Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

O Histoplasma capsulatum (H.capsulatum) é um fungo dimórfico patogênico e responsável por graves lesões pulmonares, as quais se caracterizam pelo acúmulo de leucócitos ao redor do fungo, resultando na formação de granulomas. A infecção ocorre principalmente pela inalação de conídios ou pequenos fragmentos de micélio que alcançam os alvéolos, onde se transformam em leveduras, que é a forma patogênica do fungo. Na resposta imune do hospedeiro, as integrinas participam nos mecanismos fagocíticos, essenciais na resposta à histoplasmose. As 2integrinas contêm uma cadeia 2, também conhecida como CD18, comum a várias moléculas de adesão, e uma cadeia variável. Até o momento foram identificadas quatro cadeias distintas: L, a qual forma o dímero L2, também conhecido como LFA-1 (do inglês leukocyte function antigen-1) ou CD11aCD18; m, formando m2, chamado Mac-1 (do inglês macrophage differentiation antigen 1) ou CR3 (do inglês complement receptor 3) ou CD11bCD18; x, formando x2, CD11cCD18, gp150, 95 ou CR4 (do inglês complement receptor 4) e a cadeia d, formando d2, CD11dCD18. Neste trabalho, investigamos o papel da molécula CD18 em macrófagos alveolares (MAs) e macrófagos peritoneais (MPs) nas funções efetoras contra H. capsulatum e a relação do leucotrieno B4 (LTB4) nestas respostas. Inicialmente confirmamos que MAs e MPs provenientes dos animais CD18low, expressam baixa porcentagem de CD11bCD18 (CR3). Demonstramos que, como esperado, MAs e MPs de ambos os grupos fagocitam mais leveduras opsonizadas com complemento do que não opsonizadas. Surpreendentemente, MAs de animais CD18low fagocitam 136% mais leveduras opsonizadas do que MAs de C57BL/6. Também, MPs destes animais fagocitam aproximadamente 240% mais leveduras quando infectados com H. capsulatum e opsonizados, quando comparados aos MPs de C57BL/6. A adição de LTB4 aumenta a atividade fagocítica em 520% por MAs de animais C57BL/6 e 200% por MAs de CD18low, enquanto que a adição de LTB4 aumentou a fagocitose dos MPs de animais C57BL/6 em 600% vezes quando comparado aos MPs de CD18low. Este fenômeno foi inibido pela pré-incubação destas células com antagonista específico do receptor BLT1 apenas em animais C57BL/6. A adição de LTB4 na cultura de MPs reduziu a porcentagem de morte das leveduras apenas nos animais C57BL/6. Os animais CD18low produzem espontaneamente mais LTB4 e apresentaram um grande aumento na produção de óxido nítrico quando comparados aos animais C57BL/6. Pacientes acometidos pela Doença Granulomatosa Crônica (DGC) possuem deficiência congênita da molécula CD18. Células fagocíticas isoladas do sangue periférico de pacientes com DGC foram incubadas com leveduras opsonizadas e assim como macrófagos de animais deficientes de CD18, fagocitam mais leveduras opsonizadas (900%) ou não (300%), quando comparado com células de indivíduos sadios. Sugerimos que a molécula CD18 tem importante participação nos mecanismos efetores da imunidade inata, por mecanismo dependente de mediadores lipídicos, como o LTB4, no controle dos mecanismos de defesa contra H. capsulatum. / Histoplasma capsulatum (H. capsulatum) is a pathogenic dimorphic fungus and its infection is characterized by accumulation of leukocytes and granuloma formation. Infection occurs mainly by fungal inhalation that reaches the alveoli, which became yeast (the pathogenic form). in the immune response of host, integrins participate in phagocytic mechanisms, fundamental in the response against histoplasmosis.,8^2-integrin has a 02 chain known as CD18, usual to many adhesion molecules, and a variable a chain. Until the moment, it was identified four variable a chains: aL, that constitutes the dimer aL,82, also known as LFA-1 (leukocyte function antigen) or CD11aCD18; am forming the a^m,B^2 or Mac-1 (macrophage differentiation antigen 1) and CR3 (complement receptor 3) and CD11bCD18; ax, constituting the dimer Able CD11cCD18, gp150, 95 or CR4 (complement receptor 4) and ad chain, that constitutes a^d,8^2, CD11dCD18. In the present study, we sought to investigate the effect of CD18 in alveolar (AMs) and peritoneal macrophages (PMs) effecter functions against H. capsulatum and the relation of LTB^4 in those responses. We confirm that AMs and PMs of CD18\'°^W mice have low expression of ,32-integrin compared to wild type mice (WT). We demonstrate that, as expected, AMs and PMs from WT and CD18\'°^W, phagocytosed more complement (C)-opsonized yeasts than the unopsonized yeasts. Surprisingly, AMs from CD18\'°^Wmice phagocytosed 136% more (C)-opsonized yeasts than AMs obtained from WT. Also, PMs of CD18^b°^W mice phagocytosed 240% more (C)-opsonized yeasts than PMs of WT. The addition of LTB^4, increases the phagocytic activity by AMs of WT mice in 520% and by AMs from CD18\'°^W mice in 200%, while the addition of LTB^4 only increased the phagocytosis of C-opsonized H. capsulatum by PMs of C57BL/6 mice in 600%, when compaired with PMs from CD18\'°^W mice. This phenomenon was inhibited by pretreatment of these cells with an especific BLT1 receptor antagonist only in PMs from C57BU6 mice. The addition of LTB^4 in the culture of MPs reduced the percentage of death of yeasts in animals C57BL/6. CD18\'°^W mice, spontaneously produce more LTB^4 and showed a large increase in the production of nitric oxide when compared to C57BU6. Patients affected by Chronic Granulomatous Disease (DGC) have congenital deficiency of the CD18 molecule. Phagocytic cells isolated from peripheral blood of patients with DGC were incubated with C-opsonized yeasts and as well as macrophages from CD18\'°^W, phagocytosed more C-opsonized yeasts (900%) or not (300%) when compared with cells from healthy individuals.Therefore, we suggest that the CD18 molecule has important participation in the effector mechanisms of innate immunity, a mechanism dependent on lipid mediators such as LTB^4, to control these mechanisms in defense against H. capsulatum.

