Search

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 D 3 (25(OH)D 3 ) and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) enhances bovine macrophage function. The present study aimed to investigate the role of vitamin D 3 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)D 3 or 4 ng/ml 1,25(OH) 2 D 3 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 D 3 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 D 3 reduced Rab5 expression in infected MDMs from JD- control cows, while 1,25(OH) 2 D 3 decreased Rab7 expression in JD- and JD+ subclinical animals regardless of MAP infection in vitro . Vitamin D 3 promoted phagocytosis in MDMs from JD- and JD+ clinical cows treated with either vitamin D 3 analog. Results from this study show exogenous vitamin D 3 influences macrophage M1/M2 polarization and Rab GTPase expression within MDM culture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wherry, Dassanayake, Bannantine, Mooyottu and Stabel.)

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of ruminant enteritis, targets intestinal macrophages. During infection, macrophages contribute to mucosal inflammation and development of granulomas in the small intestine which worsens as disease progression occurs. Vitamin D 3 is an immunomodulatory steroid hormone with beneficial roles in host-pathogen interactions. Few studies have investigated immunologic roles of 25-hydroxyvitamin D 3 (25(OH)D 3 ) and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) in cattle, particularly cattle infected with MAP. This study examined the effects of exogenous vitamin D 3 on immune responses of monocyte derived macrophages (MDMs) isolated from dairy cattle naturally infected with MAP. MDMs were pre-treated with ± 100 ng/ml 25(OH)D 3 or ± 4 ng/ml 1,25(OH) 2 D 3 , then incubated 24 hrs with live MAP in the presence of their respective pre-treatment concentrations. Following treatment with either vitamin D 3 analog, phagocytosis of MAP by MDMs was significantly greater in clinically infected animals, with a greater amount of live and dead bacteria. Clinical cows had significantly less CD40 surface expression on MDMs compared to subclinical cows and noninfected controls. 1,25(OH) 2 D 3 also significantly increased nitrite production in MAP infected cows. 1,25(OH) 2 D 3 treatment played a key role in upregulating secretion of pro-inflammatory cytokines IL-1β and IL-12 while downregulating IL-10, IL-6, and IFN-γ. 1,25(OH) 2 D 3 also negatively regulated transcripts of CYP24A1 , CYP27B1 , DEFB7 , NOS2 , and IL10 . Results from this study demonstrate that vitamin D 3 compounds, but mainly 1,25(OH) 2 D 3 , modulate both pro- and anti-inflammatory immune responses in dairy cattle infected with MAP, impacting the bacterial viability within the macrophage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wherry, Dassanayake, Casas, Mooyottu, Bannantine and Stabel.)