Journal: Cancer Immunology Research

Article Title: Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-γ–Dependent Manner

doi: 10.1158/2326-6066.cir-17-0218

Figure Lengend Snippet: Figure 1. St2 deficiency promotes the development of sporadic colon cancer. Sporadic colon cancer was induced by weekly administration of AOM over 6 consecutive weeks (6AOM) to WT and St2/ mice and analysis occurred 9–15 weeks after the last AOM injection depending on the onset of tumor development. A, Schematic of the experimental procedures. B, Representative whole mounts of the colons of WT and St2/ mice following 6AOM treatment 15 weeks after last AOM injection (left), and enumeration of the total number of resulting tumors per mouse (right). C and D, Representative sections from formalin-fixed and paraffin- embedded colons stained for hematoxilin–eosin (C; scale bar, 100 mm) and Ki67 or Apoptag (D; scale bar, 25 mm). E, Enumeration of positive staining cells from D expressed as the total number of Ki67þ cells (left) or Apoptagþ cells (right) per mm2 of tumor area. F and G, Quantitative RT-PCR analysis for Il33 (F) and Il1rl1 (exons 10–11, specific for membrane-bound isoform) expression (G) from tumors or adjacent normal colon tissues from 6AOM-treated WT and St2/ mice. Data were normalized against expression of Gapdh. B–G, Data were pooled from three independent experiments and shown as mean SEM, with each symbol representing an individual mouse. Data were analyzed using unpaired Student t test (E) or unpaired t test with Welsh correction (B, F). , P < 0.01; , P < 0.05; n.s., not significant.

Article Snippet: The sections were blocked with TNB Buffer (0.1 mol/L Tris–HCl, 0.15 mol/L NaCl, and 0.5% Blocking Reagent; PerkinElmer, Inc.) prior to staining with polyclonal goat anti-mouse St2 (R&D Systems, catalog # AF1004) and polyclonal goat anti-mouse IL33 (R&D Systems, catalog # AF3626).

Techniques: Injection, Staining, Quantitative RT-PCR, Membrane, Expressing

Journal: Cancer Immunology Research

Article Title: Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-γ–Dependent Manner

doi: 10.1158/2326-6066.cir-17-0218

Figure Lengend Snippet: Figure 3. IL33 stimulation induces expression of NF-kB target genes in colon-derived mesenchymal cells. A, Quantitative RT-PCR analysis of Il1rl1 mRNA expression encoding all isoforms (exons 7–8) or membrane-bound isoform (exons 10–11) in EpCAMþ/CD45 epithelial cells and EpCAM/CD45 mesenchymal cells isolated from unaffected colon ("colon") and tumors ("tumor") of WT mice undergoing the 6AOM protocol or left untreated ("UT"). Data were derived from 2 to 4 individual mice per cohort and were normalized against expression of Hprt. Data are representative of two (untreated colon) or one (colon and 6AOM colon and tumor samples) independent experiments. Mean SEM of the technical replicates are shown. B, Mesenchymal cells (CD45/EpCAM/CD90þ FSCHi) were isolated from the colon of WT mice and cultured in vitro for 16 hours. FACS plots show St2 and CD90 expression after 16 hours in vitro culture and is representative of three independents experiments. C and D, Cytokine production by mesenchymal cells, isolated as in B, and stimulated in vitro for 16 hours with IL33 (20 ng/mL) or left untreated. Quantitative RT-PCR expression analysis of indicated genes relative to expression of Hprt (C). Protein quantification following multiplex cytokine assay in the supernatant of cultured mesenchymal cells in D. Mean SEM with symbols representing independent experiments. Data were analyzed using paired Student t test. , P < 0.001; , P < 0.01; , P < 0.05. E, Immunobloting analysis of NIH-3T3 murine embryonic fibroblasts stimulated with murine IL33 (20 ng/mL).

