Fig 2: Secretion of cytokines by PBMCs isolated from naturally infected dairy cattle (subclinical n = 8, clinical n = 8) or non-infected controls (n = 8). Peripheral blood mononuclear cells were cultured 24 h with whole cell MAP sonicate (MPS) ± 4 ng/ml 1,25(OH)2D3. Cell culture supernatants were incubated overnight with cytokine specific magnetic beads for (A) IL-1β, (B) IL-6, (D) IL-17A, (E) IFN-γ, (F) TNF-α, (G) IL-10, (H) IL-36RA, and (I) MCP-1 supplied in the Milliplex bovine multiplex assay. Fluorescence was measured using the Luminex MAGPIX xMAP instrument and cytokine concentrations were determined by standard curve. Standard ELISA was used to measure (C) IL-12A concentrations. Data are presented as the mean ± SE and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.

Fig 3: 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-??Ct 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.

Fig 4: Cytokine 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. Protein concentrations were determined from cell culture supernatants using Milliplex magnetic bead assay for (A) IL-1ß, (B) IL-6, (D) IL-17A, (E) IFN-?, (F) TNF-a, (G) IL-10, (H) IL-36RA, (I) MCP-1, while (C) IL-12A was measured using standard ELISA. Data are presented as the mean ± SE and significance levels are defined as * < 0.05, ** < 0.01, *** < 0.001.

Fig 5: Cytokine gene expression from PBMCs isolated from naturally infected dairy cattle (subclinical n = 8 and clinical n = 8) or noninfected controls (n = 8). Cells were cultured 24 hrs with culture media or whole cell MAP sonicate (MPS) ± 100 ng/ml 25(OH)D3. 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) CCL5, (K) CYP24A1, and (L) CYP27B1 were determined using TaqMan assays and were normalized to eukaryotic 18S rRNA reference gene. Data were analyzed using the 2-??Ct 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.