Browsing by Author "Li, Liwu"
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- Additive effects among uterine paracrine factors in promoting bovine trophoblast cell proliferationXie, Ming (Virginia Tech, 2014-06-10)Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes.
- Alleviation of monocyte exhaustion by BCG derivative mycolic acidWu, Yajun; Caldwell, Blake; Wang, Jing; Zhang, Yao; Li, Liwu (Elsevier, 2024-01)Monocyte exhaustion with sustained pathogenic inflammation and immune-suppression, a hallmark of sepsis resulting from systemic infections, presents a challenge with limited therapeutic solutions. This study identified Methoxy-Mycolic Acid (M-MA), a branchedmycolic acid derived from Mycobacterium bovis Bacillus Calmette–Gue´ rin (BCG), as a potent agent in alleviating monocyte exhaustion and restoring immune homeostasis. Co-treatment of monocytes with M-MA effectively blocked the expansion of Ly6Chi/CD38hi/PD-L1hi monocytes induced by LPS challenges and restored the expression of immuneenhancing CD86. M-MA treatment restored mitochondrial functions of exhausted monocytes and alleviated their suppressive activities on co-cultured T cells. Independent of TREM2, M-MA blocks Src-STAT1- mediated inflammatory polarization and reduces the production of immune suppressors TAX1BP1 and PLAC8. Whole genome methylation analyses revealed M-MA’s ability to erase the methylation memory of exhausted monocytes, particularly restoring Plac8 methylation. Together, our data suggest M-MA as an effective agent in restoring monocyte homeostasis with a therapeutic potential for treating sepsis.
- Alteration of Lysosome Fusion and Low-grade Inflammation Mediated by Super-low-dose EndotoxinBaker, Bianca; Geng, Shuo; Chen, Keqiang; Diao, Na; Yuan, Ruoxi; Xu, Xiguang; Dougherty, Sean; Stephenson, Caroline; Xiong, Huabao; Chu, Hong Wei; Li, Liwu (American Society for Biochemistry and Molecular Biology, Inc, 2015-01-13)Background: Super-low-dose endotoxemia contributes to cell stress and low-grade inflammation. Results: Super-low-dose LPS removed Tollip from late endosomes/lysosomes and blocked lysosome fusion with endosomes or autophagosomes. Tollip knock-out mice had impaired wound healing. Conclusion: Super-low-dose LPS leads to cell stress through clearing Tollip and blocking lysosome fusion. Significance: Our data reveal molecular dynamics of innate immunity regulation.
- Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsisCaldwell, Blake A.; Wu, Yajun; Wang, Jing; Li, Liwu (Elsevier, 2024-03)Monocytes can develop an exhausted memory state characterized by reduced differentiation, pathogenic inflammation, and immune suppression that drives immune dysregulation during sepsis. Chromatin alterations, notably via histone modifications, underlie innate immune memory, but the contribution of DNA methylation remains poorly understood. Using an ex vivo sepsis model, we show altered DNA methylation throughout the genome of exhausted monocytes, including genes implicated in immune dysregulation during sepsis and COVID-19 infection (e.g., Plac8). These changes are recapitulated in septic mice induced by cecal slurry injection. Methylation profiles developed in septic mice are maintained during ex vivo culture, supporting the involvement of DNA methylation in stable monocyte exhaustion memory. Methylome reprogramming is driven in part by Wnt signaling inhibition in exhausted monocytes and can be reversed with DNA methyltransferase inhibitors, Wnt agonists, or immune training molecules. Our study demonstrates the significance of altered DNA methylation in the maintenance of stable monocyte exhaustion memory.
