Cell Biology & Molecular Genetics Theses and Dissertations
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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 CHARACTERIZATION OF THE ROLE OF MAPKS IN LEISHMANIA INFECTED MACROPHAGES.(2009) Yang, Ziyan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the current study, we examined the role of the Mitogen Activated Protein Kinases (MAPKs) on the biological responses of macrophages infected with Leishmania. The first section examined the role of MAPK/ERK in IL-10 production by Leishmania-infected macrophages. The macrophage-derived IL-10 has been shown to exacerbate Leishmaniasis. However, the molecular mechanisms whereby Leishmaniasis prompts IL-10 induction are poorly understood. A combination of two signals was necessary for IL-10 induction by the Leishmania amastigotes-infected macrophages. The first signal is mediated by TLR ligation whereas the second signal is mediated by FcgammaR ligation, which yields a population of regulatory macrophages that produce high levels of IL-10. Infection of macrophages with L. amazonensis amastigotes from the lesion sites sparked MAPK/ERK activation, which was required, but not sufficient for IL-10 induction. In combination with an inflammatory stimulus, LMW-HA from the extracellular matrix, these parasites triggered the macrophages to highly produce IL-10. MAPK/ERK activation initiated an epigenetic modification of chromatin at the IL-10 locus, which allowed for transcription factor Sp1 binding to drive IL-10 transcription and subsequent production. U0126, an inhibitor of MAPK/ERK activation, decreased lesion progression in Leishmania infected mice. The second section examined the role of MAPK/p38 in cytokine production and vaccination against Leishmaniasis. TLR agonists activate macrophages to produce pro-inflammatory cytokines and reactive oxygen intermediates. Inhibition of MAPK/p38 reciprocally increased IL-12 but decreased TNFa production from LPS-stimulated macrophages, which also occurred following stimulation by a variety of other TLR agonists, and using different APCs. MAPK/p38 inhibition induced IL-12p40 mRNA accumulation mainly due to enhanced mRNA stability, which was independent of IL-10. Similar results were observed by knocking down MAPK/p38 using specific siRNAs or by targeted deletion of MKK3. IL-12 production following the inhibition of MAPK/p38 skewed antigen-specific T cells to produce more IFN-gamma and less IL-4 in vitro. A MAPK/p38 inhibitor was applied as an adjuvant to vaccine mice against L. major, which resulted in smaller lesions with fewer parasites. Our findings reveal an important role of MAPKs in the Leishmania pathogenesis, and suggest that the manipulation of these kinases may provide novel therapeutics for potential clinical applications.Item The crosstalk between B-cell receptor mediated signaling and the actin cytoskeleton(2008-08-15) Sharma, Shruti; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oligomerization of the B-cell receptor (BCR) by antigen leads to both signal transduction and antigen internalization for processing and presentation. Previous studies indicate that these processes intersect at the actin cytoskeleton to coordinate the two cellular processes for the optimal activation of B-cells. The exact mechanism by which signals are transduced via the actin cytoskeleton into the efficient internalization and transport of BCR-antigen complexes is not well delineated. In this thesis, I demonstrate that Bruton's tyrosine kinase (Btk), a Tec kinase in the early signaling pathway of the BCR, is able to transduce signals from the BCR to actin regulatory proteins such as WASP and N-WASP. Upon BCR activation, Btk modulates actin dynamics by increasing the levels of phosphorylated, active WASP and N-WASP in B-cells. Btk regulates the activity of WASP and N-WASP by increasing the levels of PtdIns-4,5-P2 and phosphorylated Vav, both of which are required for WASP and N-WASP activation. Inhibition of Btk activity by a point mutation or a specific inhibitor prevents BCR-induced increases in PtdIns-4,5-P2 as well as in phosphorylated WASP, N-WASP and Vav. Furthermore, Btk deficiency or inhibition leads to a severe reduction in BCR-mediated antigen internalization, processing, and presentation to cognate T-cells. Further studies on the role of WASP show no significant effect of WASP deficiency on BCR internalization, while WASP deficiency affects B-cell development, decreasing the numbers of T1/T2 immature B-cells and marginal zone B-cells. Intriguingly, the protein expression levels of N-WASP and WAVE-2, homologues of WASP, increase in WASP-/- B-cells, implicating a compensatory role for WASP homologues in the absence of WASP. Over-expression of N-WASP's proline-rich domain inhibits BCR-mediated antigen uptake and intracellular transport. All of these data indicate that Btk, which is activated upon BCR binding to antigen, regulates actin dynamics and consequently antigen uptake and transport, by activating WASP and N-WASP via Vav and phosphatidylinositides. This presents a novel mechanism by which BCR-mediated signaling regulates BCR-mediated antigen processing and presentation.Item Regulation of calcium signaling and cellular localization of NFAT in CD8+ anergic T cells.