H3884

Distributions of the main ECM components were assessed on the mid‐substance of ACL and LDET (n = 3) using immunohistochemistry and immunofluorescence staining for different collagen types, proteoglycans and elastic fibres. The antibodies used were reactive against collagen type I, III, aggrecan, versican, decorin, biglycan, elastin, fibrillin‐1 and fibrillin‐2 (Supporting Information Table S2). All antibodies (apart from elastin, fibrillin‐1 and fibrillin‐2) were used for immunostaining of TLs as described previously (Kharaz et al. 2016), using 4‐μm paraffin‐embedded sections. Frozen sections of 5 μm were used for immunostaining of elastin, fibrillin‐1 and fibrillin‐2 with hyaluronidase (4800 IU mL^–1 in PBS, H3884, Sigma‐Aldrich) treatment as previously described (Smith et al. 2011). The distribution and arrangement of the selected collagens and proteoglycans were visualised with a Nikon Eclipse 80i. Elastic fibres were assessed with the confocal microscope (Nikon Eclipse Ti). Negative controls were included with rabbit and mouse isotope IgG and normal serum in place of primary antibody. No staining was observed in the control experiments (Supporting Information Fig. S2). Adobe photoshop CS6 software was used to measure the average staining intensity for each antibody stain in each tissue (Zamboulis et al. 2013).

The endothelial glycocalyx was degraded by the infusion of hyaluronidase from bovine testes (25 mg/kg/body weight; H3884, Sigma, St. Louis, MO, USA) and a blend of heparinase I and III from Flavobacterium heparinum (1 U/mouse; H3917, Sigma, St. Louis, MO, USA) for 30 min. Heparinase units are given as Sigma units. The enzymes were prepared in phosphate-buffered saline (PBS) and were infused through femoral vein cannulation. The control animals were infused with PBS alone for 30 min. We have successfully demonstrated in a previous study a significant degradation of the mouse retinal glycocalyx following hyaluronidase infusion at a dose of 12 mg/kg [17 (link)].

Synovial fluid was obtained from 8 RA patients, 2 Spondyloarthritis (SpA) patients and one control synovial fluid from a patient undergoing a traumatic-related surgery. Informed written consent was obtained and the study protocol was approved by the CHU Toulouse ethics committee (BioTOUL DC 2016-2804). RA patients fulfilled the criteria for the ACR/EULAR 2010 and SpA patients the criteria for the ASAS. All RA patients had active joint inflammation and were taken in charge in the Rheumatology Department of the Toulouse University Hospital. SF was collected during therapeutic arthrocentesis. Samples of SF were sent to the hospital laboratories for routine analysis. Analysis results along with patients' blood tests and medical history allowed a full assessment of inflammation taking place at the time of sample collection.
SF were transferred to heparin-treated tubes, treated with hyaluronidase 0.5 mg/mL (H3884, Sigma-Aldrich, St. Louis, MO, USA) for 10 min at room temperature and centrifuged to exclude cells and debris. Samples were then filtered on a pore size of 0.2 μm to exclude remaining macromolecules and frozen in aliquots at −80°C for later use.

Flow cytometry was used to compare between each genotype and sex (antibodies and gating strategy are detailed in Additional file 1: Supplementary data 3, 4). Whole hearts were removed and washed in PBS. Finely minced tissue was then digested for 30 min at 37 °C [Krebs–Henseleit solution containing collagenase (Clostridium histolyticum Type IA; C9891, Sigma-Aldrich), Type XI collagenase (C7657, Sigma-Aldrich), bovine pancreatic deoxyribonuclease I (D4263, Sigma-Aldrich) and hyaluronidase Type IV from bovine testes (H3884, Sigma-Aldrich)]. Digestates were then passed through a 70-micron cell strainer and reconstituted with PBS prior to density gradient centrifugation (Ficoll-Paque PLUS (17-1440-02, GE Healthcare Life Sciences). Isolated immune cells were washed again with phosphate-buffered saline, incubated with an antibody cocktail comprising CD45-FITC (clone REA737), CD8a-PerCPVio700 (clone 53-6.7), CD4-APC (clone GK1.5), Ly6G-APCVio770 (clone REA526), Ly6C-VioBlue (clone REA796), CD45B220-VioGreen (clone REA755), CD62L-Brilliant Violet 785 (clone MEL-14), F4/80-PE (clone REA126), CD11b-PEVio615 (clone REA592) and CXCR2-PEVio770 (clone REA942). Cells were fixed with 100 μL 2% paraformaldehyde.