Maternal obesity is a significant risk factor for obstetric complications such as preterm labor, preeclampsia, and chorioamnionitis. Furthermore, pregnant women with obesity are more prone to infections during pregnancy and following postpartum procedures, suggesting a compromised immune system. We therefore hypothesized that the immune adaptations associated with a healthy gestation are disrupted with obesity. Indeed, our analyses suggest that a healthy pregnancy was associated with systemic changes indicative of innate immune activation. Specifically, monocyte responses to LPS were increaed with gestational age in lean subjects, with concomitant enhancement of chromatin accessibility, which poised cells for a greater response. This trajectory was disrupted in pregnant women with obesity resulting in attenuated monocyte responses to LPS. Furthermore, epigenetic, metabolic, and functional measurements strongly support the induction of immunotolerance with pregravid obesity. These findings may explain the increased susceptibility to infections observed during pregnancy and following cesarean delivery in women with obesity.

Chronic alcohol drinking adversely affects human health and is a significant risk factor for numerous conditions including cardiac and liver disease, cancer, and sepsis. Furthermore, patients who consume alcohol chronically have increased susceptibility to viral and bacterial infections, suggesting compromised immune function. Studies to date have shown a disproportionate effect of alcohol on innate immune cells, notably monocytes and tissue resident macrophages, but the mechanisms by which chronic alcohol consumption leads to immune cell dysfunction remains poorly understood. Moreover, whether alcohol and its metabolic products are acting on immune cells in the periphery or on their progenitor cells in the bone marrow has yet to be investigated. This work aims to address the broad hypothesis that chronic alcohol drinking perturbs the epigenetic, transcriptomic, and functional landscapes of macrophages, monocytes, and their progenitor cells in the bone marrow. To that end, we utilized a non-human primate model of chronic alcohol self-administration, which is both physiologically relevant and accurately recapitulates human drinking behavior. After 12 months of daily alcohol drinking, we profiled three major cell populations from these animals: tissue-resident macrophages from the alveolar space, monocytes from the blood, and progenitor cells from the bone marrow. For each of these populations, we integrated large scale epigenomic and transcriptional data with cell-type relevant functional assays to explore alcohol-induced perturbations of immune function and the mechanisms behind them. In alveolar macrophages, we show increased baseline activation transcriptionally and epigenetically with alcohol, poising the cells towards heightened inflammatory responses to lipopolysaccharide (LPS) and respiratory syncytial virus (RSV). This was accompanied by increased ROS production but decreased ability to phagocytose bacteria. scRNA-Seq revealed oxidative stress and oxidative phosphorylation signatures, which was coupled with increased mitochondrial activation. These findings show reprogramming of the alveolar macrophages towards heightened inflammation and stress coupled with reduced functional capabilities with alcohol, which could lead to reduced ability of these cells to identify and clear infections. This may explain the increased susceptibility of patients who chronically consume alcohol to respiratory pathogens. Frequency of blood monocytes were increased in circulation with alcohol and showed a similar phenotype of hyper-inflammatory protein response to LPS. This was also complemented epigenetically with increased chromatin accessibility of loci encoding cytokine response genes at baseline state, suggesting alcohol is priming these cells for the inflammatory response. Moreover, similarly to alveolar macrophages, scRNA-Seq of monocytes revealed shifts towards oxidative stress and HIF1A induction indicating a broad impact of alcohol and its metabolites on circulating and tissue resident cells. As monocytes are short-lived cells in the blood, we determined that this hyper-inflammatory phenotype persisted after a one-month abstinence period from alcohol suggesting bone marrow progenitor involvement. Indeed, CD14+ cells in the bone marrow broadly expressed markers of stress and inflammation with alcohol. Assessment of the function of CD34+ progenitors suggested skewing of colony differentiation towards granulocyte/monocyte colonies and increased production of hyper-inflammatory monocytes in culture. scRNA-Seq of the CD34+ cells showed increased inflammatory markers in myeloid lineage subsets and broad signatures of oxidative stress with alcohol. Taken together, these data suggest an impact of alcohol on myeloid cell production and function at multiple levels: in the bone marrow, in circulation, and in the lung.

