Date of Award

January 2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Catherine A. Brissette

Second Advisor

John A. Watt

Abstract

Lyme disease, caused by the spirochete Borrelia burgdorferi (Bb), is the most commonly reported vector-borne disease in the United States – with 30,000 cases being reported to the CDC annually, though it is estimated that 300,000 individuals are infected each year in the U.S [1–4]. Due to the medical treatment of the disease, this equates to an estimated $712 million - $1.3 billion in medical costs each year [5]. Conclusively, due to the continued geographical spread and increasing incidence rate, Lyme disease is becoming a greater public health threat throughout the world.

The symptoms of Lyme disease can range from erythema migrans to more systematic disorders such as arthritis and neurological complications, termed neuroborreliosis [6,7]. Manifestations of neuroborreliosis include radiculoneuritis, meningitis, and facial palsy [8–10]. Interestingly, B. burgdorferi does not produce any known toxins, and it is thought that the resulting host immune response leads to cellular and tissue damage associated with clinical symptoms. Although many individuals will be effectively treated through the administration of antibiotics, up to 20% of patients will experience on-going symptoms termed Post-treatment Lyme Disease Syndrome (PTLDS). PTLDS is marked by persistent musculoskeletal pain and neurological complications. Inflammatory states have been associated to these symptoms with the invasion of peripheral immune cells and an increase of inflammatory cytokines.

Furthermore, the neurological complications of PTLDS has been associated with an increase in glial inflammation. It is well-documented that B. burgdorferi is capable of penetrating into the central nervous system (CNS); however, it is unknown how and where the bacterium does so. Additionally, the exact pathogenetic mechanisms of neuroborreliosis and PTLDS are poorly understood. The work within this dissertation aims to provide novel insight into these gaps in knowledge.

This dissertation is laid out into three sections relating to understanding the pathogenesis of the neurological effects of Lyme disease. In the first study, we aimed to provide an explanation for the dissemination of B. burgdorferi and peripheral immune cells into the central nervous system. Clinical presentations of neuroborreliosis is associated with an increase of peripheral immune cells, inflammatory chemokines, and live B. burgdorferi in the cerebrospinal fluid (CSF). To this end, we sought to investigate a direct route from hematogenous dissemination into the CSF. The choroid plexus (CP) is a structure within the ventricles of the brain that is responsible for the production of CSF, the formation of the blood-CSF barrier, and regulation of the immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. We hypothesize that during infection of the choroid plexus, Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state within the CNS. To investigate this hypothesis, we cultured primary human choroid plexus epithelial cells and infected with the B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.

The second and third study aim to elucidate the pathogenic mechanisms of neuroborreliosis and PTLDS, specifically the manifestations of a persistent inflammatory state. As B. burgdorferi has previously been shown to elicit an inflammatory response in astrocytes, and glial inflammation is associated with PTLDS, we sought to investigate the epigenetic modifications associated with the astrocytic response in order to determine a mechanistic explanation to these disorders. In the second study, we investigated the differential DNA methylation of astrocytes in response to three strains of B. burgdorferi – B31 MI-16, B31 e2, and 297 through the use of beadchip array. This study utilized primary human astrocytes in vitro. This study was met with a limiting factor in biological replicate variability that led to diminished results. Nevertheless, differential methylation within specific genes involving vesicle trafficking and cell communication were observed. This suggests that DNA methylation may be a mechanistic explanation for the changes in gene expression of astrocytes in response to B. burgdorferi.

In the third study, we utilized the same astrocyte model of the second study to investigate the effects of B. burgdorferi on chromatin structure of astrocytes. We performed in vitro infection of astrocytes with the B31 MI-16 strain for 24, 48, 72, and 96 hours. Following infection, ATAC-seq was performed to interrogate the chromatin structure of astrocytes in response to B. burgdorferi. We observed a robust change in chromatin accessibility at 24-hours with 25,464 differential peaks. At 48, 72, and 96 hours, these peaks were reduced to 7,266, 3,376, and 3,015 respectively. Additionally, while many of the differential peaks were associated with open chromatin at 24, 48, and 72 hours, the 96-hour time point was marked by a dramatic decrease in chromatin accessibility. Many of the peaks within gene bodies at the first three time points were associated with changes in anatomical and morphological alterations, while the 96-hour time point was highlighted by metabolic and cellular stress. This suggests that astrocytes undergo an acute response following infection observed by a large change in chromatin structure associated with inflammation and immune response genes, which later decrease in accessibility. Motif enrichment analysis provides greater insight into the overall response of astrocytes across time points. The AP-1 transcription factor is involved in the transcription of genes in response to inflammatory stimuli, stress signals, and infection. This transcription factor is made up of a heterodimer that includes the FOS, JUN, ATF, and JDP families. Motif analysis indicated significant enrichment of these family members at each time point, and in fact, analysis of peaks shared amongst all time points indicated AP-1 motif as being the most significantly enriched. These data suggest that the response of astrocytes to B. burgdorferi is in part due to the changes in chromatin accessibility that provides an environment for the transcription of genes associated with the inflammatory and immune response. Furthermore, AP-1 has been implicated as a potential transcription factor responsible for these changes in gene expression.

Together, the work within this dissertation demonstrates potential mechanisms for the pathogenesis of neuroborreliosis and PTLDS. This is highlighted by the potential of the choroid plexus as a route of dissemination for B. burgdorferi and peripheral immune cells into the CNS as an explanation for the clinical manifestations of neuroborreliosis. Additionally, these studies are the first to implicate B. burgdorferi as an epimutagen which provides insight into the mechanisms and development of the neurological and persistent symptoms of Lyme disease. In conclusion, this work provides novel insights for the pathogenesis of the neurological effects of Lyme disease which may aid in the development of future therapeutics.

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