Hyaluronan

During the first medical assessment, a venous blood sample was collected in a heparinized tube. The blood was centrifuged at 3500×g for 15 min at 18 °C. The plasma was separated into 0.5 ml aliquots and stored in a freezer at − 70 °C. Biomarkers of EG shedding were measured through commercial ELISA kits (enzyme-linked immunosorbent assay): hyaluronan (R&D, Minneapolis, MN, USA), syndecan-1 (Abcam, Cambridge, MA, USA), and biomarker of endothelial cell injury: soluble thrombomodulin (R&D, Minneapolis, MN, USA); inflammatory cytokines were measured through commercial ELISA kits: tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin 1-beta (IL-1β) (both through R&D, Minneapolis, MN, USA), and quantification of immunoglobulin G and immunoglobulin M for influenza A (IBL America, Minneapolis, MN, USA). In patients with less than 5 days after the onset of symptoms, a nasopharyngeal swab was collected for direct immunofluorescence for respiratory viruses through the D3 Ultra DFA Respiratory Virus Screening & ID Kit (Quidel Corporation, San Diego, CA, USA). For the positive results on direct immunofluorescence, a real-time polymerase chain reaction (RT-PCR) for influenza A was conducted according to the US Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA) protocol [9 ].

Proinflammatory cytokine, hyaluronan, and TWEAK levels in the culture supernatant of confluent orbital fibroblasts or in serum were determined using commercially available ELISA kits (IL-6, IL-8, MCP-1, and hyaluronan: R&D Systems; TWEAK: Bender Medsystems, Vienna, Austria) according to the manufacturers’ instructions. To investigate whether TWEAK-induced cytokine production is dependent on Fn14, cells were incubated with the Fn14-specific mAb ITEM4 (2.5 μg/ml) prior to stimulation. For comparison, GO cells were exposed to the signaling pathway inhibitors PD98059, SP600125, SB203580, and LY294002 (20 μM) and SC514 (10 μM) for 1 h prior to stimulation with TWEAK. The production of hyaluronan induced by TWEAK was analyzed in the same manner. hyaluronan concentration in the sample was determined from a standard binding curve generated with known amounts of hyaluronan. Samples were diluted 1:10 before analysis, and the mean value of triplicate samples is reported.
To analyze serum level of TWEAK, blood samples were drawn into test tubes containing 10% (v/v) sodium citrate. Platelet-free plasma was obtained by centrifugation at 3000 × g for 15 min at room temperature and stored at −80°C until analysis. Serum level of TWEAK protein (pg/ml) was measured using a commercial ELISA kit. Each sample was tested three times. All serum samples were tested in the same assay.

GO orbital fibroblasts were plated at 1 × 10⁵ cells/well in 12-well plates in DMEM containing 1% FBS and antibiotics overnight. The effect of HKMTs inhibitors for both prophylactic and treatment settings were investigated as described above. Supernatant was collected and hyaluronan (Cat# DY3614, R&D system Inc., USA), IL-6 (Cat# 430504, RRID:AB_2935703) and IL-8 (Cat# 431504, RRID:AB_2935704) (Biolegend Inc., USA) levels were measured by ELISA according to the manufacturer’s protocol. Percentages of ECM production and pro-inflammatory cytokine by orbital fibroblasts were calculated by the following formula: (test sample/unstimulated condition) *100.

CVOCA tissue lysates and culture supernatants were analyzed using ELISAs for the presence of ECM proteins. CVOCAs were homogenized and the resulting tissue lysates were analyzed using ELISA kits for type II collagen (Astarte Biologics, Bothell, WA, USA) and hyaluronan (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s protocols. CVOCAs were cultured with chondrocyte growth culture media (Lonza, Basel, Switzerland) at 37 °C and 5 % CO₂. Media collected after 1, 3, 7, and 14 days was analyzed using an aggrecan ELISA kit (DIAsource ImmunoAssays, Louvain-la-Neuve, Belgium) following the manufacturer’s protocol.