Acute inflammatory response is caused by the rapid recruitment of leukocytes, mainly neutrophils and monocytes, from blood to the tissue site. Diverse agents, including invading pathogens, injured or dead cells, and other irritants, may stimulate this response. In the ensuing inflammatory response, the recruited leukocytes and their secreted molecules help in eliminating or containing the injurious agents and promoting tissue regeneration. But often this response is imprecise and can lead to bystander tissue damage. Unchecked neutrophil activation is implicated in the pathology of many inflammatory conditions. An in-depth understanding of the pathways regulating this response, therefore, becomes critical in identifying therapeutic targets for these diseases. In this study, we investigate the role of intestinal commensal bacteria in regulating the acute inflammatory response. Furthermore, we examine the mechanism by which Interleukin-1 (IL-1) controls the inflammatory response to sterile agents. Inflammatory responses have been studied in the context of host defense against pathogens. However, we report that the innate immune system needs to be primed by intestinal flora to enable neutrophil recruitment to diverse microbial or sterile inflammatory signals. This priming requires myeloid differentiation primary response gene (88) (MyD88) signaling. In antibiotic-treated mice, which have depleted intestinal flora, we show that neutrophils get released into the blood from the bone marrow, but have a specific defect in migration into the inflammed tissue. This deficiency can be restored by pre-stimulating the mice with a purified MyD88 ligand. Despite having reduced number of infiltrating neutrophils, antibiotic-treated mice make higher levels of pro-inflammatory cytokines in the tissue, after inflammatory challenge. This suggests that antibiotic-treated mice produce some anti-inflammatory molecule(s) that counteract the effect of the pro-inflammatory cytokines. However, this effect is not due to the overproduction of the anti-inflammatory cytokine, Interleukin-10 (IL-10). In summary, our findings highlight the role of commensals in the development of acute inflammatory responses to microbial and sterile particles. The inflammatory response to sterile dead cells has been shown to be critically dependent upon IL-1. However, several key aspects of the IL-1 signaling cascade including the source of IL-1 and the cellular target of IL-1 were unresolved. We find that in most cases, the injured cells are not a major contributor of IL-1 that is required to propagate the inflammatory signal. On the contrary, we demonstrate that both the isoforms of IL-1, IL-1α/IL-1β are generated by bone marrow-derived, tissue-resident responding cells, upon sensing the injury. We also sought to determine the identity of the cellular target of IL-1 signaling. Previous studies have shown that for cell death-induced neutrophil recruitment, interleukin-1 receptor (IL-1R) expression is required on parenchymal cells. To identify this parenchymal cell, we are currently in the process of making the conditional knockout mouse of IL-1R. The latter would facilitate the parenchymal tissue-specific deletion of IL-1R. In summary, this study reports our progress in unraveling key aspects of IL-1 signaling during sterile inflammation. Taken together, we have identified key modulators of the acute inflammatory response and their mechanisms of regulation. These findings would facilitate the development of new therapies for inflammatory diseases triggered by both microbe and sterile agents.

Homeodomains (HDs) are a large family of DNA-binding domains contained in transcription factors that are most notable for regulating body development and patterning in metazoans. HDs consist of three alpha helices preceded by an N- terminal arm, where the third helix (the recognition helix) and the N-terminal arm are responsible for defining DNA-binding specificity. Here we attempted to engineer the HDs by fully randomizing positions in the recognition helix to specify each of the 64 possible 3’ triplet sites (i.e. TAANNN). We recovered HD variants that preferentially recognize or are compatible with 44 of the possible sites, a dramatic increase from the previously observed range of specificities. Many of these HD variants contain combinations of novel specificity determinants that are uncommon or absent in extant HDs, where these determinants can be grafted into alternate HD backbones with an accompanying alteration in their specificity. The identified determinates expand our understanding of HD recognition, allowing for the creation of more explicit recognition models for this family. Additionally, we demonstrate that HDs can recognize a broader range of DNA sequences than anticipated, thus raising questions about the fitness barrier that restricts the evolution HD-DNA recognition in nature. Finally, these new HD variants have utility as DNA-binding domains to direct targeting of customizable sequence-specific nuclease as demonstrated by site-specific lesions created in zebrafish. Thus HDs can guide sequence-specific enzymatic function precisely and predictably within a complex genome when used in engineered artificial enzymes.

