Graduation Date

Spring 5-7-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Interdisciplinary Graduate Program in Biomedical Sciences

First Advisor

Dr. Adam Case

Abstract

Post-traumatic stress disorder (PTSD) is a debilitating psychological disorder that affects nearly 45 million Americans. This mental disorder is characterized by behavioral symptoms such as learned helplessness, hyperarousal, withdrawal, and flashbacks. The deleterious effects of PTSD are far-reaching and go beyond behavioral dysfunction, as these individuals are at a three-fold higher risk of comorbid inflammatory diseases. Autonomic, metabolic, and cardiovascular diseases plague these individuals later in their lifetime, however, the mechanistic link connecting psychological trauma to this systemic peripheral immunological dysfunction remains elusive. T-lymphocyte-induced inflammation plays a significant role in all these disease pathologies. Previous work from our laboratory demonstrated that in in vitro model systems of PTSD, T-lymphocytes show increased pro-inflammatory cytokine production regulated by mitochondrial redox signaling. The work presented herein attempts to elucidate the driving factors potentiating mitochondrial-induced T-lymphocyte inflammation after psychological trauma. Implementing a preclinical mouse model of PTSD, repeated social defeat stress (RSDS), we observed significantly elevated levels of mitochondrial superoxide and inflammatory cytokines in T-lymphocytes in RSDS mice. Moreover, using single cell RNA sequencing technology, we were the first to report a significant elevation in gene expression for calgranulin A and calgranulin B (encoded by S100a8 and S100a9) in splenic T-lymphocytes isolated from RSDS mice. Together, these peptides form a pro-inflammatory heterodimer protein called calprotectin, which positively correlated with anxiety-like behavior, pro-inflammatory cytokine expression, and T-lymphocyte mitochondrial superoxide levels in our mice. Interestingly, previous work from our laboratory demonstrated significantly elevated gene expression of S100a8 and S100a9 in thymocytes isolated from mice lacking manganese superoxide dismutase (MnSOD) knockout mice. These mice possess increased mitochondrial superoxide, which further suggests a link between calprotectin and the redox environment. We confirmed calprotectin was upregulated in splenic T-lymphocytes from MnSOD knock-out mice and demonstrated that uncontrolled mitochondrial superoxide production potentiated dysregulation of mitochondrial and glycolytic metabolism, cytokine production, and epigenetic modifications. Taken together, I put forth the central hypothesis that psychological trauma-induced elevated mitochondrial superoxide regulates the expression of calprotectin in T-lymphocytes, which in turn, increases pro-inflammatory cytokine production from these cells. To test this hypothesis, we ran S100a9 knock-out (S100a9-/-) mice, a genetic mouse model that does not have functional calprotectin protein, through RSDS and assessed their redox and inflammatory outputs. Unexpectedly, S100a9-/- RSDS mice displayed greater elevations in pro-inflammatory cytokines in circulation and at the mRNA level in splenic T-lymphocytes, as well as elevated mitochondrial superoxide levels in splenic T-lymphocytes compared to wild-type mice. Furthermore, loss of S100a9 potentiated metabolic dysfunction, as CD8+ T-lymphocytes display elevated mitochondrial and glycolytic metabolism in these mice. Collectively, this data is highly suggestive that calprotectin may play a protective role in attenuating psychological trauma-induced T-lymphocyte inflammation through possible mitochondrial redox and metabolism-regulated mechanisms.

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