Author ORCID Identifier

https://orcid.org/0000-0003-0619-4902

Date Available

5-20-2023

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Pharmacy

Department/School/Program

Pharmaceutical Sciences

First Advisor

Dr. Barbara Nikolajczyk

Abstract

Type 2 diabetes (T2D) is a chronic low-grade inflammatory disease. A T cell cytokine profile (Th17) from PBMCs can distinguish obese T2D from obese non-diabetes subjects. Individual T cell subsets interact with each other and the diverse subsets jointly determine overall inflammation. Cellular metabolism drives cytokine production of CD4+ T cells, and therefore contributes to inflammation in T2D. However, specific changes in metabolism and function of CD4+ T cells during the progression from lean healthy to obese and diabetic stages in people have not been clarified.

We hypothesize that human regulatory T cells (Treg) impact metabolism of effector T cells (Teff), which would exhibit different functions among lean healthy, prediabetes and T2D cohorts. The metabolic influence of Treg may further alter function of Teff, which is associated with imbalanced production of pro-inflammatory and anti-inflammatory cytokines during the development of obesity and diabetes. In our study, lean healthy, obese prediabetes and obese T2D human subjects were recruited. We isolated human Teff (CD25-CD4+) and Treg (CD25+CD4+) cells from peripheral blood. Teff and Treg cells were single- or co-cultured in the presence of αCD3/CD28 dynabeads for T cell activation.

To test the functional differences in T cells from prediabetes and T2D subjects, we conducted bioplex and flow cytometry analyses to quantify the cytokine production from Teff and Treg cells in single- and co-cultured conditions. We observed that Treg increased IL-17F production in Teff from T2D subjects. Teff-derived cytokines dominantly predisposed inflammation in T2D subjects, while Treg-derived cytokines dominated inflammation in prediabetes subjects. T cell cytokine production changed during the progression of obesity and diabetes, consistent with the possibility of either a compensatory or reversible nature.

To examine the metabolic alterations in T cells from prediabetes and T2D subjects, we performed Seahorse mitochondrial stress tests, lactate measurements, western blots and confocal imaging to define metabolic processes that distinguish Teff from Treg. These approaches have shown that Treg impairs OXPHOS of Teff from prediabetes and T2D subjects, but co-cultured cells compensate by increasing utilization of non-mitochondrial glycolysis only in T2D subjects. Teff and Treg from prediabetes subjects diverted to pro-inflammatory non-mitochondrial glycolysis compared with their counterparts from the other two cohorts. The unique metabolic status of CD4+ T cells in prediabetes subjects was further indicated by changes in proteins required for fatty acid metabolism, specifically higher CD36 and lower FADS2 expression in Treg. These data indicate differences in fatty acid oxidation in addition to changes in non-mitochondrial glycolysis in Tregs, raising the possibility that these changes drive prediabetic T cells to become more inflammatory compared with counterparts from lean healthy or T2D subjects.

To determine the relationships between functional and metabolic differences observed in T cells among the three cohorts, we investigated how CD36, a long chain fatty acid transporter, mediated functional changes in T cells. Using an irreversible pharmacological inhibitor, we found that fatty acid uptake and cytokine production in Treg from prediabetes subjects were significantly lower after CD36 inhibition, while this was not the case in Treg from T2D subjects. Taken together, we conclude that Treg unexpectedly promotes inflammation in prediabetes subjects due to CD36 dysfunction.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.148

Funding Information

This study was supported by National Institutes of Health R01 grant (DK108056) from 2018-2021.

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