2.3. NLRP3 Inflammasome Inhibition

A total of 2 × 10⁶ PAMs (n = 3 pigs) were seeded per well/12-well plate and were maintained for 24 h at 37 °C with 5% CO₂. All treatments were carried out in duplicate. For MCC950 treatment: PAMs were treated with MCC950 (dissolved in DMSO; EMD Millipore, Burlington, MA, USA) at a final concentration of 10 μM for one hour at 37 °C prior to infection/transfection. Mock-treated groups were treated with an equal volume (50 µL/100 mL media) of DMSO. Following MCC950 treatment, PAMs were either infected with VR-2332 (M.O.I. 1), Ingelvac MLV (M.O.I. 1), transfected with 1 μg of poly I:C (polyinosinic-polycytidylic acid) (EMD Millipore) using Fugene6 (Promega, Madison, WI, USA) following the manufacturer’s protocol or mock infected/transfected. Poly I:C served as a positive control, as it is a well-known activator of the NLRP3 inflammasome [16]. At 3, 6, and 9 h post-infection (hpi)/transfection (hpt), PAMs were collected in Tri-Reagent (Sigma-Aldrich, St. Louis, MO, USA) and total RNA was purified and subjected to on-column DNase treatment using a RNA Clean and Concentrator kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. RNA quality was assessed using agarose electrophoresis. One microgram of DNase-treated total RNA per sample was reverse transcribed using a SuperScript III kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. Primers used for RT-qPCR are provided in Table S1. Primers were designed using primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and when possible, such that one of the primers spanned an exon–exon junction. Each RT-qPCR reaction contained 10 ng cDNA, 1X iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and 500 nM of each forward and reverse primer. RT-qPCR conditions were as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, then 58 °C for 20 s. All reactions were performed in duplicate. Melting curve analysis was utilized to confirm gene-specific amplification. Threshold cycle (Ct) values were normalized to the expression levels of Ribosomal protein L4 (RPL4). Relative expressions were determined using the 2^−∆∆Ct method [17]. Secreted IL1B levels in cell culture supernatants were quantified using an enzyme-linked immunosorbent assay kit (ELISA) for porcine IL1B (MyBioSource, San Diego, CA, USA), following the manufacturer’s instructions. Significant differences (p < 0.05) in expression were determined using analysis of variance SAS software (SAS Institute, Cary, NC, USA).

In a second experiment 5 × 10⁶ PAMs (n = 3 pigs) were seeded per well/6-well plate and were maintained for 24 h at 37 °C with 5% CO₂. PAMs were either treated with MCC950, at a final concentration of 10 µM, or mock-treated for 1 h at 37 °C. PAMs were then transfected with 1 μg of poly I:C (EMD Millipore) using Fugene6 (Promega, Madison, WI, USA) following the manufacturer’s protocol or mock transfected, resulting in four treatment groups: MCC950 and poly I:C treated, MCC950 only, poly I:C only, and mock-treated/transfected (negative control). At 24 hpt cell culture supernatants were collected. Pig-matched PAMs (2 × 10⁶ cells per/well/12-well plate) were then treated with 400 µL of cell culture supernatants from one of the four groups for 1 h at 37 °C and then infected with either VR-2332 (M.O.I. 1) or Ingelvac MLV (M.O.I. 1). At 6, 12, 24, and 48 h post-infection, PAMs were collected in Tri-Reagent (Sigma-Aldrich, St. Louis, MO, USA) and total RNA was purified and subjected to on-column DNase treatment using a RNA Clean and Concentrator kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. One microgram of DNase-treated total RNA per sample was reverse transcribed using a SuperScript III kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. PRRSV RNA levels were quantified using RT-qPCR as described above. Secreted IL1B levels in cell culture supernatants were quantified using an enzyme-linked immunosorbent assay kit (ELISA) for porcine IL1B (MyBioSource), following the manufacturer’s instructions. Significant differences (p < 0.05) in expression were determined using analysis of variance (SAS).