- Analysis of the Allergenic Potential of the Ubiquitous Airborne Fungus Alternaria Using BioinformaticsBabiceanu, Mihaela (Virginia Tech, 2011-06-15)Among the environmental airborne fungi one of the most common is Alternaria alternata. From a clinical perspective Alternaria has long been associated with IgE-mediated, histamine-dependent mold allergy, allergic rhinitis, chronic rhinosinusitis (CRS) and asthma. Recently it has been proven that an abnormal immunological response to Alternaria most likely contributes to the pathogenesis of upper respiratory airway disorders. In this body of work, we present for the first time results of several sets of experiments including, 1) the analysis of A. alternata spore germination expressed sequence tags (ESTs), 2) the survey of global allergen homologues in fungal genomes, and 3) the first microarray experiment investigating airway epithelial cell responses to this fungus. In the first project, the analyses of the EST dataset offered a first look into the gene content of A. alternata and represents the beginning of future research of this ubiquitous fungus. Annotation and classification of ESTs revealed a number of genes that could be involved in the immunomodulation process of the human immune response toward fungi. We also discovered that the majority of known allergens are expressed during the spore germination phase of A. alternata. For investigating the allergenic potential of fungi we developed a whole genome approach by querying fungal genome sequences (A. alternata, A. brassicicola, and Aspergillus fumigatus) with a database of all known allergenic proteins from a taxonomically diverse group of organisms. Interestingly, we identified homologues of diverse types of allergens in these fungal genomes and also many homologues of allergens from other organisms including those from pollen, insects, and venoms. Finally, we investigated global gene expression changes of human airway cells in response to A. alternata and an ∆alt a 1 deletion mutant. We found that wild type Alternaria spores induced significant changes in gene expression patterns in human airway epithelial cells, especially known immune response genes. Furthermore, results of these analyses revealed that Alt a 1 is a major factor in inducing epithelial inflammatory responses.
- Assembly of Inflammation-Related Genes for Pathway-Focused Genetic AnalysisLoza, Matthew J.; McCall, Charles E.; Li, Liwu; Isaacs, William B.; Xu, Jianfeng; Chang, Bao-Li (PLOS, 2007-10-17)Recent identifications of associations between novel variants in inflammation-related genes and several common diseases emphasize the need for systematic evaluations of these genes in disease susceptibility. Considering that many genes are involved in the complex inflammation responses and many genetic variants in these genes have the potential to alter the functions and expression of these genes, we assembled a list of key inflammation-related genes to facilitate the identification of genetic associations of diseases with an inflammation-related etiology. We first reviewed various phases of inflammation responses, including the development of immune cells, sensing of danger, influx of cells to sites of insult, activation and functional responses of immune and non-immune cells, and resolution of the immune response. Assisted by the Ingenuity Pathway Analysis, we then identified 17 functional sub-pathways that are involved in one or multiple phases. This organization would greatly increase the chance of detecting gene-gene interactions by hierarchical clustering of genes with their functional closeness in a pathway. Finally, as an example application, we have developed tagging single nucleotide polymorphism (tSNP) arrays for populations of European and African descent to capture all the common variants of these key inflammation-related genes. Assays of these tSNPs have been designed and assembled into two Affymetrix ParAllele customized chips, one each for European (12,011 SNPs) and African (21,542 SNPs) populations. These tSNPs have greater coverage for these inflammation-related genes compared to the existing genome-wide arrays, particularly in the African population. These tSNP arrays can facilitate systematic evaluation of inflammation pathways in disease susceptibility. For additional applications, other genotyping platforms could also be employed. For existing genome-wide association data, this list of key inflammation-related genes and associated subpathways can facilitate comprehensive inflammation pathway- focused association analyses.
- Baicalein, a novel anti-diabetic compoundFu, Yu (Virginia Tech, 2012-08-08)Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of ?-cells, leading to the impaired insulin secretion. Thus, the search for agents to protect b-cell and enhance its function is important for diabetes treatment. Studies have reported that baicalein, a flavone originally isolated from the roots of Chinese herb Scutellaria baicalensis, has various claimed beneficial effects on health, such as anti-oxidant, anti-viral, anti-thrombotic, and anti-inflammatory effects. However, it is unclear whether it exerts an anti-diabetic action. Here, we present evidence that baicalein may be a novel anti-diabetic agent. Specifically, dietary intake of baicalein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in high-fat diet (HFD)-fed middle-aged diabetic mice, which was associated with the improved isle t?-cell survival and mass. Baicalein treatment had no effect on food intake, body weight gain, circulating lipid profile, and insulin sensitivity in HFD-fed mice. In in-vitro studies, baicalein significantly augmented glucose-stimulated insulin secretion in insulin-secreting cells (INS1) and promotes viability of INS1 cells and human islets. These results demonstrate that baicalein may be a naturally occurring anti-diabetic agent by directly modulating pancreatic?-cell function.