Since the initial characterization of the human glucose transporter GLUT1, it has been observed that the presence of intracellular sugar stimulates the unidirectional rate of sugar uptake by a kinetic phenomenon known as trans-acceleration. Both GLUTs 1 and 3 catalyze transacceleration, while both GLUTs 2 and 4 do not. Although the basis for trans-acceleration is unknown, potential explanations include the requirement of a modulating cofactor, cellular context, or that the behavior is an artifact of imperfect transport measurements. This thesis examines whether trans-acceleration is a sequence-specific property intrinsic to the transporter. A method for detecting trans-acceleration in mammalian cells at physiologic temperature was developed through transport of two different glucose analogs. Homology-scanning mutagenesis was employed to exchange transmembrane domains (TMs) of GLUTs 1 and 4, and thereby test for accelerated-exchange loss- or gain-of-function. This approach was extended to GLUTs 2 and 3. The catalytic rates of these chimeric proteins were determined through transport measurements and expression measured by cell-surface biotinylation. These studies show that the sequence of putative scaffolding domain TM6 is both necessary and sufficient for trans-acceleration in scaffolds of GLUT1, GLUT2, and GLUT4. The substitution of TM6 sequence between these transporters has no effect on the turnover under exchange conditions, yet profoundly modifies turnover in the absence of intracellular sugar. We propose that the sequence-specific interaction of TM6 with other TMs structurally restrains relaxation of the empty carrier in GLUTs which catalyze trans-acceleration, and that binding of intracellular sugar affects these interactions to reduce the overall duration of the transport cycle. In addition, our model suggests that the substrate binding constant and rate of carrier relaxation are inter-dependent. In this model, the dissociation constant determined by substrate binding and dissociation rates at the endofacial sugar binding site must be larger than the equivalent constant at the exofacial site in order for trans-acceleration to occur.

Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.

Fluorescence in situ hybridization (FISH) and digital imaging microscopy were modified to allow detection of single RNA molecules. Oligodeoxynucleotide probes were synthesized with five fluorochromes per molecule and the light emitted by a single probe was calibrated. Points of light in exhaustively deconvolved images of hybridized cells gave fluorescent intensities and distances between probes consistent with single mRNA molecules. Analysis of β-actin transcription sites after serum induction revealed synchronous and cyclical transcription from single genes. The rates of transcription initiation and termination and mRNA processing could be determined by positioning probes along the transcription unit. This approach extends the power of FISH to yield quantitative molecular information on a single cell.

