DeeAnn M. Reeder, PhD - US grants

This study proposes to examine the effects of repeated or chronic stress in squirrel monkeys (Saimiri sciureus) and titi monkeys (Callicebus moloch). Chronic activation of the stress response can be induced by repeated exposure to seemingly innocuous events that pose no physical risk to the animals but to which the likelihood of habituation is small. Although many aspects of the response to acute stress has been characterized in these species, the ability to compensate for the glucocorticoid rise associated with the stress response has not been studied nor has the consequences of chronic exposure to stressors. I will separate animals from their social group and place them in a novel environment daily for eight weeks in order to examine both the transient and permanent effects of repeated psychological stress in adult monkeys. The proposed research aims first to describe the circadian rhythm of HPA activity in both species and to describe changes due to exposure to a single stressor. Next, I will examine the consequences of chronic exposure to repeated stress, which are hypothesized to include: 1) increased mean daily output of HPA hormones (including increases in free cortisol and ACTH and a reduction in CBG and bound cortisol), 2) reproductive suppression, 3) immune suppression, 4) decline in health, 5) hippocampal cell death and 6) disruption of social relationships. Finally, the research proposes to compare the effects of repeated stress in squirrel and titi monkeys: titi monkeys are predicted to show greater sensitivity to chronic stress than squirrel monkeys, especially with respect to transient changes in reproductive, immunological, and social processes. However, squirrel monkeys may be expected to exhibit more permanent changes owing to high levels of HPA hormones and a more prolonged response to each exposure to stressor. Testing both species will illuminate the respective roles of physiological predisposition and social relationships in mediating the consequences of chronic stress. To the extent that species respond similarly, I will be able to generalize the effects of chronic stress in social animals.

DESCRIPTION (provided by applicant): Reproduction requires interaction between multiple systems, including the adipocyte hormone leptin, hormones of the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes, and also immune function and social processes. We propose to explore these systems in a socially complex reproductive environment, using 2 species of seasonally breeding bats as models. Due to their lengthy gestation and lactation periods, highly seasonal breeding, high levels of glucocorticoids, and the high energetic demands of flight, these bats are ideal for studying energy balance and reproduction as mediated by leptin, the HPA axis, and the immune system. They are also good models for exploring social influences on physiology, the role of leptin in puberty and pregnancy, and potentially for understanding glucocorticoid resistance syndromes. This study aims, 1) to characterize leptin levels and HPA and MPG activity across the breeding season in both sexes and to explore the interactions between these systems, and 2) to examine social influences on reproduction (one species is much more social than the other) and variation in immune function. These goals will be achieved by observing social behavior and obtaining monthly blood samples across the breeding season to assess leptin, adrenocorticotropic hormone, glucocorticoids, and gonadal steroids and to perform complete blood counts. We will also determine differences in stress reactivity before vs. after infant birth, alterations in HPA physiology attendant to group formation, and leptin mRNA levels from fat biopsies collected pre- and postpartum and from the placenta.

ABSTRACT In line with the funding goals of the NIH and the objectives of the R21 research program, this project uses the power of transcriptomics to understand bat immune competence in relation to viral infection in a natural, variable environment. This project will be jointly led at Bucknell University by co-PD/PIs Dr. DeeAnn Reeder (internationally recognized expert in bat disease and comparative physiology) and Dr. Ken Field (classically trained immunologist with expertise in applying transcriptomic approaches to bat disease ecology). The goals of this work are to explore how intrinsic (age, sex, reproductive condition, current disease status) and extrinsic (seasonal shifts in weather and food availability) factors underlie immunological variation in African fruit bats, reservoirs for viruses of pandemic potential (including Ebola) that are becoming increasingly associated with people due to habitat modification. While important progress has been made in recent years in understanding bat immunity, much of this has been in cell culture or from limited sampling, largely from SE Asian and Australian bats; this study will fill this taxonomic and geographic gap and transform our understanding of variation in antiviral immunity by examining immune processes in the real world. To perform this work, male and female foraging bats will be collected at field sites in South Sudan during both the rainy and dry season. For Specific Aim 1, spleen tissue samples will be used to determine the differential expression (Illumina HiSeq 4000 platform and Trinity analysis pipeline) of genes involved in immune function, with an emphasis on antiviral immunity. Findings will be confirmed in subsequent qPCR studies and will be used to test the recently proposed hypothesis that bat antiviral gene expression is ?always on?, which may be related to reservoir capacity. Relationships found between intrinsic and extrinsic factors and immune gene expression will be used to describe periods of low antiviral immunity, which may increase spillover risk. For Specific Aim 2, gene expression in relation to diseased state will be analyzed for bats with exceptionally high malarial parasite (Hepatocystis) loads or with high viral loads (surveying filoviruses, coronaviruses, paramyxoviruses and orthomyxoviruses), compared to matched controls. For genes with differential expression, qPCR will be used to look for similar changes in other tissues, matched to viral findings (e.g., high viral load from oral swabs will prompt gene expression examination in salivary glands). Relationships between gene expression and disease state will be interpreted in the context of the influence of co-infection (malaria) and of viral infection on antiviral mechanisms. If our proposed specific aims are achieved, we will significantly enhance our understanding of bat immunity and the factors that influence it under natural conditions. This will improve our ability to predict when viral spillovers may be more likely and how changing environmental conditions, including the anthropogenic alteration of natural landscapes, may alter disease processes.  