- Biochemical and Microscopic Characterization of INFT-1: an Inverted Formin in C. elegansLi, Ying (Virginia Tech, 2011-03-14)Formins are potent regulators of actin dynamics that can remodel the actin cytoskeleton to control cell shape, cell cytokinesis, and cell morphogenesis. The defining feature of formins is the formin homology 2 (FH2) domain (Paul and Pollard, 2008), which promotes actin filament assembly while processively moving along the polymerizing filament barbed end. INFT-1 is one of six formin family members present in Caenorhabditis elegans (Hunt-Newbury et al., 2007) and is most closely related to vertebrate INF2, an inverted formin with regulatory domains in the C- rather than N-terminus. Nematode INFT-1 contains both formin homology 1 (FH1) and formin homology 2 (FH2) domains. However, it does not share the regulatory N-terminal Diaphanous Inhibitory Domain (DID) domain and C-terminal Diaphanous Autoregulatory Domain (DAD) domain found in mammalian INF2. In contrast to mammalian INF2, the sequence of INFT-1 starts immediately at FH1 domain and C-terminal region of INFT-1 shares little homology with INF2, suggesting that elegans INFT-1 is regulated by other mechanisms. We used fluorescence spectroscopy to determine the effect of INFT-1 FH1FH2 on actin assembly and total internal reflection fluorescence microscopy to investigate how INFT-1 formin homology 1 and formin homology 2 domains (FH1FH2) mediate filament nucleation and elongation. INFT-1 FH1FH2 nucleates actin filament and promote actin assembly. However, INFT-1 FH1FH2 reduces filament barbed-end elongation rates in the absence or presence of profilin. Evidences demonstrated that INFT-1 is non-processive, indicating a unique mechanism of nucleation. INFT-1 nucleation efficiency is similar to the efficiency of Arabidopsis FORMIN1 (AFH1), another non-processive formin. High phosphate affected the assembly activity of INFT-1 FH1FH2 in the absence or presence of profilin. INFT is thus the second example of a non-processive formin member and will allow a more detailed understanding of the mechanistic difference between processive and non-processive formins.
- Cellular Reprogramming in Skeletal Muscle after Repeated Exposures to EndotoxinDenko, Laura Michelle (Virginia Tech, 2012-06-21)Obesity-related metabolic derangements have been linked to toll-like receptor 4 (TLR4), an innate immune system receptor, due to its role in proinflammatory pathways. Lipopolysaccharide (LPS), a gram-negative bacteria cell wall component, is the ligand for TLR4, and has been shown to be elevated in states of metabolic disease. Heightened levels of circulating endotoxin is termed metabolic endotoxemia and has been linked to systemic inflammation which is associated with obesity, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD). Immune cells exhibit a protective ability to develop endotoxin tolerance. The objective of this study was to determine if endotoxin tolerance exists in skeletal muscle cells, and if a condition that mimics a state of over nutrition, such as elevated levels of fatty acids, affect this tolerance. To this end, L6 skeletal muscle cells were treated with low (50 pg/mL)- and high (500 ng/mL)-doses of LPS, with and without the presence of free fatty acids (FFAs). Tolerance was assessed by measuring: 1) changes in mRNA expression of interleukin-6 (IL-6) and monocyte chemoattractant-1 (MCP-1) as markers of a pro-inflammatory response; and 2) mRNA levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1-°) and mitochondrial oxidative capacity via an XF24 Flux Analyzer (Seahorse Bioscience) as measures of the metabolic response. Tolerance to LPS was observed in response to low- and high-doses with MCP-1 mRNA transcription but not IL-6. Changes in PGC1-° and mitochondrial OCR exhibited a tolerant effect in response to the high dose of LPS but not the low dose. The addition of free fatty acids to LPS treatments did not prevent the tolerant effects under any conditions. In conclusion, LPS tolerance exists in skeletal muscle cells but appears to differ depending on pro-inflammatory target and LPS concentration. Additionally, fatty acids, in the current model, have no effect on LPS tolerance.