Article Snippet: The sections were blocked with TNB Buffer (0.1 mol/L Tris–HCl, 0.15 mol/L NaCl, and 0.5% Blocking Reagent; PerkinElmer, Inc.) prior to staining with polyclonal goat anti-mouse St2 (R&D Systems, catalog # AF1004) and polyclonal goat anti-mouse IL33 (R&D Systems, catalog # AF3626).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Membrane, Isolation, Cell Culture, In Vitro, Multiplex Assay, Cytokine Assay, Western Blot

Journal: Cancer Immunology Research

Article Title: Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-γ–Dependent Manner

doi: 10.1158/2326-6066.cir-17-0218

Figure Lengend Snippet: Figure 4. Loss of IL33 signaling in both the radiosensitive and the radioresistant cell compartments promotes Treg infiltration within colon tumors. A–C, Flow cytometric analysis of enzymatically digested tumors collected from 6AOM-treated BM chimeric mice generated as in Fig. 2. Representative FACS plots show St2 and Foxp3 expression on gated CD4þ/CD3þ/CD90High/CD45þ/EpCAM T cells (A). Data show Foxp3þ/CD4þ Treg among CD45þ/EpCAM hematopoietic cells (B, left), and ratio of Treg to EpCAMþ/CD45 tumor cells (B, right). Frequency of St2þ cells among Foxp3þ/CD4þ Tregs within the spleen or tumors from the indicated BM chimeras (C). A–C, Bars show mean SEM; symbols represent individual mice. D–G, Flow cytometric analysis of enzymatically digested colon tumors from 6AOM-treated WT or St2/ mice. Representative FACS plots showing intracellular expression of Gata3 and Rorgt in Foxp3þ/CD4þ Tregs (D). Frequency of Gata3þ

Article Snippet: The sections were blocked with TNB Buffer (0.1 mol/L Tris–HCl, 0.15 mol/L NaCl, and 0.5% Blocking Reagent; PerkinElmer, Inc.) prior to staining with polyclonal goat anti-mouse St2 (R&D Systems, catalog # AF1004) and polyclonal goat anti-mouse IL33 (R&D Systems, catalog # AF3626).

Techniques: Generated, Expressing

Journal: Cancer Immunology Research

Article Title: Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-γ–Dependent Manner

doi: 10.1158/2326-6066.cir-17-0218

Figure Lengend Snippet: Figure 5. Loss of IL33 signaling in the radioresistant cell compartment leads to a decrease in IFN gene signature and immune activation within colon tumors. Whole genome expression analysis of tumors collected from BM chimeras generated as in Fig. 2. A, Distribution of commonly and uniquely differentially expressed genes between tumors collected from the indicated BM chimeras (red—upregulated, blue—downregulated). B, Relative expression level (z score) of the 65 DE genes commonly regulated in BM chimeras in which St2 deficiency was restricted to the radioresistant compartment as indicated in A. C, Metacore analysis of biological processes for the 65 DE genes selected as in B. D, Normalized expression counts of selected genes in tumors from the indicated BM chimeras as determined by RNAseq. Mean SEM normalized expression counts with each symbol representing an individual mouse.

Article Snippet: The sections were blocked with TNB Buffer (0.1 mol/L Tris–HCl, 0.15 mol/L NaCl, and 0.5% Blocking Reagent; PerkinElmer, Inc.) prior to staining with polyclonal goat anti-mouse St2 (R&D Systems, catalog # AF1004) and polyclonal goat anti-mouse IL33 (R&D Systems, catalog # AF3626).

Techniques: Activation Assay, Expressing, Generated

Journal: Mucosal immunology

Article Title: Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis

doi: 10.1038/s41385-019-0138-4

Figure Lengend Snippet: ( a, b ) The mRNA expression of IL-33 ( Il33 ) and its receptor ST2 ( Il1rl1 ) in the colonic mucosa Salmonella / E.coli co-colonization (day 21) ( a ) and DSS + E. coli (day 21) ( b ) models. ( c ) mRNA expression of ST2 ( IL1RL1 ) and IL-33 ( IL33 ) genes in the ileal tissue from control subjects (N=11), patients with inactive CD (N=16) and active CD (N=51). Data were derived from Gene Expression Omnibus (GEO) dataset GSE75214. Bars represent median. N.S.; not significant, *; P < 0.05, **; P < 0.01, ***; P < 0.001 by Kruskal-Wallis test. ( d ) Mice pre-colonized with AIEC strain LF82 were infected with S. Typhimurium Δ spiA (ST). Anti-ST2 blocking antibody and isotype control IgG (50µg/mouse) were injected intra-peritoneally 3 times per week starting on day 7 post Salmonella infection (6 times total). Mice were sacrificed 21 days following ST infection. ( e ) Intestinal colonization (feces) of S. Typhimurium Δ spiA (left) and pre-colonized E. coli LF82 (right) on indicated days following ST infection. ( f ) mRNA expression of indicated cytokines 21 days post ST infection. ( g ) Expression of fibrosis-related genes in the colonic mucosa (day 21). ( h, i ) Accumulation of collagen was quantified by Picro-sirius red staining ( h ) and collagen assay ( i ). Dots represent individual mice. Bars represent median ( e ) or mean ( f , g , i ). N.S.; not significant, *; P < 0.05, **; P < 0.01, ***; P < 0.001 by 1-Way ANOVA with Bonferroni post-hoc test.

