Recombinant mouse tnfα

Bone marrow cells were collected from the tibia and femur of WT or Mincle^-/- mice and treated with red blood cell lysis buffer (Sigma Aldrich). MΦ were generated by incubating bone marrow cells at 37°C with 40 ng/ml M-CSF (Biolegend, San Diego, CA) in complete RPMI 1640 medium (Gibco), as described before [18 (link)]. Cell medium was replenished at day 3, and cells were collected at day 7. After collection and quantification, 5 x 10⁵ viable cells were seeded into 12- or 24-well plates and allowed to adhere overnight prior to infection. For TNFα-treated MΦs, recombinant mouse TNFα (Biolegend) was added 30 min prior to infection at a final concentration of 25 ng/mL [29 (link)]. Bacteria were added at a multiplicity of infection (MOI) of 2, 5, or 10 and centrifuged at 2,000 RPM for 5 min to synchronize infection. For experiments utilizing heat-killed bacteria, bacterial stocks were incubated at 56°C for 30 min [30 (link)] and used at a MOI equivalent of 10.

MC-38 cells were dissociated with 2 mM EDTA in PBS (pH 7.4) and resuspended in DMEM supplemented with 5% FBS. In total, 1.5 x 10⁴ cells in 100 µl of the medium were seeded per well of 96-well plate followed by incubation for 3 h at 37°C to allow cells to adhere to the well surface. Afterward, 50 µl of cell medium in every well were replaced with 50 µl of DMEM containing 2x concentrations of either lectin, in the presence or absence of cycloheximide (Santa Cruz Biotechnology), or corresponding controls followed by incubation at 37°C for 20 h. Final concentrations of MAL I, AAL, WGA were 50 µg/ml, 60 µg/ml, and 4 µg/ml, respectively. cycloheximide was used at 2.5 µg/ml final concentration. An equivalent amount of lectin resuspension buffer was used as a negative control, recombinant mouse TNF-α (BioLegend) at 20 ng/ml and 80 µM cisplatin (Santa Cruz Biotechnology) were used as positive controls for apoptotic cell death. Cell viability was assessed in a fluorescence-based assay using double staining with Hoechst 33342 (Thermo Fischer Scientific) and propidium iodide (Stemcell Technologies). In experiments, in which cycloheximide was used, cytotoxicity assay based on lactate dehydrogenase (LDH) release was performed instead. All lectins were from Vector Laboratories (Burlingame, California, USA).

The BioFlux 200 48-well plate 0-20 dyn (Fluxion, USA) microcapillaries were coated with 1% gelatin (ThermoFisher, USA) and 25 μg/ml human fibronectin (ThermoFisher, USA) solution in growth medium for 24 h at 37°C. Single cell suspension of MVECs (2.5 × 10⁶/ml) in growth medium was loaded into capillaries using the BioFlux 200 system (Fluxion, USA), and cells were cultured inside the microcapillaries until the formation of a confluent monolayer. Cells were stimulated with either 5 ng/ml recombinant mouse TNF-α (BioLegend), T cell-derived exosomes, or a combination of both for 16 hours. For the adhesion assay, single cell suspension of freshly isolated mouse splenocytes was labelled with the CellTrace CFSE (ThermoFisher, USA) and used at concentration of 3 × 10⁵ cells/ml. The flow of splenocytes at 1 dyn/cm³ through the microcapillaries coated with cMVECs was induced for 30 minutes, and adhesion of CFSE-labeled splenocytes was analyzed with a fluorescence microscope (LS720, Etaluma, USA).

All animal care and use were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NRC) and experimental protocols were approved by the Institutional Ethical Committee on Animal Experimentation, according to applicable regional law. Male NMRI mice were purchased from Charles River Laboratories (France), male wild-type FVB mice were purchased from Janvier (France), and male transgenic Gfap-luc mice (FVB/N-Tg(Gfap-luc)-Xen) were obtained from Taconic Laboratories (USA). The latter animals express luciferase under the transcriptional control of the glial fibrillary acidic protein (Gfap) promoter [32 (link)] and are commonly used as a model system for noninvasive quantification of astrocyte activation in living animals over time [27 (link), 33 (link), 34 (link)]. Unless mentioned otherwise, animals were housed in groups of 4 per cage under a normal 12:12 h light-dark cycle (lights on at 06:00 a.m. with a 30 min dim and rise phase). Food and water were available ad libitum.
Recombinant mouse TNF-α was purchased from Biolegend (product ID 575208) and dissolved in sterile phosphate buffered saline prior to injection.