Journal: Clinical & Translational Immunology

Article Title: Blockade of IL‐33 signalling attenuates osteoarthritis

doi: 10.1002/cti2.1187

Figure Lengend Snippet: Increasing IL‐33 levels exacerbates OA in vivo . (a) protein and (b) mRNA expression of cartilage‐degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in isolated human chondrocytes from Non‐OA ( n = 20) and OA patients ( n = 20) treated with either PBS (vehicle control) or rIL‐33 (30 ng mL −1 , 24 h). (c) MMP‐13 and (d) MMP‐3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non‐OA ( n = 12) and OA patients ( n = 12) treated with either PBS (vehicle control) or rIL‐33 (30 ng mL −1 ; 24 h). (e, f) OARSI scoring of cartilage tissue, (g) synovitis scoring and (h) osteophyte maturity scoring of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL‐33 (33 μg kg −1 ; daily for 12 weeks post‐surgery). (i) von Frey pain assessment of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL‐33 (33 μg kg −1 ; daily for 12 weeks post‐surgery). (j) mRNA expression of cartilage‐degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in whole knee joints of DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either PBS (vehicle control) or rIL‐33 (33 μg kg −1 ; daily for 12 weeks post‐surgery). All RT‐qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with unpaired 2‐tailed Student’s t ‐tests ( c, d ), two‐way analysis of variance followed by the Tukey‐Kramer test ( f, g, h ), or repeated measures 2‐way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point (i; DMM PBS vs DMM rIL‐33). n indicates the number of human specimens or mice per group. NS = non‐significant. P < 0.01, P < 0.001 or P < 0.0001 are represented as **, *** or ****, respectively.

Article Snippet: For experiments which increased IL‐33 levels in vivo , mouse rIL‐33 (Enzo Life Sciences, Farmingdale) was administered intraperitoneally (i.p) on a daily basis at 33 μg kg −1 for 12 weeks post‐surgery.

Techniques: In Vivo, Expressing, Isolation, Quantitative RT-PCR

Journal: Clinical & Translational Immunology

Article Title: Blockade of IL‐33 signalling attenuates osteoarthritis

doi: 10.1002/cti2.1187

Figure Lengend Snippet: Neutralising ST2 attenuates OA. (a, b) OARSI scoring of cartilage tissue, (c) synovitis scoring and (d) osteophyte maturity scoring of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 50 µg per mouse; daily for 12 weeks post‐surgery) or αST2 (50 µg per mouse; daily for 12 weeks post‐surgery). (e) von Frey pain assessment of sham‐ ( n = 20) or DMM‐ ( n = 20) operated WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 50 µg per mouse; daily for 12 weeks post‐surgery) or αST2 (50 µg per mouse; daily for 12 weeks post‐surgery). (f) mRNA expression of cartilage‐degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in whole knee joints of DMM‐operated ( n = 20) treated intraperitoneally with either IgG1 (vehicle control; 50 µg per mouse; daily for 12 weeks post‐surgery) or αST2 (50 µg per mouse; daily for 12 weeks post‐surgery). (g) protein and (h) mRNA expression of cartilage degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in isolated human chondrocytes from Non‐OA ( n = 20) and OA patients ( n = 20) treated with rIL‐33 (30 ng mL −1 , 24 h) and IgG1 (vehicle control; 3 μg mL −1 , 24 h) or αST2 (3 μg mL −1 , 24 h). (i) MMP‐13 (j) MMP‐3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non‐OA ( n = 12) and OA patients ( n = 12) treated with rIL‐33 (30 ng mL −1 ; 24 h) and IgG1 (vehicle control; 3 μg mL −1 , 24 h) or αST2 (3 μg mL −1 , 24 h). All RT‐qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with two‐way analysis of variance followed by the Tukey‐Kramer test (b, c, d) or repeated measures 2‐way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point ( e ; DMM IgG1 control mice vs DMM αST2 mice) or with unpaired 2‐tailed Student’s t ‐tests (i, j) . n indicates the number of human specimens or mice per group. NS = non‐significant. P < 0.001 or P < 0.0001 are represented as *** or ****, respectively.

Article Snippet: For experiments which increased IL‐33 levels in vivo , mouse rIL‐33 (Enzo Life Sciences, Farmingdale) was administered intraperitoneally (i.p) on a daily basis at 33 μg kg −1 for 12 weeks post‐surgery.

