The fruit fly Drosophila melanogaster has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the Drosophila immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G-) bacteria and some Gram-positive (G+) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the Drosophila immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the Drosophila PRRs can recognize G- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G+ bacteria such as Micrococcus luteus and Bacillus megaterium and fungal PAMPs from Beauveria bassiana and Geotrichum candidum. This thesis also investigates the expression of the antimicrobial peptide genes, Diptericin B and Attacin C, and the putative intracellular antimicrobial peptide gene Attacin D, and explores a potential evolutionary link between them.

No description available.

<p>Diacylglycerol (DAG), a lipid messenger generated upon T cell receptor (TCR) engagement, mediates signaling through the IKK/NF-&#954;B and Ras/ERK pathways. Further downstream of the Ras/ERK pathway are mammalian target of rapamycin (mTOR) and MAP kinase signal integrating kinases Mnk1 and Mnk2. While mTOR acts as a critical regulator of T cell metabolism, homeostasis and function, Mnk1 and Mnk2 phosphorylate the initiation factor eIF4E that plays an important role in cap-dependent mRNA translation. Diacylglycerol kinases (DGKs) terminate DAG-mediated signals by phosphorylating DAG into phosphatidic acid. T cells that lack both &#945; and &#950; isoforms of DGK accumulate excess DAG upon activation, resulting in hyper-activation of the IKK/NF-&#954;B, Ras/ERK and mTOR pathways, hypersensitivity to TCR stimulation, and loss of self-tolerance. Here, we have examined the mechanisms by which dysregulated DAG-mediated signaling affects T cell function. To this end, we studied the effects of hyper-activating individual DAG-mediated pathways (IKK/NF-&#954;B and TSC/mTOR) on T cell function. We also examined the role of ERK-activated kinases Mnk1 and Mnk2 in T cell function.</p><p>Using mice with T cell-specific expression of a constitutively active form of IKK&#946; (`IKK' mice), we found that uncontrolled IKK&#946;/NF-&#954;B signaling promotes T cell apoptosis and attenuates responsiveness to TCR stimulation. Defective IL-2 production and increased FasL expression contributed to enhanced IKK T cell apoptosis. Impaired IKK T cell activation and proliferation were associated with defects in TCR signaling, and upregulation of the cell surface inhibitory receptor PD1. In vivo, IKK T cells mounted a compromised antigen-specific CD8 T cell response with curtailed expansion and exaggerated contraction phases. Notably, expression of transcriptional repressor Blimp1 (a regulator of T cell exhaustion) was increased in IKK T cells, and conditionally deleting Blimp1 was able to largely restore responsiveness to TCR stimulation.</p><p>Investigating Mnk1/2 double knockout (DKO) mice, we found that Mnk1 and Mnk2 are dispensable for T cell development and function, but important for the pathogenesis of experimental autoimmune encephalomyelitis (EAE). TCR engagement activated Mnk1/2 in a Ras/ERK-dependent manner in primary T cells, and was inhibited by DGK &#945; and &#950;. Mnk1/2 deficiency did not affect the development of conventional &#945;&#946; T cells, regulatory T cells, or invariant NKT cells. Mature T cells from DKO mice showed normal activation and CD4 TH differentiation ex vivo, but DKO mice developed lower clinical scores than WT counterparts in an EAE model, correlating with a smaller pool of MOG-reactive IL-17-producing and IFN&#947;-producing CD4 cells. These results suggest that Mnk1/2 may play a minimal role in T cell development and function but may control non-T cell lineages to regulate TH1 and TH17 differentiation in vivo. </p><p>To determine the effect of constitutive mTOR complex 1 activity on anti-bacterial CD8 responses, we investigated mice with T cell-specific deletion of TSC1, a suppressor of mTOR complex 1 activity. Using an established model system of transgenic (OT1) CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 responses. Fewer TSC1-deficient OT1 cells were present in the peripheral blood and spleen at the peak of the response and fewer memory cells were found at later time points, in individual and competitive adoptive transfer experiments with WT counterparts. Weak expansion of TSC1-deficient cells was correlated with defects in survival and proliferation in vivo, while exaggerated contraction was associated with an increased ratio of SLECs to MPECs in the effector cell population. This perturbation in effector-memory differentiation was concomitant with enhanced T-bet expression and decreased Eomes expression among activated TSC1 KO cells. Upon competitive adoptive transfer with WT counterparts and antigen re-challenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 cell-intrinsic role for TSC1 in regulating antigen-specific primary and memory responses.</p><p>In sum, findings from these studies provide deeper insight into the regulation of T cell function by DAG-mediated pathways, and may have implications for the design of immune-modulation strategies during vaccination, autoimmunity and cancer immunotherapy.</p> / Dissertation

