Immunosuppressive drugs are the cornerstone of treating immune-mediated diseases in dogs. Currently, dosage adjustments are often based on resolution of clinical signs and monitoring for adverse effects. Recently, a validated pharmacodynamic assay (PD) has been used to monitor dogs treated with cyclosporine by assessing interleukin-2 (IL-2) and interferon-gamma (INFγ). There is no study to date looking at cytokine secretion and detection in dogs receiving other immunosuppressive drugs.

The aim of this study was to evaluate multiple immunosuppressive drugs and determine individually whether they affected secretion of IL-2, IL-6, IL-10, INFγ or tumour necrosis factor alpha (TNFα) in healthy dogs using a multiplex assay.

Blood was obtained from healthy adult Walker hounds before and after immunosuppressive treatment at standard doses using a randomised crossover study design. Treatments included azathioprine, cyclosporine, mycophenolate mofetil, leflunomide, or prednisone. Blood was sampled prior to and following 7 days of treatment. Four dogs were tested for each drug with a minimum wash out period of three weeks. Whole blood was activated with either phorbol 12-myristate 13-1 acetate and ionomycin (PMA/I) or lipopolysaccharide (LPS). Cytokines in the supernatant were analysed using a multiplex assay. The Kruskall-Wallis test was used to test for carryover effects of the drugs. The Wilcoxon signed-rank test was used to assess each activator effect on cytokine production.

Pre-treatment, stimulation of blood with PMA/I significantly increased INFγ, IL-10, and TNFα, whereas stimulation with LPS significantly increased IL-6, IL-10, and TNFα production (p<0.001 for all cytokines). There was no significant increase in IL-2 with either activator. No significant carry over effect was detected for any of the drugs used.

Azathioprine, leflunomide, and mycophenolate treatments did not reliably change cytokine production for any of the cytokines tested. Dogs treated with cyclosporine had no detectable IL-10, TNFα, and INFγ after treatment when blood was stimulated with PMA/I. All dogs treated with prednisone had marked reduction in their TNFα production (<30% of pre-treatment concentration) after PMA/I stimulation. All dogs, and 3 out of 4 dogs, had a reduction of at least 80% in TNFα and IL-6 concentrations respectively after LPS stimulation.

Specific patterns of cytokine suppression may be useful to monitor efficacy of immunosuppression in dogs treated with cyclosporine and prednisone. No clear trend was seen with the other immunosuppressive drugs tested.

Disclosures

Disclosures to report:

Funding from the co-authors include funding from the Australian research Council, Canine Health Foundation, Canine Research Foundation, Hills Pet Nutrition, NexVet Biologics and PlassVacc. None of this funding is directly related to this project. None of the authors have shares, receive consulting fees or have financial interests with any company. Neither do family members.