- Characterization of an in vitro exercise model and the effects of a metabolic endotoxemia on skeletal muscle adaptation to electric pulse stimulationHarvey, Mordecai Micah (Virginia Tech, 2017-06-20)The prevalence of obesity and type II diabetes is increasing. Although exercise is widely accepted for prevention and treatment, evidence of resistance to exercise in patients with these diseases is also mounting. Muscle contraction during exercise stimulate cellular responses important for adaptation. These responses include the release of myokines and the subsequent increase in substrate metabolism. This study aimed to define a culture model for simulating exercise in human primary skeletal muscle cells. We hypothesized that chronic electric pulse stimulation (EPS) of human myotubes in vitro would emulate cellular and molecular responses to exercise observed in vivo. To define this model, we applied EPS to human myotubes for varied lengths of time and measured interleukin-6 (Il-6), peroxisome proliferator-activated receptor gamma coactivator 1- (PGC1-), superoxide dismutase 2 (SOD2), substrate metabolism, metabolic enzyme activity, heat stress markers, and pH. To recreate the inflammatory milieu observed in metabolic disease states we treated the myotubes with a low dose of 20 EU lipopolysaccharide (LPS). Following the 24-hour stimulation we observed significant increases in transcription of Il-6, PGC1-, and SOD2. Basal glucose and fatty acid oxidation were also markedly increased in the cells after EPS. Cells treated with LPS elicited a blunted transcriptional, metabolic, and enzymatic response to EPS. These findings suggest that EPS is a viable model for simulating the effects of exercise. Our observations also indicate that an inflammatory environment could play a role in interfering with the adaptations to exercise.
- Characterization of Biomedical and Incidental Nanoparticles in the Lungs and Their Effects on HealthMcDaniel, Dylan K. (Virginia Tech, 2018-11-20)Nanomaterials are defined as any material with at least one external dimension less than 100 nm. Recently, nanomaterials have become more common in medicine, technology, and engineering. One reason for their increased interest is due to nanomaterials having unique properties that allow them to interact effectively with biological systems. In terms of drug delivery, the lungs are a highly desirable site to administer therapeutic nanoparticles. Indeed, inflammatory diseases such as asthma and emphysema could potentially benefit from nanoparticle-mediated delivery. However, the lungs are also in constant contact with airborne particulate matter. Thus, harmful nanoparticles can enter the lungs and cause or even exacerbate inflammatory diseases. Our work focused on characterization of both therapeutic and potentially harmful nanoparticles in the lungs. We found that fluorescently-labeled nanoparticles were phagocytosed by macrophages and did not induce apoptosis or inflammation in the lungs, making them potentially useful as a therapeutic for inflammatory diseases. We also characterized a rare form of titanium-based particles called Magnéli phases, which have been shown to be produced via coal burning. We found that while these particles are non-inflammatory in the lungs of mice, they lead to apoptosis of macrophages as well as a change in gene expression associated with increased fibrosis. Ultimately, this was shown to lead to a decrease in lung function parameters and airway hyperresponsiveness, indicating increased lung stiffness after long-term nanoparticle exposure. Our data adds significant contributions to the field by assessing two nanoparticles with vastly different compositions in the lungs. Overall, we found that the unique properties of both particle types allows for interactions with cells and tissues. These interactions can have important outcomes on health, both in terms of disease treatment and exacerbation.
- Characterizing the Innate Immune Response of Human Airway Cells to the Unique Fungal Allergen Alt a 1Hayes, Tristan Alonzo (Virginia Tech, 2017-04-25)Allergic airway diseases such as rhinitis, asthma, and chronic rhinosinusitis are responsible for causing a huge economic burden on patients and society. Patients suffering from asthma often have allergies to pollen, dust mite, and mold. Interestingly, studies have shown that there is a correlation between severe asthma and sensitization to fungi including Aspergillus, Alternaria, Cladosporium, and Penicillium. This project has been focused on studying the innate immunomodulatory activities of the major allergen Alt a 1, from the ubiquitous airborne fungus, Alternaria alternata. In several studies, 90-100% of allergic patients who are sensitized to Alternaria, have Alt a 1 specific IgE antibodies indicating that it is a major and clinically relevant allergen. Although progress has been made over the past few decades regarding elucidating the mechanistic underpinnings of allergic inflammation, more research needs to be done, especially in regards to innate immunity and its role in the sensitization and exacerbation aspects of allergic diseases. Published studies have increasingly made it clear that Toll-like receptors (TLRs) are key players in innate immunity to several allergens. For example, the dust mite allergen, Der p 2, has been shown to mimic the activity of human and mouse MD2 in the presence of LPS to trigger a response through TLR4. Bet v 1, an allergen from Birch tree, has been shown to enter and be transported through lung epithelium in patient cells. It is hypothesized that transcytosis of allergens like Bet v 1 may contribute to sensitization and exacerbation in atopic individuals. This project was focused on two primary aims; (1) Characterize the innate immune response of Alt a 1 in human airway epithelial cells, and (2) Identify if and how Alt a 1 can enter human airway cells. We found that Alt a 1 was able to stimulate innate immune responses in bronchial epithelial cells and this was dependent upon TLR2, TLR4 and the downstream adaptor proteins MyD88 and TIRAP. We also found in our studies that Alt a 1 rapidly enters bronchial epithelial cells. Furthermore, our data suggests that endocytosis of Alt a 1 may be partially dependent upon interaction with phosphatidyl-inositol-3-phosphate (PI-3-P).
