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A Network Approach to Understanding miRNA Regulation in Adipose Tissue
Get PDFAdipose tissue is a complex organ that is essential for lipid storage and energy homeostasis in mammals. While transcription factors that govern adipogenesis, inflammation, non-shivering thermogenesis, and endocrine function in fat tissue have been well-described, comparatively less attention has been given to post-transcriptional regulators. Here, we employ two contrasting approaches to identify miRNAs that influence adipocyte phenotype. First, miRNAs were sequenced from 6 fat pads, primary preadipocytes, and primary mature adipocytes. Comparative analyses of abundance across adipose tissue type and stage of differentiation revealed a number of miRNAs that influence key metabolic processes. miR-335 inhibited adipogenesis, miR-192 promoted pro-thermogenic and pro-inflammatory pathways, and miR-338 upregulated a variety of genes involved in both lipogenesis and lipolysis/β-oxidation. Although most effects on mRNA abundance were modest, these candidates and others identified in the analysis provide a foundation for further study. The second portion of this thesis focuses on network-based approaches to describe miRNA regulators in adipose tissue with an emphasis on miRNA binding activity. Most notably, we used Ago HITS-CLIP to comprehensively map mRNA:miRNA interactions in brown and white fat, revealing 21,281 unique miRNA binding sites in 6,717 genes. Most binding sites were shared between brown and white fat, although reads per binding site and reads per gene varied substantially. Targets for each miRNA were ranked to generate a catalog of miRNA binding activity. The miR-29 family emerged as a top regulator of adipose tissue phenotype with multiple binding sites in the leptin 3\u27-UTR that were confirmed with luciferase assay validations. miR-29 gain and loss-of-function modulated leptin mRNA and protein levels in primary adipocytes, and miR-29 abundance inversely correlated with leptin levels in adipose tissues. This work represents the only experimentally generated miRNA targetome in adipose tissue and identifies the first known post-transcriptional regulator of leptin
Sympathy for the Microbiota: How Changes in Gut Microbial Composition Influence the Immune System and Basic Physiology by Way of the Sympathetic Nervous System
Get PDFThe gastro-intestinal tract is the most extensive mucosal surface in mammals and harbors the most numerous immune cell reservoir as well as a complex and autonomous nervous system. The GI tract is continuously exposed to foreign antigen and potentially toxic chemicals through the ingestion of food. In addition, the lumen of the human GI tract is populated with approximately 1013 bacteria, which is equivalent to the number of cells in the entire human body (Sender et al., 2016). The intestinal microbiome, which aids in host metabolism, represents a diverse population of microorganisms separated from the inside of the body by only a single layer of epithelial cells. Thus, in this highly inflammatory and ever-changing environment, the intestine must be able to balance tolerance to dietary antigens and microbes, while remaining vigilant when exposed to potentially toxic substances or pathogens. Sensing of the microbiota has been previously shown to impact the intestinal immune and nervous systems, including crosstalk between the two. However, how microbes influence the enteric associated nervous system and whether these effects play a role in mediating host physiology, inclusive of immune responses, remains unanswered. We previously uncovered neuro-immune cross-talk in the intestine (Muller et al., 2014), whereby muscularis macrophages (MM) that reside in the muscle layers of the intestine express bone morphogenetic protein 2 (BMP2) that can directly impact intrinsic enteric associated neurons (iEAN) and intestinal motility. Reciprocally, iEAN express colony-stimulating factor 1 (CSF1), which may play a role in the local maintenance and differentiation of MM. Both axes of this crosstalk occur during normal, steady-state conditions and are dependent upon the commensal microbiota. To determine whether these macrophages played a role during the course of an intestinal infection, we first aimed to better characterize how MM differed from their counterparts that reside in the intestinal lamina propria, in close proximity to the microbialrich lumen. Through two-photon intravital microscopy, cleared tissue light-sheet imaging, and RNA-sequencing of purified macrophage populations, we found that MM are morphologically, anatomically, dynamically, and transcriptionally distinct from LpM. These MM were skewed towards a more anti-inflammatory transcriptional profile and were in close apposition to actively firing iEAN. Utilizing an attenuated form of Salmonella typhimurium, spiB, we found that although MM are not in direct contact with luminal contents, they were rapidly polarized towards an enhanced anti-inflammatory profile in response to pathogenic infection. Furthermore, polarization was dependent upon sympathetic neuronal activation in the