Fig 1: Reversal of IL-17A-mediated inflammatory mediator release, structural degeneration and functional impairment and reversal thereof with an IL-17A antagonist of isolated rat tail tendon fascicles ex vivo. (A) IL-17A antagonist inhibition (0.625 µg/mL) of IL-17A mediated protein release of CXCL1 and IL-6 in fascicle culture supernatant analysed by ELISA (n=3 rats). (B) Representative histological sections of tendon fascicles freshly harvested and at day 10 of culture with IL-17A (30 ng/mL) without and with IL-17A antagonist (1 µM) as well as with PBS (control). Paraffin-embedded tendon fascicles were cut along the longitudinal axis and 5 µm thick slices were obtained from the fascicle core and stained with Alcian blue and Nuclear Fast Red. (C) Time course of IL-17A-mediated degeneration measured as decline in fascicle mechanical stiffness (Young’s modulus) (n=6 rats). (D) IL-17A antagonist inhibition (0.625 µg/mL) of IL-17A mediated degeneration measured as decline in fascicle mechanical stiffness (Young’s modulus) and tensile strength (failure stress) of fascicles freshly harvested and at day 10 of culture with IL-17A and PBS (n=6 rats). Ordinary ANOVA (Šídák’s multiple comparisons test) was applied for statistical analysis, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as individual values (symbol) and as mean±SD (bar). ANOVA, analysis of variance; CXCL1, chemokine (C-X-C motif) ligand 1; PBS, phosphate buffered saline; ROUT, robust non-linear regression and outlier removal.
Fig 2: Expression of selected IL-17A pathway signature genes and inhibition thereof with the anti-IL-17A IgG1 mAb secukinumab in cells derived from healthy human hamstring and gracilis tendon. (A) Expression levels of NFKBIZ, ZC3H12A, IL6, CXCL1 and MMP3 measured by AmpliSeq RNA after stimulation with IL-17A and TNF-α (0.15 ng/mL) (n=4–6 subjects/1–3 hamstring and three gracilis tendons, in total three outlier data points (CXCL1) were identified and removed using ROUT with Q=1%). (B) Anti-IL-17A mAb secukinumab inhibition (100 µg/mL) of IL-17A stimulated release of IL-6 (ELISA) (n=3–6 subjects/hamstring tendons). (C) Anti-IL-17A mAb secukinumab inhibition (100 µg/mL) of IL-17A stimulated expression of CXCL1 and MMP3 (RT qPCR) (n=4–8 subjects/hamstring tendons, in total 2 outlier data points (MMP3) were identified and removed using ROUT with Q=1%). Ordinary ANOVA (Šídák’s multiple comparisons test) or Kruskal-Wallis test (Dunn’s multiple comparisons test) was applied for statistical analysis, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as individual values (symbol) and as mean±SD (bar). ANOVA, analysis of variance; CXCL1, chemokine (C-X-C motif) ligand 1 mRNA; IL-17A, recombinant human interleukin 17A; mAb, monoclonal antibody; NFKBIZ, NF-kappa-B inhibitor zeta mRNA; ROUT, robust nonlinear regression and outlier removal; RPM, reads per million mapped reads; TNF-α, tumour necrosis factor alpha; ZC3H12A, zinc finger CCCH-type containing 12A mRNA.
Fig 3: Effects of IL-17A blockade with the anti-IL17A IgG1 mAb on inflammation, gait imbalance and tendon mechanical function in a rat disease model of rotator cuff tendinopathy in vivo. (A) Representative MRI of healthy and injured rotator cuff at day 7 and day 21 postinjury and weekly subcutaneous dosing of anti-IL17A mAb and PBS (control). Arrows indicate the anatomical location of the supraspinatus tendon. (B) Anti-IL-17A mAb reversal of rotator cuff inflammation quantified by MRI relaxation time T2 (n=6–12 rats). (C) Anti-IL-17A mAb reversal of shoulder dysfunction assessed by gait analysis of paw maximum contact area and maximum paw contact intensity (n=12–16 rats, in total four outlier data points were identified and removed using ROUT with Q=1%). (D) Anti-IL-17A mAb attenuation of supraspinatus tendon swelling assessed as tendon cross-sectional area (n=6 rats). (E) Anti-IL-17A mAb attenuation of supraspinatus tendon impaired mechanical stiffness (dynamic modulus) and tensile strength (failure stress) (n=6 rats). Ordinary and two-way ANOVA (Šídák’s, Tukey’s and Dunnett’s multiple comparisons test) was applied for statistical analysis, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data are presented as individual values (symbol) and as mean±SD (bar). ANOVA, analysis of variance; IL-17A, interleukin 17A; T2, relaxation time; mAb, monoclonal antibody; PBS, phosphate buffered saline; sc, subcutaneous.
Fig 4: Expression of IL17A and its receptor heterodimer subunits IL17RA and IL17RC compared with other IL-17 family member ligands and receptors in tendon biopsies of human rotator cuff tendinopathy. (A) Expression of IL17A, F and IL17RA, RC (RT qPCR) (n=6–10 subjects). (B) Expression of IL17B, C, D, E (RT qPCR) (n=6–10 subjects). (C) Expression of IL17RB, RD, RE (RT qPCR) (n=6–10 subjects). (D) Expression of TNFA (RT qPCR) (n=6–10 subjects). The relative mRNA expression of the gene of interest was calculated by the ΔCT method relative to the GAPDH housekeeping gene. Ordinary ANOVA (Šídák’s multiple comparisons test) was applied for statistical analysis, nsp>0.05, **p<0.01. Data are presented as individual values (symbol) and as mean (line). Data are presented as individual values (symbols) and mean (line). Healthy (subscapularis tendon, no rotator cuff tear, Bonar score 1); Early-stage tendinopathy (subscapularis tendon, rotator cuff tear of adjacent supraspinatus tendon, Bonar score 2–3); Late-stage tendinopathy (supraspinatus tendon with tear, Bonar score 4). ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL17A to F, interleukin 17 A to F mRNA; IL17RA to RE, interleukin 17 receptor A to E mRNA; ns, not significant; TNFA, tumour necrosis factor alpha mRNA.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Rat IL-17A Protein, CF