Figure 8.

Cytokine gene expression from monocyte-derived macrophages (MDMs) co-cultured with peripheral blood mononuclear cells (PBMCs) from naturally infected dairy cattle (clinical n=7, subclinical n=7) or noninfected controls (n=9). PBMCs isolated from whole blood were cultured 5-6 days to generate MDMs, pre-treated with vitamin D₃ as detailed in methods, then incubated 24 hrs with live MAP (10:1 MOI) ± 25(OH)D₃ or ± 1,25(OH)₂D₃. Extraction and purification of RNA was performed using Qiagen RNeasy Mini kits and was reverse transcribed with Superscript IV. Gene expression for (A) IL1B, (B) IL12A, (C) IL17A, (D) IFNG, (E) NOS2, (F) TNF, (G) DEFB7, (H) DEFB10, (I) IL10, (J) RANTES, (K) CYP24A1, and (L) CYP27B1 were determined using TaqMan assays and was normalized to the eukaryotic 18S rRNA reference gene. Data were transformed using the 2^-ΔΔCt method and are presented as the mean relative gene expression (RQ) ± SE compared to each sample’s respective non-stimulated (NS) control. Statistics were performed on ∆∆Ct values and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.