celiac-superior mesenteric ganglion (CG-SMG), which enabled communication from the lumen to MM through adrenergic receptor beta 2 (ADBR2) engagement (Gabanyi & Muller et al., 2016). Together, these results established that neurotransmitter signaling can swiftly coordinate a response to infection through anatomically-associated immune cells. To gain insights into how sympathetic neurons, and more broadly, all extrinsic enteric associated neurons (eEAN) might detect and/or be influenced by changes in the microbiota, we performed unbiased RNA translational profiling comparing eEAN sensory and effector nodes from germ free and specific pathogen free mice. RNA sequencing revealed changes in activity-dependent transcripts in afferent sensory neurons in the nodose ganglion and efferent sympathetic neurons in the CG-SMG. These results served as an entry point to identify which microbial signals and neuronal populations transmit microbial information to the central nervous system (CNS), and which CNS populations integrate this information to control gut physiology. We identified a subset of distal intestine-projecting vagal neurons positioned to play an afferent role in luminal detection through chemogenetic manipulation, translational profiling and anterograde tracing. Using retrograde polysynaptic neuronal tracing from the intestinal wall, we identified brainstem sensory nuclei activated by specific bacterial metabolites, including short chain fatty acids. Finally, chemogenetic modulation demonstrated that activation of sympathetic premotor glutamatergic neurons is sufficient to regulate gastrointestinal transit. In sum, we established an anatomical, molecular, and functional framework for the complex circuit(s) monitoring microbial content (Muller et al., 2019a). Equipped with a more complete understanding of how the gut sympathetic nervous system functions, we then asked whether pathogen-induced adrenergic changes in MM are important for a proper tissue-protective response. Infection with multiple pathogens, including spiB, led to a significant and persistent loss in iEAN numbers and chronic dysmotility. Intersectional genetics, pharmacological intervention, and chemogenetic sympathetic activation was used to demonstrate that MM-specific Β2AR signaling is required to confer neuronal protection during the course of infection and prevent further pathogen-induced damage. Furthermore, translational profiling of iEAN led to the discovery that these neurons express a unique combination of inflammasome pathway components as compared to other peripheral neuronal sub-populations. These inflammasome components are activated in iEAN during infection, and neuron-specific gene targeting was sufficient to prevent pathogen-induced neuronal loss. Thus, we found that MM-adrenergic signaling can mitigate EAN inflammasome-dependent cell-death during enteric infection (Matheis & Muller et al., 2019). Based upon our discovery of iEAN dysbiosis detection via the inflammasome, we next probed how changes in the microbiota might further impact these neurons. Using translational profiling, we found that distinct regions of the intestine have unique gene expression programs in the presence of the microbiome. By comparing this profile to germ free mice, we found that the microbiota is a principal driving force in establishing these regional differences and that specific neuropeptides populations are decreased in the distal intestine in the absence of a microbiota. Chemogenetic characterization of microbiota-influenced iEAN identified a subset of viscerofugal CART+ neurons that modulate feeding behavior through insulin-glucose levels independent of the central nervous system. We discovered that CART+ iEAN numbers decrease in the absence of the microbiota through the same iEAN inflammasome pathway we previously identified. Finally, genetic ablation of this inflammasome pathway was sufficient to prevent insulinglucose level changes normally seen in antibiotic-treated conditions (Muller et al., 2019b). Through these studies we found that multiple signals generated by changes in the gut microbial composition are integrated by the sympathetic nervous system to control gastrointestinal motility, enteric immunity, and blood glucose. Cumulatively, these findings have increased our understanding of how the enteric associated nervous system responds to changes in the microbiota and consequently regulates local tissue and overall mammalian homeostasis
Rosamunde String Quartet
No full textRosamunde String Quartet: Noah Bendix-Balgley and Shanshan Yao, violins; Teng Li, viola; Nathan Vickery, cello performing Felix Mendelssohn: Quartet No. 6 in F Minor, Op. 80; Franz Schubert: String Quartet No. 13 in A Minor, D 804 “Rosamunde”.https://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1002/thumbnail.jp
Effective Adaptive Immunity to Tumors in Mice
No full textSlide 5-28: Effective adaptive immunity to tumors in mice that have rejected tumors via NKT and NK cellshttps://digitalcommons.rockefeller.edu/nkt-cells/1026/thumbnail.jp
CD11c Promoter-EYFP Mice Visualizing Dendritic Cells In Vivo