In the United States, approximately 64% of women of childbearing age are either overweight or obese. Maternal obesity during pregnancy is associated with a greater risk for adverse maternal-fetal outcomes. Adverse health outcomes for the offspring can persist into adulthood, increasing the incidence of several chronic conditions including cardiovascular disease, diabetes, and asthma. Since these diseases have a significant inflammatory component, these observations are indicative of perturbation of the normal development and maturation of the immune system of the offspring in utero. This hypothesis is strongly supported by data from several rodent studies. In this thesis we examine the impact of maternal obesity on the neonatal immune system.

Varicella Zoster Virus is neurotropic alpha herpesvirus that causes varicella (chickenpox) and establishes latency in the sensory ganglia. Reactivation results in herpes zoster (HZ, shingles), a painful and debilitating disease that affects 1 million people in the US each year. FDA approved vaccines reduce the incidence of both varicella and HZ; however, they induce short-lived immunity and are contraindicated for individuals who are immunosuppressed. The development of more efficacious vaccines has been hampered by the fact the VZV is a strictly human pathogen which has hindered the development of a robust translational animal model. Previous studies from our laboratory have shown that intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV), the homolog of VZV, recapitulates the hallmarks of VZV infection in humans. In this dissertation, we use this animal model to investigate the host-pathogen interactions in the lung and sensory ganglia (sites of primary infection and latency) as well as the role that T cells play in SVV tropism and reactivation. First, we show that SVV infection causes severe lung damage characterized initially by significant immune infiltration, an up-regulation of genes involved in antiviral immunity and a down-regulation of genes involved in lung development. This robust host response correlates with a decrease in viral loads and is then followed by an increase in genes important for tissue repair. Next, we show that SVV DNA and transcripts are detected in the ganglia 3 days post-infection (DPI), before the development of varicella. Interestingly, CD4 and CD8 T cells were also shown to infiltrate the sensory ganglia 3 DPI; before the development of cell mediated immunity, which suggests a role for T cells in SVV tropism into the ganglia. SVV acute infection of the ganglia induces robust gene expression changes indicative of an innate antiviral immune response along with a down-regulation of genes that play a role in the nervous system function. Interestingly, genes important for neuron development remained down-regulated 100 days post infection suggesting that SVV latency leads to a long-lived remodeling of the ganglia transcriptional profile. Next, given the crucial role that T cell play in SVV dissemination, we characterized the transcriptional changes that SVV infection induces within them. This analysis revealed that SVV alters expression of several genes that may support viral replication and dissemination. Lastly, we report that the decline of both CD4 and CD8 T cell immunity followed by stress can lead to SVV reactivation, which also causes long-lasting gene expression changes in the ganglia transcriptome, indicative of neuronal damage. These findings provide novel insights into the host pathogen interactions of VZV during acute infection and guide efforts toward more efficacious vaccines and therapeutics.

Diarrheal disease is the second leading cause of death in children under five, killing over 500,000 children every year, and disproportionally impacting low- and middle-income countries (LMIC). Additionally, diarrheal diseases contribute to long-lasting morbidities for millions of others reducing quality of life through malnutrition, poor growth rates, and reduced vaccine efficacy. A wide range of enteric pathogens (Rotavirus, Norovirus, Campylobacter, Shigella, Giardia, etc.) are responsible for diarrheal diseases in children under five. However, these known pathogens only account for 40-50% of cases in LMIC leaving a substantial number that cannot be attributed to a specific pathogen and making targeted intervention impossible. It also remains unclear why children who grow up in the same environment have different susceptibility to enteric pathogens. One potential explanation is that the resident commensal microbes, which are largely inherited from mothers at least early in life, play a protective or detrimental role in disease susceptibility. Either through direct interaction with pathogens or influence on immune development. However, the role of the commensal gut microbiome in the susceptibility to and severity of diarrheal disease is unclear. This is in part due to high variability within the “Healthy” human gut microbiome, a lack of longitudinal studies that include pre-symptom samples, and difficulty in collecting samples in resource-limited settings. In this dissertation, we describe and utilize an infant macaque model of naturally acquired diarrheal disease to investigate questions that are either difficult or impossible to study in humans. We use a combination of clinical, microbiological, and immunological assays on primary samples to describe novel findings regarding the causes and consequences of diarrheal disease in infants. We first show that the gut microbiome of infant macaques develops predictably and with similar features to humans in LMIC suggesting that our model could provide valuable insight into a disease that primarily impacts the developing world. Second, using samples from host sites not accessible in humans we found that growth faltering independent of acute diarrhea was associated with immune dysfunction in the colon and mixing of small and large intestine microbial communities. Next, we identify and genomically characterize four previously unknown and yet to be cultured campylobacter species that are likely to contribute to idiopathic disease in humans. Finally, we examine both the fecal and vaginal microbiome of reproductive-age female macaques to shed light on the dynamics of these communities while exploring their potential impact on infant health. Together these experiments have yielded valuable insight into the causes and consequences of diarrheal disease and have led to potential targets for both pathogen detection and treatments.

