Humoral Factors and Circulating Extracellular Vesicles in Type 1 Diabetes Induce Beta Cell Cytotoxicity

Nagesha Guthalu Kondegowda (Arthur Riggs Diabetes and Metabolism Institute)

Nagesha Guthalu Kondegowda, Joanna Filipowska, Nancy Leon-Rivera, Rollie Hampton, Rosemary Li, Selassie Ogyaadu, Clive Wasserfall, Mark Atkinson, Helna Reijonen, Yuan Yate-Ching, Navneet Dogra, Daniel Roeth, Markus Kalkum, Carol Levy, Susmita Sahoo, Rupangi C. Vasavada

The goal of these studies is to examine the role of humoral factors and circulating extracellular vesicles (cEVs) in the pathogenesis of Type 1 diabetes (T1D). T1D is an autoimmune disease in which beta cell loss and dysfunction play a vital role in its pathogenesis. EVs, small membrane bound structures, are secreted into the circulation by almost every cell type. Due to their distinct cargo, EVs act as molecular messengers and inter-organ communicators, and play an important role in both normal and pathophysiological conditions. EV cargo content is based on the environment of the tissue it is being secreted from and can alter the phenotype of recipient cells. Humoral factors in the serum from T1D subjects can be cytotoxic to rodent beta cell lines and islets. Also, serum EVs of long term T1D patients carry distinct miRNA cargo and cause defect in β-cell secretion. Therefore, we hypothesized that cEVs from T1D subjects, at different stages of the disease, are detrimental to islet health, through their differential RNA and protein cargo, with the potential to serve as disease biomarkers.

Serum and plasma samples were obtained from early (1-5 years since diagnosis) and late (>10 years since diagnosis) stage T1D patients, from autoantibody positive (Aab+) donors, and age, sex and ethnicity matched non-diabetic healthy donors (HD). cEVs and EV-depleted fractions prepared from T1D and HD plasma using ultracentrifugation and exoquick methods, were characterized by Dynamic light scattering (DLS), Nanoparticle Tracking Analysis, Western blotting and transmission electron microscopy. PKH26 labeled cEVs were used for uptake studies in human islet cells. Cytotoxicity assays were performed on rat insulinoma cell line (INS1), mouse and human islet cells cultured for 24h in media in which fetal calf serum (FCS) was substituted for human serum (10%v/v), or in islet media treated with cEVs, from the donors indicated above. Cleaved caspase 3 staining, TUNEL with insulin and glucagon co-staining, were used to assess cell death. Mouse serum from T1D NOD female mice at different stages of the disease was examined similarly. Seahorse analysis was used to assess the effects on mitochondrial function in human islets. RNAseq and proteomic analyses of cEV cargo and the functional contribution of candidate miRNAs towards b-cell cytotoxicity was assessed.

Summary of Results
Our initial findings show that there were no obvious differences in physical characteristics, cell surface markers, size and number, in cEVs from T1D versus HD plasma. cEVs from T1D and HD donors tagged with PKH26 dye showed uptake in INS1 cells and human beta cells. Serum and plasma from T1D donors (both early and late-stage) (n=5-9) significantly increased cell death in INS1 cells, and in human beta cells, comparable to cell death levels induced by pro-inflammatory cytokines, suggesting that humoral cytotoxicity may persist with disease progression. The same findings hold true when serum from early and late stage of the disease from female NOD mice was used. Our initial data indicate that serum from Aab+ subjects (n=8) induce cytotoxicity in human beta cells, similar to that induced by serum from T1D subjects, suggesting that the humoral beta cell cytotoxicity occurs early, before disease onset. Seahorse analysis suggests that T1D serum-treatment reduces the mitochondrial respiratory spare capacity of human islets compared to HD serum (n=4).
We then tested our hypothesis that the humoral cytotoxicity against beta cells seen in the serum and plasma of T1D patients is mediated by cEVs. Indeed, cEVs from T1D donors, but not HD (n=8/group), significantly induced human beta cell death, and this was not observed with the EV-depleted fraction. Relevant to disease pathology, T1D cEV-induced cytotoxic effect was specific to human beta cells but did not induce cell death in human alpha cells in the same islet prep. To investigate the cargo and molecular mechanisms, we performed a pilot RNASeq and proteomic analysis on cEVs (n=5) from T1D and HD subjects. We identified differential miRNA, lncRNA, mRNA and protein cargo in cEVs from T1D vs HD subjects. A functional analysis of candidate miRNAs in T1D cEVs showed specific miRNAs are detrimental to beta cells.

Serum, plasma, and plasma-derived cEVs from human T1D subjects induce human beta cell but not alpha cell death in vitro. Humoral cytotoxicity on beta cells may initiate early in pre-disease at-risk Aab+ individuals, and likely persist in late-stage disease. The differential RNA and protein cargo in T1D cEVs likely mediates the detrimental effects on human beta cells. Our findings suggest that cEVs, at various stages of the disease, play a role in the pathogenesis of T1D as related to the beta cell. Our ongoing studies are investigating the effects of the cEVs on the immune system, their molecular mechanisms of action, as well as the differential RNA and protein cargo in cEVs from different stages of the disease, with the potential to develop new biomarkers and therapeutics for T1D.