Selenium is toxic at high doses, yet metabolically essential in trace amounts, and therefore provides an excellent illustration of the rule of paracelsus that "the dose alone determines the poison". The only mammalian selenoprotein of known function is glutathione peroxidase (GPx). This enzyme is expressed ubiquitously, and is responsible for detoxifying peroxides and hydroperoxides which, if left unchecked, may damage important biomolecules such as DNA and membrane fatty-acids. GPx is a homotetramer; each subunit contains one mole atom of selenium incorporated as a selenocysteine residue in the active site of the enzyme. Using oligonucleotides generated against the known bovine GPx amino-acid sequence, cDNA clones were isolated corresponding to the human GPx mRNA. Sequence analysis indicated that the selenocysteine in the active site of the enzyme was incorporated at an opal terminator (UGA) codon. Therefore the glutathione peroxidase mRNA constitutes the first example of natural suppression of a terminator codon in human cells. Regulation of human GPx gene expression by selenium was examined. Selenium replete HL-60 cells possessed approximately 30-fold more enzymatic GPx activity than selenium deficient cells. However steady-state GPx mRNA levels and rate of transcription of the GPx gene differed by less than 1.5-fold. Cycloheximide abolished the increase in enzymatic activity observed upon selenium replenishment. Cellular immunoreactive GPx protein levels correlated with enzymatic activity, indicating that the human GPx gene is regulated post-transcriptionally by selenium. The mechanism of this post-transcriptional regulation was investigated. A selenium labelled tRNA species was identified which exhibited features in common with a previously characterized tRNAUGA. This data suggested that selenium may be incorporated into GPx via a co-translational mechanism using a selenocysteinyl tRNA intermediate. Selenium did not alter cytoplasmic levels of the tRNAUGA, indicating that accumulation of cytoplasmic suppressor tRNA was not the point of regulation of GPx by selenium. A model is proposed for the co-translational insertion of selenocysteine into GPx mediated by a charged tRNA species present in selenium replete but absent from selenium deficient cells. Models are also proposed to explain the discrimination between the selenocysteine UGA codon and authentic UGA terminator codons. The regulation of the GPx gene was examined during mono-myelocytic differentiation of HL-60 cells in vitro and also during interferon-gamma activation of human peripheral blood macrophages and PMN. During phagocyte cell differentiation or activation, the ability to generate peroxide developed, however the peroxide-destroying capacities of GPx did not increase concomitantly. Complex regulatory patterns involving both transcriptional and translational controls were observed. The association of GPx gene expression with chronic granulomatous disease was explored. No correlation was found with either the autosomal or X-linked forms of the disease, a finding contradictory to previously published material.

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance due to frequent natural exposure and their increasing use in translational applications. Depending on the preparation studied and the endpoint measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. Highly purified chitosan and chitin were prepared and the capacity of these glycans to stimulate the release of the inflammasomeassociated cytokine IL-1β was examined. Chitosan was shown to be a potent inflammasome activator in mouse bone marrow macrophages, macrophages polarized towards a M1 or M2 phenotype, dendritic cells, peritoneal cells, and human PBMCs. Acetylation of the chitosan to chitin resulted in a near total loss of IL-1β activity in all cell types tested. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL- 1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. The reason for chitin’s inability to elicit IL-1β is unknown, but it does not appear to be due to active inhibition of the inflammasome and while chitin appears to be more readily digested by macrophage cell lysates, it does not occur at a rate which would likely impact inflammasome activation. There are three proposed mechanisms for NLRP3 inflammasome activation: K+ efflux, ROS, and lysosomal destabilization. The contributions of these mechanisms were tested and it was revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, the laminin receptor was evaluated as a potential chitin receptor. These studies provide insight into the activating properties of chitin and chitosan and highlight the importance of matching particle size and degree of acetylation to the level of activity desired for translational applications.

Fireflies are beetles that generate yellow-green light when their luciferase enzyme activates and oxidizes its substrate, D-luciferin. This bioluminescent reaction is widely used as a sensitive reporter both in vitro and in vivo. However, the light-emitting chemistry is limited by the properties of the small molecule D-luciferin. Our lab has developed a panel of synthetic luciferin analogs that improve on the inherent characteristics of D-luciferin. My thesis work focuses on harnessing these novel substrates to further expand the utility and molecular understanding of firefly bioluminescence. The first part of my thesis focuses on using synthetic luciferins to improve bioluminescence imaging beyond what is possible with D-luciferin. Our substrates emit red-shifted light compared to D-luciferin, bringing the wavelength to a range that is more able to penetrate through tissue, but at a cost of lower signal intensity. I developed mutant luciferases that increase the maximal photon flux with the synthetic luciferins over what is achievable with the wild-type luciferase, and furthermore discriminate between substrates based on their chemical structures. Additionally, I have expanded the bioluminescence toolkit by harnessing the intrinsic properties of the luciferins to non-invasively and specifically assay the activity of a single enzyme (fatty acid amide hydrolase) in live mice. Therefore, my work presents an effective way to generally improve upon bioluminescent reporters, but also to measure the activity of a specific enzyme of interest in the context of a living organism. The second part of my thesis employs synthetic luciferins to more deeply probe the light-emitting chemistry of bioluminescence. Our synthetic substrates reveal latent luciferase activity from multiple luciferase homologs that are inactive with D-luciferin. These enzymes, the fatty acyl-CoA synthetases, are predicted to be luciferase’s evolutionary predecessors, but it was not clear how the light emitting chemistry originated. My work shows that the luciferase must activate the luciferin and provide oxygen access, but the light emitting chemistry is a fundamental property of that activated intermediate. In summary, the work described herein not only expands our understanding of firefly bioluminescence, but also broadens its practical applications to shine bioluminescent light on the dark corners of biology.

