Vitamin D3 alters macrophage phenotype and endosomal trafficking markers in dairy cattle naturally infected with Mycobacterium avium subsp. paratuberculosis

Macrophages are important host defense cells in ruminant paratuberculosis (Johne’s Disease; JD), a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). Classical macrophage functions of pathogen trafficking, degradation, and antigen presentation are interrupted in mycobacterial infection. Immunologic stimulation by 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D3 on macrophage phenotype and endosomal trafficking of MAP in monocyte-derived macrophages (MDMs) cultured from JD-, JD+ subclinical, and JD+ clinically infected cattle. MDMs were pre-treated 100 ng/ml 25(OH)D3 or 4 ng/ml 1,25(OH)2D3 and incubated 24 hrs with MAP at 10:1 multiplicity of infection (MOI). In vitro MAP infection upregulated pro-inflammatory (M1) CD80 and downregulated resolution/repair (M2) CD163. Vitamin D3 generally decreased CD80 and increased CD163 expression. Furthermore, early endosomal marker Rab5 was upregulated 140× across all stages of paratuberculosis infection following in vitro MAP infection; however, Rab5 was reduced in MAP-activated MDMs from JD+ subclinical and JD+ clinical cows compared to healthy controls. Rab7 expression decreased in control and clinical cows following MDM infection with MAP. Both forms of vitamin D3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH)2D3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro. Vitamin D3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D3 analog. Results from this study show exogenous vitamin D3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture.