Collagenase

Embryonic hearts were collected under a stereomicroscope, and the 3 anatomic heart structures (At, GV-AVJ, and Vt) were microdissected. Heart tissue was minced into 1 mm³ fragments and incubated for 15 minutes at 37°C in the enzymatic solution: for E 13.5 and E 17.5 hearts, 0.2 mg/mL collagenase (Sigma-Aldrich) in Hank's Balanced Salt Solution with calcium and magnesium (HBSS^+/+, Invitrogen); for E 9.5 hearts, 0.1 mg/mL collagenase in HBSS^+/+; for postnatal hearts 20 mM 2,3-Butanedione monoxime (BDM; Sigma-Aldrich) was added in all isolation steps to DPBS without Ca²⁺ and Mg²⁺(DPBS^−/−, Invitrogen); and for adult hearts, 0.2 mg/mL collagenase with 20 mM BDM (Sigma-Aldrich) and with 60 U/mL DNase I (Roche, Switzerland). At the end of each round of digestion, tissue fragments were resuspended using a P1000 pipette (approximately 20 times). The remaining tissue was allowed to sediment, and the supernatant was collected in a tube containing the same volume of 10% FCS (Life Technologies)-HBSS^−/− and kept on ice while the digestion protocol continued. Digestion was repeated until no macroscopic tissue was detected. After digestion, cell suspensions were centrifuged 10 minutes, 290g at 4°C, resuspended in 1% FCS HBSS^−/−, and filtered with a 70 μm mesh strainer (Fisher Scientific).

To prepare single cells for RNA expression analysis, draining LN, mesenteric LN and spleen were digested with 200 U ml⁻¹ collagenase (Sigma) for 30 min. Joints were digested with 200 Uml⁻¹ collagenase (Sigma) and 2.4 mgml⁻¹ hyaluronidase (Sigma). Next, these cells were filtered through a cell strainer with a 70-μm nylon mesh (Becton Dickinson). RNA from joint-, dLN-, mLN- and spleen-derived single cells and RNA from cultured cells were extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma). Tissue total RNA from joint, draining LN, spleen, thymus, colon, lung and liver was extracted using the Sepasol reagent (Nacalai Tesque, Kyoto, Japan). All RNA were denatured in the presence of an oligo dT primer and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was performed with a SYBR Green qPCR kit (Invitrogen, Carlsbad, CA, USA) and an iCycler system (Bio-Rad, Hercules, CA, USA) with the sets of primers described in Supplementary Table 3.

Primary ATII-LCs were isolated as described by Driscoll B. et al. 2012 [22 (link)]. Briefly, mice were euthanized and left kidney was excised. Lungs were perfused with 5 ml of PBS through the right ventricle of the heart. One ml collagenase (Sigma-Aldrich) was instilled into the lungs via tracheal catheter followed by 0.5 ml 1% low-melt agarose (Sigma-Aldrich) warmed to 45°C. Lungs were immediately covered with ice for 2 minutes. Lungs were then dissected, placed in a culture tube containing 1 ml of collagenase (Sigma-Aldrich), and incubated for 45 minutes at room temperature. Lung tissue was teased apart, transferred to a culture dish containing 7 ml of DMEM and 100 μl of DNase (Sigma-Aldrich) and then filtered through 100-, 40- and 25 μm nylon meshes. Single cells were stained with a mixture of APC labelled anti-mouse antibodies (CD11b, CD11c, CD16/32, CD19, CD31, CD45, and F4/80). ATII-LCs were isolated by negative selection with anti-APC coated magnetic beads using the Magnetic Activated Cell Sorting (MACS) system (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.

The AMPs targeted antimicrobial activity was investigated in co-cultures by using 3D spheroids and S. aureus as a reference strain for articular cartilage infections as we have previously shown (Bonifacio et al., 2018 (link), 2020 (link)). Briefly, spheroids were allowed to equilibrate for 48 h in the complete medium; then, the medium was replaced by 1 ml of the antibiotic-free DMEM containing 1 × 10⁵ bacteria. Spheroids and bacteria were cultivated in direct contact for 48 h and then the numbers of viable cells and bacteria were determined. Specimens were collected into 15 ml tubes and enzymatically disaggregated by 1 ml of a collagenase/trypsin solution (1 mg/ml collagenase, 0.25% trypsin in PBS, all from Sigma-Aldrich) for 10 min at 37°C. Then, the tubes were vortexed for 3 times 30 s to favor cells separation and the trypan blue assay and the CFU count were used to evaluate the number of viable cells and bacteria, respectively. The experiment was carried using three different samples.

The enzymatic degradation of GelMA hydrogels was evaluated using collagenase, as reported elsewhere [48 (link)]. Briefly, the GelMA solution (200 μL) was transferred to a PDMS mold (d = 6 mm and th = 3 mm) and polymerized to yield disc-shaped constructs. The discs were then equilibrated overnight in PBS solution. They were then soaked in 5 mL of 0.1 M Tris–HCl buffer (pH 7.4) containing 5 mM CaCl₂ for 1 h to reabsorb the water. Subsequently, collagenase (Sigma-Aldrich, USA) solution was added to give a final concentration of 1 mg/mL. The collagenase solution was changed every 8 h, and the residue was carefully removed from the solution, gently blotted on filter paper, and weighed. The degradation rate (n = 3) was calculated using the following equation: Lost Weight (%) = 100 (Wt − Wi/Wi × 100, where Wt is the residual wet weight at a specific time point and Wi is the initial wet weight.