Figure 4.

Macrophage expression of activation markers (A) CD40, (B) MHCII, and pan-macrophage marker (C) CD68. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of dairy cattle naturally infected with Mycobacterium avium subsp. paratuberculosis (MAP) (clinical n=7, subclinical n=8) or noninfected controls (n=6). Cells were cultured 5-6 days to generate monocyte-derived macrophages (MDMs), pre-treated with vitamin D₃ as detailed in methods, then incubated 24 hrs with live MAP ± 25(OH)D₃ or ± 1,25(OH)₂D₃. The mean fluorescence intensity (MFI) for each target was measured using marker specific primary antibodies coupled with an Alexa Fluor labeled secondary antibody (MHCII[AF488]; CD68[AF594]), or biotin labeled secondary and NeutrAvidin-DyLight 650 tertiary antibody (CD40). Data are presented as the MFI ± SE and significance levels are as follows: *< 0.05, **< 0.01, ***< 0.001.