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Clinical & Translational Immunology

Article Title: Blockade of IL‐33 signalling attenuates osteoarthritis

doi: 10.1002/cti2.1187

Figure Lengend Snippet: Neutralising IL‐33 attenuates OA. (a, b) OARSI scoring of cartilage tissue, (c) synovitis scoring and (d) osteophyte maturity scoring of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 15 µg per mouse; daily for 12 weeks post‐surgery) or αIL‐33 (15 µg per mouse; daily for 12 weeks post‐surgery). (e) von Frey pain assessment of sham‐operated ( n = 20) or DMM‐operated ( n = 20) WT mice (12 weeks post‐surgery end timepoint) treated intraperitoneally with either IgG1 (vehicle control; 15 µg per mouse; daily for 12 weeks post‐surgery) or αIL‐33 (15 µg per mouse; daily for 12 weeks post‐surgery). (f) mRNA expression of cartilage degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in whole knee joints of DMM‐operated ( n = 20) treated intraperitoneally with either IgG1 (vehicle control; 15 µg per mouse; daily for 12 weeks post‐surgery) or αIL‐33 (15 µg per mouse; daily for 12 weeks post‐surgery). (g) protein and (h) mRNA expression of cartilage degrading proteases (MMP‐13, ADAMTS‐5 and MMP‐3) and chondrogenic markers (COL2A1, SOX‐9 and Aggrecan) in isolated human chondrocytes from Non‐OA ( n = 20) and OA patients ( n = 20) treated with rIL‐33 (30 ng mL −1 , 24 h) and IgG1 (vehicle control; 10 μg mL −1 , 24 h) or αIL‐33 (10 μg mL −1 , 24 h). (i) MMP‐13 ( j) MMP‐3 protein level in cell media supernatants obtained from isolated human chondrocytes from Non‐OA ( n = 12) and OA patients ( n = 12) treated with rIL‐33 (30 ng mL −1 ; 24 h) and IgG1 (vehicle control; 10 μg mL −1 , 24 h) or αIL‐33 (10 μg mL −1 , 24 h). All RT‐qPCR gene expressions were normalised to the endogenous level of 18 s in respective groups. Data are expressed as mean ± S.E.M. with two‐way analysis of variance followed by the Tukey‐Kramer test ( b, c, d ) or repeated measures 2‐way ANOVA with Bonferroni’s post hoc tests was used to compare groups at each time point ( e ; DMM IgG1 control mice vs DMM αIL‐33 mice) or with unpaired 2‐tailed Student’s t ‐tests ( i, j ). n indicates the number of human specimens or mice per group. NS = non‐significant. P < 0.0001 is represented as ****.

Article Snippet: For experiments which increased IL‐33 levels in vivo , mouse rIL‐33 (Enzo Life Sciences, Farmingdale) was administered intraperitoneally (i.p) on a daily basis at 33 μg kg −1 for 12 weeks post‐surgery.

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A transgenic mutant mouse line accompanied by the complete deletion of interleukin-33 showed insulin and leptin resistances

doi: 10.1101/416529

Figure Lengend Snippet: The Il33 gene is completely deleted in the mutant mice and recombinant IL-33 protein restored insulin resistance in the muscle of the preobese mutant mice. ( A ) Schematic representation of the control and mutant allele surrounding the transgene integration site. Transgene integration caused the deletion of a 124.2-kb genomic DNA fragment (a blue region) at the left integration (LI) site of the mutant allele. It also caused the inversion of a 38.2-kb genomic DNA fragment with one break in the right breakpoint (RB) and the other break in the right transgene integration (RI) site (a red arrow) and the deletion of a 4.6-kb DNA fragment (a yellow region) at the right end of the mutant allele. The orientation of transcription is indicated by black arrows. White arrowheads indicate PCR primers (a1 and a3, b1-b3, and c1-c3) used for confirmation of the transgene insertion sites and breakpoint region. The orientation of the genomic DNA with respect to the centromere (cen) and telomere (tel) of chromosome 19 is indicated by black arrowheads. Flp, Flp recombinase expression vector pEF-NFLP. ( B ) PCR analysis of genomic DNA from the control and mutant mice using primers based on the inserted transgene and the flanking endogenous genomic DNA (left and center panels) or the flanking right breakpoint (right panel). Lane M contains size markers. ( C ) Real-time quantitative RT-PCR analysis in the various tissue of the control (white bars) and preobese mutant (mint green bars) mice (n = 7-10 per group). n.d., not detectable. ( D ) Insulin tolerance test (0.75 mU/g of body weight) results of the male control (white circles), the preobese mutant (mint green circles) and recombinant IL-33 (rIL-33) protein treated preobese mutant (light blue triangles) mice (n = 8-9 per group). The preobese mutant mice received an intraperitoneal injection of vehicle or rIL-33 at 1 hour before the insulin tolerance test. The control versus preobese mutant mice with vehicle: *P < 0.05, **P < 0.01; Vehicle versus rIL-33 treated preobese mutant mice: †P < 0.05, ††P < 0.01. ( E ), GIR (left), EGP (center), and R d (right) of the male control (white bars), preobese mutant (mint green bars) and rIL-33 treated preobese mutant (light blue bars) mice in the hyperinsulinemic-euglycemic clamp studies (n = 6-12 per group). *P < 0.05; **P < 0.01; n.s., not significant. ( F ) Real-time quantitative RT-PCR analysis in the muscle (left) and the liver (right) of the control (white bars) and preobese mutant (mint green bars) mice (n = 7-10 per group). n.d., not detectable. Means ± SEM.

Article Snippet: We injected 2.0 µg mouse rIL-33 (Adipogen, San Diego, California, USA) or saline as the control intraperitoneally at 1 hour before the insulin tolerance test.

Techniques: Mutagenesis, Recombinant, Expressing, Plasmid Preparation, Quantitative RT-PCR, Injection