Article Snippet: IL-33-ST2 signaling in vivo was blocked by the intraperitoneal administration of 50 μg of an anti-mouse ST2 antibody or control rat IgG (Genentech.

Techniques: Expressing, Control, Derivative Assay, Gene Expression, Infection, Blocking Assay, Injection, Staining, Collagen Assay

Journal: Mucosal immunology

Article Title: Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis

doi: 10.1038/s41385-019-0138-4

Figure Lengend Snippet: ( a ) Human IEC T84 cells and primary colonic fibroblasts (1 × 10 5 cells/500 µl/well; 24 well plate) were stimulated with E. coli HS or LF82 at MOI=10 for 3 hr followed by 15 hrs of additional culture in the presence of gentamycin (100 µg/ml) to prevent bacterial overgrowth. mRNA expression of IL-33 ( IL33 ) and ST2 ( IL1RL1 ) was quantified by qPCR. Data shown are mean ± SD (N=6). N.S.; not significant, *; P < 0.05 by 1-Way ANOVA with Bonferroni post-hoc test. ( b ) Strep-treated mice were then inoculated with human commensal E. coli strain HS or human AIEC strain LF82 (1 × 10 9 CFU). The control and ST groups only received PBS. Seven days following E. coli colonization, mice were infected with 2×10 7 CFU of S. Typhimurium Δ spiA (ST). At day 21 post ST infection, the colonic tissues were harvested and ST2 was strained. Representative images of ST2 immunohistochemistry are shown.

Article Snippet: IL-33-ST2 signaling in vivo was blocked by the intraperitoneal administration of 50 μg of an anti-mouse ST2 antibody or control rat IgG (Genentech.

Techniques: Expressing, Control, Infection, Immunohistochemistry

Journal: Mucosal immunology

Article Title: Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis

doi: 10.1038/s41385-019-0138-4

Figure Lengend Snippet: ( a ) T84 cells were stimulated with human commensal E. coli strain HS or E. coli strains isolated from CD patients. Gray bars indicate non-AIEC strains and black bars indicate AIEC strains based on adhesion/invasion assay. ( b ) T84 cells were stimulated with AIEC LF82 or various mutants of LF82 at MOI=10 for 3 hr followed by 15 hrs of additional culture in the presence of gentamycin (100 µg/ml) to prevent bacterial overgrowth. mRNA expression of IL-33 ( IL33 ) and ST2 ( IL1RL1 ) was quantified by qPCR. Data shown are mean ± SD (N=6). N.S.; not significant, *; P < 0.05 by Dunnett test (vs control). ( c ) TLR5 and NLRC4 mRNA were knocked-down in T84 cells using siRNA specific to human TLR5 or NLRC4 . Relative expression to Ctrl Non-targeting pool siRNA are shown. Data shown are mean ± SD (N=3). **; P < 0.01 by Student-t test. ( d ) TLR5 and NLRC4 mRNA were knocked-down in T84 cells using siRNA. Cells were then stimulated with AIEC LF82 at MOI=10 or medium (untreated; UT) for 3 hr followed by 15 hrs of additional culture in the presence of gentamycin (100 mg/ml) to prevent bacterial overgrowth. mRNA expression of ST2 ( IL1RL1 ) and IL-33 ( IL33 ) was quantified by qPCR. Data shown are mean ± SD (N=3). N.S.; not significant, *; P < 0.05, **; P < 0.01 by 1-Way ANOVA with Bonferroni post-hoc test.

Article Snippet: IL-33-ST2 signaling in vivo was blocked by the intraperitoneal administration of 50 μg of an anti-mouse ST2 antibody or control rat IgG (Genentech.

