Analysis of the immunomodulatory properties of mycobacterium cell wall fraction on the cytokine production of peripheral blood mononuclear cells of healthy dogs.

Abstract

Mycobacterium cell wall fraction (MCWF) is derived from nonpathogenic Mycobacterium phlei and is used as an immunomodulatory compound in clinical practice, yet its mode-of-action requires further research. To evaluate the host response to MCWF in canine peripheral blood mononuclear cells (PBMCs) by using enzyme-linked immunosorbent assays (ELISA) and quantitative reverse transcription (qRT)-PCR for assessment of cytokines. Eight healthy Labrador retrievers. PBMCs were isolated from whole blood using density centrifugation. The cells were cultured with different concentrations of MCWF or a potent stimulator of cytokine production, phorbol 12-myristate 13-acetate/ionomycin, or left in cell culture medium for 24, 48 and 72 h. Cytokines were measured by ELISA for interleukin (IL)-4, IL-10 and interferon-gamma (IFN-γ), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-γ, tumour necrosis factor alpha (TNF-α) and transforming growth factor-beta. A significant increase of IL-10 messenger ribonucleic acid (mRNA) was detected at all time points for all concentrations of MCWF (p < 0.05). Protein analysis reflected this finding, with a maximum IL-10 concentration of 300.6 ± 38.3 μg/mL. Compared to the negative control, post-stimulation elevation of IFN-γ mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-α mRNA was increased for 0.5 μg/dL MCWF only at 72 h (p < 0.05). MCWF stimulation of PBMCs results in the elevation of both proinflammatory and regulatory cytokine mRNA. Further research into the role of MCWF as a systemically administered regulatory immunomodulator or adjuvant to allergen-specific immunotherapy should be considered.