B virus is an alphaherpesvirus, endemic to macaque monkeys, capable of deadly human zoonosis with an 80% mortality rate in untreated cases. The macaque monkey is widely used in biomedical research and the threat of B virus poses an occupational hazard to researchers, veterinarians, and animal handlers. B virus establishes a life-long latent infection in sensory neurons of the peripheral nervous system (PNS) in the natural host. In human infections, B virus readily transits to the central nervous system (CNS) and destroys brain tissues. Identifying immune correlates of B virus infection in the PNS of the natural host is critical in understanding viral lethality in the human host. The lack of an accurate animal model and restrictions on handling potentially infected nervous tissue previously limited studies of B virus infection in macaque ganglia. To address this barrier, a long-lived mixed neuron/glia cell culture model was established from macaque DRG explants using a novel methodology that relied on cellular migration from whole tissues. Utilizing this model, the hypothesis tested was that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation. Chemokines were upregulated in B virus-infected cultures and infected cell media induced leukocyte chemotaxis. Leukocytes were less effectively activated by media from infected cells when compared to media from mock-infected cells. To identify factors responsible for this, focused microarrays were performed and cytokine profiles were quantified from B virus and mock-infected culture supernatants. IL-6 protein levels were significantly reduced in B virus infected cultures. This observation led to the hypothesis that IL-6 downregulation impairs leukocyte activation and, indeed, when IL-6 was added to B virus-infected culture supernatants to control levels, these cultures were far more effective at eliciting leukocyte activation when compared with mock-infected cultures. Collectively, these data support the hypothesis that acute B virus infection of macaque ganglia triggers cellular defense networks to promote leukocyte recruitment and impact leukocyte activation and identifies a potential viral mechanism to impair leukocyte functionality. Additionally, this work presents a novel methodology for establishing long-lived mixed neuron/glia cultures from postnatal/adult macaque DRGs.

Le système nerveux central est isolé de l’environnement grâce à un ensemble de barrières, incluant la barrière osseuse et la barrière hémato-encéphalique. Il existe cependant des zones où ces barrières sont absentes ou affaiblies, et c’est notamment le cas au niveau des nerfs olfactifs qui ont pour origine les neurones présents dans la cavité nasale. Ces neurones participent à la détection des odeurs et leurs axones contactent directement le système nerveux central au niveau des bulbes olfactifs en traversant la lame criblée de l’éthmoïde. Cette « voie olfactive » représente ainsi un site d’entrée privilégié de certains pathogènes vers le cerveau. La muqueuse olfactive, du fait de son positionnement à l’interface entre l’environnement et le système nerveux central, constitue donc une zone particulièrement sensible sur le plan immunologique. Si cette muqueuse est connue pour produire des composants antimicrobiens, les mécanismes cellulaires et moléculaires mobilisés dans le cadre d’infections par des pathogènes respiratoires restent peu décrits.Au cours de ma thèse, nous nous sommes tout d’abord focalisés sur l’interleukine 17c, connue comme puissant médiateur des réponses immunitaires innées épithéliales respiratoires et dont les récepteurs sont exprimés dans la muqueuse olfactive. Nous avons notamment pu montrer qu’elle était mobilisée in vivo dans un contexte mimant une infection virale et qu’elle favorisait le renouvellement épithélial ainsi que l’infiltration de cellules immunitaires. En voulant caractériser son action dans un contexte viral, nous avons été amenés à étudier les effets de deux virus respiratoires sur la muqueuse olfactive (le virus influenza et le virus respiratoire syncytial). Nous avons observé que les deux virus pouvaient infecter efficacement les neurones sensoriels olfactifs, mais avec une charge virale plus élevée pour influenza. A dose équivalente, le virus de la grippe provoque d'importants dégâts dans la muqueuse olfactive mais ne s’établit pas durablement dans la muqueuse, ce qui suggère que ce virus est éliminé très efficacement et rapidement. En nous focalisant sur les processus d'élimination des neurones sensoriels olfactifs infectés, nous avons identifié un nouveau mécanisme antiviral précoce basé sur l'élastase, une enzyme précédemment décrite comme sécrétée par les neutrophiles, principaux acteurs du système immunitaire inné.Dans l’ensemble, ces travaux de thèse mettent en lumière les défenses immunitaires présentes dans la cavité nasale contre les virus respiratoires et apportent de nouvelles perspectives dans le contrôle des virus infectant le système nerveux central par la voie olfactive. / The central nervous system is sheltered from the environment thanks to cranial bones and the blood brain barrier. Some parts of these barriers are weaker, especially around olfactory nerves originating from olfactory sensory neurons in the nasal cavity. These neurons detect odorants and their axons cross the cribriform plate to project directly into the brain at the level of the olfactory bulbs. The cribriform plate is a thin and perforated area of the cranial bones allowing the crossing of the olfactory nerves. This “olfactory pathway” constitutes a privileged entry site for viruses toward the central nervous system. Hence, the olfactory mucosa represents a particularly sensitive area for the immune system. While the olfactory mucosa is known to produce various anti-microbial compounds, the described molecular and cellular mechanism of immune system defenses against viruses remains sparse.The interleukin 17c (IL-17c) is known as an innate immunity response actor in the respiratory epithelium. While its receptors are expressed in the olfactory mucosa, its role in this tissue was unknown. We found that IL-17c is involved in olfactory mucosa responses to Poly(I:C) mimicking virus presence. We observed that nasal instillation of IL-17c accelerated the olfactory mucosa turn-over and induced its infiltration by immune cells. In attempt to characterize the role of IL-17c in a real viral context, we started to focus on the impact of two viruses of the respiratory tract: influenza and the respiratory syncytial virus. We observed that both viruses could effectively infect olfactory sensory neurons but with a higher virus load for influenza. Indeed, at similar doses, influenza induced important damages in the olfactory mucosa but was not present, indicating that influenza virus is very effectively and rapidly eliminated from the olfactory mucosa. By focusing on the elimination processes of infected olfactory sensory neurons, we identified a novel early anti-viral mechanism based on elastase, an enzyme previously described as secreted by neutrophils, main actors of the innate immunity system.Overall, my PhD results provide new insights on the immune defenses present in the olfactory mucosa against respiratory viruses and could bring new perspectives in the control of virus infecting the central nervous system.

