Pascal Lavoie

The full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.

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Premature birth is a major contributor to under-five mortality, causing approximately 1 million deaths every year. This vulnerability stems from the high susceptibility of neonates, particularly those born prematurely, to severe life-threatening infections. The overarching goal of my thesis work was to study the immunological basis for preterm neonates’ vulnerability to infections. It was previously known that monocytes isolated from preterm cord blood show suppressed responses to innate immune activation. In Chapter 2, we employed untargeted transcriptomics comparing monocytes at rest and after LPS stimulation, from neonates born preterm (37 weeks gestation) and from adults. Results establish cellular energy metabolism as a major regulator of innate immune responsiveness during fetal life, perhaps to purposely limit overt inflammation in utero. Additionally, our experiments suggested a central role for mTORC1, and its negative regulator DDIT4L in suppressing innate immune responses in preterm monocytes. In Chapter 3, I developed a monocytic cell line model to study the impact of DDIT4L on innate immune function. DDIT4L overexpression limited protein synthesis, cellular proliferation and LPS-induced cytokine production. Additionally, experiments in primary neonatal monocytes revealed developmental changes in mitochondrial function. Lastly, data linking reduced DDIT4L expression to more severe inflammatory lung disease in preterm neonates point towards a putative role in preventing inflammatory-mediated tissue damage in neonates. Beyond the immediate postnatal period, preterm infants remain particularly vulnerable to respiratory viral infections. In Chapter 4, I studied infants at high-risk for respiratory syncytial virus (RSV) infections. I showed that most infants have been exposed to RSV, as evidenced by increased IgM titers, Fc receptor binding and antibody-mediated phagocytosis by the end of their first winter season. Overall, my study suggests that most high-risk preterm infants develop antibody responses against RSV by one year of age, in the absence of overt clinical signs. This challenges the dogma that preterm infants are unable to fight respiratory illnesses without getting sick and provide insights into the highest period of vulnerability to these viruses. In sum, my work provides fundamental insights into the maturation of immune responses in the first year of life in premature babies.

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Newborns lack educated adaptive immunity and therefore rely on innate immune defenses to protect themselves from infections. Premature babies are at high risk of severe infections due to the immaturity of their immune system. Umbilical cord blood is readily accessible to study the premature neonate’s immune system, but it does not capture important maturation events that may occur during the neonatal period. The overall goal of my PhD was to investigate the immune system of premature infants during the neonatal period.In chapter 2, I examined the whole blood response to immune stimulation of two prototypic Toll-like receptors: TLR4 and TLR7/8, in preterm infants aged 1-42 days in the neonatal intensive care unit. I identified major functional deficits in pro-inflammatory cytokine levels compared to term cord blood, which were not due to a lack of immune cells. These findings support previous observations made from preterm cord blood studies. To the best of our knowledge, we were the first at the time to study functional TLR responses during this critical development period.In chapter 3, I used RNA-sequencing methods to investigate how these responses are developmentally regulated. Using deconvolution algorithms, I found that preterm cord blood is distinct from preterm postnatal blood and term cord blood, with a gradual transition from an immature immune system enriched in hematopoietic stem cells, myeloid and erythroid progenitor cells, to a more mature immune cell composition in term cord blood. I also provide the first data directly linking immaturity of the preterm immune system to the risk of sepsis. In chapter 4, I examined innate-like characteristics of naïve CD4 T cells in term cord blood, as a means whereby newborns may compensate for a lack of adaptive memory immune cells. I demonstrate that activation of the antigen presenting cell is important to enhance innate-like IL-8 production in naïve CD4 T cells. The work within this thesis further characterized the immune function of newborns across the gestational age spectrum, providing novel insights into their immune development and factors underlying the high susceptibility of premature infants to infection.

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Innate immunity is the first line of defense against infection, and is particularly important in newborns, as they lack immunological memory. During fetal development, innate immunity must be carefully regulated to prevent miscarriage. In contrast, upon reaching term, the innate immune system of the newborn must rapidly become operational to provide protection against exposure to the extra-uterine microbial environment. Human immune responses are generally highly variable among individuals of all ages. According to current models, immune reactivity is highly influenced by an individual’s genetic make-up. However, studies in my thesis suggest that immune reactivity is also drastically influenced by non-genetic factors. My overall goal during my PhD was to understand the mechanisms regulating innate immune reactivity in humans, across the development spectrum, to better understand why some individuals are more susceptible to significant clinical infections. I hypothesized that examining responses at the systems level would inform me on how innate immune reactivity is regulated throughout life.Candida species (spp) are common neonatal pathogens. Despite the clinical importance of these pathogens, relatively little is known about the maturation of anti-fungal innate immune defenses in newborns. In Chapter 2 of my thesis, I examined innate immune responses to Candida spp. in preterm infants. I discovered that cellular metabolism plays a major role in regulating immune reactivity during fetal life, via regulation of protein translation.In Chapter 3 of my thesis, I applied similar methods to understand the factors driving the variability in innate immune responses in healthy adults. As expected, I found a large diversity in immune responses between individuals. Surprisingly, some of these protein level responses were largely independent of gene transcription events. I provide evidence that metabolic pathways also modulate immune reactivity in healthy adults.Overall, my findings enhance our understanding of the factors regulating immune responses in the highly genetically diverse human population, providing insight into the development of these pathways in the late fetal/early neonatal period, and support a major role for metabolism in regulating immune reactivity in the general population and during ontogeny.

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Almost four million neonates die of infectious and prematurity-related causes across the world annually. The innate immune system provides evolutionarily ancient first-line protection against most microbial pathogens. In contrast, the adaptive immune system is capable of developing an immunological memory that provides enhanced protection in vertebrates. A mechanistic understanding of the maturation of the human preterm neonatal immune system is lacking and this may limit our ability to develop more age-appropriate immunological therapies. In Chapter 1, I analyzed prototypic anti-microbial receptor responses in a clinically well-characterized cohort of premature infants to provide evidence that responses develop asynchronously and follow a developmental pattern that is independent of perinatal factors linked to the premature delivery. In Chapter 2, I dissected molecular rate-limiting steps along a major inflammatory pathway leading to the production of the interleukin-1β (IL-1β) cytokine, across development. The IL-1β cytokine is particularly important as its production serves to amplify innate immune responses. I show that premature neonates born early in the third trimester of gestation lack IL-1β responses due to a lack of activity in the caspase-1 enzyme. In the final chapter, I developed an efficient purification method to study the phenotype of neonatal invariant Natural Killer T (iNKT) cells. Using this method, I show that neonatal iNKT cells display heightened proliferative capacity compared to conventional T cells, consistent with an innate-like phenotype. Altogether, my work provides important insights into mechanisms and developmental characteristics of the fetus and early newborn immune system. This work forms the basis for future studies aimed at understanding how functional characteristics of the neonatal immune system can be therapeutically modulated to prevent neonatal infections.

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