 values determined by the AlphaLISA are shown in parentheses. Compounds belonging to chemotypes I, II, and III and unclassified are grouped together. CB6114052, CB5107562, and NAT13-343201 are 3 initial hits. The average IC50 values of 17 compounds at 63.7 μM were used as references for setting the maximum concentration at 66 μM. (B) Titration curves for iST2-1, iST2-2, and iST2-4 measured with the AlphaLISA and the HEK-Blue IL-33 assay. (C) In vivo dose-escalation toxicity evaluation of 7 selected compounds in healthy mice. Data represent mean ± SEM (n = 3 per compound). hPBMC, human peripheral blood mononuclear cell.

Article Snippet: HEK-Blue IL-33 cells purchased from InvivoGen (catalog hkb-hil33) were designed to detect bioactive IL-33 by monitoring activation of the NF-κB and AP-1 pathways.

Techniques: Incubation, Concentration Assay, Titration, In Vivo

Journal: JCI Insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: (A) Chemical structures of racemic iST2-1, (R)-iST2-1, (S)-iST2-1, analogs of iST2-1, and structural alignment of (R)-iST2-1 (purple) with (S)-iST2-1 (green). IC50 values of the inhibitors are shown in parentheses. (B) Inhibition curves and IC50 values (in parentheses) of 4 representative compounds. Each data point is an average of triplicate measurements ± SD. (C) SAXS profiles, the residual plot between the SAXS profiles of apo-ST2 and ST2/iST2-1, comparison of the Kratky plot based on the SAXS profiles of apo-ST2 and ST2/iST2-1, the pair-wise distance distribution [P(r)] of apo-ST2 and ST2/iST2-1 calculated from the SAXS profiles. Dmax values are shown in parentheses. (D) Ab initio shape reconstruction of apo-ST2 (gray) and ST2/iST2-1 (purple). (E) Mapping of the (R)-iST2-1 and (S)-iST2-1 binding sites (surface envelop) in ST2 based on 32-ns MD simulations. Maps detected from 13- and 15-Å octahedron boxes are colored in purple and green, respectively. Consensus binding sites (S1r, S2r and S1s) are circled. The D1 and D2 domains of ST2 are labeled. (F) Binding sites of (R)-iST2-1 (green mesh) and (S)-iST2-1 (red mesh) that block interaction between ST2 and IL-33 (orange) are highlighted. (G) Binding sites of (R)-, (S)-, (S,S)-iST2-1 in the glycosylated ST2 from the MD simulations. (S,S)-iST2-1 is (S)-iST2-1 in which the proton on the pyrrolidine group is in the S form and its mapped site is shown in cyan mesh. SAXS, small-angle X-ray scattering.

Article Snippet: HEK-Blue IL-33 cells purchased from InvivoGen (catalog hkb-hil33) were designed to detect bioactive IL-33 by monitoring activation of the NF-κB and AP-1 pathways.

Techniques: Inhibition, Comparison, Binding Assay, Labeling, Blocking Assay

Journal: JCI Insight

Article Title: From proteomics to discovery of first-in-class ST2 inhibitors active in vivo

doi: 10.1172/jci.insight.99208

Figure Lengend Snippet: (A) The dosages were calculated using the body weight of each mouse at 20 mg and correspond to twice the IC50 values of the inhibitors determined by the HEK-Blue IL-33 assay. (B) Number of T cells infiltrating the gut in the B6C3H.SW GVHD model at day 14. Flow cytometric analysis of intestinal CD4+IFN-γ+ and FoxP3+CD4+ T cell populations on day 14 in the (C) hu-T cells → NSG model and day 21 in the (D) hu-T cells → NSG, (E) B6 → C3H.SW GVHD models treated with iST2-1, iST2-2, or iST2-3. n = 2 per group at day 14 and 21 respectively, and for each model. Data represent mean ± SEM (n = 2). TBI, total body irradiation; hPBMC, human peripheral blood mononuclear cell. *P < 0.05, **P < 0.01 by unpaired t test.

