Effects of 1,25-Dihydroxyvitamin D3 and 25-Hydroxyvitamin D3 on PBMCs From Dairy Cattle Naturally Infected With Mycobacterium avium subsp. paratuberculosis

Abstract

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.

Keywords: Johne's disease; Mycobacterium avium subsp. paratuberculosis; PBMC; cattle; immune responses; vitamin D.

Figure 1

Serum 25-hydroxyvitamin D₃ concentrations (ng/ml) for dairy cattle grouped by different stages of infection with Mycobacterium avium ssp. paratuberculosis for two separate experiments including either (A) 1,25(OH)₂D₃ or (B) 25(OH)D₃ treatment. Each infection status group is comprised of n = 8. Whole blood was collected via jugular venipuncture into serum separation vacutainer tubes, allowed to clot, and centrifuged to collect serum. Data are presented as the mean ± SE and significance levels are as follows: * < 0.05, ** < 0.01.

Figure 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.

Figure 3

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) ± 100 ng/ml 25(OH)D₃. 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.

Figure 4

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) ± 4 ng/ml 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) 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^−ΔΔCt 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 ΔΔCt values and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.

Figure 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^−ΔΔCt 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 ΔΔCt values and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.

Figure 6

Expression of activation and memory markers on the surface of (A) CD4+, (B) CD8+, (C) γδ T cells, (D) monocytes, and total B cells. Peripheral blood mononuclear cells were isolated from naturally infected dairy cattle (subclinical n = 8, clinical n = 8) or non-infected controls (n = 8) and cultured 144 h with whole cell MAP sonicate (MPS) ± 4 ng/ml 1,25(OH)₂D₃. Surface markers were detected using fluorescently labeled antibodies and a flow cytometer. Data are presented as the mean percentage ± SE and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.

Figure 7

Expression of activation and memory markers on the surface of (A) CD4+, (B) CD8+, (C) γδ T cells, (D) monocytes, and total B cells. Peripheral blood mononuclear cells were isolated from naturally infected dairy cattle (subclinical n = 8, clinical n = 8) or non-infected controls (n = 8) and cultured 144 h with whole cell MAP sonicate (MPS) ± 100 ng/ml 25(OH)D₃. Surface markers were detected using fluorescently labeled antibodies and a flow cytometer. Data are presented as the mean percentage ± SE and significance levels are as follows: * < 0.05, ** < 0.01, *** < 0.001.