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IntroductionMacrophages are the preferential target of Mycobacterium avium subsp. paratuberculosis (MAP), the etiologic agent of ruminant paratuberculosis. Uptake of pathogens by intestinal macrophages results in their trafficking through endosomal compartments, ultimately leading to fusion with an acidic lysosome to destroy the pathogen. MAP possesses virulence factors which disrupt these endosomal pathways. Additionally, levels of serum vitamin D3 have proven relevant to host immunity. Dynamics of endosomal trafficking and vitamin D3 metabolism have been largely unexplored in bovine paratuberculosis.MethodsThis study aimed to characterize expression of early and late endosomal markers Rab5 and Rab7, respectively, within CD68+ macrophages in frozen mid-ileum sections harvested from cows at different stages of natural paratuberculosis infection. Additionally, factors of vitamin D3 signaling and metabolism were characterized through expression of vitamin D3 activating enzyme 1α-hydroxylase (CYP27B1), vitamin D3 inactivating enzyme 24-hydroxylase (CYP24A1), and vitamin D3 receptor (VDR) within CD68+ ileal macrophages.Results and discussionCows with clinical paratuberculosis had significantly greater macrophage and MAP burden overall, as well as intracellular MAP. Total expression of Rab5 within macrophages was reduced in clinical cows; however, Rab5 and MAP colocalization was significantly greater in this group. Intracellular Rab7 colocalization with MAP was not detected in subclinical or Johne's Disease negative (JD-) control cows but was present in clinical cows. Additionally, macrophage CYP27B1 expression was significantly reduced in clinical cows. Taken together, the results from this study show disparate patterns of expression for key mediators in intracellular MAP trafficking and vitamin D metabolism for cows at different stages of 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.

The role of vitamin D3 in modulating immune responses has been well-established for over two decades; however, its specific functions have not been extensively detailed in cattle, particularly cattle in different stages of infection with Mycobacterium avium subspecies paratuberculosis (MAP). Consistent with previous work in our lab, the present study showed that infected cattle in the clinical stage of disease have reduced serum 25-hydroxyvitamin D3 [25(OH)D3]. Additionally, effects of vitamin D3 on peripheral blood mononuclear cells (PBMCs) from naturally infected dairy cattle in subclinical (n = 8) or clinical (n = 8) stages of infection were compared to non-infected control cows (n = 8). Briefly, PBMCs were isolated and cultured in vitro with 4 ng/ml 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or 100 ng/ml 25(OH)D3. Treatment with 1,25(OH)2D3 resulted in decreased secretion for some pro-inflammatory cytokines in clinical animals, including IL-1β, IL-6, and IFN-γ. Similar responses for IL-1β and IL-6 were noted with the addition of 25(OH)D3. Additionally, pro-inflammatory cytokine gene expression tended to be upregulated in PBMCs from clinical animals after treatment with 1,25(OH)2D3. In contrast, PBMCs from clinical animals treated with 25(OH)D3 showed downregulation of pro-inflammatory cytokine gene expression, although only significant for IL1B. Following 25(OH)D3 treatment, clinical animals showed significant reduction in CD4+CD25+ T cells. CYP27B1 gene expression was notably decreased in clinical and control animals following 25(OH)D3 treatment but increased in subclinical cows. 1,25(OH)2D3 treatment reduced CYP24A1 gene expression in all groups, while 25(OH)D3 treatment only significantly reduced expression for control cows. Lastly, serum 25(OH)D3 levels were significantly lower in clinical animals. Taken together, these data show vitamin D3 modulates cytokine signaling in cattle at different stages of MAP infection and, therefore, may have implications on disease progression.

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 D3 is an immunomodulatory steroid hormone with beneficial roles in host-pathogen interactions. Few studies have investigated immunologic roles of 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in cattle, particularly cattle infected with MAP. This study examined the effects of exogenous vitamin D3 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)D3 or ± 4 ng/ml 1,25(OH)2D3, then incubated 24 hrs with live MAP in the presence of their respective pre-treatment concentrations. Following treatment with either vitamin D3 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)2D3 also significantly increased nitrite production in MAP infected cows. 1,25(OH)2D3 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)2D3 also negatively regulated transcripts of CYP24A1, CYP27B1, DEFB7, NOS2, and IL10. Results from this study demonstrate that vitamin D3 compounds, but mainly 1,25(OH)2D3, modulate both pro- and anti-inflammatory immune responses in dairy cattle infected with MAP, impacting the bacterial viability within the macrophage.

Mycobacterium avium subspecies paratuberculosis (MAP) is an environmentally hardy pathogen of ruminants that plagues the dairy industry. Hallmark clinical symptoms include granulomatous enteritis, watery diarrhea, and significant loss of body condition. Transition from subclinical to clinical infection is a dynamic process led by MAP which resides in host macrophages. Clinical stage disease is accompanied by dysfunctional immune responses and a reduction in circulating vitamin D3. The immunomodulatory role of vitamin D3 in infectious disease has been well established in humans, particularly in Mycobacterium tuberculosis infection. However, significant species differences exist between the immune system of humans and bovines, including effects induced by vitamin D3. This fact highlights the need for continued study of the relationship between vitamin D3 and bovine immunity, especially during different stages of paratuberculosis.