Bats appear to have co-evolved with coronaviruses (CoVs) for millennia. The CoVs that bats carry include the closest relatives of SARS-COV-2, the causative agent of COVID-19. The relationship between the bat host and CoV virus appears to have selected for immune tolerance that enables bats to control CoV replication and yet avoid immune damage. Although some novel CoV hosts (humans) are able to manage and clear the virus without significant consequence, many do not, falling victim instead to a pathological inflammatory response that results from overly-exuberant immune signaling. Understanding how bats avoid this deleterious path may provide insight into new disease mitigation strategies. The researchers supported by this award will leverage a large existing set of bat samples to better understand how bats respond to infection with CoVs. Beyond these direct COVID-19 societal benefits, this project will benefit society by training young scientists in disease ecology and in bioinformatics, preparing them for future careers in the transdisciplinary STEM workforce. Data from this study will be published in peer-reviewed journals, presented at scientific meetings, and shared through public data repositories.

The purpose of this study is to identify immune mechanisms associated with tolerance of Coronavirus (CoV) infections in bats. Gaining information on bat responses to CoV infections will shed light on the mechanisms of effective immune control. Parallel study of (1) the CoV virome, and (2) the accompanying gene up- or down-regulation in response to infection will reveal immune system signatures of viral tolerance and advance fundamental understandings of antiviral immunity. By comparing responses in the African little epauletted fruit bat (Epomophorus labiatus), which host beta-CoVs, and the North American little brown myotis (Myotis lucifugus), which host alpha-CoVs, the common mechanisms of tolerance to both alpha- and beta-CoVs will be determined. A powerful dual RNA sequencing approach will be deployed to simultaneously sequence the virome and the host transcriptome. Using weighted gene correlation network analysis, gene expression counts will be used to determine gene networks in the host that are most tightly correlated to each CoV infection. These correlated gene networks will be analyzed functionally to determine which immune pathways are associated with CoV tolerance by either blocking inflammation and immune activation or by dampening tissue damage. Finally, the functions of these co-regulated genes will be compared to those documented in novel hosts (human and other animals). This RAPID award is made by the Physiological and Structural Systems Cluster in the BIO Division of Integrative Organismal Systems, using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

ABSTRACT This project focuses on understanding the role that the unique physiology of bats plays in their ability to act as host reservoirs for diseases that can spill over to humans. The project will be carried out under field conditions in Uganda on three species of bats that have varying links to the spread of Ebola virus (EBOV) to humans. By comparing the ability of these three species of bats to respond to Ebola-like immune challenges, this work will help identify the characteristics that contribute to spillover risk. In the long term, this work will help identify host species for EBOV and other related viruses that present risk to humans. It will also help explain how different species of bats respond to different types of viral infections. The main focus of this project will be to identify behaviors and molecular pathways that enable reservoir hosts to tolerate infections, providing critical insight into one of the mechanisms that leads to spillover. This work is driven by the hypothesis that some bat species have coevolved with particular types of viral infections and, therefore, have adapted mechanisms to minimize pathology during infection. Bats are globally biodiverse and have many unique ecological and physiological adaptations, including flight and the ability to employ both hypo- and hyperthermic body temperature regulation. This project focuses on three bat species chosen because they are in close contact with humans, their habitats cover the range of EBOV exposure risk, and they have divergent coevolutionary histories with viral pathogens; two of the three species have significant ties to EBOV epidemiology. This project addresses these questions under natural conditions in the field by taking the innovative approach of using EBOV virus-like particles as a proxy for experimental infection with biohazardous pathogens. This project has three specific aims that will allow the achievement of its goals. First, the project tests the hypothesis that specific African bat species will display signatures of EBOV disease tolerance in response to challenge with EBOV virus-like particles, and thus are likely to be natural reservoir hosts. These experiments will provide significant insight into disease tolerance in bats and the potential identity of EBOV reservoir(s). Second, this project tests the hypothesis that bats display variable levels of disease tolerance that depend upon innate immune pathways that have undergone unique evolutionary selection in bats. Third, this project explores whether tolerance of and resistance to viral infection are facilitated by the unique metabolic behaviors of bats, namely that they can depress metabolism and enter torpor to conserve energy and can elevate metabolism and thus temperature during flight. The role of changes in body temperature is poorly understood and these experiments will identify whether these physiological responses contribute to immunological tolerance and resistance in important disease reservoirs. Together, the successful completion of these goals will help determine whether infection tolerance confers on African bat species the ability to serve as reservoir hosts for virulent zoonotic viruses and will identify molecular, physiological, and behavioral mechanisms that contribute to tolerance phenotypes.