While the success of combination antiretroviral therapy (ART) has extended the lifespan of people with human immunodeficiency virus (HIV)(PWH), approximately half of PWH on suppressive ART will experience HIV-associated neurological dysfunction. While ART has decreased the incidence of severe neurological disease and dementia in PWH, the incidence of milder neurological and cognitive complications remains stable. Despite the frequency of HIV neurological disease, contributing factors and inflammatory pathogenesis are difficult to observe in PWH over time. Extracellular vesicles (EVs) constitute an understudied method of intercellular communication and molecule delivery in viral infections. EVs carry inflammatory mediators to areas of the periphery during ART suppression but are understudied in the brain. In this dissertation, we use a biologically relevant simian-human immunodeficiency virus (SHIV)-infected non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) to investigate the formation of a myeloid viral reservoir, inflammation during ART-mediated viral suppression, and the roles of myeloid EVs in persistent SHIV neuropathogenesis.In Chapter 2, we characterized viral and immune persistence in the CNS using SHIV.D, a novel model of HIV-1 in rhesus macaques (RM). Here, we demonstrate viral replication in the brain and neuropathogenesis after ART in RM using novel macrophage-tropic transmitted/founder (TF) SHIV.D.191859. Using quantitative immunohistochemistry (IHC) and DNA/RNAscope, we demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These findings were replicated in a second cohort of RM necropsied after 6 months of suppressive ART. We concluded that TF SHIV.D models HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques, and is a biologically relevant model to study HIV neuropathogenesis. In Chapter 3, we investigated EVs in a SHIV.D/RM model of HIV. To determine the potential roles of different cell-derived EV populations in SHIV/HIV neuropathogenesis, we developed a method to investigate changes in the cellular origin of EVs in vivo in RM. EVs that are released by neural and glial cells into the blood circulatory system can serve as biomarkers for injury and illness as well as give insight into CNS dysfunction and other disease processes in a non-invasive manner. Here, we present a bead-free multiparameter conventional flow cytometry method to phenotype, characterize, and determine cellular origin of plasma extracellular vesicles. Using RM plasma and two four-parameter panels, we identified the following subsets of plasma EVs: tetraspanin CD81+, CD11b+ macrophage-derived, CD14+ monocyte-derived, TMEM119+ microglia-derived, CD171+ neuron-derived, CD3+ T cell-derived, and CD31+ endothelium-derived EVs. EVs were isolated from RM plasma before infection with SHIV.D, during acute viremia, and after ART suppression. EV flow cytometry on these samples revealed a significant increase in TMEM119+ microglial EVs and CD171+ neuronal EVs in RM plasma during viremia and ART suppression. In Chapter 4, we investigate myeloid-specific EVs in an in vitro SHIV.D/RM model. Using primary RM monocyte-derived macrophages (MDM), we determined that MDMs increased EV production after SHIV.D infection. Whole proteomic analysis was conducted on EVs from SHIV-infected and uninfected MDM. Gene ontology pathway analysis and gene set enrichment analysis reveal pathways associated with overrepresented proteins in myeloid EVs. Finally, differential abundance analysis demonstrated that myeloid EVs isolated from SHIV.D-infected MDMs carried significantly increased levels of neuropathogenic and inflammatory proteins. Altogether, these studies improve our understanding of SHIV.D viral persistence and persistent neuropathogenesis in the RM brain as a model for HIV-1 chronic neuropathogenesis and describe the contribution of myeloid EVs to neurological disease during SHIV/HIV infection. / Biomedical Sciences