Elevation of VEGFR-3, the primary mediator of lymphangiogenesis (i.e., new lymphatic vessel formation), is frequently associated with inflammation related to chronic disease and cancer. In the latter case, VEGFR-3 dependent lymphangiogenesis induced by inflamed tumors increases the incidence of distant metastasis, leading to decreased patient survival. However, the molecular mechanisms underlying inflammation-induced VEGFR-3 elevation and lymphangiogenesis are currently unknown. Two potential candidate genes that may regulate expression of VEGFR-3 are Prox1, the primary mediator of embryonic lymphangiogenesis, and NF-κB, the key intracellular regulator of inflammation-induced transcription. We hypothesized that the key inflammatory mediator, NF-κB, regulates transcription of key mediators of lymphangiogenesis, VEGFR-3 and Prox1. We further hypothesized that inflammation-induced elevation of VEGFR-3 and Prox1 are essential steps required for robust lymphangiogenesis in response to inflammation. The three primary goals of this study were to (1) delineate the time-course of events leading to inflammation-induced lymphangiogenesis in vivo; (2) clone and characterize the VEGFR-3 promoter and identify factors regulating VEGFR-3 expression in vitro; and (3) characterize the lymphatic phenotype of NF-κB p50 knockout mice. To begin testing these hypotheses, we used a mouse model of peritonitis to characterize induction of lymphangiogenesis and expression kinetics of NF-κB, Prox1 and VEGFR-3. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-κB followed by sequential upregulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Characterization of the VEGFR-3 promoter by luciferase-reporter and ChIP assays showed direct activation by Prox1, NF-κB p50 and p65 transcription factors. This also revealed that Prox1 and NF-κB p50 bind in close proximity and synergistically activate the VEGFR-3 promoter. Characterization of p50 knockout mice revealed significantly decreased lymphatic vessel density in several organs that corresponded to reduced VEGFR-3 and Prox1 expression. Activation of NF-κB by inflammatory stimuli also elevated expression of NF-κB, Prox1 and VEGFR-3 in cultured lymphatic endothelial cells, which enhanced proliferation and migration in response to the VEGFR-3-specific ligand, VEGF-C152S. Collectively, our findings suggest that induction of the NF-κB pathway by inflammatory stimuli activates Prox1, and both NF-κB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of pre-existing lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.

No description available.