- Chronic Treatment of TMAO Undermines Mouse Cardiac Structure and Function in a Sex-specific MannerDing, Hanzhang (Virginia Tech, 2023-12-19)Cardiovascular disease (CVD) is a major cause of mortality and morbidity worldwide, often with heart failure as the terminal stage. Clinical studies have associated elevated levels of trimethylamine N-oxide (TMAO), a gut-derived metabolite, with adverse outcomes of CVD. As of today, TMAO's effects on cardiac structure and function are not well understood. In this study, both male and female TMAO-treated hearts showed functional deficits based on electrocardiography and echocardiography results. Immunohistochemistry results showed signs of hypertrophic cardiomyopathy in TMAO-treated male hearts while female TMAO-treated hearts showed signs of dilated cardiomyopathy. Neither TMAO group showed signs of fibrosis. Overproduction of reactive oxygen species was only observed in male TMAO-treated hearts. At the level of individual cardiomyocytes, significant delays in time to reach maximum contraction and dilation were only seen in TMAO-treated male hearts along with higher contractile force. Overall, TMAO-treated hearts show significant functional deficits with altered structure in a sex-specific way. Our study utilizes a variety of methods to comprehensively characterize features of TMAO-induced heart failure in both males and females which extends our current knowledge from human clinical associations.
- Computational Analysis of Gene Expression Regulation from Cross Species Comparison to Single Cell ResolutionLee, Jiyoung (Virginia Tech, 2020-08-31)Gene expression regulation is dynamic and specific to various factors such as developmental stages, environmental conditions, and stimulation of pathogens. Nowadays, a tremendous amount of transcriptome data sets are available from diverse species. This trend enables us to perform comparative transcriptome analysis that identifies conserved or diverged gene expression responses across species using transcriptome data. The goal of this dissertation is to develop and apply approaches of comparative transcriptomics to transfer knowledge from model species to non-model species with the hope that such an approach can contribute to the improvement of crop yield and human health. First, we presented a comprehensive method to identify cross-species modules between two plant species. We adapted the unsupervised network-based module finding method to identify conserved patterns of co-expression and functional conservation between Arabidopsis, a model species, and soybean, a crop species. Second, we compared drought-responsive genes across Arabidopsis, soybean, rice, corn, and Populus in order to explore the genomic characteristics that are conserved under drought stress across species. We identified hundreds of common gene families and conserved regulatory motifs between monocots and dicots. We also presented a BLS-based clustering method which takes into account evolutionary relationships among species to identify conserved co-expression genes. Last, we analyzed single-cell RNA-seq data from monocytes to attempt to understand regulatory mechanism of innate immune system under low-grade inflammation. We identified novel subpopulations of cells treated with lipopolysaccharide (LPS), that show distinct expression patterns from pro-inflammatory genes. The data revealed that a promising therapeutic reagent, sodium 4-phenylbutyrate, masked the effect of LPS. We inferred the existence of specific cellular transitions under different treatments and prioritized important motifs that modulate the transitions using feature selection by a random forest method. There has been a transition in genomics research from bulk RNA-seq to single-cell RNA-seq, and scRNA-seq has become a widely used approach for transcriptome analysis. With the experience we gained by analyzing scRNA-seq data, we plan to conduct comparative single-cell transcriptome analysis across multiple species.
