Journal: Nature Communications

Article Title: MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease

doi: 10.1038/ncomms7774

Figure Lengend Snippet: ( a ) IL-33 , ( b ) soluble ST2 ( sST2 ) and ( c ) mST2 mRNA expression in human tendon samples. Fold change in gene expression of IL-33, sST2 and mST2 in control (semi-membranosus tendon, n =10), torn supraspinatus tendon (established pathology) and matched subscapularis human tendon samples (early pathology; n =17). Data are mean±s.d. relative to the housekeeping gene18S (mean of duplicate analysis). * P <0.05, ** P <0.01, *** P <0.001 versus control (Student's t- test). ( d ) Immunostaining of IL-33 and ST2 in control ( n =10), torn tendon ( n =17) and early tendinopathy ( n =17). Graphs illustrate modified Bonar scoring based on 10 high-power fields. Data are mean±s.d. * P <0.05, ** P <0.01 versus control (Student's t -test). Scale bar, 65 μm. ( e ) Fold change in gene expression of IL33 and ST2 24 h post incubation with tumour necrosis factor (TNF-α), IL-1β or in combination depicting relative expression to media alone utilizing housekeeping gene GAPDH . Data are mean±s.d. of triplicate samples, representative of three individual patient samples. * P <0.05, ** P <0.01 versus control (media) (Student's t -test). ( f ) IL-33 immunostaining of human tendon explants cultured for 24 h with medium (control), 100 ng ml −1 TNFα or 100 ng ml −1 TNFα+100 ng ml −1 IL-1β. ( g ) Fold change in COL1 and COL3 mRNA expression in human tendon explants cultured for 24 h with rhIL-33, relative to housekeeping gene GAPDH . ( h ) Time course of COL1 and COL3 mRNA expression following incubation with rhIL-33, relative to housekeeping gene GAPDH . ( i ) Collagen 1 and 3 protein expression in human tendon explants 24 h post incubation with rhIL-33. For ( g – i ) data are mean±s.d. of triplicate samples, representative of three individual patients. * P <0.05, ** P <0.01 versus control (Student's t -test).

Article Snippet: We also tested neutralizing antibodies to IL-33 (0.5 μg ml −1 , R&D systems) by injecting (i.p) the antibody or normal IgG control immediately post injury in WT and St2 −/− mice (four per group/treatment/experiment). miR-29a mimic (50 ng ml −1 in 0.1 ml) was delivered via direct injection to injured (immediately post injury) and uninjured mice tendons ( n =6 per group/treatment/experiment).

Techniques: Expressing, Immunostaining, Modification, Incubation, Cell Culture

Journal: Nature Communications

Article Title: MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease

doi: 10.1038/ncomms7774

Figure Lengend Snippet: Kinetics of ( a ) Il33 and ( b ) soluble St2 gene expression post injury. Data are mean fold change±s.d. showing relative expression to 18s housekeeping gene (pooled data from four mice per group performed on four sequential occasions, n =16 per condition). * P <0.05, ** P <0.01 versus control (WT) mice (ANOVA) + P <0.05,++ P <0.01, NS not significant; control (WT) injured versus St2 −/− injured. ( c ) Immunohistochemistry showing IL-33 and ST2 expression in tendon biopsies of WT and St2 −/− mice day 1 post injury. IgG control shown in bottom left of pictures. Black horizontal line indicates 50 μm. ( d ) Col3 mRNA and ( e ) Collagen 3 protein levels in tendon biopsies of WT and St2 −/− mice post injury. ( f ) Col1 mRNA and ( g ) Collagen 1 protein levels in tendon biopsies of WT and St2 −/− mice post injury. For ( d – g ) data are mean±s.d. of duplicate samples, representative of four mice per condition ( n =16). * P <0.05, ** P <0.01 versus control (WT) mice. + P <0.05, ++ P <0.01 control (WT) injured versus St2 −/− injured mice (ANOVA). mRNA graphs show relative expression to 18s housekeeping gene. ( h ) Percentage change in tendon strength for WT and St2 −/− mice post injury. Data are mean±s.d., representative of four mice per condition ( n =16). * P <0.05, ** P <0.01 versus control mice. # P <0.05 St2 −/− injured versus WT injured mice (Mann–Whitney U -test).

Article Snippet: We also tested neutralizing antibodies to IL-33 (0.5 μg ml −1 , R&D systems) by injecting (i.p) the antibody or normal IgG control immediately post injury in WT and St2 −/− mice (four per group/treatment/experiment). miR-29a mimic (50 ng ml −1 in 0.1 ml) was delivered via direct injection to injured (immediately post injury) and uninjured mice tendons ( n =6 per group/treatment/experiment).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Nature Communications

