Anti-MAP Triple Therapy Supports Immunomodulatory Therapeutic Response in Crohn's Disease through Downregulation of NF-κB Activation in the Absence of MAP Detection.

Abstract

We previously reported that the triple antibiotic formulation, known as anti-MAP therapy, exhibits unique synergistic antimicrobial activity and should be effective for treatment of Crohn’s disease (CD) associated with Mycobacterium avium subspecies paratuberculosis (MAP). The absence of MAP detection in some CD cases may be linked to poor diagnostics or lack of association with the disease. To understand the therapeutic response of some CD patients to anti-MAP therapy in absence of MAP detection, we investigated the immunomodulatory potency of anti-MAP therapy and its major ingredients, clarithromycin (CLA) and rifabutin (RIF), in THP-1, Caco-2, and Jurkat T-cells. Anti-MAP formulation at 2.0 μg/mL decreased MAP viability in macrophages by 18-fold over 72 h. Additionally, M1/M2 macrophage polarization ratio was reduced by 6.7-fold, and expression and protein levels of TNF-α and IL-6 were reduced by 2.9-fold, whereas IL-10 increased by 5.0-fold in these cells. Mechanistically, the effect of anti-MAP formulation on NF-κB p65 activation was dose-dependent and decreased to 13.4% at 2.0 μg/mL. Most importantly, anti-MAP therapy also reversed pro-inflammatory response in lipopolysaccharide (LPS)-induced macrophages, which shows that the anti-inflammatory effect of the treatment is not just due to a decrease in MAP viability. To study the anti-cytotoxic effects of anti-MAP therapy in Caco-2 monolayers infected with MAP or treated with dextran sodium sulfate (DSS), we showed a 45% decrease in lactate dehydrogenase (LDH) activity and an 84% increase in glutathione (GSH) activity, which supports anti-apoptotic activity of the drug. In Jurkat T-cells, anti-MAP therapy decreased T-cell proliferation by 4.8-fold following treatment with phytohemagglutinin (PHA) and by 2.9-fold with MAP purified protein derivative (PPD). Overall, the data demonstrate that anti-MAP therapy plays a significant role in modulating and eliciting a protective immune response in macrophages, endothelial cells, and T lymphocytes, even in absence of infection. This may explain the therapeutic response of some CD patients to treatment, even in absence of MAP detection, infection, or total eradication. The study supports anti-MAP therapy as an alternate treatment option in CD patients, especially in absence of reliable MAP diagnostics.