Total RNA was isolated from PLF WBCs using a commercially available kit, RNeasy Plus Mini Kit, supplied by Qiagen (Valencia, CA). Total RNA was quantified using a NanoDrop 2000 apparatus (ThermoFisher Scientific, Waltham, MA). Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the high capacity RNA to cDNA kit supplied by Applied Biosystems Quantitative polymerase chain reaction (qPCR) and performed using TaqMan® probe-based gene expression assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA). Individual TaqMan gene expression assays were selected for proinflammatory and profibrotic cytokines and cytokine receptors (IL-1ß, IL-6, TNFα, HMGB1, TGFß1, TNFαR1 and TGFßR1), for inducible nitric oxide synthase and for relevant cytoprotective and antioxidant enzymes [heme oxygenase-1 (HO-1), NADPH:quinone oxidoreductase-1 (Nqo1) and glutathione s-transferase mu 1 (Gstm1)]. Quantitative real-time PCR was performed using 25ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Gene expression data were normalized to 18S ribosomal RNA housekeeping gene and calibrated to the control samples according to the ΔΔCT method as described previously (15 (link)).