Ifn gamma

IL-1β and IFNγ respectively induce NF-κB and IFN target genes expressed in diseased shoulder tendon tissues8 11 (link) and were also identified in diseased Achilles tendon tissues in the current study. We therefore investigated if treatment of cultured tendon cells with these cytokines induced more profound expression of SFA markers and NF-κB and IFN target genes in diseased relative to healthy tendon-derived cells. Tendon-derived cells were isolated from healthy hamstring and diseased Achilles tendons using previously described protocols11 (link); passage 1–3 cells were used for all experiments. Cells were grown until 80% confluence prior to stimulation with IL-1β (10 ng/mL, Sigma) or IFN gamma (20 ng/mL, BioLegend) in medium (DMEM F12, Lonza) containing 1% heat-inactivated human serum (Sigma). Non-treated (vehicle only) cells served as experimental controls. After cytokine/vehicle treatment, cells were incubated at 37°C and 5% CO₂ for 24 hours until experimental harvest for mRNA or flow cytometry.

Human monocytes isolated by Rosette Sep © negative selection were plated at 5 x 10⁴ cells/ well in 96-well u-bottom plates. Tv was counted, and then reconstituted in complete RPMI media, and rendered dead, but intact as described above. Dead, intact Tv were then added to monocytes, or human monocyte derived macrophages (HMDM) at an MOI (multiplicity of infection) of 1, and allowed to incubate overnight (16 hours). Positive controls were 100 ng/ml LPS (sigma) 10 μg/ml poly (I:C) (Tocris) or 1000 U/ml IFN gamma (Biolegend). Subsequently plates were centrifuged and supernatants were harvested and frozen at -80°C. Supernatants were then thawed on ice and analyzed using Cytometric Bead Array (Becton-Dickenson) for IL-8, IL-6, IL-1β, TNFα, and IL-12 according to the manufacturer’s instructions. IL-6, IL-1β, and IL-12 were multiplexed, and IL-8 was measured separately on supernatants diluted 1:100. Data were analyzed using FlowJo (Treestar) to determine MFI, which was normalized to absolute concentrations according to a standard curve generated using lyophilized protein provided with the kit. IL-23 was measured using Legend Max Human IL-23 (p19/p40) ELISA kit with pre-coated wells (Biolegend) according to the manufacturer’s instructions. Wells were read using a Victor³ 1420 plate reader (Perkin-Elmer), and MFI was normalized as described above.

Foxp3/Transcription Factor Staining Buffer Set, True‐Phos™ Perm Buffer, Cell Staining Buffer, and recombinant human cytokines for in vitro stimulation (IFN‐gamma, interleukin (IL)‐6, IL‐2, IL‐4; all carrier‐free) were purchased from Biolegend.
The following antibodies were used to analyse Tregs via flow cytometry: anti‐CD4 (clone OKT4; APC‐Cy7‐labeled), anti‐CD3 (clone HIT3a; PE‐Cy7) anti‐CD25 (clone M‐A251; PerCP‐labeled), anti‐CD127 (clone A019D5; BV‐421‐labeled), anti‐Foxp3 (clone 206D; AlexaFluor647‐labeled), anti‐CD39 (clone A1; FITC‐labeled), and isotype controls (all from Biolegend). STAT activation was studied using commercially available PE‐labeled antibodies against phospho‐STAT1 (clone A17012A.Rec), phospho‐STAT3 (clone 13A3‐1), phospho‐STAT5 (clone A17016B.Rec), and phospho‐STAT6 (clone A15137E) (all Biolegend).