The Role of Vimentin and the NLRP3 Inflammasome in Influenza A Infection A Senior Thesis Presented to The Faculty of the Department of Molecular Biology, Colorado College By Maggie Turner Bachelor of Arts Degree in Molecular Biology April 28, 2017 ________________________ Dr. Olivia Hatton Primary Thesis Advisor __________________________ Dr. Sara Hanson Secondary Thesis Advisor ABSTRACT Infection with Influenza A virus (IAV) continues to cause morbidity and mortality in children across the globe, in part due to the excessive inflammatory response during pathogen clearance. Using a murine model of IAV infection, this study focuses on the role of the innate immune system in IAV infection through the scope of NLRP3 inflammasome protein activation and assembly. We were able to detect the presence of the NLRP3-inflammasome target proteins ASC, Pro-caspase-1, NLRP3, RIG-1, and IL- 1β in both juvenile and adult mice. Notably, we found significantly increased levels of ASC and RIG-1 protein in juveniles compared to adults. This suggests that ASC and RIG-1 are related to the observed excessive inflammatory response upon IAV infection in juveniles. To examine NLRP3-inflammasome assembly, we created multiple mutant constructs of the inflammasome scaffolding protein Vimentin as well as vimentin-/- cells. IL-1β production was greatly inhibited in vimentin-/- cells compared to the wild type upon activation of the NLRP3 inflammasome. The same trend was seen when only the head region of the protein was present. We suggest that the intermediate filament (IF) Vimentin serves as a protein scaffold for inflammasome assembly, and that expression of Vimentin is a necessary checkpoint in the innate immune response. Furthermore, we propose the Vimentin-targeting drug withaferin A as a potential treatment for IAV induced acute lung injury. Further studies are to be conducted to compare age-related differences in expression of other inflammasome-related proteins as well as the effect of other Vimentin constructs on IL-1β production. A better understanding of age-related differences in innate immune signaling as well as the overall structure of the 2 inflammasome will be essential to improve care of and treatment for this high-risk population. INTRODUCTION Influenza A virus (IAV) is a highly contagious RNA virus that causes upper and lower respiratory tract infections in humans, resulting in 200,000 hospitalizations and 36,000 deaths per year in the United States alone (Nair et al., 2011). Furthermore, severe influenza infection is seen worldwide and is estimated to cause 3 to 5 million cases and 250,000 to 500,000 deaths annually (Coates et al., 2015). Mild IAV infection is generally characterized by symptoms such as fever, headache, and sore throat. Severity of infection increases as infection progresses to the lower respiratory tract where coughing, wheezing, breathing impairment, and respiratory failure can arise. IAV infection can cause alveolar damage, capillary damage, and pulmonary edema or fluid accumulation in the lungs that leads to acute lung injury (ALI), which is often one of the biggest contributors to flu- related deaths (Taubenberger and Morens, 2008). Furthermore, IAV infection is often seen to precede pneumonia diagnoses, suggesting that the IAV virus contributes to the development of this disease. As an RNA virus without proofreading mechanisms, IAV mutates its surface proteins hemagglutinin (HA) and neurominidase (NA) rapidly, causing the emergence of novel strains each year that can result in recurring epidemics and pandemics with significant attributable morbidity and mortality. The influenza pandemic of 1918, for example, resulted in approximately 100 million deaths (Taubenberger and Morens, 2008). Flu shots aim to predict these viral mutations to protect the general population before infections begin; however, genetic recombination and genetic drift can make these 3 predictions extremely difficult. Thus, understanding the infection mechanism of IAV could unveil potential antiviral treatment targets to more successfully keep infectious spread of IAV under control. Although IAV infection is a global concern, the greatest burden of IAV infection falls on children, the elderly, and those with chronic medical conditions. Specifically, 40% of children under the age of 5 are affected by IAV infection annually in the United States, with numbers reaching 90 million worldwide (Nair et al., 2011). Of these 90 million children, 20 million will develop lower respiratory tract infections and 1 million progresses to life-threatening stages of infection. Approximately half of these cases occur in children that would otherwise be considered healthy, whereas severely infected adults more often have a preexisting condition that