Modulation of Mycobacterium bovis-specific responses of bovine peripheral blood mononuclear cells by 1,25-dihydroxyvitamin D(3)

Abstract

Historically, administration of vitamin D has been considered beneficial in the treatment of tuberculosis. The interaction of this vitamin [i.e., 1,25-dihdroxyvitamin D(3) [1,25(OH)(2)D(3)]] with the antitubercular immune response, however, is not clear. In the present study, in vitro recall responses of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis were used to study the immune-modulatory effects of 1,25(OH)(2)D(3) on M. bovis-specific responses in vitro. Addition of 1 or 10 nM 1,25(OH)(2)D(3) inhibited M. bovis-specific proliferative responses of PBMC from M. bovis-infected cattle, affecting predominantly the CD4(+) cell subset. In addition, 1,25(OH)(2)D(3) inhibited M. bovis-specific gamma interferon (IFN-gamma) production yet enhanced M. bovis-specific nitric oxide (NO) production. Lymphocyte apoptosis, measured by flow cytometry using annexin-V staining, was diminished by addition of 1,25(OH)(2)D(3) to PBMC cultures. These findings support the current hypothesis that 1,25(OH)(2)D(3) enhances mycobacterial killing by increasing NO production, a potent antimicrobial mechanism of activated macrophages, and suggest that 1,25(OH)(2)D(3) limits host damage by decreasing M. bovis-induced IFN-gamma production.

FIG. 1

Addition of 1,25(OH)₂D₃ decreases the percentage of proliferating CD4⁺ cells within the eldest generations. PBMC were cultured with no stimulation (A to C) or with M. bovis PPD (5 μg/ml) (D to F). In addition, cultures received either 0 (A and D), 1 (B and E), or 10 (C and F) nM 1,25(OH)₂D₃. After a 6-day incubation, cells were harvested, stained with either CACT138A, anti-CD4; CACT80C, anti-CD8α; or BAQ4A, anti-WC1 and analyzed by flow cytometry for PKH67 intensity and cell surface marker expression. After flow cytometric analysis, data were analyzed by using the Modfit Proliferation Wizard to determine the number of cells that had proliferated (grey peaks). A representative response from a single M. bovis-infected animal is depicted. Gates for this particular sample were set on live (e.g., based upon light scatter properties) and CD4⁺ cells. Black peaks depict the parent generations (e.g., PKH67 bright), whereas daughter generations are depicted with peaks in various shades of grey.

FIG. 2

Antigen-specific IFN-γ and NO responses of M. bovis-infected cattle. PBMC were cultured with no stimulation (NS) or with either M. bovis strain 1315 CF (5 μg/ml), M. bovis PPD (5 μg/ml), ESAT-6 (1 μg/m), or PWM (2 μg/ml). Supernatants were harvested after 72 h for detection of IFN-γ by ELISA (a) and detection of nitrite by Griess reaction (b). PBMC were obtained from noninfected cattle (n = 3 [closed bars]) and M. bovis-infected cattle (n = 2 [hatched bars]). Addition of L-NMMA (at a concentration equimolar to the amount of l-arginine in the culture medium), a competitive inhibitor of the enzyme NOS, inhibited nitrite production to levels detected in medium alone (e.g., background levels; data not shown). For a specific stimulant, responses of infected cattle differ from responses of controls. Symbols: ∗, P < 0.01; ∗∗, P < 0.05; ∗∗∗, P < 0.001. Error bars, SEM.

FIG. 3

Addition of 1,25(OH)₂D₃ increases M. bovis-specific and PWM-stimulated production of nitrite by PBMC from M. bovis-infected cattle (n = 5). Mononuclear cells were cultured or with either M. bovis strain 1315 CF (5 μg/ml) (a), rESAT-6 (1 μg/ml) (b), M. bovis PPD (5 μg/ml) (c), or PWM (2 μg/ml) (d). To each of these treatments either no vitamin D, 1 nM 1,25(OH)₂D₃, or 10 nM 1,25(OH)₂D₃ was added as described in Materials and Methods. Supernatants were harvested after 24, 48, or 72 h for detection of nitrite by the Griess reaction as an indication of NO production. Symbols: ∗, P < 0.1; ∗∗, P < 0.05; ∗∗∗, P < 0.001 (differs from unsupplemented [no vitamin D] cultures at specific times). Error bars, SEM.

FIG. 4

Addition of 1,25(OH)₂D₃ decreases M. bovis-specific production of IFN-γ by PBMC from M. bovis-infected cattle (n = 5). Mononuclear cells were cultured with either M. bovis strain 1315 CF (5 μg/ml) (a), rESAT-6 (1 μg/ml) (b), M. bovis PPD (5 μg/ml) (c), or PWM (2 μg/ml) (d). To each of these treatments either no vitamin D, 1 nM 1,25(OH)₂D₃, or 10 nM 1,25(OH)₂D₃ was added as described in Materials and Methods. Supernatants were harvested after 24, 48, or 72 h for detection of IFN-γ by ELISA (Bovigam assay; CSL Limited). Symbols: ∗, P < 0.1; ∗∗, P < 0.05 (differs from unsupplemented [no vitamin D] cultures at specific times). Error bars, SEM.