Article Title: MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease

doi: 10.1038/ncomms7774

Figure Lengend Snippet: ( a ) Col1 mRNA, ( b ) Collagen 1 protein, ( c ) Col3 mRNA and ( d ) Collagen 3 protein in WT and St2 −/− mice on day 1 post injury. mRNA graphs show relative expression to 18s housekeeping gene. Data are mean±s.d. of duplicate samples, representative of four mice per condition ( n =16). * P <0.05,** P <0.01, injured versus uninjured mice. + P <0.05 WT versus ST2 −/− mice. (ANOVA). ( e ) Percentage change in tendon strength in WT and St2 −/− mice on day 1 post injury with or without rhIL-33 treatment. Data are mean±s.d., representative of four mice per group ( n =16). ** P <0.01, injured versus injured+IL-33 mice (Mann–Whitney U -test). ( f ) Col1 mRNA, ( g ) Collagen 1 protein, ( h ) Col3 mRNA, ( i ) Collagen 3 protein levels and ( j ) percentage change in tendon strength post tendon injury in WT mice with or without anti-IL-33 treatment. Data for mRNA show relative expression to 18s housekeeping gene. Data are mean±s.d., representative of four mice per condition ( n =16). * P <0.05, ** P <0.01, injured versus uninjured mice (Mann–Whitney U -test).

Article Snippet: We also tested neutralizing antibodies to IL-33 (0.5 μg ml −1 , R&D systems) by injecting (i.p) the antibody or normal IgG control immediately post injury in WT and St2 −/− mice (four per group/treatment/experiment). miR-29a mimic (50 ng ml −1 in 0.1 ml) was delivered via direct injection to injured (immediately post injury) and uninjured mice tendons ( n =6 per group/treatment/experiment).

Techniques: Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease

doi: 10.1038/ncomms7774

Figure Lengend Snippet: ( a ) Luciferase activity assay of HEK 293 cells co-transferred with pre- miR-29a containing 3′UTR of WT or mutated soluble ST2 and MREs of 3′UTR of soluble ST2 . ( n =3). ** P <0.01, *** P <0.001 versus scrambled control. (Student's t -test). ( b ) sST2 and mST2 mRNA levels following addition of scrambled mimic, miR-29a mimic or miR-29a antagomir in human tenocyte cultures. ( n =5), ** P <0.01 versus scrambled control (Student's t -test). ( c ) sST2 protein production by tenocytes following incubation with miR-29a mimic/antagomir. ( n =5), * P <0.05 versus scrambled control. (Student's t -test). ( d ) qPCR showing fold change in miR-29a in tendon of WT mice post injury. ( e ) qPCR showing fold change in miR-29a in WT and St2 −/− mice following treatment with rhIL-33 or PBS on Day 1 post injury. ( f ) miR-29a expression in the tendon of WT mice post injury with or without anti-IL-33 treatment. Data in d – f are mean fold change±s.d. of duplicate samples and are representative of four mice per group ( n =16), * P <0.05, ** P <0.01 versus control (ANOVA). Data for mRNA show expression relative to u6 housekeeping gene. ( g ) Immunofluorescence stain of mimic (red) and counterstained with phalloidin (green, for cytoskeletal structure) showing localization of mimic around tenocytes at 24 h post injection of miR-29a mimic in WT mice. Scale bar, 80 nm. ( h ) Col3 mRNA, ( i ) Collagen 3 protein, ( j ) Col1 mRNA and ( k ) Collagen 1 protein levels post treatment with miR-29a mimic after tendon injury in WT mice. Data for mRNA are total copy number of gene versus 18S housekeeping gene in duplicate samples. Data in h – k are mean±s.d. of duplicate samples, representative of six mice per group, * P <0.05, ** P <0.01 versus control (ANOVA).

Article Snippet: We also tested neutralizing antibodies to IL-33 (0.5 μg ml −1 , R&D systems) by injecting (i.p) the antibody or normal IgG control immediately post injury in WT and St2 −/− mice (four per group/treatment/experiment). miR-29a mimic (50 ng ml −1 in 0.1 ml) was delivered via direct injection to injured (immediately post injury) and uninjured mice tendons ( n =6 per group/treatment/experiment).

Techniques: Luciferase, Activity Assay, Incubation, Expressing, Immunofluorescence, Staining, Injection

Journal: Nature Communications

Article Title: MicroRNA29a regulates IL-33-mediated tissue remodelling in tendon disease

doi: 10.1038/ncomms7774

Figure Lengend Snippet: Tendon injuries or repetitive micro tears causing stress of tendon cells result in the release of IL-33 from tenocytes or immune cells and the downstream phosphorylation of NFκB which in turn represses miR-29a causing an increase in collagen type 3 and sST2 production. IL-33 may also increase collagen synthesis in an ERK-dependent manner. The increased presence of collagen 3 reduces the tendon tensile strength lending to early tendon failure. However, elevated sST2 may act, in a feedback loop fashion, as a protective mechanism by removing excess IL-33 from the system.

Article Snippet: We also tested neutralizing antibodies to IL-33 (0.5 μg ml −1 , R&D systems) by injecting (i.p) the antibody or normal IgG control immediately post injury in WT and St2 −/− mice (four per group/treatment/experiment). miR-29a mimic (50 ng ml −1 in 0.1 ml) was delivered via direct injection to injured (immediately post injury) and uninjured mice tendons ( n =6 per group/treatment/experiment).

Techniques: