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

Background: 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. Objective: 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. Animals: Eight healthy Labrador retrievers. Materials and Methods: 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-gamma), and by qRT-PCR for IL-4, IL-10, IL-13, IFN-gamma, tumour necrosis factor alpha (TNF-alpha) and transforming growth factor-beta. Results: 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 mu g/mL. Compared to the negative control, post-stimulation elevation of IFN-gamma mRNA was noted at 24 h with all concentrations of MCWF (p < 0.01), and TNF-alpha mRNA was increased for 0.5 mu g/dL MCWF only at 72 h (p < 0.05). Conclusions and Clinical Relevance: 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.