Osteocalcin (OC) is a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix. The Osteocalcin gene is transcriptionally regulated by several basal, hormone- and cytokine-responsive elements. To address the potential role of TGF-β1 regulation and tissue-specific expression of the OC gene, we defined regulatory elements and factors mediating the transcriptional activity of the rat OC (rOC) promoter. TGF-β1 modulates the differentiation of cells of the osteogenic lineage and downregulates the osteoblast-specific expression of OC. By promoter deletion and mutational analyses, a TGF-β1 responsive element at nt -146 to -139 and a contiguous tissue-specific enhancer element at nt -136 to -130 on the rOC promoter were identified. These studies show that Fra-2, a member of the AP-1 family of proteins, binds to the TGF-β1 responsive element and activates basal OC expression. TGF-β1 induced phosphorylation of Fra-2 inhibits this activation, resulting in repression of OC gene transcription. The tissue-specific enhancer element contiguous to the TGF-β1 responsive element contains an AML (Cbfa) binding sequence. This element, designated OC Box II, contributes to 75% of the basal OC promoter activity and forms an osteoblast-specific protein-DNA complex in in-vitro assays. The activation potential of this binding sequence was established by overexpressing AML (Cbfa) transcription activator proteins in osteoblastic as well as in non-osseous cell lines. Interestingly, overexpression not only enhances rOC promoter activity in osteoblasts but also mediates promoter activity in a non-osseous human fibroblastic cell-line. Subsequently, we identified AML-3 (Cbfa1) as the major AML family member present in the osteoblast specific complex and demonstrate that AML-3 (Cbfa1) is expressed predominantly as a 5.4 kb transcript in rat bone tissues. Finally, to establish the functional involvement of AML (Cbfa) transcription factors in osteoblast differentiation, we utilized antisense strategies to demonstrate that blocking expression of all AML (Cbfa) related genes in primary osteoblast cultures significantly decreased several parameters which are linked to differentiation of normal diploid osteoblasts. These results indicate that AML-3 (Cbfa1) is a key transcription factor in bone cells and that the activity of the AML (Cbfa) family of proteins is required for completion of osteoblast differentiation.