Techniques: Isolation, Invasion Assay, Expressing, Control

Journal: Mucosal immunology

Article Title: Flagellin-mediated activation of IL-33-ST2 signaling by a pathobiont promotes intestinal fibrosis

doi: 10.1038/s41385-019-0138-4

Figure Lengend Snippet: ( a ) SPF C57BL/6 mice were pre-treated with 20mg of streptomycin (Strep) 1 day prior to E. coli inoculation. A CD-associated AIEC strain LF82 (WT or Δ fliC mutant) strains (1 × 10 9 CFU) were then inoculated to Strep-treated mice. Seven days after AIEC colonization, mice were infected with 2×10 7 CFU of Salmonella typhimurium Δ spiA (ST). ( b ) Intestinal colonization (in feces) of ST and AIEC LF82 (WT or Δ fliC mutant) at indicated days post ST infection. Dots represent individual mice. Bars represent median. ( c ) Expression of inflammation-related genes in the colonic mucosa (day 21 ST). ( d ) The mRNA expression of IL-33 ( Il33 ) and its receptor ST2 ( Il1rl1 ) in the colonic mucosa. ( e ) Representative images of ST2 immunohistochemistry (day 21). ( f ) Expression of fibrosis-related genes in the colonic mucosa (day 21 ST). (g, h) Accumulation of collagen was examined by Sirius red staining ( g ) and collagen assay ( h ). Dots represent individual mice. Bars represent mean. N.S.; not significant, *; P < 0.05, **; P < 0.01, ***; P < 0.001 by 1-Way ANOVA with Bonferroni post-hoc test.

Article Snippet: IL-33-ST2 signaling in vivo was blocked by the intraperitoneal administration of 50 μg of an anti-mouse ST2 antibody or control rat IgG (Genentech.

Techniques: Mutagenesis, Infection, Expressing, Immunohistochemistry, Staining, Collagen Assay

Journal: The Journal of Experimental Medicine

Article Title: IL-33 amplifies an innate immune response in the degenerating retina

doi: 10.1084/jem.20150894

Figure Lengend Snippet: ST2 is expressed on activated Müller cells after phototoxic stress and blocking ST2 signaling protects photoreceptors. (A) Expression of membrane-bound (ST2L) and soluble (sST2) ST2 following CLE. Retina RNA from BALB/c mice exposed to light for various days was analyzed by qPCR using probes specific for ST2L and sST2, and normalized to 18s rRNA. ST2 expression in nonexposed mice (d0) was set as 1. Each data point represents an individual mouse ( n = 5–6/time point). (B) Expression of ST2 on activated (GFAP + vimentin + ), resting (GFAP − vimentin + ) Müller cells (MC), and microglia (CD45 lo CD11b + ; MGL) after 7 d light exposure. (C) OCT analysis of retina thickness at baseline (d0) and after 7 d exposure to light in ST2 +/+ and ST2 −/− mice. Delta retina thickness was calculated by subtracting retina thickness of day 0 by that of day 7 for each mouse ( n = 10/genotype). Bars, 100 µm. (D) Rods and cones in ST2 +/+ and ST2 −/− mice were quantified by flow cytometry. FACS plots indicate gating strategy and percentages, as well as absolute numbers (×10 5 ; between parentheses) of rods and cones in ST2 +/+ and ST2 −/− mice. Each data point represents an individual mouse ( n = 5–6/group). Rho, rhodopsin; CAR, cone arrestin. (E) Morphometric analysis of ONL thickness of ST2 +/+ and ST2 −/− mice at baseline (d0) and after 14 d exposure to light plotted as a function of distance from the optic nerve head (ONH). Data shown are means ± SEM ( n = 5–7/group). (F) ERG of ST2 +/+ and ST2 −/− mice at baseline (d0) and after 7 d CLE. (top) Representative ERG recordings at 25 cd/s/m 2 light intensity of baseline and after 7 d CLE. Data shown are means ± SEM ( n = 10/genotype). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant; one-way ANOVA with Dunnett’s post-tests (A), two-way ANOVA with Tukey’s post-tests (F), or unpaired two-tailed Student’s t tests (C, D, and E). Data are representative of at least two independent experiments with similar results.

Article Snippet: Culture supernatant was harvested 6 d after infection and analyzed for sST2 secretion by ELISA (R&D Systems) and Western blotting using the goat anti–mouse ST2 Ab (R&D Systems).

