IL-10-producing Regulatory B Cell Development in Human Autoimmune Disease

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of costimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of “regulatory B cells” has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported with interleukin-10 (IL-10) being the most studied. IL-10-producing regulatory B cells predominantly localize within a rare CD1dhiCD5+ B cell subset in mice and the CD24hiCD27+ B cell subset in adult humans. This specific IL-10-producing subset of regulatory B cells have been named “B10 cells” to highlight that the regulatory function of these rare B cells is primarily mediated by IL-10, and to distinguish them from other regulatory B cell subsets that regulate immune responses through different mechanisms. B10 cells have been studies in a variety of animal models with autoimmune disease and clinical settings of human autoimmunity. There are many unsolved questions related to B10 cells including their surface phenotype, their origin and development in vivo, and their role in autoimmunity.

In Chapter 3 of this dissertation, the role of the B cell receptor (BCR) in B10 cell development is highlighted. First, the BCR repertoire of mouse peritoneal cavity B10 cells is examined by single cell sequencing; peritoneal cavity B10 cells have clonally diverse germline BCRs that are predominantly unmutated. Second, mouse B10 cells are shown to have higher frequencies of λ+ BCRs compared to non-B10 cells which may indicate the involvement of BCR light chain editing early in the process of B10 cell development in vivo. Third, human peripheral blood B10 cells are examined and are also found to express higher frequencies of λ chains compared to non-b10 cells. Therefore, B10 cell BCRs are clonally diverse and enriched for unmutated germline sequences and λ light chains.

In Chapter 4 of this dissertation, B10 cells are examined in the healthy developing human across the entire age range of infancy, childhood and adolescence, and in a large cohort of children with autoimmunity. The study of B10 cells in the developing human documents a massive transient expansion during middle childhood when up to 30% of blood B cells were competent to produce IL-10. The surface phenotype of pediatric B10 cells was variable and reflective of overall B cell development. B10 cells down-regulated CD4+ T cell interferon-gamma (IFN-γ) production through IL-10-dependent pathways and IFN-γ inhibited whereas interleukin-21 (IL-21) promoted B cell IL-10 competency in vitro. Children with autoimmunity had a contracted B10 cell compartment, along with increased IFN-γ and decreased IL-21 serum levels compared to age-matched healthy controls. The decreased B10 cell frequencies and numbers in children with autoimmunity may be partially explained by the differential regulation of B10 cell development by IFN-γ and IL-21 and alterations in serum cytokine levels. The age-related changes of the B10 cell compartment during normal human development provide new insights into immune tolerance mechanisms involved in inflammation and autoimmunity.

These studies collectively demonstrate that BCR signals are the most important early determinant of B10 cell development in vivo, that human B10 cells are not a surface phenotype defined developmental B cell subset but a functionally defined regulatory B cell subset that regulates CD4+ T IFN-γ production through IL-10-dependent pathways and that human B10 cell development can be regulated by soluble factors in vivo such as the cytokine milieu. The findings of these studies provide new insights into immune tolerance mechanisms involved in human autoimmunity and the potent effects of IL-21 on human B cell IL-10 competence in vitro open new horizons in the development of autologous B10 cell-based therapies as an approach to treat human autoimmune disease in the future.

The Effects of Brominated Flame Retardants on Thyroid Hormone Homeostasis in Human Placenta Tissues and Cell Culture

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.

In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.

In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation.

In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure.

This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.

Putting together phylogenetic and ontogenetic perspectives on empathy.

The ontogeny of human empathy is better understood with reference to the evolutionary history of the social brain. Empathy has deep evolutionary, biochemical, and neurological underpinnings. Even the most advanced forms of empathy in humans are built on more basic forms and remain connected to core mechanisms associated with affective communication, social attachment, and parental care. In this paper, we argue that it is essential to consider empathy within a neurodevelopmental framework that recognizes both the continuities and changes in socioemotional understanding from infancy to adulthood. We bring together neuroevolutionary and developmental perspectives on the information processing and neural mechanisms underlying empathy and caring, and show that they are grounded in multiple interacting systems and processes. Moreover, empathy in humans is assisted by other abstract and domain-general high-level cognitive abilities such as executive functions, mentalizing and language, as well as the ability to differentiate another's mental states from one's own, which expand the range of behaviors that can be driven by empathy.

Distribution and Conservation of the Antillean Manatee in Hispaniola

Antillean manatees (Trichechus manatus manatus) were heavily hunted in the past throughout the Wider Caribbean Region (WCR), and are currently listed as endangered on the IUCN Red List of Threatened Species. In most WCR countries, including Haiti and the Dominican Republic, remaining manatee populations are believed to be small and declining, but current information is needed on their status, distribution, and local threats to the species.