Article Snippet: HEK-Blue IL-33 cells purchased from InvivoGen (catalog hkb-hil33) were designed to detect bioactive IL-33 by monitoring activation of the NF-κB and AP-1 pathways.

Techniques: Irradiation

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: Journal of Inflammation (London, England)

Article Title: Association of intestinal and systemic inflammatory biomarkers with immune reconstitution in HIV+ patients on ART

doi: 10.1186/s12950-020-00262-4

Figure Lengend Snippet: Systemic immune activation, inflammation, microbial translocation, proinflammatory cytokines, intestinal damage and inflammation biomarkers

Article Snippet: Serum concentration of Intestinal Fatty Acids-Binding Protein (I-FABP) and the soluble IL-33 receptor sST2 were measured by ELISA (Human Intestinal Fatty Acid Binding Protein I-FABP ELISA kit; CUSABIO, Houston, TX, USA and Human ST2/IL-33R Quantikine ELISA kit, R&D Systems, Minneapolis, MN, USA), following each manufacturer protocols.

Techniques: Activation Assay, Translocation Assay, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: Association of intestinal and systemic inflammatory biomarkers with immune reconstitution in HIV+ patients on ART

doi: 10.1186/s12950-020-00262-4

Figure Lengend Snippet: Determination of gut damage and inflammation. a I-FABP, b sST2, c Fecal lactoferrin, d Fecal calprotectin, e sIgA, f Association between I-FABP levels and Lipopolysaccharide concentration, g Association between fecal calprotectin levels and absolute CD4 + T-cells count, h Association between fecal calprotectin levels and duration of HIV infection and i Association between fecal calprotectin levels and duration of antiretroviral treatment. Kruskall-Wallis test with Bonferroni correction. Data show as median (IQR); * p < 0.05 . Spearman correlation test

Article Snippet: Serum concentration of Intestinal Fatty Acids-Binding Protein (I-FABP) and the soluble IL-33 receptor sST2 were measured by ELISA (Human Intestinal Fatty Acid Binding Protein I-FABP ELISA kit; CUSABIO, Houston, TX, USA and Human ST2/IL-33R Quantikine ELISA kit, R&D Systems, Minneapolis, MN, USA), following each manufacturer protocols.

Techniques: Concentration Assay, Infection

Journal: The international journal of biochemistry & cell biology

Article Title: TLR-mediated Induction of Pro-allergic Cytokine IL-33 in Ocular Mucosal Epithelium

doi: 10.1016/j.biocel.2011.06.003

Figure Lengend Snippet: TLR-dependent induction of IL-33 by microbial ligands in primary HCECs. A. Levels of IL-33 mRNA at 8 hours after stimulation by 50μg/ml polyI:C or 10μg/ml of Pam3CSK4, PGN, polyI:C, LPS, flagellin, FSL-1, R-837, ssRNA40 or C-CpG-ODN. B. The concentrations of IL-33 protein detected by ELISA in cell lysates treated with various TLR ligands (X-axis) for 48 hours. C. Levels of IL-33 mRNA at different time periods (4-24 h) in cells treated with 50μg/ml polyI:C (left), or by increasing concentration (10, 25, or 50μg/ml) of polyI:C at 8 hours (right), evaluated by quantitative RT and real-time PCR. Results shown are the mean ± SD of three to five independent experiments. *P< 0.05; **P < 0.01.