(2008-05-30) Srinivasan, Mathangi; Frauwirth, Kenneth A; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Anergy is an important mechanism of maintaining peripheral immune tolerance. T cells rendered anergic, are refractory to further stimulation and are characterized by defective proliferation and IL-2 production. I have used a model of in vivo anergy induction in murine CD8+ T cells to analyze the initial signaling events in anergic T cells. Tolerant T cells displayed reduced PLCγ activation and calcium mobilization, indicating a defect in calcium signaling. This defect could be overcome by facilitating complete release of calcium from the ER. Reduced calcium signaling in CD8+ anergic T cells is therefore predominantly regulated by modulations in signaling events upstream of ER store release of calcium. Impaired calcium mobilization correlated with a block in nuclear localization of NFAT1 in anergic cells, upon stimulation. However, I found that stimulation of anergic, but not naïve T cells induced nuclear translocation of NFAT2. This suggested that NFAT2 is activated preferentially by reduced calcium signaling, and I confirmed this hypothesis by stimulating naïve T cells under conditions of calcium limitation (by addition of the calcium chelator EGTA), or partial calcineurin inhibition. This phenomenon was independent of the phosphorylation activity of the NFAT1 and 2 kinases p38 and JNK, and resultant nuclear exit, as cellular translocation assays using specific kinase and nuclear export inhibitors caused no change in pattern of NFAT1 and NFAT2 nuclear localization. Proliferative and transcriptional changes were also observed under conditions of calcium limitation or sub optimal calcineurin activity. Thus, my work provides new insight into how T cell stimulation conditions might dictate activation of a specific member of the NFAT family and aid in differential transcriptional activation. My results have also led to the hypothesis that NFAT1 and NFAT2 transcriptional activation profiles in anergic T cells are different from responsive T cells. The preferential nuclear accumulation of NFAT2 in anergic T cells might be important for the regulation of genes related to anergy maintenance.Item The Role of Mammalian Actin Binding Protein 1 in Coupling BCR Signaling and Antigen Transport Functions(2008-04-23) Onabajo, Olusegun Okelola; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell receptor (BCR) serves as both signal-transducer and antigen-transporter. Binding of antigens to the BCR induces signaling cascades and antigen-processing and presentation, two essential cellular events for B cell activation. BCR-initiated signaling increases BCR-mediated antigen-processing efficiency by increasing the rate and specificity of antigen transport. Previous studies showed a critical role for the actin cytoskeleton in these two processes. Here I found that actin-binding protein 1 (Abp1/HIP-55/SH3P7) functioned as an actin-binding adaptor protein, coupling BCR signaling and antigen-processing pathways with the actin cytoskeleton. Gene knockout of Abp1 and over-expression of the SH3 domain of Abp1 inhibited BCR-mediated antigen internalization, consequently reducing the rate of antigen transport to processing compartments and the efficiency of BCR-mediated antigen-processing and presentation. BCR activation induced tyrosine phosphorylation of Abp1 and translocation of both Abp1 and dynamin 2 from the cytoplasm to the plasma membrane, where they colocalized with the BCR and cortical F-actin. The inhibitory effect of a dynamin PRD deletion mutant on the recruitment of Abp1 to the plasma membrane and the internalization of the BCR, co-immunoprecipitation of dynamin with Abp1, and co-precipitation of Abp1 with GST fusion of the dynamin PRD, demonstrate the interaction of Abp1 with dynamin 2. In addition to its role in antigen transport and processing, Abp1 is also important for BCR signal transduction. Splenic B cells from Abp1 knockout mice and A20 B cell line with Abp1 knockdown displayed higher levels of protein tyrosine phosphorylation after BCR crosslinking when compared with wild type mice. BCR-triggered ERK phosphorylation in Abp1-deficient splenic B cells occurred sooner and for a much shorter duration than the wild type B cells, while both Abp1 knockout and knockdown significantly reduced BCR-induced phosphorylation of JNK. These results demonstrate that the BCR regulates the function of Abp1 by inducing Abp1 phosphorylation and actin cytoskeleton rearrangement, and that Abp1 facilitates BCR-mediated antigen-processing by simultaneously interacting with dynamin and the actin cytoskeleton. My results further suggest a negative regulatory role for Abp1 in BCR signal transduction.Item Characterization of the Type II Activated Macrophage and the Regulation of Heparin Binding EGF-like Growth Factor(2008-03-28) Edwards, Justin Philip; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three populations of activated macrophages were generated in vitro and characterized each. These include the classically (Ca-MΦ), alternatively (AA-MΦ), and type II activated (MΦ-II) macrophages. Here, a side-by-side comparison of the three cell types