Date of Award

11-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Molecular and Systems Pharmacology

Research Advisor

Francesca-Fang Liao, PhD

Committee

Hao Chen, PhD Tauheed Ishrat, PhD Santosh Kumar, PhD Tony N. Marion, PhD Junming Yue, PhD

Keywords

Acute Neuroinflammation, Cerebral Vasculature, Exosomes and Neuroinflammation, Panendothelitis

Abstract

Sepsis-associated encephalopathy (SAE) is an acutely progressing brain dysfunction induced by systemic inflammation. The mechanism of initiation of neuroinflammation during SAE, which ultimately leads to delirium and cognitive dysfunction, remains elusive. The goal of this project was to study the molecular events of SAE to capture its onset and progression into the central nervous system (CNS), and further identify the cellular players involved in mediating acute inflammatory signaling. Gene expression profiling on the cerebral vessels isolated from the brains of the mice treated with peripheral lipopolysaccharide (LPS) revealed that the cerebral vasculature responds within minutes to acute systemic inflammation by upregulating the expression of immediate early response genes, followed by activation of the NF-κB pathway. To identify the earliest responding cell type, fluorescence-activated cell sorting (FACS) was utilized to sort the immunolabelled glial and vascular cells from the brains of the mice treated with LPS at different time points and gene expression profiling was performed. Bioinformatic analysis of the sequencing data followed by further validation revealed that the cerebral endothelial cells (CECs) activation is the earliest event in the CNS and that they are the most likely source of proinflammatory mediators that could further initiate glial cell activation. This is further followed by the activation of apoptotic signaling in the CECs which is known to lead to blood brain barrier (BBB) disruption and allow the peripheral cytokines to leak into the CNS, exacerbate the gliosis and result in neuroinflammatory cascade. Together, these results model the sequential events during the advancement of systemic inflammation into the CNS, and facilitate better understanding of the interplay between the vascular and glial cells in initiating and driving acute neuroinflammation during SAE.

Systemic inflammation does lead to neuroinflammation, thereby linking the peripheral inflammatory conditions to the CNS. However, the mechanisms through which systemic inflammation exerts its effect on the CNS are poorly understood. Exosomes are small (30 to 100 nanometers) membrane bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell to cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs and cytokines both locally and systemically to the neighboring as well as distant cells to further modulate their transcriptional and/or translational states and affect the functional phenotype of those cells which have taken up these exosomes. The role of circulating blood exosomes in mediating neuroinflammation during systemic inflammation was thus studied. Serum derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice which were earlier treated with LPS and used to study SDEL effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as control. In vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the cell lines of microglia (BV2), astrocytes (C8-D1A) and cerebral microvascular endothelial cells (Bend.3). To further study their effects in vivo, SDEL were then intravenously injected into normal adult mice. The recipient mice that received SDEL exhibited elevated microglial activation. Moreover, increased astrogliosis, and elevated CNS expression of pro-inflammatory cytokine mRNA were observed in SDEL recipient mice. Additionally, SDEL injected directly into the cerebral ventricles resulted in significant microgliosis as well as, astrogliosis. Together, these results demonstrate a novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0001-6023-1060

DOI

10.21007/etd.cghs.2020.0522

Additional Files
2020-027-Kodali-DOA.pdf (317 kB)
Declaration of Authorship
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