No full textSlide 6-35: CD11c promoter-EYFP mice visualizing dendritic cells in vivohttps://digitalcommons.rockefeller.edu/targeted-vaccines/1034/thumbnail.jp
The Role of Adipocytes in the Tumor Microenvironment in Obesity-Driven Breast Cancer Progression
Get PDFObesity affects more than 1 in 3 adults in the United States and has been shown to increase the relative risk of death for women with breast cancer and to increase the risk of developing breast cancer in post-menopausal women. Breast cancer develops in an environment containing white adipose tissue (WAT) which predominately consists of mature white adipocytes. In obesity, WAT undergoes hypertrophy and hyperplasia and can ultimately develop hypoxia, insulin resistance, inflammation, and dysregulated endocrine function. Since obesity directly affects WAT, we hypothesized that molecular changes in white adipocytes in the tumor microenvironment contribute to breast cancer progression. We developed mouse models of obesity-driven breast cancer using diet induced obesity (DIO) and orthotopic models of breast cancer. From one of these models, we performed RNA sequencing of peritumoral mammary fat pads and uninvolved contralateral fat pads. Pathway analysis showed an upregulation in transcripts involved in polyamine and creatine biosynthesis in the obese tumor microenvironment. We have found that the rate limiting enzyme in creatine synthesis, Gatm, is upregulated in adipocytes in the tumor microenvironment of obese mice and plays a key role in breast tumor progression. Using an adipocyte-specific knockout of Gatm (Adipo-Gatm KO), we found a significant reduction in obesity-driven breast tumor growth relative to littermate controls. We have also found that knocking down the creatine transporter (Slc6a8) in breast cancer cells fully attenuates obesitydriven tumor progression. These data support a central role for creatine metabolism in breast tumor progression in mice and suggest that adipocyte-derived creatine is utilized by the tumor to support its growth. We also examined mammary adipose tissue from human breast cancer patients and found that Gatm is increased in the mammary adipose tissue of obese/overweight relative to normal weight breast cancer patients, supporting the translational relevance of our findings. Overall, we have established models of obesity-driven breast cancer, identified key gene candidates and pathways that affect obesity-dependent breast cancer, and have investigated underlying mechanisms
Analyzing Resistance to Design Chemical Inhibitors of AAA Proteins
Get PDFChemotype-specific resistance is a major factor limiting the efficacy of molecularly targeted therapeutics. Analyses of resistance mechanisms to drugs reveal that mutations conferring resistance often arise in the drug\u27s binding sites. As selective binding of inhibitors to their protein targets is mediated by specific interactions of the inhibitor with the protein backbone and side chains, mutations that disrupt these interactions can lead to resistance. Identifying resistance-conferring alleles can thus reveal and suggest the biochemical determinants of the drug\u27s selectivity and potency. In this thesis, I explore how mutations in active sites of proteins can be leveraged to understand inhibitor binding and to design selective chemical inhibitors. In my thesis work I focus on the design of chemical probes for proteins from the AAA (ATPase Associated with diverse cellular Activities) superfamily, for which only a few inhibitors are available. As the number of inhibitor-bound models of AAA proteins is also limited, the key protein-inhibitor interactions needed for design of probes for these proteins are not known. I have developed an approach - Resistance Analysis During Design - that involves testing selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations that retain enzymatic activity. These analyses, along with computational docking, can guide optimization of inhibitor potency and selectivity. We used this approach to design the pyrazolylpyrrolopyrimidine-based spastazoline - the first potent and selective chemical probe for spastin, a microtubule-severing AAA protein needed for cell division and intracellular vesicle transport. I confirmed the predicted binding mode of spastazoline analogs by X-ray crystallography and used these high-resolution structural models, along with biochemical analyses of spastin mutant alleles, to design an allele-specific inhibitor of spastin. Further, I show that RADD can be used to analyze the binding modes of diaminotriazole-based chemical inhibitors that are chemically unrelated to spastazoline. I also identified a more potent, diaminotriazole-based analog and used our approach to show that it binds spastin\u27s active site in a different orientation in comparison to the starting compound. The distinct binding modes of these compounds predicted by RADD also match the high-resolution models I generated using X-ray crystallography. Together, these data show how analyses of resistance can be useful at the early stages of the inhibitor design process. In summary, the work in my thesis outlines how mutations in active sites of proteins can be identified and how they could facilitate inhibitor design. I discuss how the binding models of spastin inhibitors I developed can inform on the design of new inhibitors for other AAA proteins and suggest experiments that could be valuable to advance these efforts. I also propose how analyses of resistance to chemical inhibitors could be valuable for designing drugs against which resistance might be less likely to arise
