Recombinant mouse il 4

16 Alzet, model 2006 miniature osmotic pumps (Durect Corporation, Cupertino, CA) were loaded with mouse recombinant IL-4 (R&D Systems, Minneapolis, MN) at a concentration of 2 µg/ml diluted in carrier solution comprising of 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) and phosphate buffered saline (PBS). 5 additional pumps were loaded with IL-4 with carrier solution and 15 mg/ml UHMWPE particles (mean diameter 1µm ± 0.1 µm as assessed by electron microscopy) isolated from knee replacement simulators as previously described [22 (link)]. The particles were tested negative for endotoxin using Limulus Amebocyte Lysate Kit (BioWhittaker, Walkersville, MD). Pumps were connected via 6cm long vinyl tubing (Durect Corporation), pre-filled with their corresponding solutions, to a collection vessel containing 500µl of a mouse bone marrow macrophage (mBMM) augmented media. The whole assembly was placed inside 50ml centrifuge tube partially filled with PBS, and then incubated at +37°C for 4 weeks. The conditioned mBMM media was initially collected at day 3, 5 and 7 after the beginning of the infusion and then at seven day intervals (week 1, 2, 3 and 4 samples) with fresh media added to the collection vessel after each sample collection. Conditioned media was stored at −80°C until used for subsequent experiments.

Conventional UHMWPE particles were generated in total knee replacement simulator and isolated from the test supernatants following previously established protocol.^{16 (link),17 (link),19 (link)} Particles were then washed twice in ethanol (96 and 70%), resuspended in phosphate buffered saline (PBS), and stored in −80°C until used. Electron microscopy showed that most of the particles were spherical with a mean diameter of 0.48 ± 0.10 μm (range 0.26–0.81 μm). Particles did not contain a significant endotoxin contamination as tested with Limulus Amebocyte Lysate Kit (BioWhittaker, Walkersville, MD). Alzet osmotic pumps (Model 2006, Durect Corporation, Cupertino, CA) were loaded either with (a) carrier solution (1% BSA-PBS); (b) carrier solution with 15 mg/ml UHMWPE particles; or (c) carrier solution with UHMWPE particles and 10 μg/ml mouse recombinant IL-4 (R&D Systems, Minneapolis, MN). Six-centimeter long vinyl tubing (Durect Corporation) was prefilled with the appropriate solution, connected to the pumps, and subsequently used to connect the pumps to hollow A-40 titanium rods (6 mm long, 21 g, New England Small Tube, Litchfield, NH).

Mouse splenic B cells were purified from Slfn5^−/− mice (10–12-week-old) and age- and sex-matched littermate controls using an EasySep Mouse B Cell Isolation Kit (Stemcell Tech) according to the manufacturer’s instructions. Purified B cells were cultured in RPMI 1640 supplemented with 10% FBS and 50 μM β-mercaptoethanol. 2 × 10⁶ cells were stimulated with 10 μg mL⁻¹ LPS (Sigma) or 10 μg mL⁻¹ LPS plus 10 ng mL⁻¹ mouse recombinant IL-4 (R&D systems) for 96 h. Stimulated B cells were analyzed on a CytoFLEX flow cytometry (Beckman) and processed using FlowJo.

Naïve B cells were MACS-purified by negative selection from splenocytes of WT, Dnase1l3^−/−, Ifnar1^−/− or Dnase1l3^−/−Ifnar1^−/− mice using CD43 microbeads. Purified B cells were labelled with 3 μM Cell trace CFSE (Invitrogen), in PBS+ 2% BSA for 10 mins at RT. CFSE labelled cells were washed twice with PBS and subsequently suspended in RPMI + 10% FBS + 2mM L-glutamine + 10μM β-Mercaptoethanol and plated in round bottom 96 well plates at a concentration of 0.5×10⁶ cells/ well in 200μl media. Cells were either left unactivated or activated with: (1) 5 μg/ml anti-IgM (Jackson Immunoresearch) + anti-CD40 (LEAF-purified, BioLegend) and 5 ng/ml recombinant mouse IL-4 (R&D systems) OR (2) 1μg/ml LPS (LPS-EK Ultrapure, InvivoGen) + 5 ng/ml recombinant mouse IL-4. Unactivated (UA) and activated (A) B cells were supplemented or not with recombinant mouse IFNα1 (BioLegend) at indicated concentrations. B cells were analyzed for activation, proliferation and differentiation after 3 days of culture.