Article Snippet: The real-time PCR was performed in a Mx3005P™ system (Stratagene) with 20μl reaction volume containing 5μl of cDNA, 1μl of TaqMan® Gene Expression Assay for IL-33 (TaqMan Assay ID Hs00369211 m1) or GAPDH (Hs99999905 m1) and 10μl Master Mix.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction

Journal: The international journal of biochemistry & cell biology

Article Title: TLR-mediated Induction of Pro-allergic Cytokine IL-33 in Ocular Mucosal Epithelium

doi: 10.1016/j.biocel.2011.06.003

Figure Lengend Snippet: Stimulated secretion of IL-33 cellular protein to culture medium by ATP in primary HCECs. The HCECs were exposed to microbial ligand 50μg/ml polyI:C (A, C, E, G) or 10μg/ml flagellin (B, D, F, H) for 48 hours without or with addition of 5mM ATP to media 30min prior to measurement. The IL-33 protein concentrations in culture media (A & B) and in cell lysates were detected by ELISA (C & D), and the level of cellular IL-33 protein was also evaluated by Western blot analysis using β-actin as control (E & F) with quantitative ratio of IL-33/β-actin (G & H). Results shown are the mean ± SD of three to five independent experiments. *P< 0.05; **P < 0.01.

Article Snippet: The real-time PCR was performed in a Mx3005P™ system (Stratagene) with 20μl reaction volume containing 5μl of cDNA, 1μl of TaqMan® Gene Expression Assay for IL-33 (TaqMan Assay ID Hs00369211 m1) or GAPDH (Hs99999905 m1) and 10μl Master Mix.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The international journal of biochemistry & cell biology

Article Title: TLR-mediated Induction of Pro-allergic Cytokine IL-33 in Ocular Mucosal Epithelium

doi: 10.1016/j.biocel.2011.06.003

Figure Lengend Snippet: IL-33 induction in an ex vivo human corneal tissues evaluated by immunohistochemical staining. A fresh corneoscleral tissue was cut into four equal-sized pieces. Each quarter was placed into a well of eight-chamber slides, epithelial side up in 150μL of serum-free SHEM medium, without or with polyI:C (50μg/ml) or flagellin (10μg/ml) for 24 hours in a 37°C incubator. Frozen sections of corneal tissues were used for IL-33 immunohistochemical staining with isotype IgG as the negative control. Magnification 400x.

Article Snippet: The real-time PCR was performed in a Mx3005P™ system (Stratagene) with 20μl reaction volume containing 5μl of cDNA, 1μl of TaqMan® Gene Expression Assay for IL-33 (TaqMan Assay ID Hs00369211 m1) or GAPDH (Hs99999905 m1) and 10μl Master Mix.

Techniques: Ex Vivo, Immunohistochemical staining, Staining, Negative Control

Journal: The international journal of biochemistry & cell biology

Article Title: TLR-mediated Induction of Pro-allergic Cytokine IL-33 in Ocular Mucosal Epithelium

doi: 10.1016/j.biocel.2011.06.003

Figure Lengend Snippet: TLR and NF-κB signaling pathways are involved in IL-33 induction by polyI:C or flagellin. The HCECs exposed to polyI:C (50μg/mL) or flagellin (10μg/mL) were preincubated in the absence or presence of rabbit TLR3Ab (10μg/mL), TLR5Ab (10μg/mL), BAY11-7082 (10μM) or NF-kB activation inhibitor quinazoline (NF-kB-I, 10μM) for 1 hour, and Pepinh-MYD (40μM) or Pepinh-TRIF (40μM) for 6 hours. The cultures treated by ligands for 8 hours were subjected to RT and real-time PCR to measure IL-33 mRNA (A & B), the cultures treated for 48 hours were used to evaluate IL-33 protein in cell lysates by ELISA (C & D) and Western blot using β-actin as control (E & F) with quantitative ratio of IL-33/β-actin (G & H).. Results shown are the mean ± SD of three to five independent experiments. *P< 0.05; **P < 0.01.