Molecular Mechanisms and Antigen Receptor Requirements for Lymphocyte Adaptation to Intestinal Tissues
Get PDFThe intestine plays a crucial role in food digestion, nutrient absorption, water retention, and waste excretion. It contains the most populous immune cell reservoir in the body and is continuously exposed to a large and diverse number of diet- and microbiota- derived antigens. The highly stimulating luminal environment is separated from the core of the body, the lamina propria (LP), by just a single layer of epithelial cells. The intestinal immune system is thus tasked with being able to tolerate innocuous stimuli while mounting an effective response against potential pathogens in a controlled manner. To ensure appropriate balance between tolerance and resistance, T cells undergo tissue adaptation upon migrating from the gut-draining mesenteric lymph nodes (mLN) to the intestinal lamina propria and epithelium (IE). We sought to elucidate the transcriptional mechanisms and T cell receptor (TCR) signaling requirements of CD4+ T cell plasticity and adaptation in the intestinal tissues. Within the intestine, peripherally induced Foxp3+ regulatory T cells (iTregs), which are instrumental in limiting inflammatory responses to non-self antigen, are located primarily in the lamina propria. However, CD8αα-expressing intraepithelial CD4+ T cells (CD4- IELs), which also exhibit anti-inflammatory properties and depend on similar environmental cues, reside in the epithelium. Using intravital microscopy, we find distinct cell dynamics of intestinal Tregs and CD4-IELs. We addressed the molecular imprinting of the gut epithelium on T cells by integrating mouse genetics with single-cell RNAsequencing analyses. Transcriptionally, CD4+ T cells from mLN, LP and IE segregate based on the intestinal layer they occupy; trajectory analysis suggests a stepwise loss of CD4-programming and acquisition of an intraepithelial profile as CD4+ T cells adapt to the epithelium and convert to CD4-IELs. We found that upon migration to the epithelium, Tregs can lose Foxp3 expression and convert to CD4-IELs in a microbiota-dependent fashion, an effect in part attributed to loss of the CD4 lineage-defining transcription factor ThPOK. Treg fate-mapping coupled with RNA- and ATAC-sequencing revealed that the Treg program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of Thpok results in premature acquisition of an IEL profile by mLN Tregs, partially recapitulating epithelium imprinting. Furthermore, we demonstrate that iTregs and CD4-IELs perform complementary roles in the regulation of intestinal inflammation in response to dietary antigen. To uncover the specific role of the T cell receptor in the process of CD4-IEL development, we combined in vivo fate-mapping and gene ablation models with single cell TCRsequencing. Single-cell TCR repertoire and transcriptomic analysis of intraepithelial CD4+ T cells revealed different extents of clonal expansion and TCR overlap between cell states; fully differentiated CD4-IELs from regulatory or conventional CD4+ T cells were the least diverse. Conditional deletion of TCR on differentiating CD4+ T cells or of MHCII on intestinal epithelial cells prevented CD4-IEL differentiation. However, TCR ablation on developed CD4-IELs did not affect their accumulation. Overall, our results reveal an inter- and intra-tissue specialization of anti-inflammatory CD4+ T cells shaped by discrete niches of the intestine. We uncovered the stepwise molecular mechanisms and TCR-signaling requirements for T cells to adapt to the intestinal epithelium. We found that the coordinated replacement of the circulating lymphocyte program with site–specific transcriptional and chromatin changes is necessary for tissue imprinting. Furthermore, our results indicate that local recognition of possibly a limited set of antigens is an essential signal for the differentiation and adaptation of T cells to the epithelium. Taken together, the work presented in this thesis demonstrates that a combination of genetic, TCR, and environmental triggers is crucial in driving T cell plasticity and adaptation to the tissues within the intestine
STudents Physcial Chemistry Laboratory. View no. 2, 1962
No full textStudents Physical Chemistry laboratory in the Smith Hall, Room C-14, 1962https://digitalcommons.rockefeller.edu/laboratories-and-equipment/1062/thumbnail.jp
Paul Huang, Violin and Helen Huang, Piano
No full textPaul Huang, violin and Helen Huang, piano, performed Brahms: Violin Sonata No.1 G Major, Op. 78, “Regensonate”; Ysaÿe: Violin Sonata in D Minor, Op. 27, No. 3, “Ballade”; Mendelssohn: Violin Sonata F Major.
Photo by Ludwig Vanhttps://digitalcommons.rockefeller.edu/tri-institutional-noon-recitals/1006/thumbnail.jp