All animal experiments were performed in accordance with protocols approved by the Institute of Animal Care and Use Committee of the Hokkaido University Graduate School of Medicine. C57BL/6 mice were obtained from Japan SLC, Inc. (Shizuoka, Japan) and used as wild-type (WT) mice. GM3S null mice were generated as previously described (23). This type of null mouse was backcrossed with C57BL/6 mice for >11 generations. The GM3S genotypes of the mice were analyzed via PCR as described previously (23).

Peritoneal macrophages were isolated from six- to eight-week-old C57BL/6 mice (24). Briefly, one milliliter of 4% Brewer thioglycolate medium was injected into the peritoneal cavity, allowing the inflammatory response to proceed for four days. Then, the cells were harvested by injecting 10 mL of ice-cold PBS into this cavity and collecting the fluid from the peritoneum. Thereafter, the cells were washed three times and freshly used in further experiments.

Primary articular chondrocytes were isolated from five-day-old C57BL/6 N mice via collagenase D according to a standard protocol (22, 25). In brief, we isolated cartilage from the femoral condyles and tibial plateau. We took care to remove the hypertrophic zone from the collected tissues. Then, we treated the tissues with collagenase D in DMEM at 37°C overnight. The isolated cells were seeded into dishes or flasks. Primary chondrocytes were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich), 2 mM L-glutamine, and 25 mg/L penicillin/streptomycin. The cells were used between passages 0 and 3.

Murine peritoneal macrophages were seeded on 24-well plates or 48-well plates at a ratio of 2×10⁵. After three hours, the medium was changed to remove the nonadherent cells, and the cells were incubated overnight at 37°C and 5% CO₂. Mouse cultured cells were prepared as described above and resuspended at 4.0×10⁵ cells/ml. Then, 500 µl of the cell suspension was placed directly upon the monolayer of the macrophages and incubated at 37°C for 48 hours. Thereafter, the cells and supernatants were harvested for further analyses. All experiments were repeated at least two times to obtain reproducible data.

Murine macrophages and cultured cells were prepared in the same manner as those used for direct coculture. The cultured cells were seeded (2 × 10⁵ cells) onto transwell insert cell cultures (Falcon cell culture inserts, BD, Franklin Lakes, NJ, USA) and placed on a 24-well plate of macrophage monolayer cultures. After 48 hours of incubation, the cells and supernatants were harvested for further analyses.

The concentrations of IL-6 in the culture supernatants were measured via mouse IL-6 DuoSet ELISA (R&D Systems, Inc., USA) according to the manufacturer’s instructions.

NO in the culture supernatant was quantified on the basis of the amount of nitrite, the product generated upon degradation of NO (26). A Griess reagent system was used for this assay according to the manufacturer’s recommendations (Promega, Tokyo, Japan).

Isolated murine macrophages and primary or passaged chondrocytes were seeded onto a 15 mm micro cover glass (MASATANI, Japan) and cultured in a 24-well plate for six hours. The cells were fixed with 10% formalin (Wako, Japan), treated with 0.1% Triton X-100 for 3 min, and then incubated with 5% FBS (Sigma)-PBS (blocking buffer) for one hour at RT. The cells were incubated with primary antibodies against murine F4/80 and IL-6 (BioLegend) at 1:200 for one hour at 37°C. The bound antibody was detected by a specific secondary antibody conjugated with Alexa Fluor®594 (Jackson ImmunoResearch, West Grove, PA, USA). The cell nuclei were stained with DAPI (Dojindo Molecular Technologies, Kumamoto, Japan). Imaging was performed by using a Keyence all-in-one microscope (Itasca, IL 60143, USA).

Total RNA was extracted from each sample via TRIzol reagent (Thermo Fisher Scientific). For the synthesis of cDNA, reverse transcription was performed via the GoScript TM reverse transcriptase kit (Pro-mega, Madison, USA). The cDNA samples were subjected to quantitative real-time PCR via TB Green^® Premix Ex Taq™ II (Takara, Japan) on a Thermal Cycler Dice Real-Time System II (model TP900; Takara Bio, Shiga, Japan) with gene-specific primers ( Supplementary Table 1 ). The relative expression of each targeted gene to the expression of GAPDH was calculated via the 2^-ΔΔCt method.

Cultured chondrocytes (> 1 × 10⁶ cells) were washed five times with PBS and collected via a scraper (Sumilon, Japan). The collected cells were resuspended in 100 mL of PBS and homogenized via an ultrasonic homogenizer (TAITEC, Saitama, Japan). The cell lysates were precipitated with EtOH, and subsequently, the proteinous pellet and supernatant fractions were separated via centrifugation (22, 27, 28). The resulting pellet was dissolved in H₂O, and the cellular protein concentration was measured via a BCA protein assay kit (Thermo Fisher Scientific). The pellet fractions corresponding to 25 μg and 50 μg of protein were used for N-glycan and O-glycan analyses, respectively. The supernatant fraction corresponding to 100 μg of protein was concentrated for GSL analysis. Glycomic analyses of N-glycans and GSLs were performed by glycoblotting methods combined with the SALSA procedure (29, 30). O-glycome analysis was performed via β-elimination in the presence of pyrazolone analogs (BEP) as previously described (31). This methodology allows a comparative analysis of glycomes.

The data were statistically analyzed via an unpaired two-tailed Student’s t test and one-way analysis of variance followed by the Kruskal−Wallis test followed by Dunn’s multiple comparison test via GraphPad software (GraphPad Software, La Jolla, CA, USA). Differences between groups with a P value less than 0.05 were considered statistically significant. The results are presented as the means ± standard deviations (SDs).

Direct coculture of macrophages with passaged chondrocytes increased the production of proinflammatory cytokines, including IL-6 and NO, as the number of passages increased ( Figures 1A, B ). However, indirect coculture did not activate macrophages ( Supplementary Figure 1 ). NO levels were below the datable limits of the kit used. In cocultured macrophages with passaged chondrocytes, IL-6 was predominantly detected in F4/80 macrophages ( Figure 1C ).

To determine whether gangliosides influence immunological behavior cocultured macrophages with passaged chondrocytes, we established mice with a disrupted gene (ST3Gal-5) encoding GM3 synthase (GM3S) (23), an a2,3 sialyltransferase that transfers a sialic acid residue to lactosylceramide to yield GM3 ganglioside ( Figure 4A ). We performed genotyping PCR using Yamashita’s primer sets ( Supplementary Figure 2 ). The expression level of the GM3S genes was assessed at the mRNA level in primary chondrocytes via qRT−PCR, which revealed efficient knockout of the target ST3Gal-4 gene in the cells ( Figure 4B ). Coculturing passaged GM3S knockout chondrocytes with macrophages significantly suppressed IL-6 expression ( Figure 4C ), indicating the reduction of macrophages activation.