- Computational Systems Biology Analysis of Cell Reprogramming and Activation DynamicsFu, Yan (Virginia Tech, 2012-07-17)In the past two decades, molecular cell biology has transitioned from a traditional descriptive science into a quantitative science that systematically measures cellular dynamics on different levels of genome, transcriptome and proteome. Along with this transition emerges the interdisciplinary field of systems biology, which aims to unravel complex interactions in biological systems through integrating experimental data into qualitative or quantitative models and computer simulations. In this dissertation, we applied various systems biology tools to investigate two important problems with respect to cellular activation dynamics and reprograming. Specifically, in the first section of the dissertation, we focused on lipopolysaccharide (LPS)-mediated priming and tolerance: a reprogramming in cytokine production in macrophages pretreated with specific doses of LPS. Though both priming and tolerance are important in the immune system's response to pathogens, the molecular mechanisms still remain unclear. We computationally investigated all network topologies and dynamics that are able to generate priming or tolerance in a generic three-node model. Accordingly, we found three basic priming mechanisms and one tolerance mechanism. Existing experimental evidence support these in silico found mechanisms. In the second part of the dissertation, we applied stochastic modeling and simulations to investigate the phenotypic transition of bacteria E.coli between normally-growing cells and persister cells (growth-arrested phenotype), and how this process can contribute to drug resistance. We built up a complex computational model capturing the molecular mechanism on both single cell level and population level. The paper also proposed a novel way to accelerate the phenotypic transition from persister cells to normally growing cell under resonance activation. The general picture of phenotypic transitions should be applicable to a broader context of biological systems, such as T cell differentiation and stem cell reprogramming.
- Control of lupus nephritis by changes of gut microbiotaMu, Qinghui; Zhang, Husen; Liao, Xiaofeng; Lin, Kaisen; Liu, Hualan; Edwards, Michael R.; Ahmed, Sattar Ansar; Yuan, Ruoxi; Li, Liwu; Cecere, Thomas E.; Branson, David B.; Kirby, Jay L.; Goswami, Poorna; Leeth, Caroline M.; Read, Kaitlin A.; Oestreich, Kenneth J.; Vieson, Miranda D.; Reilly, Christopher M.; Luo, Xin M. (2017-07-11)Background: Systemic lupus erythematosus, characterized by persistent inflammation, is a complex autoimmune disorder with no known cure. Immunosuppressants used in treatment put patients at a higher risk of infections. New knowledge of disease modulators, such as symbiotic bacteria, can enable fine-tuning of parts of the immune system, rather than suppressing it altogether. Results: Dysbiosis of gut microbiota promotes autoimmune disorders that damage extraintestinal organs. Here we report a role of gut microbiota in the pathogenesis of renal dysfunction in lupus. Using a classical model of lupus nephritis, MRL/lpr, we found a marked depletion of Lactobacillales in the gut microbiota. Increasing Lactobacillales in the gut improved renal function of these mice and prolonged their survival. We used a mixture of 5 Lactobacillus strains (Lactobacillus oris, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus johnsonii, and Lactobacillus gasseri), but L. reuteri and an uncultured Lactobacillus sp. accounted for most of the observed effects. Further studies revealed that MRL/lpr mice possessed a “leaky” gut, which was reversed by increased Lactobacillus colonization. Lactobacillus treatment contributed to an anti-inflammatory environment by decreasing IL-6 and increasing IL-10 production in the gut. In the circulation, Lactobacillus treatment increased IL-10 and decreased IgG2a that is considered to be a major immune deposit in the kidney of MRL/lpr mice. Inside the kidney, Lactobacillus treatment also skewed the Treg-Th17 balance towards a Treg phenotype. These beneficial effects were present in female and castrated male mice, but not in intact males, suggesting that the gut microbiota controls lupus nephritis in a sex hormone-dependent manner. Conclusions: This work demonstrates essential mechanisms on how changes of the gut microbiota regulate lupusassociated immune responses in mice. Future studies are warranted to determine if these results can be replicated in human subjects.