Article Snippet: The real-time PCR was performed in a Mx3005P™ system (Stratagene) with 20μl reaction volume containing 5μl of cDNA, 1μl of TaqMan® Gene Expression Assay for IL-33 (TaqMan Assay ID Hs00369211 m1) or GAPDH (Hs99999905 m1) and 10μl Master Mix.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Nuclear localization of the IL-33 and reduced with the onset of labor. The sections fixed paraffin-embedded of human myometrium were immune-stained with anti-IL-33 antibody. (A) Immunohistochemistry results showed that whether in TNL or PNL tissue, IL-33 mostly located in the nuclear while reduced sharply and emerged in the cytoplasm with the initiation of labor as shown in figure TL and PTL. Quantification of IL-33 expression within the nucleus region showed apparent differences between labor and non-labor tissue (n=4). (B) The sections of human myometrium were visualized by an Alexa Flour 594 secondary antibody labeled with IL-33 (Red), and nuclei were stained with DAPI (Blue). Analysis of nuclei was performed by confocal microscopy, fluorescence signals of IL-33 and nuclei were superimposed. Immunofluorescence staining results reflected the same phenomenon. (C) From Western blots, we also can see that non-labor groups had more nuclear expression and less cytoplasmic expression of IL-33 compared to labor groups while the total IL-33 had no obvious differences between groups. QT-PCR discovered no apparent alteration in the levels of IL-33 mRNA. Each value represents the mean ± standard deviation (SD) of three independent experiments. ** P<0.01, *** P<0.001, bar 50 μm.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Staining, Immunohistochemistry, Expressing, Labeling, Confocal Microscopy, Fluorescence, Immunofluorescence, Western Blot, Standard Deviation

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Dynamic changes of IL-33 position during LPS stimulation. (A) Localization analysis of confocal laser scanning microscopy images of IL-33 with the stimulation of 10 μg/ml LPS, we could see that IL-33 in the nucleus declined sharply compared with the control group, especially in 3 hours. However, with the longer time of the LPS treatment, the expression of IL-33 commenced to rise again from 6 hours and reached the peak at 12 hours. The lower panel is the percentage of cells of which IL-33 expressed mainly in the nucleus (n=3). (B) After the primary cells were loaded with LPS for different times, cytoplasmic and nuclear proteins were isolated. In the cytoplasmic and nucleus fraction IL-33 were quantified by Western blotting, the experiment was performed as described for . Each experiment was performed at least three times while shown are representative results (n=3). From the blots, we known that whether cytoplasmic or nucleus fraction IL-33 levels presented the same change trend as immunofluorescence. * P<0.05, ** P<0.01, bar 50 μm.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Confocal Laser Scanning Microscopy, Expressing, Isolation, Western Blot, Immunofluorescence

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 silencing enhanced LPS-induced expression of calcium channels and the intracellular calcium concentration. Primary myometrium cells were transfected with IL-33 or non-targeting (as control) siRNAs and treated with 10 μg/ml LPS or PBS (as control) for 6 hours. (A) Immunofluorescence staining analysis performed with fluo-3AM (green), nuclei were stained with DAPI (blue) (n=3). (B) Western blot analysis for expression levels of Cav 3.1 and Cav 3.2 in stably transfected and non-transfected myometrium cells with treatment as indicated (n=5). * P<0.05, ** P <0.01, bar 50 μm.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Expressing, Concentration Assay, Transfection, Immunofluorescence, Staining, Western Blot, Stable Transfection