Techniques: Blocking Assay, Expressing, Membrane, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-33 amplifies an innate immune response in the degenerating retina

doi: 10.1084/jem.20150894

Figure Lengend Snippet: Soluble ST2 protects photoreceptors from phototoxic stress. (A) BALB/c mice were subretinally injected with an AAV (AAV2/5) expressing soluble ST2 (AAV-sST2) or AAV empty vector (AAV-EV) which served as a negative control. Expression of sST2 in the retina and RPE/choroid 3 wk after infection was analyzed by ELISA and Western blotting (10 µg of retina lysates and 3 µg of RPE/choroid lysates). rsST2, recombinant soluble ST2-His. (B) Mice were subretinally injected with AAV-sST2 or AAV-EV and exposed to light 3 wk after infection. Rods and cones were quantified by flow cytometry before (d0) and after 7 d CLE. Each data point represents an individual mouse ( n = 10/group). ***, P < 0.001; ****, P < 0.0001; unpaired two-tailed Student’s t tests. Data are representative of two independent experiments with similar results.

Article Snippet: Culture supernatant was harvested 6 d after infection and analyzed for sST2 secretion by ELISA (R&D Systems) and Western blotting using the goat anti–mouse ST2 Ab (R&D Systems).

Techniques: Injection, Expressing, Plasmid Preparation, Negative Control, Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: IL-33 amplifies an innate immune response in the degenerating retina

doi: 10.1084/jem.20150894

Figure Lengend Snippet: IL-33 induces chemokine and cytokine expression through autocrine activation of Müller cells. (A) qPCR analysis of CCL2, IL-6, and IL-1β expression in the retina of ST2 +/+ and ST2 −/− mice after CLE ( n = 5–6/group). Gene expression in d0 ST2 +/+ mice was set as 1. CCL2 protein expression in the retina was measured by ELISA. (B) IL-33 induces CCL2 secretion from rMC-1 cells. ST2 expression on the surface of rMC-1 cells was detected by flow cytometry. CCL2 levels in 24-h culture supernatants were measured by ELISA. Data shown are means ± SEM of triplicate wells. (C) Autocrine induction of CCL2 from rMC-1 cells is blocked with IL-33 TRAP. rMC-1 cells were cultured in HG-containing medium in the presence of IL-33 TRAP or a control protein for up to 72 h. CCL2 expression and secretion were measured by qPCR and ELISA, respectively. Data shown are means ± SEM of triplicate experiments. (D) CCR2 expression on retinal CD45 lo CD11b + myeloid cells before (d0) and after CLE (d7) was analyzed by flow cytometry. (E and F) Immunohistochemistry analysis of Iba1 + cells in the retina of ST2 +/+ and ST2 −/− mice. Iba1 + cells (arrows) in the ONL, OS, GCL, and OPL of the entire superior and inferior retina before (d0) and after CLE (d14) were quantified. Each data point represents an individual mouse ( n = 10/genotype). Bars, 50 µm. (G) Effect of monocyte depletion on photoreceptor survival in ST2 +/+ and ST2 −/− mice exposed to light. ST2 +/+ and ST2 −/− mice were treated intravenously with clodronate-liposomes (Clod) or control liposomes (Ctrl) daily starting 2 d before CLE as shown in the diagram. Retinal cells were quantified by flow cytometry 7 d after CLE. n = 4–6/group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant; one-way (A, F, and G) or two-way (B and C) ANOVA with Tukey’s post-tests. Data are representative of at least two independent experiments with similar results.

Article Snippet: Culture supernatant was harvested 6 d after infection and analyzed for sST2 secretion by ELISA (R&D Systems) and Western blotting using the goat anti–mouse ST2 Ab (R&D Systems).

