The Effects of the Gut Microbiota on the Host Chromatin Landscape

The human gut microbiota is home to tens of trillions of microbes belonging to all three domains of life. The structure and expressed functions of this community have myriad effects on host physiology, metabolism, and immune function. My studies focused on a facet of host-microbial interactions and mutualism that has not been explored to a significant degree in part because of the absence of suitable tools: namely, if, when, and how the gut microbiota produces durable effects on host biology through its impact on the epigenome. To address this area, I turned to gnotobiotic mice and developed a variety of experimental, methodological, and computational approaches to characterize the chromatin landscape of various host cell populations. I selected small populations of T cells likely to be exposed to the gut microbiota and its products, principally TCR αβ+ and TCR γδ+ small intestinal intraepithelial lymphocytes (IELs). I also chose to study circulating CD4+ and CD8+ T cells, with the advantage that these populations can be isolated from donors without using highly invasive techniques. I designed a series of approaches to enrich, isolate, and purify each of these cell populations from single animals, and to subsequently compare their chromatin landscape within and across mice using a recently described Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq), focusing on enhancer and super-enhancer loci within the mouse genome. These analyses revealed a conserved set of super-enhancer loci between αβ and γδ IELs, including super-enhancers near genes responsible for phospholipid binding and T cell receptor signaling. In comparing C57BL/6J male mice reared under germ-free (GF) conditions to age- and sex-matched conventionally raised (CONV-R) mice (i.e., animals that acquired microbes from their environment beginning at birth), I was able to directly assess the impact of colonization on chromatin ‘state’ in these different purified cell populations and to identify colonizationdependent effects in IELs on enhancers associated with genes involved in a number of metabolic and signaling pathways. I then compared the results to GF mice that had been colonized following the end of the weaning period with an intact cecal microbiota from a CONV-R C57BL/6J donor. The resulting conventionalized (CONV-D) animals allowed me to identify modifications to host chromatin landscape that are ‘induced’ following the suckling-weaning transition and, in doing so, ascertain whether there were developmental windows that could constrain the durable effects of the microbiota on chromatin accessibility. In doing so, I observed changes in chromatin accessibility with colonization that may reveal a functional maturation of IEL populations that is related to the timing of exposure to the microbiota during postnatal development. My thesis project involved development of an elaborate, multi-faceted computational pipeline for the analysis of these novel, large datasets, including the prediction and characterization of putative enhancers and super-enhancers, proximally associated genes, and metabolic pathways influenced by those elements. As a whole, this work defines the impact of gut microbial colonization on host chromatin landscape and provides an analytical toolkit for further studies

Longitudinal Predictors of Diabetic Ketoacidosis Hospitalizations and Hemoglobin A1c: Examining Adaptation to Type-1 Diabetes in Adolescents

Diabetic Ketoacidosis (DKA) and elevated hemoglobin A1c (HbA1c) in youth with type-1 diabetes (T1D) can result in significant morbidity and mortality. Elucidating the risk factors for poor glycemic control and DKA hospitalizations is crucial for the refinement and development of prevention and treatment efforts. Based on a conceptual framework, this study used path analysis to evaluate individual and family characteristics, psychosocial responses, and individual and family responses that prospectively predict number of DKA hospitalizations and HbA1c approximately one year after assessment, accounting for socio-demographics. 174 Youth 12-18 years old with T1D (M = 14.78, SD = 1.65) and their caregivers completed measures assessing demographics, internalizing symptoms, diabetes stress, diabetes-related family conflict, and adherence. Medical records were reviewed to obtain the number of episodes of DKA and the HbA1c closest to 1-year follow-up. Thirty-one participants had at least one episode of DKA based on chart review. Identifying as Black/ African American, a younger age, and higher baseline HbA1c significantly predicted higher HbA1c at follow-up (p \u3c .05). For DKA Count, greater duration of diabetes, higher baseline HbA1c, lower income, and identifying as Non-Hispanic White and higher Youth report of internalizing symptoms were significant predictors (p \u3c 0.05). Utilizing screeners for internalizing symptoms in endocrinology clinics may be critical for early detection of youth with T1D at risk for DKA

Functional deficits precede structural lesions in mice with high-fat diet-induced diabetic retinopathy

Obesity predisposes to human type 2 diabetes, the most common cause of diabetic retinopathy. To determine if high-fat diet–induced diabetes in mice can model retinal disease, we weaned mice to chow or a high-fat diet and tested the hypothesis that diet-induced metabolic disease promotes retinopathy. Compared with controls, mice fed a diet providing 42% of energy as fat developed obesity-related glucose intolerance by 6 months. There was no evidence of microvascular disease until 12 months, when trypsin digests and dye leakage assays showed high fat–fed mice had greater atrophic capillaries, pericyte ghosts, and permeability than controls. However, electroretinographic dysfunction began at 6 months in high fat–fed mice, manifested by increased latencies and reduced amplitudes of oscillatory potentials compared with controls. These electroretinographic abnormalities were correlated with glucose intolerance. Unexpectedly, retinas from high fat–fed mice manifested striking induction of stress kinase and neural inflammasome activation at 3 months, before the development of systemic glucose intolerance, electroretinographic defects, or microvascular disease. These results suggest that retinal disease in the diabetic milieu may progress through inflammatory and neuroretinal stages long before the development of vascular lesions representing the classic hallmark of diabetic retinopathy, establishing a model for assessing novel interventions to treat eye disease

PexRAP inhibits PRDM16-mediated thermogenic gene expression

How the nuclear receptor PPARγ regulates the development of two functionally distinct types of adipose tissue, brown and white fat, as well as the browning of white fat, remains unclear. Our previous studies suggest that PexRAP, a peroxisomal lipid synthetic enzyme, regulates PPARγ signaling and white adipogenesis. Here, we show that PexRAP is an inhibitor of brown adipocyte gene expression. PexRAP inactivation promoted adipocyte browning, increased energy expenditure, and decreased adiposity. Identification of PexRAP-interacting proteins suggests that PexRAP function extends beyond its role as a lipid synthetic enzyme. Notably, PexRAP interacts with importin-β1, a nuclear import factor, and knockdown of PexRAP in adipocytes reduced the levels of nuclear phospholipids. PexRAP also interacts with PPARγ, as well as PRDM16, a critical transcriptional regulator of thermogenesis, and disrupts the PRDM16-PPARγ complex, providing a potential mechanism for PexRAP-mediated inhibition of adipocyte browning. These results identify PexRAP as an important regulator of adipose tissue remodeling

Targeting cellular calcium homeostasis to prevent cytokine-mediated beta cell death

AbstractPro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.</jats:p

Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19