Theses and Dissertations from UMD
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Item INFLAMMATORY MACROPHAGE REGULATION OF ANGIOGENESIS AND SKELETAL MUSCLE PHENOTYPES(2023) Evans, William Stuart; Prior, Steven J; Kinesiology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Chronic inflammation is a hallmark of cardiovascular disease; however, there is a lack of understanding of how systemic inflammation affects the peripheral skeletal muscle to potentially hasten frailty and functional declines in patients. The overarching objective of this dissertation was to determine whether this systemic inflammation is accompanied by macrophage infiltration of skeletal muscle and reductions in skeletal muscle capillarization and fiber size. Using animal models of a) heart failure (HF) induced by transverse aortic constriction (TAC), and b) skeletal muscle ischemia, this work illuminates changes that occur in skeletal muscle with cardiovascular disease-related inflammation. The first study demonstrated that pressure overload resulted in cardiac hypertrophy in male rats consistent with heart failure with preserved ejection fraction (HFpEF), while females did not show cardiac hypertrophy or HF. The second study demonstrated sex-specific differences in skeletal muscle, with TAC male rats exhibiting smaller fiber sizes and greater capillarization, and female TAC rats exhibiting lower capillarization than Sham counterparts. This study then investigated skeletal muscle macrophages to determine whether they might underly or contribute to these differences. There were fewer macrophages in the skeletal muscle of male TAC rats than male Sham rats, and macrophage conditioned medium from TAC rats produced less-developed capillary networks in an ex vivo, experimental assay. Finally, the third study investigated whether an acute bout of systemic inflammation, in the absence of HF, could alter the infiltration of macrophages, or skeletal muscle fiber size or capillarization. Hindlimb ischemia was used to induce acute, systemic inflammation that peaked after 1 day. This systemic inflammation increased the infiltration of macrophages into remote, non-ischemic skeletal muscle by day 7; however, muscle structure was preserved over this short time course. This dissertation demonstrates that cardiovascular disease-associated inflammation is linked with tissue-level changes in macrophages in a sex-specific manner. These changes accompany and may, over time, contribute to skeletal muscle fiber atrophy and changes in capillarization in cardiovascular disease patients.Item Identification and functional analysis of a biflavone as a novel inhibitor of TRPV4-dependent atherogenic process in macrophages(2021) Alharbi, Mazen Obaid; Rahaman, Shaik O.; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cardiovascular disease is the major cause of death throughout the world. Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity throughout the world. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden macrophage foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Transient receptor potential vanilloid 4 (TRPV4), a non-selective Ca2+-permeant ion channel and a mechanosensor, is widely expressed in diverse cell types including macrophages. Accumulating reports from our laboratory and others on TRPV4 recognized this plasma membrane receptor/channel as an essential modulator of various physiological functions in cardiac, pulmonary, urinary, skeletal, digestive system, and central and peripheral nervous systems. Thus, it is expected that aberrant regulation of TRPV4 activity may lead to multiple pathological conditions such as cardiovascular disease, pulmonary disease, inflammation, neurological disorders, inflammatory bowel disease and wound healing. Previous studies by our group and others have reported that TRPV4 can be activated by numerous mechanical and biochemical stimuli including shear stress, osmolarity, temperature, and growth factors, as well as by alterations in matrix stiffness in vitro and in vivo. Recently, we reported that oxidized low-density lipoprotein-mediated and matrix stiffness-induced macrophage foam cell formation, a critical pathological process in atherosclerosis, is regulated in a TRPV4-dependent manner. Given that TRPV4 is a mechanosensitive channels and mechanical factors like hypertension, disrupted laminar flow of blood, and matrix stiffening are recognized pro-atherogenic factors, makes TRPV4 an important target for therapeutic intervention of atherosclerosis. The objectives of this proposal is to: i) identify natural inhibitor (s) of TRPV4 utilizing a fluorometric imaging plate reader-supported Ca2+ influx assay, ii) functionally characterize the identified compound, and iii) determine the mechanisms by which the identified compound blocks pro-atherogenic/inflammatory TRPV4 activity in macrophages. We expect that the results of this study may strengthen the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.Item THE PROATHEROGENIC ROLE OF THE TRPV4 CALCIUM-PERMEABLE CHANNEL IN MACROPHAGES(2016) Merth, Michael; Rahaman, Shaid O; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Atherosclerosis is a multi-faceted chronic disease and one of the leading causes of cardiovascular diseases which attribute to 28.2% of all-cause mortality worldwide. Central tenets of atherogenesis are macrophage passage and transformation to foam cells. It is known that numerous macrophage membrane proteins regulate this process by controlling properties such as migration and binding and uptake of oxidized lipids. The focus of this study is to identify mechanisms by which the transient receptor potential (TRP) channel of the vanilloid subfamily, TRPV4, a calcium-permeable channel, regulates proatherogenic macrophage functions. The findings show TRPV4 is expressed and functional in macrophages, TRPV4 modulates macrophage migratory characteristics, oxidized low-density lipoprotein (oxLDL) uptake and foam cell formation is reliant on TRPV4-elicited Ca2+ influx, and the physiological inflammatory stimulus of lipopolysaccharide can mediate TRPV4 function. These results identified previously unknown components to macrophage migration and foam cell development. Collectively, these discoveries associate the TRPV4 channel with atherogenesis by identifying new regulators for macrophage phagocytosis and migration that are essential to atherosclerosis development.Item ALTERED PORCINE ALVEOLAR MACROPHAGE PHENOTYPE BY ALL-TRANS RETINOIC ACID AND ASCARIS SUUM INFECTION(2011) Perry, Trinity Lynn; Mosser, David M; Urban, Joseph F; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ascaris suum is known to cause ascariasis of both