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 silencing highlighted LPS-induced endoplasmic reticulum stress response. Based on the discovery that calcium ions affected endoplasmic reticulum stress, Western blot and QT-PCR were we further to explore the expression of endoplasmic reticulum stress in tissues from protein and mRNA level. (A) The protein levels of P-IRE1α and XBP1s in the TL and PTL groups were higher than those in the TNL and PNL groups while there was no alteration in the level of GRP78 protein(n=6). (B) The mRNA level of XBP1s in the PTL groups was higher than that in the PNL groups (n=6). Furthermore, in order to illustrate the protein level changes of endoplasmic reticulum stress during labor, LPS was used to stimulate primary uterine smooth muscle cells for different time course and then Western blot was used to detect the alteration of endoplasmic reticulum stress. (C) It was found that the protein level of pIRE1α reached its peak at 10 minutes while XBP1s at 15 minutes, while the protein level of GRP78 did not change significantly (n=5). We also detected apparent alteration in endoplasmic reticulum stress protein during cells stimulated based knockdown experiments targeting IL-33. (D) It revealed that the endoplasmic reticulum stress response in the siRNA-based group was more obvious compared with the LPS stimulated directly especially at 30 minutes (n=5). * P<0.05, ** P<0.01, *** P<0.005, ****P<0.001

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Western Blot, Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 siRNA and ER stress response affected COX-2 expression in myometrium cells. (A) In the process of studying whether IL-33 affects COX-2, we found that the COX-2 expression in the siRNA-mediated group was significantly increased compared with the LPS alone group (n=5). (B) Western blot analyses showing protein expression of COX-2 in myometrium cells was decreased following treatment with LPS for 12 hours (n=5). * P<0.05, ** P<0.01. Each value represents the mean ± standard deviation (SD) of three independent experiments.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 knockdown enhanced LPS-induced NF-κB and p38/MAPK signaling pathways. (A) Relative levels of p-P38, P38, p-NF-κB and NF-κB were assessed by western blot analysis at the indicated time point after LPS (10 μg/ml) stimulation. Phosphorylation levels of P38 and NF-κB increased gradually with LPS stimulation and peaked at 15 minutes and 1 hour, respectively(n=5). (B) Western blot analysis of p-P38, P38, p-NF-κB and NF-κB expression in cells transfected with siRNA targeting IL-33 after treatment with LPS for 30minutes. Compared with the LPS group, the protein expression of phosphorylated P38 and NF-κB were increased in the LPS + siRNA IL-33 group(n=5). (C) The protein level of COX-2 was decreased when SB-202190 and JSH-23 blocked p38/MAPK and NF-KB signaling pathway, respectively(n=5). * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Western Blot, Expressing, Transfection

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: IL-33 siRNA and cytoplasmic calcium influence the expression of IL-8 and IL-6. Compared with the LPS group, the expression of IL-8 and IL-6 was increased in the LPS+IL-33 siRNA group and decreased in the LPS+BAPTA-AM group (n=5). * P <0.05.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

Techniques: Expressing

Journal: medRxiv

Article Title: Interleukin-33 stimulates stress in the endoplasmic reticulum of the human myometrium via an influx of calcium during initiation of labor

doi: 10.1101/2021.11.05.21265965

Figure Lengend Snippet: Model for the role of IL-33 in the myometrium participates in maintaining a uterine quiescent state at the tissue-to-cellular level during late pregnancy.

Article Snippet: Then samples were incubated overnight at 4□ with goat anti-human IL-33 monoclonal antibody (MAB36253, R&D) at a concentration of 0.1 µg/mL and then 1-3 drops of biotinylated secondary antibodies for 2 hours at room temperature.