put them at an increased risk of death (Nair et al., 2011). Such clinical observations have also been seen in various animal models (Sun et al., 2010). In mouse models of IAV-induced pneumonia, increased mortality and pulmonary injury are seen in juvenile mice compared with adults upon first exposure to the virus (Coates et al., 2015). These results suggest age-related differences in the pathogenesis of IAV infection that leave children at particularly high risk of severe disease. However, little research has been conducted on the molecular reasons for variations in disease severity. Both anatomical and molecular mechanisms have been proposed to explain these observed age-related differences in IAV infection. From an anatomical standpoint, pediatric airways are smaller and thus prone to obstruction by the fluids produced upon infection, putting greater respiratory stress on the lungs as compared to adults (Coates et al., 2015). Infants also have a more cartilaginous chest wall that increases the likelihood 4 of lung collapse and creates difficulty in the reflation process (D’Agelis et al., 2011). The combination of these anatomic factors may explain the increased severity of IAV symptoms during infection in children, but they fail to explain why high numbers of healthy children develop severe infections when the same trend is not seen in adults. Research into molecular mechanisms suggests a shift in the intensity and mode of immune response with age, as young children are much more reliant on their innate immune responses as the adaptive immunity is developing (Prendergast et al., 2012). This excessive inflammatory response could then cause harmful tissue damage or predispose children to secondary bacterial infections. In this study, we will explore the innate immune response to IAV infection as a molecular mechanism underlying age-related differences in IAV infection. Ultimately, we aim to uncover specific therapeutic targets to improve vaccine design to support these at risk populations. IAV Detection Mechanism Influenza A virus is a typically single-stranded, enveloped RNA virus with 8 RNA segments that encode 12 proteins. However, the virus can also be found to contain double-stranded RNA (Son et al., 2015). IAV first targets the respiratory epithelium by binding to sialic acid receptors on the host cell surface with the viral HA protein, triggering endocytosis. The virus can survive in the endosome until acidification occurs and viral components are released into the host cell cytoplasm (Samji, 2009). These components are transported to the nucleus where replication occurs. The new virons are assembled and leave the cell via budding, where the NA surface protein cleaves the viron connection to the host cell and infectious spread continues (Samji, 2009). 5 Once inside the cell, the virus may be detected at any point in its replication process. Researchers are making significant progress in elucidating various mechanisms of IAV detection by the host. One mode of IAV detection involves pattern recognition receptors (PRRs). PRRs are mainly found on antigen presenting cells (APCs) such as macrophages and dendritic cells, but they have also been seen in other immune and non- immune cells such as epithelial cells, endothelial cells, and fibroblasts (Takeuchi and Osamu, 2010). The main role of PRRs is to detect invading pathogens or foreign microorganisms to alert other immune cells and initiate a defense response. Four families of PRRs have been discovered to date. These families include the transmembrane protein Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), as well as the cytoplasmic protein Retinoic acid-inducible gene (RIG)-I-like helicase (RLH) receptors and NOD-like receptors (NLRs) (Takeuchi and Osamu, 2010). Each of these families has been further divided into subtypes. The main immune cells targeted in the case of IAV infection are macrophages and epithelial cells. IAV infection in these cell types often begins in the lungs, as the virus is easily transmitted by inhalation of virus-laden aerosols (Brankston et al., 2007). Thus, pulmonary macrophages and epithelial cells play a large role in the first line of detection of and defense against the virus. Infection with IAV results in activation of three of the four families of PRRs in pulmonary macrophages and epithelial cells that initiate a defense mechanism against the virus: the NLRs, the RLH receptors and the TLRs (Iwasaki and Pillai, 2014). PRRs function similarly by detecting pathogen associated molecular patterns (PAMPs), but the location of these PRRs differs by class as mentioned above. TLRs recognize PAMPs on the IAV surface, while Nod-like receptors (NLRs) detect IAV RNA-related PAMPs in 6 the cytoplasm. Eleven TLRs in total have been