pigs and humans. Ascariasis is associated with a reduced control of intracellular pathogens and decreased vaccine efficacy. All-trans retinoic acid (ATRA), an active metabolite of Vitamin A (VA), is known to mediate a diverse array of physiological and biological processes including immune and inflammatory responses. The phenotype of alveolar macrophages (AM), the most abundant cell in the lung, has not been critically examined during chronic infection with A. suum nor has the effect of administration of dietary ATRA alone or during infection. This combination of treatments is likely common in underdeveloped regions where infection with Ascaris is prevalent and public health initiatives to control nutrient deficiencies often include VA supplementation. In this study we showed that a low dose and repeated "trickle" infection with A. suum alone and with ATRA administration altered the AM cell surface antigen phenotype.Item The Role of Interleukin-19 in Interleukin-10 Production by Regulatory Macrophages(2010) Yahil, Ron Jonathan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interleukin-19 (IL-19) is a recently discovered member of the IL-10 family of Class II cytokines. Although it is known to be secreted by monocytes and has been associated with various models of disease, the biological function of IL-19 remains largely unknown. IL-19 does share many important characteristics with IL-10. Because of this, we hypothesized that IL-19 may be regulated in a manner similar to IL-10, and may provide insight into the molecular mechanism of IL-10 regulation. In addition, IL-19 has been reported to increase IL-10 production in monocytes, and we theorized that it may be able to do the same in macrophages. Like IL-10, IL-19 is expressed in regulatory macrophages. Also, IL-19 is itself able to increase IL-10 production in regulatory macrophages, and the mechanism is independent of ERK phosphorylation. This work suggests that IL-19 can play a central role in the anti-inflammatory processes of IL-10.Item Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.Item THE FUNCTIONAL REGULATION OF FCRN EXPRESSION AND FCRN-MEDIATED ANTIGEN PRESENTATION(2009) Liu, Xindong; Zhu, Xiaoping; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The neonatal Fc receptor for IgG (FcRn), a major histocompatibility complex (MHC) class I-related molecule, plays an important role in IgG transport and protection. The transport of IgG across epithelial and endothelial barriers and the IgG homeostasis maintained by FcRn contributes to the effective humoral immunity. Thus, the level of FcRn itself will affect the IgG-associated immune responses. Although FcRn is expressed in a variety of tissues and cell types, the extent to which FcRn expression is regulated by immunological and inflammatory events remains unknown.I showed here that FcRn was up-regulated by the stimulation of inflammatory cytokines or Toll-like receptor ligands in human peripheral blood mononuclear cell (PBMC) and THP-1 cell line. By chromatin immunoprecipitation, I identified three NF-κB binding sites within introns 2 and 4 of the human FcRn gene. These intronic binding sites boost FcRn transcription activities through looping with the promoter region. In contrast, FcRn expression was down-regulated by Th1 cytokine IFN-γ, and the down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. It has been demonstrated that IFN-γ activated STAT1 bound with GAS sequence in human FcRn promoter, and which blocked the transcriptional machinery. Fc gamma receptors (FcγRs) expressed in macrophages (MФ) and dendritic cells (DCs) can mediate antigen presentation in both MHC class II and MHC class I pathways. We tested here the role for FcRn in antigen presentation of IgG-restricted Immune complexes (ICs). It was observed that the expression of FcRn in MФ, but not in DC enhanced the phagosomal ICs antigen presentation to CD4 T cells. A low pH value in phgosome of MФ facilitated FcRn binding to ICs, stabilizing the antigens and promoting the efficient MHC II-peptide assembly. However, the alkalized phagosomes in DC failed FcRn to enhance the antigen presentation of ICs.Item The Influence of Parasite-derived Chemokines in Leishmaniasis(2006-12-13) Conrad, Sean Martin; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Transgenic chemokine-secreting parasites were generated and used to actively recruit immune cells into Leishmania lesions. It was hypothesized that the chemokine induced cell migration would influence the magnitude and character of the immune response and thereby effect the outcome of disease. Two different transgenic chemokine-secreting parasites were engineered. One transgenic parasite secretes murine MCP-1, a CC chemokine primarily responsible for macrophage recruitment. The other transgenic parasite secretes murine IP-10, a CXC chemokine known to attract activated T-cells. Both transgenic parasites transcribed murine chemokine mRNA, translated murine chemokine protein, and infected and replicated inside resting peritoneal macrophages similar to wild-type parasites. However, the two transgenic parasites caused diverse phenotypes in infected mice. The MCP-1 secreting parasites caused little or no detectable lesions, while the IP-10 secreting parasites caused lesions that were significantly larger than the wild-type infected mice. The healing phenotype caused by MCP-1 secreting parasites was further analyzed. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with MCP-1 secreting parasites resulted in minimal lesion development compared to mice infected with wild-type parasites. MCP-1 secreting parasites caused substantial lesions with relatively high numbers of parasites in CCR2 KO mice indicating that the parasites are viable and healthy, and that the lack of lesion development is CCR2- dependent. The enumeration of transgenic MCP-1 parasites in lesions demonstrated a significant reduction in parasite numbers, which coincided with an increase in CCR2-positive macrophage migration on day 7. CCR2-positive macrophages isolated from ears of mice infected with transgenic MCP-1 parasites contained virtually no parasites, whereas infection with wild-type parasites resulted in heavily-infected macrophages in lesions. The lack of parasite survival in mice infected with MCP-1 secreting parasites suggests that parasite-derived MCP-1 is recruiting a population of CCR2-positive macrophages to the lesion that efficiently kill Leishmania parasites. In-vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages followed by stimulation with a combination of both MCP-1 and IFN-g. This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-g to efficiently kill Leishmania parasites.