Techniques: Expressing, Activation Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Culture, Control, Immunohistochemistry, Liposomes

Journal: The Journal of Experimental Medicine

Article Title: IL-33 amplifies an innate immune response in the degenerating retina

doi: 10.1084/jem.20150894

Figure Lengend Snippet: IL-33 lacking the N-terminus induces ST2-dependent cytokine and chemokine expression and retinal cell loss. (A) IL33 tm1/tm1 mice lacking the N-terminal nuclear localization sequence and chromatin binding domain (shown in diagram) but retaining the C-terminal cytokine domain show localization of dsRed-IL-33 C-term in cytoplasm of Müller cells. Images shown are Z-section views of confocal images of an IL33 tm1/tm1 retina flat-mount. Arrows, IL-33 + Müller cells. Bars, 5 µm. Expression of dsRed-IL-33 C-term in Müller cells was verified by flow cytometry. IL-33 mRNA in the retinas of IL33 +/+ , IL33 tm1/+ , and IL33 tm1/tm1 mice was analyzed by qPCR. IL-33 protein levels in the retina and serum were measured by ELISA. (B) ST2-dependent increases of CCL2 and IL-6 expression and loss of cones and RGC in IL33 tm1/tm1 mice. Retinas from IL33 +/+ , IL33 tm1/+ , and IL33 tm1/tm1 mice bred on ST2 +/− or ST2 −/− background were analyzed by qPCR and flow cytometry. Each data point represents an individual mouse ( n = 3–7/genotype). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant; one-way ANOVA with Tukey’s post-tests. Data are representative of two independent experiments with similar results.

Article Snippet: Culture supernatant was harvested 6 d after infection and analyzed for sST2 secretion by ELISA (R&D Systems) and Western blotting using the goat anti–mouse ST2 Ab (R&D Systems).

Techniques: Expressing, Sequencing, Binding Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: IL-33 amplifies an innate immune response in the degenerating retina

doi: 10.1084/jem.20150894

Figure Lengend Snippet: Mononuclear phagocytes are required for IL-33/ST2-induced photoreceptor cell loss in a model of RPE cell loss. (A) GFAP expression (red) in Müller cells (arrows) of mice treated with saline or NaIO 3 . Bar, 50 µm. (B) IL-33 processing in the retina of mice treated with saline or NaIO 3 was analyzed by Western blotting. The ratio of IL-33 p19 versus IL-33 p30 in the retina was quantitated with Image J software. (C) CCL2 expression in the retina of ST2 +/+ and ST2 −/− mice treated with saline or NaIO 3 was measured by ELISA. n = 6–7/group. (D) Macrophage infiltration in the retina in NaIO 3 -treated ST2 +/+ and ST2 −/− mice. Macrophages (CD45 hi CD11b + ) in the retina were quantified by flow cytometry. Retina macrophages express higher level of CCR2 compared with microglia (CD45 lo CD11b + ). n = 6–7/group. MΦ, macrophage; MGL, microglia. (E) Immunohistochemistry analysis of Iba1 + cells in the retina of NaIO 3 -treated ST2 +/+ and ST2 −/− mice. Iba1 + cells in the OS and ONL of the entire superior and inferior retina in saline- or NaIO 3 -treated mice (d3) were quantified. n = 6/group. (F) Protection of photoreceptors in NaIO 3 -treated ST2 −/− mice. (top) Retina thickness of ST2 +/+ and ST2 −/− mice before (d0) and after NaIO 3 treatment (d7) was measured by OCT. Representative cross-sectional OCT images are shown. Delta retina thickness was calculated by subtracting the retina thickness of d0 by that of d7 for an individual mouse. Bars, 100 µm. (bottom) Rods and cones of ST2 +/+ and ST2 −/− mice treated with saline or NaIO 3 (d3 and d7) were quantified by flow cytometry. Each data point represents an individual mouse ( n = 6–8/group). (G) Effect of monocyte depletion on photoreceptor survival in ST2 +/+ and ST2 −/− mice treated with NaIO 3 . ST2 +/+ and ST2 −/− mice were treated intravenously with clodronate-liposomes (Clod) or control liposomes (Ctrl) daily starting 1 d before NaIO 3 treatment as shown in the diagram. Retinal cells were quantified by flow cytometry 3 d after saline or NaIO 3 treatment. Each data point represents an individual mouse ( n = 5/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant; one-way ANOVA with Tukey’s post-tests (C, D, E, and G) or unpaired two-tailed Student’s t tests (F). Data are representative of at least two independent experiments with similar results.

Article Snippet: Culture supernatant was harvested 6 d after infection and analyzed for sST2 secretion by ELISA (R&D Systems) and Western blotting using the goat anti–mouse ST2 Ab (R&D Systems).

