Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Fall 1-1-2017 The neonatal anti-viral response fails to control measles virus spread in neurons despite interferon- gamma expression and a Th1-like cytokine profile Priya Ganesan Follow this and additional works at: https://dsc.duq.edu/etd Part of the Developmental Neuroscience Commons, Immunology and Infectious Disease Commons, and the Virology Commons Recommended Citation Ganesan, P. (2017). The neonatal anti-viral response fails to control measles virus spread in neurons despite interferon-gamma expression and a Th1-like cytokine profile (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/ 230 This One-year Embargo is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. THE NEONATAL ANTI-VIRAL RESPONSE FAILS TO CONTROL MEASLES VIRUS SPREAD IN NEURONS DESPITE INTERFERON-GAMMA EXPRESSION AND A TH1-LIKE CYTOKINE PROFILE A Dissertation Submitted to the Graduate School of Pharmaceutical Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Priya Ganesan December 2017 Copyright by Priya Ganesan 2017 THE NEONATAL ANTI-VIRAL RESPONSE FAILS TO CONTROL MEASLES VIRUS SPREAD IN NEURONS DESPITE INTERFERON-GAMMA EXPRESSION AND A TH1-LIKE CYTOKINE PROFILE By Priya Ganesan Approved November 12, 2017 Lauren A. O’Donnell, Ph.D. Paula A. Witt-Enderby, Ph.D. Associate Professor of Pharmacology Professor of Pharmacology Graduate School of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences Duquesne University, Pittsburgh, PA Duquesne University, Pittsburgh, PA (Committee Chair) (Committee Member) Wilson S. Meng, Ph.D. Dr. Christopher Surratt Associate Professor of Pharmaceutics Professor of Pharmacology Graduate School of Pharmaceutical Sciences Graduate School of Pharmaceutical Sciences Duquesne University, Pittsburgh, PA Duquesne University, Pittsburgh, PA (Committee Member) (Committee Member) Dr. Kerry Empey James K. Drennen, III, Ph.D. Assistant Professor Associate Dean and Associate Professor Graduate School of Pharmaceutical Sciences of Pharmaceutics University of Pittsburgh, Pittsburgh, PA Duquesne University Graduate School of (Committee Member) Pharmaceutical Sciences J. Douglas Bricker, Ph.D. Dean, Duquesne University School of Pharmacy and the Graduate School of Pharmaceutical Sciences iii ABSTRACT THE NEONATAL ANTI-VIRAL RESPONSE FAILS TO CONTROL MEASLES VIRUS SPREAD IN NEURONS DESPITE INTERFERON-GAMMA EXPRESSION AND A TH1-LIKE CYTOKINE PROFILE By Priya Ganesan December 2017 Dissertation supervised by Dr. Lauren A. O’Donnell Neonates are highly susceptible to infections in the central nervous system (CNS) and have a greater risk of viral infections and encephalopathies. Neurotropic viral infections can lead to blindness, hearing loss and neurological deficiencies such as cognitive impairment, epilepsy, and even death in the neonatal and pediatric populations. Viral infections also are hypothesized to indirectly contribute to neurodegenerative and neuropsychiatric diseases such as Schizophrenia and Parkinson’s disease later in life due to early neuronal damage or stress. Many diverse viruses are capable of invading the neonatal CNS including Borna Disease Virus, Coxsackievirus (CV), Herpes simplex viruses (HSV), and measles virus. Although, we understand that many viruses can cause CNS disease, the mechanisms of viral pathogenesis in the brain and the character of the neonatal anti-viral immune response are not well understood. However, it is hypothesized that neurological damage results from the combined effect of the virus and the immune response. iv Therefore, it is critical to develop immune-mediated strategies to promote viral clearance from the CNS while preventing neuronal damage or loss. This remains a challenge during neonatal CNS infections because of the uniquely immature nature of the neonatal immune system and the sensitivity of developing neurons to inflammation. In order to better understand how the neonatal immune response behaves in the brain, we use neurotropic measles virus (MV) as a model to understand the deficits in the neonatal immunity. Measles is a single-stranded, negative-sense RNA virus that is highly contagious in humans. Typical infection involves inhalation of infected respiratory droplets, infection of dendritic cells and macrophages in the respiratory tract resulting in transient immunosuppression, and a characteristic fever and rash. However, in some cases, MV also causes severe neurological diseases such as Post-infectious encephalomyelitis (PIE), Subacute sclerosing panencephalitis (SSPE), and Measles inclusion body encephalitis (MIBE). Currently, there