Ebolavirus (EBOV) is amongst the deadliest human viruses that cause hemorrhagic fever. The Zaire species (ZEBOV) is responsible for most outbreaks and highest mortality rates, with the newly identified variant, Makona (ZEBOV-Makona) responsible for the recent epidemic in West Africa. The high virulence of ZEBOV is attributed to its ability to subvert innate immune responses, notably type I interferon (IFN) production, and causes excessive inflammation. However, studies investigating how ZEBOV modulates innate immunity has mostly been extrapolated from in vitro experiments. Therefore, ZEBOV-host interactions in vivo are not fully understood. In this dissertation, I address this gap in knowledge by investigating longitudinal host responses in peripheral blood collected from cynomolgus macaques infected with ZEBOV using both immunological assays and transcriptomic analysis. We first comprehensively analyzed ZEBOV-Makona pathogenesis and discovered correlations between: increased IFNα and interferon stimulated genes (ISGs); higher inflammatory transcripts and non-classical monocytes; decreased expression of lymphocyte related genes and lymphopenia. This data also showed ZEBOV-Makona infection results in less severe and delayed appearance of clinical symptoms compared to previously identified variants. Second, transcriptomic analysis of purified immune cell subsets isolated from ZEBOV-Makona infected animals revealed that monocytes, but not T or B cells, support ZEBOV infection. Importantly, monocytes are one of the key players in mediating ZEBOV pathogenesis by initiating events that contribute to the characteristic symptoms of Ebola virus disease (EVD). Next, gene expression changes in animals infected with a recombinant ZEBOV containing mutations in VP35, which abrogate its ability to inhibit type I IFN, were indicative of a regulated innate immune response and successful development of adaptive immunity, which effectively immunized these animals against subsequent ZEBOV-Makona challenge. This data highlights the importance of type I IFN signaling and VP35 as a key virulence factor. Lastly, a promising vaccine candidate, rVSV-ZEBOV, has been shown to provide rapid protection to macaques against ZEBOV challenge. Transcriptomic analysis of animals vaccinated 7 or 3 days before challenge indicate that early expression of antiviral ISGs engendered by the vaccine can rapidly protect animals while paving the way for development of an adaptive immune response.

Chronic heavy alcohol consumption results in intestinal injury characterized by increased permeability, dysbiosis, nutrient malabsorption, increased risk of colorectal cancer, and functional alterations in mucosal immune cells. Studies have implicated dysregulation of tight junction proteins as well as quantitative and qualitative changes in gut microbiota as key contributors to gut injury. Recently, we showed using a macaque model of voluntary ethanol self-administration that moderate consumption resulted in a more robust T-cell and antibody vaccine response to Modified Vaccinia Ankara (MVA), while heavy drinkers generated blunted T-cell and antibody response compared to controls. First, we show that chronic alcohol consumption modulates host defense and response to booster vaccination by altering gene expression in circulating leukocytes. Using this model, we previously examined the effect of chronic ethanol consumption on cytokine production using intracellular cytokine staining, and found that colonic T cells exhibited a disruption of inflammatory cytokine production. Next, we show the modulation of transcriptome and microbiota profiles by chronic alcohol consumption within the different segments of the gastrointestinal tract. Lastly, we report alterations in the transcriptional profiles of intestinal lamina propria lymphocytes following chronic alcohol consumption. Since our animals lack overt organ damage, the alterations in gene expression and microbial composition precede clinical disease and could potentially serve as early biomarkers before detrimental effects occur. These findings provide novel insight into the mechanisms of ethanol-induced mucosal damage and lay the foundation for follow up studies to develop new interventions to reverse mucosal damage and improve immunity in heavy alcohol abusers.

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