Lymphotactin Mediates Antiviral T Cell Trafficking Within
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LYMPHOTACTIN MEDIATES ANTIVIRAL T CELL TRAFFICKING WITHIN THE CENTRAL NERVOUS SYSTEM DURING WEST NILE VIRUS ENCEPHALITIS A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Biological Sciences By Sharese Tronti 2019 SIGNATURE PAGE THESIS: LYMPHOTACTIN MEDIATES ANTIVIRAL T CELL TRAFFICKING WITHIN THE CENTRAL NERVOUS SYSTEM DURING WEST NILE VIRUS ENCEPHALITIS AUTHOR: Sharese Tronti DATE SUBMITTED: Spring 2019 Department of Biological Sciences Dr. Douglas Durrant Thesis Committee Chair Biological Sciences Dr. Andrew Steele Biological Sciences Dr. Jamie Snyder Biological Science ii ABSTRACT West Nile Virus (WNV), a neurotropic flavivirus, can cause neuroinvasive disease in humans. After peripheral infection, WNV is able to enter the central nervous system (CNS) and infect neurons causing neuronal injury and inflammation that potentially may result in fatality. In order to restrict viral replication and pathogenesis within the CNS during WNV encephalitis, virus-specific CD8+ T cells are critically dependent on dendritic cell (DC) mediated reactivation at this site. However, the mechanism by which DCs are recruited to the brain to ensure their interaction with infiltrating virus-specific CD8+ T cells remains unknown. Previous studies have demonstrated that, upon activation, CD8+ T cells rapidly produce the chemokine lymphotactin when activated. The receptor for lymphotactin, XCR1, is exclusively expressed on a subset of DCs, CD8+ DCs, which have been shown to be essential in establishing protective peripheral immunity against viruses and intracellular bacteria. In this study, we show that lymphotactin regulates the CNS entry of T lymphocytes, potentially promoting virologic control within the CNS and limiting neuronal cell death. Although, no apparent differences were obtained in survival and clinical disease progression, WNV-infected mice, that had been treated with an XCL1-neutralizing antibody, displayed increased parenchymal localization of T cells within the brain compared to control mice. These data indicate that lymphotactin contributes to stabilizing the cellular interactions between infiltrating T cells and CNS-localized DCs with may impart protective CNS inflammation by regulating the parenchymal entry of T cells during WNV encephalitis. iii Table of Contents Signature Page ………………………………………………………………………… ii Abstract ………………………………………………………………………………... iii List of Tables ………………………………………………………………………….. vi List of Figures …………………………………………………………………………. vii Introduction ……………………………………………………………………………. 1 West Nile Virus ………………………………………………………………………… 1 West Nile Virus Neuroinvasive Disease ……………………………………………..3 West Nile Virus Immunity ……………………………………………………………. 5 Dendititic Cells and Lymphotactin …………………………………………………. 8 Hypothesis ……………………………………………………………………………... 10 Materials and Methods ………………………………………………………………… 12 Ethics Statement ……………………………………………………………………….. 12 Virus …………………………………………………………………………………….. 12 Mouse Infection ……………………………………………………………………….. 12 Lymphotactin Neutralization ………………………………………………………… 13 Tissue Collection and Preparation …………………………………………………. 13 RNA Isolation and qRT-PCR ………………………………………………………… 13 Lymphotactin Protein Analysis ……………………………………………………… 14 Immunohistochemistry ……………………………………………………………….. 14 Statistical Analysis ……………………………………………………….…………… 15 Results …………………………………………………………………………………. 16 Lymphotactin Expression and its Protective Role During WNV Neuroinvasive iv Disease ……………………………………………………………………………...….. 16 Lymphotactin Is Effectively Neutralized In Peripheral Tissues via Anti-XCL1.. 18 Lymphotactin Plays a Role in Control of WNV Burden within the CNS .……... 19 Lymphotactin Plays a Protective Role Against Virus-Associated Neuronal Cell Death ………………………………………………………………………………. ..… 19 Lymphotactin Impacts the Localization of T cells During WNV Encephalitis… 20 Discussion …………………………………………………………………………….. 31 References …………………………………………………………………………….. 37 v LIST OF TABLES Table 1: Health Status Assessment Results on day 5. ………………………………….22 vi LIST OF FIGURES Figure 1: CD8+ T cells arrested within the perivascular space without antigen- recognition……………………………………………………………………………… 23 Figure 2: Lymphotactin levels during WNV encephalitis …………………………….. 24 Figure 3: Percent of weight change in mice from days -1 to 5 post West Nile infection..25 Figure 4: Percent of survival to day 5 post West Nile infection ………………………. 25 Figure 5: Lymphotactin is effectively neutralized in peripheral tissues via anti- XCL1…………………………………………………………………………………… 26 Figure 6: Lymphotactin plays a role in control of WNV burden within the CNS. ...….. 27 Figure 7: Lymphotactin plays a protective role against virus-associated neuronal cell death ……………………………………………………………………………….. ….. 