Techniques: Expressing, Saline, Western Blot, Software, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunohistochemistry, Liposomes, Control, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

doi: 10.1167/iovs.09-3983

Figure Lengend Snippet: Primer Sequences for Real-time PCR

Article Snippet: After blocking with 0.01 M phosphate buffer containing 2.5% BSA and goat IgG (1:100) for 3 minutes at room temperature, sections were incubated with primary antibodies, rat anti-mouse Mφ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse NOS2 (1:100; Santa Cruz Biotechnology), rabbit anti-mouse Arg1 (1:100; Santa Cruz Biotechnology), or rabbit anti-mouse ST2 (2 μg/mL; ProSci Incorporated, Poway, CA) for 1 hour, followed by Alexa Fluor 546-conjugated goat anti-rat antibody (1:1500; Invitrogen) or Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1500; Invitrogen) for another hour.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

doi: 10.1167/iovs.09-3983

Figure Lengend Snippet: Modulation of IL-33 in vitro. Real-time RT-PCR showed ST2 expression on RAW cells was significantly upregulated over constitutive levels after stimulation with LPS after 2 (P < 0.05), 4, 6, and 8 hours (each at P < 0.01) (A). After transfection with a vector overexpressing IL-33, RAW cells significantly downregulated mRNA for pro-inflammatory mediators (B) IL-1β (P < 0.05), MIP-2 (P < 0.01), IL-6 (P < 0.01), and TNF-α (P < 0.01). In addition, Th1-type cytokines such as IFN-γ (P < 0.01) and IL-12 (P < 0.01) (C) and Th2-type cytokines such as IL-5 (P = 0.01) and IL-10 (P < 0.01) (D) were upregulated. Data are the mean ± SEM of two similar experiments (5 samples/group/time).

Article Snippet: After blocking with 0.01 M phosphate buffer containing 2.5% BSA and goat IgG (1:100) for 3 minutes at room temperature, sections were incubated with primary antibodies, rat anti-mouse Mφ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse NOS2 (1:100; Santa Cruz Biotechnology), rabbit anti-mouse Arg1 (1:100; Santa Cruz Biotechnology), or rabbit anti-mouse ST2 (2 μg/mL; ProSci Incorporated, Poway, CA) for 1 hour, followed by Alexa Fluor 546-conjugated goat anti-rat antibody (1:1500; Invitrogen) or Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1500; Invitrogen) for another hour.

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation

Journal: Investigative Ophthalmology & Visual Science

Article Title: IL-33 Shifts Macrophage Polarization, Promoting Resistance against Pseudomonas aeruginosa Keratitis

doi: 10.1167/iovs.09-3983

Figure Lengend Snippet: Immunostaining and real-time RT-PCR. Dual immunostaining for F4/80 (Mφ) and ST2 in the cornea of rmIL-33 compared with PBS-treated B6 mice. More staining for ST2 (blue) was associated with Mφ (red) in rmIL-33–-treated mice (A) than in PBS-treated mice (B) cornea. Substitution of the primary antibodies with IgG showed no detectable staining (C). Original magnification, ×1000. Real-time RT-PCR analysis of LPS-stimulated peritoneal-elicited Mφ, with or without rmIL-33, showed a significant upregulation of ST2 on Mφ after LPS stimulation in the presence of rmIL-33 (P = 0.04) (D). Upregulation of IL-10 (E; P = 0.003) and downregulation of IL-12 (F; P = 0.003) were observed after LPS, together with rmIL-33 treatment. Data for RT-PCR are the mean ± SEM of two similar experiments (5 samples/group/time).

Article Snippet: After blocking with 0.01 M phosphate buffer containing 2.5% BSA and goat IgG (1:100) for 3 minutes at room temperature, sections were incubated with primary antibodies, rat anti-mouse Mφ (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-mouse NOS2 (1:100; Santa Cruz Biotechnology), rabbit anti-mouse Arg1 (1:100; Santa Cruz Biotechnology), or rabbit anti-mouse ST2 (2 μg/mL; ProSci Incorporated, Poway, CA) for 1 hour, followed by Alexa Fluor 546-conjugated goat anti-rat antibody (1:1500; Invitrogen) or Alexa Fluor 633-conjugated goat anti-rabbit antibody (1:1500; Invitrogen) for another hour.

Techniques: Immunostaining, Quantitative RT-PCR, Staining, Reverse Transcription Polymerase Chain Reaction