Th17 cells and their cytokine products, IL-17A and IL-17F have been implicated in chronic inflammatory diseases including cystic fibrosis, COPD and steroid resistant asthma. Furthermore these cytokines have been implicated in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Anti-IL-12p40 antibodies which suppress Th17 cytokine elaboration has been recently been approved for the treatment of psoriasis however this drug can also suppress Th1 immunity and theoretically increase the risk of infection with intracellular pathogens. To identify small molecules which may be selective for Th17 cells as opposed to Th1 cells, we recently conducted a compound library screen to indentify small molecules that would suppress Th17 cells but not Th1 cells. In this screen we included know compounds such as calcineurin inhibitors (Cyclosporine A), FK-506, and rapamycin as known suppressors of T-cell function. CyA, FK-506, and rapamycin suppressed both Th1 and Th17 cells and thus were non-selective. However macrolides, specifically Azithromycin showed dose dependent suppression of Th17 development with an ED50 of approximately 100 uM and little suppression of Th1 responses. Based on this we also examined the activities of clarithromycin and roxithromycin. The latter compound also showed significant activity in inhibiting Th17 development with an ED50 of 200 uM. Based on these data, we hypothesize that these macrolides represent potential inhibitors of Th17 effector function and that structure activity relationships (SAR0 can be used to improve the activity of these compounds. We will test this hypothesis with two Specific Aims.
Specific Aim 1. Perform SAR on azithromycin and roxithromycin to identity structures required for activity as well as more potent inhibitors of Th17 cytokine elaboration. These compounds will be incubated with Th17 or Th1 cells at varying doses prior to activation with anti-CD3/anti-CD28. Outcome measures will include intracellular IL-17 and IFNgamma as well as cell viability by FACS. Cell supernatants will be harvested and the level of secreted IL-17 or IFNgamma will be measured by Luminex.
Specific Aim 2. To screen and azithromycin and roxithromycin as well as new compound identify in Aim 1 in vivo using a Th17/Th1 adoptive transfer system of lung inflammation. Under this aim, we will perform adoptive transfers of ovalbumin specific Th17 or Th1 cells IV. Azithromycin, roxithromycin or derivatives identified in Aim 1 ill be administered IP one hour prior to intrapulmonary ovalbumin challenge. Outcome measures will include BAL neutrophilia and macrophage emigration as well as lung levels of IL-17, IFN gamma as well as the downstream Th17 and Th1 target genes (KC, G-CSF, MCP-1 and MIG, IP-10 respectively).
Interleukin-17 can cause lung inflammation and asthma. This project is to identify drugs that can inhibit IL-17 and lung inflammation.
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