In DNA immunization a plasmid expressing an antigen of interest is inoculated into an animal and antigen-specific humoral and cellular immune responses are raised. In this dissertation we sought to further our understanding of antibody responses raised following DNA inoculation. Specifically, we examined the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, the role of CD4+ and CD8+ T cells in the DNA-raised antibody response, the longevity of functional antigen expression, and the nature of the antigen presenting cell. In all of these studies mice were immunized with an influenza hemagglutinin-expressing plasmid and plasmid was delivered by either the gene gun or intramuscular routes of inoculation. To examine the role of lymphoid tissue in the initiation and maintenance of the long-term antibody response, responses raised in gene gun immunized mice were compared to responses raised in mice primed with an influenza infection. Antibody and antibody secreting cell (ASC) responses were analyzed at various times following immunization or sublethal infection for as long as 1.5 years. We found that the antibody response raised with a single gene gun immunization was similar in longevity to that raised in infection-primed mice. The long-term maintenance of the antibody response was associated with the localization of the majority of antibody secreting cells to the bone marrow. The kinetics of ASC bone marrow localization was 4-to-8 weeks slower in DNA-immunized than infection primed mice. This corresponded to a slower rise in the antibody response to plateau levels in DNA-immunized mice. We hypothesize that it is possible that the difference in kinetics may be linked to differences in the time course and dose of antigen expression following DNA immunization and a natural infection. Antibody and ASC responses were also compared following a challenge influenza virus infection. We found that DNA-immunized and infection-primed mice responded similarly in the acute post challenge phase with increases in antibody secreting cells in the mediastinal lymph nodes. While only DNA-immunized mice had post challenge increases in antibody, the antibody response remained 3-to-4 fold lower than post challenge responses in infection primed mice. We suggest that despite post challenge increases in these responses in DNA-immunized mice that the immune response raised with DNA immunization efficiently limited replication of the challenge virus and thus limited the post challenge antibody response. We also addressed the role that CD4+ and CD8+ T cells played in the ability to prime and boost the DNA-raised antibody response. To answer this question mice were in vivo depleted of CD4+ or CD8+ T cells for 3 weeks prior to through 2 weeks following DNA immunization or boost. Antibody responses were measured 4 and 8 weeks after DNA prime and 2 weeks after DNA boost. For both the gene gun and intramuscular routes of inoculation, the antibody response was independent of CD8+ T cells, but dependent on CD4+ T cells. The presence of CD4+ T cells was required at the time of DNA immunization, but not at the time of DNA boost. The absence of CD4+ T cells at the time of DNA delivery resulted in a four week delay in the appearance of antibody. Since influenza hemagglutinin has been characterized as a T-dependent antigen the requirement for CD4+ T cells at the time of DNA prime was not surprising, but the appearance of a delayed H1-specific antibody response suggested that DNA-expressed antigen had continued to be available to prime CD4+ T cells as they reappeared following the disappearance of depleting antibody. The independence of the antibody response on the presence of CD8+ T cells suggested that DNA-primed H1-specific CD8+ T cells did not limit the plateau level of response or the ability to boost a suboptimal response. The results from our CD4+ T cell depletion experiment suggested that DNA-expressed antigen continued to be available for an extended period of time following immunization. To examine the duration of functional antigen expression for raising an antibody response, mice lacking α/β T cells (TCR-/-) were immunized with DNA or immunized with hemagglutinin protein. Naive T cells from TCR+/+ mice were transferred into the immunized TCR-/- mice on various days post DNA or protein immunization. The results from these studies show that antigen is available to raise antibody longer following DNA immunization than following a protein immunization. This result is likely due to continued expression of plasmid DNA. We found differences in the longevity of antigen expression following gene gun and intramuscular routes of inoculation. For gene gun immunizations, not intramuscular immunizations, approximately 90% of functional antigen was lost within one week of immunization. We suggest that this is consistent with a role for antigen expression by transfected cells within the target site, the epidermis, which is largely lost by 1-2 weeks following gene gun immunization. We also found that following intramuscular immunization the dominant IgG isotype changed with time of TCR+/+ T cell transfer. By contrast, there was no change in the dominant isotype following gene gun immunizations. These results suggest that the factor(s) that contribute to the development of the Th1-bias seen following intramuscular DNA immunizations are lost early. To examine the nature of the antigen presenting cell following DNA immunization, dendritic cells were sorted from the inguinal lymph nodes and spleens of gene gun or intramuscularly immunized mice on various days following DNA delivery. The dendritic cell (CD11c+) and non-dendritic cell (CD11c-) populations were used in restimulation assays with H1-specific T cell clones. Despite similar titers of raised antibody in gene gun and intramuscularly immunized mice, H1-specific antigen presenting dendritic cells were isolated from the inguinal lymph nodes and spleens of gene gun, but not intramuscularly immunized mice. Antigen presentation by dendritic cells was detected for as long as 21 days following gene gun delivery. We hypothesize that the inability to detect dendritic cell presentation of antigen following intramuscular DNA delivery may be due to a more broad distribution of antigen presenting cells, different properties of antigen presenting cells, and/or the contribution of other non-dendritic cells to antigen presentation following intramuscular, but not gene gun, immunizations. We present our results within a model for the initiation and maintenance of DNA-raised antibody responses. Within this model our data specifically contribute to understanding the initiation and generation of the DNA-raised antibody response within lymphoid tissue and the maintenance of the DNA-raised antibody response.