Functional Analysis of Complement Variants in a Genotyped iPSC Epithelial Cell Model System - Complement, a part of innate immunity, eliminates invading microorganisms, apoptotic cells, and cellular debris to maintain the homeostasis of the cellular environment. The complement pathway is tightly regulated to prevent the consequences of inflammation. This regulation can be disrupted by disease, infection, aging, genetic variation or any combination. The long-term objective of this proposal is to uncover the effect of Alternative Pathway (AP) genetic variations on the local complement response in epithelial cells. Our experiments will be done on Retinal Pigment Epithelial (RPE) cells differentiated from Induced Pluripotent Stem Cells (iPSCs) obtained from patients with intermediate Age-related Macular Degeneration. In our first aim we will differentiate 8 iPSCs lines to RPE, four of which have CFH risk alleles and another four which have the risk alleles corrected to non-risk (isogenic controls). After the iPSCs have reached RPE maturity we will quantitate mRNA and protein levels and functional capabilities of complement system players (components, regulators and receptors) synthesized by iPSC-RPE cells. In addition, intracellular AP levels will be determined for iPSC-RPE and their isogenic controls. These results will inform the influence of genetic variation on secretion of complement proteins into the extracellular environment, their intracellular content and pathway differences. Because extracellular vesicles (EVs) are an important cargo for complement and non-complement proteins we will investigate in the second aim the differences in EV protein content secreted from iPSC-RPE cells from apical and basal membrane locations. Our working hypothesis is that the EV protein content and quantity will differ between cells which have the CFH risk variants and their isogenic controls which carry non-risk CFH variants. Secreted proteins also contribute to deposit formation under the cells, which we will investigate by scanning electron microscopy and immunostaining. Cells and their isogenic controls will be exposed to smoke extract and cytokines to determine if the EVs, deposit composition and complement system players (components, regulators and receptors) are influenced by CFH risk variants. Our preliminary results of single cell RNA sequencing (scRNA-Seq) of RPE have led to our central hypothesis that complement plays a crucial role in maintenance of healthy RPE. But the GWAS results on AMD suggest a dysfunction in the complement system. Overall, these studies will provide unique knowledge about the influence of genetic variation on the complement pathway by its focus on functional assays. Our interdisciplinary team is uniquely poised to address this fundamental question by combining our broad background of GWAS and genetic variants of AMD (Stambolian), complement genetics (Atkinson) and RPE complement and extracellular vesicle analysis (Rohrer). The proposal’s outcomes will fill a critical gap in our understanding of genetic variants on RPE complement activity and identify new potential therapeutic targets directed at the RPE. We predict that these results will also have implications for other epithelial cells that serve as barriers in the kidney and lung, also targets of complement activation in human diseases.