is presented, focusing primarily on differences between MΦ-II and AA-MΦ, because both have previously been classified as M2 macrophages, distinct from Ca-MΦ. I show that MΦ-II are similar to Ca-MΦ in that MΦ-II and Ca-MΦ, but not AA-MΦ, produce high levels of NO and have low arginase activity. MΦ-II and Ca-MΦ express relatively high levels of CD86, whereas AA-MΦ are virtually devoid of this co-stimulatory molecule. Ca-MΦ and MΦ-II are efficient antigen presenting cells (APCs), whereas AA-MΦ fail to stimulate efficient T-cell proliferation. The differences between Ca-MΦ and MΦ-II are more subtle. Ca-MΦ produce IL-12/23 and give rise to Th1 cells when used as APCs. MΦ-II produce high levels of IL-10 and low IL-12 and thus give rise to Th2 cells secreting IL-4 and IL-10. I identify two new markers for the MΦ-II, sphingosine kinase and LIGHT. Thus, classically, type II, and alternatively activated macrophages represent three distinct populations of cells with different biological functions. The expression of Heparin-Binding Epidermal Growth Factor-like Growth Factor (HB-EGF) by MΦ-II is then characterized. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. MΦ-II exhibit increased HB-EGF mRNA levels and protein secretion relative to resting and Ca-MΦ. HB-EGF induction is dependent upon the activation of the MAPKs ERK1/2 and p38. The transcription factor, Sp1, was recruited to 3 sites within the first 2kb of the HB-EGF promoter following stimulation, and the site located at -83/-54 was required for HB-EGF promoter activity. These regions of the promoter became more accessible to endonuclease activity following activation, and this accessibility was contingent upon activation of ERK. We show that several conditions which enhanced macrophage IL-10 production also resulted in HB-EGF induction, suggesting that these two genes may be coordinately regulated. These observations indicate that in addition to the secretion of the anti-inflammatory IL-10, another characteristic of MΦ-II is their production of angiogenic HB-EGF.Item The development and characterization of transgenic Leishmania major expressing murine CD40L(2007-05-18) Field, Ann Elizabeth; Mosser, David M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leishmanization is the inoculation of live Leishmania into the host to vaccinate against subsequent infections. This approach has been largely discontinued due to safety concerns. We have previously shown that combining CD40L with Leishmania antigen preferentially induces a type 1 immune response and provides some protection to vaccinated mice. In the present study, we developed transgenic L. major which express and secrete the extracellular portion of CD40L (L. major CD40LE). We hypothesized that these organisms would be less virulent but more immunogenic than wild-type organisms, and therefore be more effective at leishmanization. Transgenic parasites expressing CD40L mRNA and protein were developed. These parasites had similar growth characteristics to wild-type organisms. Susceptible BALB/c mice infected with these parasites developed significantly smaller lesions containing fewer parasites than animals infected with wild-type organisms. Infection of C57BL/6 CD40L-/- mice with transgenic L. major resulted in significantly smaller lesions than infection with wild-type L. major, indicating in vivo biological activity of the transgenic protein. Infection of resistant C57BL/6 mice with low doses of transgenic parasites induced a significant amount of protection against subsequent high dose infection with wild-type organisms. These results demonstrate that transgenic organisms expressing CD40L are less virulent than wild-type organisms while retaining full immunogenicity. The implications of this study are that parasites expressing immune-modulatory molecules may be improved alternatives to traditional leishmanization.Item The Effects of Antigen Valency and CpG ODN on B Cells(2007-03-27) Arunkumar, Nandini; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)B cells express toll-like receptor 9 (TLR9), that recognizes microbial DNA containing unmethylated cytosyl guanosyl (CpG) sequences, induces innate immune responses and facilitates antigen-specific adaptive immunity. Studies indicate that in addition to stimulating innate immunity, TLR9 ligands can induce apoptosis in TLR9 expressing cancer cells. To understand the mechanism for TLR9-induced apoptosis, we compared the effects of CpG containing oligodeoxynucleotides (CpG ODN) on mouse primary, splenic B cells and a mouse lymphoma B cell line, CH27. CpG ODN stimulated the proliferation of primary B cells but inhibited cell proliferation and induced apoptosis in CH27 lymphoma B cells in a sequence-specific, TLR9-dependent fashion. While CpG ODN induced sustained activation of NF-B and increase in c-myc protein levels in primary B cells, NF-B activation was transient in the lymphoma B cells. These data suggest that the differential effects of CpG DNA on primary and lymphoma B cells occur due to differences in NF-B activation. The CpG ODN-induced impaired NF-B activation in the lymphoma B cells results in an imbalance between NF-B and c-myc activities, inducing apoptosis in TLR9-expressing B lymphoma cells. The