The aim of this thesis was to verify the hypothesis implying that n-3 polyunsaturated fatty acids inhibit the development of mild chronic inflammation. The experiment was conducted on rats with different diets (control, with the addition of safflower oils, fish oils and oils from algae Schizochytrium). For processing the results of the collected tissues was used determination of expression of selected genes by qRT-PCR, detection of proteins and in the cytosol and nuclear fractions by Western blot and the quantification of cytokines by ELISA. Feeding oil from algae Schizochytrium has led to lowering the final weight and blood glucose, further to enhance expression of PPAR-gama and increasing the production of anti-inflammatory cytokines IL-10 and TGF-beta1. In kontrast, no difference was observed in the expression of GPR120 and adiponectin receptors or proteins in an amount of NF-kappaB and PPAR-gama between diets. Elevated plasma levels of adiponectin were found. The results of the experiment shows that it is possible to recommend oil from algae Schizochytrium as a useful supplement of human diet as prevention of chronic degenerative diseases.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Philip R. Hardwidge / Diarrheagenic Escherichia coli (E. coli) strains are common etiological agents of diarrhea. Diarrheagenic E. coli are classified into enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC or enterohemorrhagic E. coli [EHEC]), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and adherent invasive E. coli (AIEC). In addition to encoding toxins that cause diarrhea, diarrheagenic E. coli have evolved numerous strategies to interfere with host defenses. In the first project, we identified an ETEC-secreted factor (ESF) that blocked TNF-induced NF-[kappa]B activation. One of the consequences of TNF-induced NF-[kappa]B activation is the production of pro-inflammatory cytokines that help to eliminate pathogens. Modulation of NF-[kappa]B signaling may promote ETEC colonization of the host small intestine. In this study, we fractionated ETEC supernatants and identified flagellin as necessary and sufficient for blocking the degradation of the NF-[kappa]B inhibitor I[kappa]B[alpha] in response to TNF[alpha]. In the second project, we attempted to identify an ETEC cAMP importer. ETEC diarrhea leads to cAMP release into the lumen of the small intestine. cAMP is a key secondary messenger that regulates ETEC adhesin expression. We hypothesized that a cAMP importer is present in ETEC, accounting for its hypersensitivity to extracellular cAMP. We used Tn5 transposome-mediated mutagenesis to construct a mutant library and screen for cAMP-hyporesponsive mutants. However, none of the 17,956 mutants we screened were cAMP-hyporesponsive. In the third project, we focused on gut microbiota and the T3SS effector NleH. We used the mouse-specific pathogen C. rodentium and transplanted performed microbiota between different mouse strains. We evaluated microbiota populations as a function of infection with WT and [Delta]nleH C. rodentium strains before and after microbiota transplantation. Microbiota transfer altered the resistance to WT C. rodentium infection in C57BL/10ScNJ mice and the NleH effector promoted host resistance to C. rodentium.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22742号 / 医博第4660号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武田 俊一, 教授 武藤 学, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Over the past 30 years, transcription factor nuclear factor kappa B (NF-κB) has been extensively characterized in organisms ranging from flies to humans, where it is known to play key roles in developmental and immune-related processes. More recently, DNA sequencing approaches have identified homologs of NF-κB and many upstream signaling components in basal phyla, including Cnidaria (sea anemones, corals, hydras, and jellyfish), Porifera (sponges), and single-celled protists, including Capsaspora owczarzaki and some choanoflagellates. However, little is known about the activity and regulation of NF-κB proteins in these basal organisms. In this dissertation, the structure, activity, and biology of NF-κB in three basal phyla is examined and the extent of conservation with more derived organisms as well as phylum-specific properties are investigated. In the coral Orbicella faveolata (Of) a simplified but nearly complete Toll-like receptor (TLR)-to-NF-κB pathway exists, but basal to cnidarians, there are fewer upstream signaling molecules present. For example, in the poriferan Amphimedon queenslandica (Aq) and the protist Capsaspora owczarzaki (Co), singular NF-κBs and some upstream signaling proteins are encoded in their genomes, but no canonical TLRs exist. In contrast, the expanded family of choanoflagellates, including the choanoflagellate Acanthoeca spectabilis (As), contains TLR-like and up to three NF-κB-like homologs, although their domain structures differ from NF-κB pathway members of higher organisms. Of-NF-κB, Aq-NF-κB, and Co-NF-κB all resemble the mammalian NF-κB protein p100 in that they contain an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and similar DNA binding-site profiles. C-terminal truncation results in translocation of these basal NF-κBs to the nucleus and increases their DNA-binding and transcriptional activation activities. Nevertheless, unlike mammalian NF-κB p100, the C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. The three As-NF-κB-like proteins all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ from one another in DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Expression of IKKs induce proteasome-dependent C-terminal processing of Of-NF-κB and Aq-NF-κB in human cells, and processing requires C-terminal serines. In contrast, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells and no IKK homolog exists in the Co genome, suggesting that IKK-mediated processing of NF-κB is a mechanism that evolved solely in animals. Treatment of Of and sponge tissue with lipopolysaccharide (LPS), a ligand for mammalian innate immunity, results in gene expression changes consistent with NF-κB pathway mobilization in Of and increases both DNA-binding activity and processing of sponge NF-κB. Furthermore, sponge tissue contains constitutive NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Moreover, exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. Together, these data suggest that the mechanism as well as level of activation of NF-κB in basal organisms is different from what is observed in higher organisms. Additionally, NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. Overall, these data represent the first functional characterization of NF-κB signaling proteins in an endangered coral, in any organisms basal to cnidarians (i.e., an evolutionary important sponge), and outside the Kingdom Animalia (protists). These findings suggest that these seemingly simple organisms contain conserved innate immune-like pathways that may be regulated by NF-κB and provide information about the evolution and diversification of this biologically important transcription factor.

Protein-protein interactions (PPI) mediate numerous biological processes, but inhibiting these interactions with small molecules has been difficult to achieve in drug discovery. A small number of successes have shown that some PPIs are amenable to inhibition. Computational algorithms designed to measure the druggability of PPIs have been developed based on these successes. These algorithms have identified the interaction between the NF-κB essential modulator (NEMO) and I𝜅B kinase β (IKKβ) as a candidate for inhibition. Furthermore, in vivo peptide-based inhibition of the NEMO-IKKβ interface has shown benefits in attenuating the NF-𝜅B response in cellular and animal models. In addition to its intrinsic interest as a drug target, developing inhibitors against the NEMO/IKKβ interaction may help in the development of improved methods for PPI inhibition. In this thesis, the production of full-length, recombinant forms of soluble NEMO is described. This protein was used in a variety of biochemical assays to advance our understanding of NEMO structure and function. Furthermore, a fluorescence anisotropy (FA) assay was developed to screen for compounds inhibiting the NEMO/IKKβ PPI. Hits from the FA assay were tested by several methods to confirm true inhibition. Additionally, the FA assay was used to accurately measure the affinity of NEMO for IKKβ and to assess the degree of cooperativity in IKKβ binding. The oligomeric state of NEMO has been characterized through the development of a panel of NEMO cysteine to alanine mutants, using polyacrylamide gel electrophoresis analysis, analytical ultracentrifugation, and fluorescence anisotropy. These data represent the first comprehensive characterization of full-length human NEMO, and may provide a path toward development of drug-like inhibitors of the NEMO/IKKβ interaction.