28 Figure 8: Lymphotactin impacts the localization of T cells during WNV encephalitis.. 29 Figure 9: Lymphotactin-mediated recruitment of DCs to the perivascular space ……. 30 vii INTRODUCTION West Nile Virus (WNV), a mosquito-borne neurotropic flavivirus, has emerged globally as a significant cause of viral encephalitis and meningitis. Due to human and environmental factors, WNV has disseminated throughout the Western world and continues to spread causing increased outbreaks and WNV disease in humans. Currently, there remains no vaccine or specific therapy approved to treat or prevent WNV infection. Due to the fact that WNV continues to pose a significant public health risk, there remains a pressing need to understand the viral and host factors that determine viral pathogenesis and outcome of WNV infection. West Nile Virus West Nile Virus (WNV) is spread by the bite of a mosquito and is maintained in a cycle between birds and mosquitoes, but can also be spread to humans and other mammals which serve as dead-end hosts (Lim et al, 2011). WNV is endemic in parts of Africa, Asia, Europe, and the Middle East and since its emergence in the United States in 1999, reports have been made of its presence in the Caribbean, Mexico, and South America (Dauphin et al, 2004). According to the CDC, there were 2544 cases of WNV infections in the US in 2018, 1594 of them being neuroinvasive resulting in 137 deaths (CDC, 2019). West Nile infection in humans can range from a flu-like disease to encephalitis, acute flaccid paralysis, and death (Cho and Diamond, 2012). Prevention of WNV infection is limited to the prevention of mosquito bites, including insect repellent, long clothing, and mosquito population control. There is currently no specific therapy or vaccine approved for human use (DeFilette et al, 2012). WNV is a neurotropic flavivirus with a single-stranded positive-sense RNA 1 genome (Donadieu et al, 2013). The ssrRNA genome encodes for three structural proteins; including, the capsid protein (C), the envelope protein (E), and the membrane protein (prM), and at least seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) (Kramer et al, 2007). Upon infection, WNV initially targets primarily dendritic cells and endothelial cells as well as macrophages and monocytes (Pierson et al, 2013). Viral dissemination occurs during the lytic infection of these cells, which leads to increased viremia. Although, the receptor necessary for virion attachment and entry have not yet been fully identified, it is known that WNV gains entry into cells by receptor-mediated endocytosis via clatherin-coated pits and delivery of the RNA genome occurs following membrane fusion within the early endosome (Chambers et al, 1990). After, the viral genome is then released into the cytoplasm where translation of the nonstructural protein occurs as well as RNA synthesis. The viral RNA then associates with the endoplasmic reticulum, with the aid of NS2A, NS2B, NS4A, and NS4B, where replication of the viral genome RNA occurs (Brinton, 2014). Genomic viral RNA is packaged into progeny virions which then bud into the endoplasmic reticulum to form enveloped immature virions. Further maturation occurs in the trans Golgi network, where the NS3-NS2B complex is required for efficient polyprotein processing. Finally, the virions are secreted into the extracellular space by exocytosis. West Nile virions begin to be released from infected cells 8 hours after infection and peak around 24 hours (Cho and Diamond, 2012). WNV is transferred after a mosquito intradermally inoculates a subject. The virus initially replicates in keratinocytes, newly recruited neutrophils, and skin dendritic cells (Langerhans cells) which then migrate to the regional lymph nodes and the bloodstream 2 (Lim et al, 2011). This leads to primary viremia during days 3-4 after infection which can then lead to infection of the kidneys, liver, and spleen (Samuel and Diamond, 2006). In the periphery, infection is countered by the development of an early immune response including type I and II IFN production as was as the effector functions of innate immune cells such as IgM secreting B cells, NK cells, neutrophils, and macrophages (Cho and Diamond, 2012). Within the draining lymph nodes, viral antigen presentation to naïve T cells, complement activation, and antiviral cytokine and chemokine expression occurs. By the end of the first week post-infection, WNV is cleared from the peripheral organs and infection of the central nervous system (CNS) can be observed starting on day five after infection, with the probability of neuroinvasion correlated with the duration and level of viremia (Cho and Diamond, 2012). The mechanisms in which WNV enters