Selective microRNA modification to enhance regulatory T cell function in IBD. Preliminary longitudinal studies of a TNF-driven model of ileiti (i.e. TNF?ARE) that recapitulates many features of Crohn's disease;demonstrated expansion of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the inflamed ileum along the disease timecourse by both flow cytometry and RT-PCR. This expansion recapitulates evidence from the intestine of IBD patients. The expansion is clearly insufficient to protect mice from chronic disease in this T cell-dependent model, as severe inflammation is still visible in 20wk-old mice. Phenotypic evaluation of Tregs from TNF?ARE mice uncovered a significant deficiency in their capacity to produce IL-10 that could be reversed by in vivo administration of anti-TNF antibodies. Moreover these effects could be recapitulated in vitro by treating WT Tregs with recombinant TNF?. This effect was independent of changes in IL-10 mRNA suggesting a post-transcriptional modification mechanism may play a role. Copy number assessment of microRNA expression from isolated wildtype and TNF?ARE Foxp3+-GFP+ Tregs identified a differential pattern of expression of mir106a that appears to account for the decreased IL-10 protein synthesis. Mir106a has been proposed previously as a microRNA upregulated by TNF? that suppresses IL-10 expression and be preferentially upregulated in IBD patients. Initial targeted inhibition of mir106a with a lentiviral antimir resulted in increased IL-10 output measured by flow cytometry and ELISA, as well as enhanced wildtype Treg suppressive function. This project will now assess the consequences of lentiviral suppression of mir106a within inflamed Tregs both in vitro and in vivo. Newly converted Tregs from WT and TNF?ARE mice will be spinoculated with a lentimir or control lentivirus prior to assessment of IL-10 production and suppressive function using the classical fluorescence-based proliferation assay. Additional in vitro studies using an IL-10 3'UTR reporter construct will also be employed to better define exactly how mir106a targets IL-10 translation. These studies will then be translated to the TNF?ARE model, a preclinical model of Crohn's disease. To further improve the selectivity and specificity of lentiviral antimir delivery in mice, fluorescently labeled nanoparticles coated with anti-CD4+ antibodies will be used to limit delivery of the lentivirus to the target cells. These nanoparticle will then be administered to 20-week-old inflamed TNF?ARE mice and the therapeutic potential to attenuate ileitis will be assessed postmortem. Preliminary data demonstrate that antibody conjugation can be used successfully to enhance intestinal delivery of fluorescent nanoparticles to the inflamed lamina propria. While these studies will focus on targeting a specific microRNA in a single cell type, this approach has the potential to be transferred to a whole host of microRNA that are differentially expressed in disease across a range of cell types.
Inflammatory bowel disease incidence is increasing worldwide with approximately 1.4 million patients in the U.S. There is a need for better treatments, as most IBD patients require surgery. This project will assess an alternative avenue of research as well as alternative approach to drug deliver in IBD.
|Collins, Colm B; Strassheim, Derek; Aherne, Carol M et al. (2014) Targeted inhibition of heat shock protein 90 suppresses tumor necrosis factor-? and ameliorates murine intestinal inflammation. Inflamm Bowel Dis 20:685-94|