B cell antigen receptor (BCR) binds to antigens in their native form. The BCR can distinguish subtle differences in antigen structure and trigger differential responses. Here, we analyzed the effects of antigen valency on the functions of the BCR using three different antigen systems - anti-BCR antibody -based antigens, phosphorylcholine (PC)-based antigens, and hen egg lysozyme (HEL)-based antigens. While both paucivalent and polyvalent antigens induced the redistribution of surface BCR into microdomains, polyvalent antigen-induced BCR microdomains persisted. Significantly, this trend was consistently observed in all three antigen systems studied. Ganglioside GM1, tyrosine-phosphorylated proteins and phosphorylated ERK colocalized with BCR microdomains, suggesting these function as surface signaling microdomains. Co-receptor, CD19 and MHC class II molecules, but not CD45 and transferrin receptor, concentrated in the BCR surface microdomains. Prolonged BCR caps were also concomitant with a reduction in BCR movement to late endosomes/lysosomes. Thus, antigen valency influences B cell responses by modulating the stability of BCR-signaling microdomains and BCR-mediated antigen transport.Item Active and Passive Immunization Strategies for Protection of Mice and Monkeys Against Orthopoxvirus Infection(2006-10-18) Fogg, Christiana; Simon, Anne; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Poxviridae are large DNA viruses that replicate in the cytoplasm of vertebrates or invertebrates. The genus Orthopoxvirus includes variola virus, the cause of smallpox, and vaccinia virus (VACV), the prototypal family member used in the licensed smallpox vaccine. Interest in the development of an alternative smallpox vaccine emerged because of complications associated with recent vaccination efforts and the growing number of people excluded from vaccination. Antibody therapies are also of interest for Orthopoxvirus infection treatment instead of vaccinia immune globulin from human donors. Essential to these efforts are studies that elucidate aspects of the immune response required for protection against disease. Two infectious forms of virus exist, intracellular mature virus (IMV), which mediates spread between hosts, and extracellular virus (EV), which is required for efficient spread within a host. IMV and EV each possess an outer membrane with viral proteins targeted by the adaptive immune response. I have used soluble baculovirus-expressed forms of VACV proteins from the IMV and EV in order to understand the role of immunity to these particles during infection. Subcutaneous immunization of mice multiple times with the EV proteins A33 and B5 and the IMV protein L1 either individually or in combinations induced specific antibody responses and protected against weight loss and death caused by virus infection, especially following immunization with A33+B5+L1 or A33+L1. Similar patterns of protection were observed by passive immunization of mice with polyclonal or monoclonal antibodies against A33, B5, or L1 prior to or after intranasal challenge. A27 was investigated as an alternative IMV protein to L1, but proved less effective alone or in combination with A33. Potent and more rapid immune responses to the A33 and L1 proteins were stimulated by the use of the adjuvants QS-21, or alum mixed with CpG oligodeoxynucleotides. Protection against a lethal challenge was observed in a small study with monkeys that were immunized with A33, B5, and L1 and challenged with monkeypox. My data indicate protection against orthopoxviruses is seen in animal models so long as a good antibody response is made to both the IMV and EV forms.Item The Innate Immune Response of Drosophila melanogaster against the Birnavirus Drosophila X Virus.(2005-12-01) Zambon, Robert Anthony; Wu, Louisa P; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The immune responses of Drosophila melanogaster to bacterial and fungal infections has been extensively studied. Here we expand from these two groups of pathogens and examined the immune response of Drosophila against a viral pathogen, specifically the birnavirus Drosophila X virus (DXV). We initially developed a screening system utilizing the anoxia sensitivity which is induced by DXV infection. This system allowed us to examine the effect of various mutations, in previously identified innate immune pathways as well as other possible antiviral pathways, on resistance to viral infection. Using this initial screening method we identified both the Toll pathway and RNAi pathway as possible antiviral responses in Drosophila. Furthermore, we found that increased susceptibility to viral infection by alteration of either of these pathways was generally associated with increased viral load in infected flies. Additionally, we developed cDNA clones of the entire DXV genome to use as the basis for examination of the effects of RNAi on DXV infection and for the development of a reverse genetics system. Using these clones, we show that DXV is sensitive to RNAi. Although RNAi does not result in clearing of virus infection, it inhibits viral replication. Our results indicate that both the Toll and RNAi pathways are playing roles in Drosophila's immune response to DXV infections.