Lipid mediated oral tolerance - Project Summary: Autoimmune conditions, allergies and immunogenicity against therapeutic proteins are caused by unwanted immune responses against proteins. Our long-term goal is to rationally develop an immunotherapy platform using protein delivery strategies to treat these clinical conditions. We observed that an oral pre-administration of antigens loaded in lyso-phosphatidylserine (lysoPS), but not in double chain PS, based nanoparticles effectively reduced unwanted immune response against several protein antigens, forming the basis for an innovative immunotherapy. The overall objective of this application is to elucidate the cellular and molecular mechanisms of oral delivery and tolerance mediated by lysoPS based nanoparticles. The rationale for the proposed research is that once we gain a mechanistic understanding of the tolerogenic property of lysoPS, it would result in an innovative immunotherapy approach and clinical strategies to prevent and reverse immunogenicity of therapeutic proteins and to treat/cure autoimmune conditions and allergies. We also found that there are differences in biophysical characteristics such as PS exposure of the nanoparticles comprised of lyso and double chain PS. Based on these observations, our working hypothesis is that lysoPS mediated tolerance is due to its distinct structural/biophysical characteristics that influences its access to gut associated lymphoid tissue, receptor mediated cellular uptake/signaling and underlying molecular and cellular mechanism of oral tolerance. Three specific aims (SA) are proposed to test our hypothesis and accomplish our objective using a multidisciplinary approach. In SA 1, we will investigate the impact of biophysical properties of lysoPS based lipidic particles and its impact on M-cell mediated particulate uptake. Biophysical characteristics such as PS exposure, clustering, and lamellar properties will be investigated using orthogonal techniques. We will adopt in vitro, ex vivo and in vivo studies using M-cell deficient mice to investigate the impact of M-cell engagement of PS based particles containing model antigen Ovalbumin OVA. In SA 2, we will investigate the impact of PS exposure on receptor binding and intracellular signaling. Based on our observation that functional blocking antibody against Tim-4 receptor reversed PS mediated tolerance, we will investigate the role of this receptor binding in tolerance using advanced cell-biological and imaging techniques and also using knock-out mice models. In SA 3, we will investigate the mechanism of lysoPS mediated oral tolerance towards OVA, in particular, the effects on gut resident dendritic cells, generation of regulatory T-cells and B-cell response. The generation of Tregs will be investigated using OT-II transgenic mice and by adoptive transfer approaches. We will investigate the impact of Tregs on B-cells by investigating the impact of lysoPS on the expression of IL-10, TGFb and neuritin. Overall, our combined approach investigating the biophysical, cell biological, innate and adaptive mechanisms of lysoPS tolerance will lay the foundation for a comprehensive clinical approach to reduce immunogenicity of therapeutic proteins as well as prevent and reverse autoimmunity and allergies.