is no cure for these MV-related neurological conditions, which occur overwhelmingly in newborns and children. Thus, the goal of this project is to define how neonatal immunity responds to MV infection in the unique microenvironment of the brain. The role of interferon-gamma (IFN), a key anti-viral cytokine in controlling adult CNS infections, was explored during a neuronally-restricted MV infection in the neonatal brain. We hypothesized that neonatal mice would be deficient in either IFN production or in the infiltration of IFN-producing immune cells in the brain. In order to address this question, we utilized the CD46+ mouse model, in which the human CD46+ receptor for MV is expressed only in mature neurons of the CNS. We explored the differences in the neonatal immune response, where the mice succumb to MV infection, and compared that to the CD46+ adults, which successfully control MV and survive. Our findings suggest that IFN, which is critical for viral control and survival in v adults, only delays mortality in CD46+ neonates. The neonatal brains also show the infiltration of natural killer cells, neutrophils, infiltrating monocytes and T cells in an IFN-independent manner, all of which are capable of contributing to the IFN pool. However, neonates and adults differentially express pathogen recognition receptors (e.g. Toll-like receptors) and Type I interferons during infection in the CNS, which suggests that the initial recognition of the virus by the immune system may differ in an age-dependent manner. Both neonates and adults expressed IFN, CXCL10, IL-1, and IL-1RA, among other cytokines/chemokines. Regardless, CD46+ neonates succumb to infection despite mounting a Th1-like, but apparently defective, inflammatory response. We further explored whether there are age-dependent differences in IFN signalling given that both ages of mice expressed this critical cytokine. Both neonatal and adult CD46+ mice express similar levels of IFN but only adults show robust induction of the IFN- responsive genes CIITA and CXCL9. This suggests that IFN signaling may be defective in the induction of IFN-responsive genes in neonates compared to adults. To dissect the role of individual components of the immune system, we utilized CD46+ mice crossed to specific immune knockouts: CD46+/IFN-KO mice, which lack IFN, and CD46+/RAG2-KO mice, which lack mature B and T cells. We found that neonates lacking IFN succumbed more rapidly than wildtype CD46+ mice, while neonates lacking mature B and T cells showed delayed morbidity and mortality. Neonates without IFN show high infiltration of neutrophils and inflammatory monocytes but similar numbers of NK cell infiltration compared to CD46+ neonates. CD46+/IFN-KO neonatal brains also show high infiltration of CD4 and CD8 T cells at the later stages of MV infection. Additionally, IFNα is significantly upregulated in the absence of IFN in the brain post-MV infection. Thus, compensatory cytokines and high immune vi cell infiltration may contribute to uncontrolled inflammation and earlier death in CD46+/IFN-KO neonates. CD46+ mice deficient in T-cells and B-cells (CD46+/RAG2-KO) show prolonged survival and mount a robust IFNβ response post-MV infection. This suggests that the adaptive immune response may be detrimental during the neonatal period, potentially leading to greater tissue damage. Additionally, CD46+/RAG2-KO neonates alone upregulate unique genes such as bone morphogenetic proteins (BMPs), which may mediate neuroprotection. Thus, these results suggest age-dependent expression of cytokine profiles in the brain and distinct dynamic interplays between lymphocyte populations and cytokines/chemokines in MV-infected neonates. vii DEDICATION To my parents, Savithri and Ganesan Rajamani, and my sister Gowri Ganesan whose continuous support, sacrifices, love and strength throughout my life made this possible. viii ACKNOWLEDGEMENTS I thank Dr. Lauren A. O’Donnell, for giving me the opportunity to purse research in her lab, for her constant support, enthusiasm, and for being an amazing mentor. Your advice and patience have been pivotal to my journey as a researcher. I am indebted for all the lessons I learnt from you during this journey. I would like to thank my committee members Drs. Paula Witt-Endery, Wilson Meng, Chris Surratt, and Kerry Empey for their support and invaluable guidance. I want to especially thank Dr. Witt-Enderby and Dr. Meng for continuous encouragement during tough times and job search. I would like to thank Sanket Anaokar and Harsha Sree Pulugulla for their help with qRT-PCR and PCR. I also