- Corticosteroid-Encapsulated Nanoparticles in Thermoreversible Gels for the Amelioration of Choroidal Neovascularization in Age-Related Macular DegenerationHirani, Anjali A. (Virginia Tech, 2015-04-30)Age-related macular degeneration (AMD) is one of the leading causes of blindness in adults over the age of 60. Currently, at least 11 million patients in the United States have some form of macular degeneration and this number is projected to grow as the population ages. The more severe form of the disease – neovascular (wet) AMD, is characterized by intraocular neovascularization, inflammation, and retinal damage; however, the disease progression can be deterred through intraocular injections of anti-angiogenic agents. The complications and burden that arise from repetitive injections as well as the difficulty posed by targeting the posterior segment of the eye make this an interesting territory for the development of novel drug delivery systems. New methods for drug delivery are being investigated exploring the use of nanoparticles and other polymeric materials. The goal of this project is to study the potential use of poly(lactide-co-glycolic acid)-polyethylene glycol (PLGA-PEG) nanoparticles in thermoreversible gels as localized sustained intraocular drug delivery. We prepared stable and reproducible corticosteroid-encapsulated nanoparticles in thermoreversible gels to inhibit vascular endothelial growth factor (VEGF) overexpression characteristic of neovascular AMD. We characterized the drug delivery system by obtaining size, shape, and drug encapsulation data. We also demonstrated that the polymer could be injected into the vitreous as a solution and transition to a gel phase based on the temperature difference between regular indoor environment and the vitreous body. The drug delivery system was tested on human retinal pigment epithelial cells (ARPE-19), for cytotoxicity, uptake and VEGF expression. We also examined the drug delivery system's ability to mitigate the disease progression in a mouse model of choroidal neovascularization (CNV). The effect on blood vessel area was shown and the changes in the mRNA expression of angiogenesis mediators were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). These results indicate that the proposed drug delivery systems has the promise to be developed for retinal diseases, involving CNV, including neovascular AMD. Further studies are warranted in developing this promising intraocular drug delivery system for wet AMD and similar ophthalmic diseases.
- Deficiency in Toll-interacting protein (Tollip) skews inflamed yet incompetent innate leukocytes in vivo during DSS-induced septic ColitisDiao, Na; Zhang, Yao; Chen, Keqiang; Yuan, Ruoxi; Lee, Christina K.; Geng, Shuo; Kowalski, Elizabeth A.; Li, Liwu (Nature, 2016-10-05)Functionally compromised neutrophils contribute to adverse clinical outcomes in patients with severe inflammation and injury such as colitis and sepsis. However, the ontogeny of dysfunctional neutrophil during septic colitis remain poorly understood. We report that the dysfunctional neutrophil may be derived by the suppression of Toll-interacting-protein (Tollip). We observed that Tollip deficient neutrophils had compromised migratory capacity toward bacterial product fMLF due to reduced activity of AKT and reduction of FPR2, reduced potential to generate bacterial-killing neutrophil extra-cellular trap (NET), and compromised bacterial killing activity. On the other hand, Tollip deficient neutrophils had elevated levels of CCR5, responsible for their homing to sterile inflamed tissues. The inflamed and incompetent neutrophil phenotype was also observed in vivo in Tollip deficient mice subjected to DSSinduced colitis. We observed that TUDCA, a compound capable of restoring Tollip cellular function, can potently alleviate the severity of DSS-induced colitis. In humans, we observed significantly reduced Tollip levels in peripheral blood collected from human colitis patients as compared to blood samples from healthy donors. Collectively, our data reveal a novel mechanism in Tollip alteration that underlies the inflamed and incompetent polarization of neutrophils leading to severe outcomes of colitis.
- Detecting intracellular translocation of native proteins quantitatively at the single cell levelCao, Zhenning; Geng, Shuo; Li, Liwu; Lu, Chang (The Royal Society of Chemistry, 2014-04-07)The intracellular localization and movement (i.e. translocation) of proteins are critically correlated with the functions and activation states of these proteins. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. In this report, we demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. We sequentially conducted chemical release of cytosolic proteins and fluorescence immunostaining of a targeted protein. The detected fluorescence intensity of cells was shown to be quantitatively correlated to the cytosolic/nuclear localization of the protein. We used our approach to detect the translocation of native NF-_B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. The incorporation of fluorescence immunostaining makes our approach compatible with the analysis of cell samples from lab animals and patients. Our method will dramatically lower the technological hurdle for studying subcellular localization of proteins.
- Development of Exhausted Memory Monocytes and Underlying MechanismsPradhan, Kisha; Yi, Ziyue; Geng, Shuo; Li, Liwu (2021-10-28)Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6C(hi) population, and deplete the homeostatic non-classical Ly6C(lo) population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6C(hi) monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD(+); elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.