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 (Ustekinumab) which suppress Th17 cytokine elaboration have 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 known compounds such as calcineurin inhibitors (Cyclosporine A), FK-506, and rapamycin, which are 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 5M and little suppression of Th1 responses. Based on this we also examined the activities of clarithromycin and roxithromycin. The latter compounds also showed significant activity in inhibiting Th17 development with an ED50 of 200 5M. Based on these data, we hypothesize that these macrolides represent potential inhibitors of Th17 effector function and that structure activity relationships (SAR) can be used to improve the activity of these compounds. We will test this hypothesis with two specific aims.
Specific Aim 1. Synthesis and SAR analysis of macrolide analogs to identify structures required for activity and more potent inhibitors of Th17 cytokine elaboration. A series of macrolide analogs will be synthesized using erythromycin as the starting material. These compounds will then 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 IFN? as well as cell viability by FACS. Cell supernatants will be harvested and the level of secreted IL-17 or IFN? will be measured by Luminex.
Specific Aim 2. In vivo screening of potent compounds identified in Aim 1 using a Th17/Th1 model of lung inflammation. Selected compounds with potent IC50s will be tested in vivo in an adoptive transfer model using ovalbumin specific Th17 or Th1 cells IV as previously described. Outcome measures will include BAL neutrophilia and macrophage emigration as well as lung levels of IL-17, IFN? as well as the downstream Th17 and Th1 target genes (KC, G-CSF, MCP-1 and MIG, IP-10 respectively). The series of proposed experiments should greatly increase our knowledge of the potential of small molecule inhibitors of Th17 cells. These compounds may improve treatment for chronic lung diseases such as COPD or steroid resistant asthma.
Interleukin-17 can cause lung inflammation and asthma. This project is to indentify drugs that can inhibit IL-17 and lung inflammation.
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