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: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Estimation of IL-17A in whole lung homogenates from mock- (open) or influenza virus-(solid)- infected wild-type (black) and TCRδ−/− (red) neonates by ELISA at 1 day after infection. Samples are pooled from at least two independent experiments and data are shown as mean ± SEM (Mann-Whitney test).B. Survival rate of influenza virus-infected TCRδ−/− neonates administered with low levels of recombinant mouse IL-17A (rmIL-17A, green, n=37, 100pg/mouse) or PBS control (red, n=19) at the time of infection. Data are combined from six independent trials, which individually showed the same trend, and data are presented as mean ± SEM.C. Schema outlining wild-type and Il17a−/− γδ T cell transfers to TCRδ−/− neonates and subsequent infection.D and E. Body weight profile (D) and survival rate (E) of influenza-infected TCRδ−/− neonates receiving wild-type (black, n=15) or Il17a−/− (red, n=13) γδ T cells intranasally or no cell transfer (grey, n=10). Data are combined from four independent trials, which individually showed the same trend. Weight profile data are presented as mean ± SEM.F. Protein levels of IL-33 assessed by ELISA in influenza virus-infected wild-type (black, n=15) and TCRδ−/− lungs (red, n=9) at 1 day following infection. Samples are pooled from three independent experiments, and data are shown as mean ± SEM.G. Protein levels of IL-33 detected by ELISA in the lungs of TCRδ−/− neonates that had been infected with influenza virus and simultaneously treated with either a low level of rmIL-17A (green, n=7, 100pg/mouse) or PBS control (red, n=6). Data were collected 1 day after infection/treatment. Samples are pooled from at least two independent experiments, and data are shown as mean ± SEM.H. Survival rate of influenza virus-infected wild-type (black, n=20) and Il33−/− neonates (blue, n=17) with intranasal influenza virus infection. Data are combined from ten independent experiments and shown as mean ± SEM in weight change.I. Survival rate of influenza virus-infected TCRδ−/− neonates administered with recombinant mouse IL-33 (rmIL-33, brown, n=18, 10ng/mouse) or PBS control (red, n=18) at the time of infection. Data are combined from six individual experiments.*p<0.05, **p<0.01, n.s., not significant.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Recombinant

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A. Relative expression of Il33 mRNA, as assessed by quantitative real-time PCR, in mouse lung epithelial cells (LET1s) treated with medium (white), A/x31 influenza virus (blue, 3 MOI), rmIL-17A (green, 50ng/ml), or virus+rmIL-17A (red) at 24hrs and 48hrs after treatment. Data are shown as mean ± SEM and combined from three separate experiments that independently showed the same trend.B. Immunoblot assay of IL-33 protein in stimulated LET1s for the same conditions as in (A) at 48 hrs after stimulation.C. Quantitative real-time PCR analysis of Il33 mRNA in LET1 cells treated as in A and B with and without STAT3 phosphorylation inhibitor S3I-201 (100μM in 0.05% DMSO) at 48hrs after treatment. Data are shown as mean ± SEM and combined from two separate experiments that independently showed the same trend.D. Immunoblot analysis of IL-33 and phosphorylated STAT3 (pSTAT3) in the LET1 lysates treated as before (A, B, and C). Total STAT3 (tSTAT3) and β-actin are also shown. Immunoblots were repeated twice showing similar results.*p<0.05, ****p<0.0001

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: A- C. Correlation between concentrations of human IL-17A and IL-33 (A), IFN-γ and IL-33 (B) and IL-33 and Areg protein levels (C) in the nasal aspirates of influenza-infected children (< 8 years old, n=51).D. Concentration of IL-17A at Day 0 after enrollment in the nasal aspirates of children with mild (black, n=11) and severe (red, n=12) influenza disease outcomes.E. Concentration of IFN-γ at Day 0 after enrollment in the nasal aspirates of children with mild (black, n=11) and severe (red, n=12) disease outcomes.Data are presented as mean ± SEM, and in each case cytokine values (pg/ml) were log10 transformed for visualization only. *p<0.05.

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Infection, Concentration Assay, Transformation Assay

Journal: Immunity

Article Title: Lung γδ T cells mediate protective responses during neonatal influenza infection that are associated with Type-2 immunity

doi: 10.1016/j.immuni.2018.07.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cytokines were measured in supernatants as follows: IL-17A, IL-33, and amphiregulin were assayed by Human IL-17A, IL-33, and amphiregulin DuoSet ELISA kits (R&D), and the level of IFN-γ was detected by Human Milliplex assays (Millipore) according to manufacturer instructions.

Techniques: Plasmid Preparation, Recombinant, Cell Stimulation, Protease Inhibitor, Blocking Assay, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Sequencing, Software