To assess the past and current distribution and conservation status of the Antillean manatee in Hispaniola, I conducted a systematic review of documentary archives dating from the pre-Columbian era to 2013. I then surveyed more than 670 artisanal fishers from Haiti and the Dominican Republic in 2013-2014 using a standardized questionnaire. Finally, to identify important areas for manatees in the Dominican Republic, I developed a country-wide ensemble model of manatee distribution, and compared modeled hotspots with those identified by fishers.

Manatees were historically abundant in Hispaniola, but were hunted for their meat and became relatively rare by the end of the 19th century. The use of manatee body parts diversified with time to include their oil, skin, and bones. Traditional uses for folk medicine and handcrafts persist today in coastal communities in the Dominican Republic. Most threats to Antillean manatees in Hispaniola are anthropogenic in nature, and most mortality is caused by fisheries. I estimated a minimum island-wide annual mortality of approximately 20 animals. To understand the impact of this level of mortality, and to provide a baseline for measuring the success of future conservation actions, the Dominican Republic and Haiti should work together to obtain a reliable estimate of the current population size of manatees in Hispaniola.

In Haiti, the survey of fishers showed a wider distribution range of the species than suggested by the documentary archive review: fishers reported recent manatee sightings in seven of nine coastal departments, and three manatee hotspot areas were identified in the north, central, and south coasts. Thus, the contracted manatee distribution range suggested by the documentary archive review likely reflects a lack of research in Haiti. Both the review and the interviews agreed that manatees no longer occupy freshwater habitats in the country. In general, more dedicated manatee studies are needed in Haiti, employing aerial, land, or boat surveys.

In the Dominican Republic, the documentary archive review and the survey of fishers showed that manatees still occur throughout the country, and occasionally occupy freshwater habitats. Monte Cristi province in the north coast, and Barahona province in the south coast, were identified as focal areas. Sighting reports of manatees decreased from Monte Cristi eastwards to the adjacent province in the Dominican Republic, and westwards into Haiti. Along the north coast of Haiti, the number of manatee sighting and capture reports decreased with increasing distance to Monte Cristi province. There was good agreement among the modeled manatee hotspots, hotspots identified by fishers, and hotspots identified during previous dedicated manatee studies. The concordance of these results suggests that the distribution and patterns of habitat use of manatees in the Dominican Republic have not changed dramatically in over 30 years, and that the remaining manatees exhibit some degree of site fidelity. The ensemble modeling approach used in the present study produced accurate and detailed maps of manatee distribution with minimum data requirements. This modeling strategy is replicable and readily transferable to other countries in the Caribbean or elsewhere with limited data on a species of interest.

The intrinsic value of manatees was stronger for artisanal fishers in the Dominican Republic than in Haiti, and most Dominican fishers showed a positive attitude towards manatee conservation. The Dominican Republic is an upper middle income country with a high Human Development Index. It possesses a legal framework that specifically protects manatees, and has a greater number of marine protected areas, more dedicated manatee studies, and more manatee education and awareness campaigns than Haiti. The constant presence of manatees in specific coastal segments of the Dominican Republic, the perceived decline in the number of manatee captures, and a more conservation-minded public, offer hope for manatee conservation, as non-consumptive uses of manatees become more popular. I recommend a series of conservation actions in the Dominican Republic, including: reducing risks to manatees from harmful fishing gear and watercraft at confirmed manatee hotspots; providing alternative economic alternatives for displaced fishers, and developing responsible ecotourism ventures for manatee watching; improving law enforcement to reduce fisheries-related manatee deaths, stop the illegal trade in manatee body parts, and better protect manatee habitat; and continuing education and awareness campaigns for coastal communities near manatee hotspots.

In contrast, most fishers in Haiti continue to value manatees as a source of food and income, and showed a generally negative attitude towards manatee conservation. Haiti is a low income country with a low Human Development Index. Only a single dedicated manatee study has been conducted in Haiti, and manatees are not officially protected. Positive initiatives for manatees in Haiti include: protected areas declared in 2013 and 2014 that enclose two of the manatee hotspots identified in the present study; and local organizations that are currently working on coastal and marine environmental issues, including research and education on marine mammals. Future conservation efforts for manatees in Haiti should focus on addressing poverty and providing viable economic alternatives for coastal communities. I recommend a community partnership approach for manatee conservation, paired with education and awareness campaigns to inform coastal communities about the conservation situation of manatees in Haiti, and to help change their perceived value. Haiti should also provide legal protection for manatees and their habitat.