Abstract Type XIII collagen is a transmembrane protein consisting of a short intracellular portion, a transmembrane anchor, and a long extracellular domain with a mainly collagenous sequence. Histochemical and cell biological studies have revealed that type XIII collagen has a wide distribution in various tissues and that it is mostly localized to cell-cell and cell-matrix contacts. In order to study type XIII collagen at the molecular level, the protein was expressed in insect cells as a homotrimer. The recombinant protein was found to reside in the plasma membrane of insect cells with its N-terminus intracellular and C-terminal part extracellular, i. e. in a type II orientation. The trimerization of type XIII collagen chains was initiated by 21 amino acid residues adjacent to the transmembrane domain on the extracellular side, and this sequence was found to be conserved in several other collagenous transmembrane proteins. In addition to the transmembrane form, the ectodomain of type XIII collagen was secreted into the cell culture medium, a result of proteolytic cleavage by furin-like proteases at the non-collagenous NC1 domain. The ectodomain was purified from the insect cell culture medium with a typical collagenous composition and conformation, and it showed as a 150 nm-long rod in rotary shadowing electron microscopy. Furthermore, the recombinant ectodomain showed high affinity binding to several extracellular matrix proteins, e. g. fibronectin, nidogen-2, and perlecan, as well as to heparin. The type XIII collagen ectodomain also showed selective recognition to collagen receptor integrins. Integrin α1 and α11 I domains bind to type XIII collagen with a high affinity, and both integrins α1β1 and α11β1 mediate cell attachment to type XIII collagen. The present results suggest that type XIII collagen shares common aspects with other collagenous transmembrane proteins in terms of chain association and ectodomain shedding. However, it is notably distinct in its structure and binding specificity compared to other types of collagen and cell-surface proteins. The data imply that type XIII collagen might participate in multiple cell-cell and cell-matrix interactions.

During chronic and severe acute liver injury, regeneration is thought to occur through hepatic progenitor cells (HPCs). Understanding the regulation of HPCs may offer therapeutic opportunities to enhance liver regeneration. HPCs are associated with an increase in laminins in the extracellular matrix. Laminins are heterotrimeric proteins, composed of an alpha, beta and gamma chain. There are 5 alpha chains with different distributions and functions, but the relative contributions of these in HPC-mediated liver regeneration are not known. My aims were to describe the laminin alpha chains associated with the HPC response and to define the functional effects of specific laminin chains on HPCs. I examined the laminin alpha chains in two mouse models of HPC activation: a transgenic model using conditional deletion of Mdm2 in hepatocytes, and a dietary model using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). The laminin alpha 5 (Lama5) chain is significantly upregulated in both models and forms a basement membrane which surrounds the progenitor cells. I have also demonstrated Lama5 expression in the ductular reaction seen in human liver disease. Using primary mouse cell cultures, I have shown that Lama5 is produced predominantly by the HPCs themselves, rather than by stellate cells. The HPCs express the cell surface receptor alpha-6 beta-1 integrin, a binding partner of Lama5. I then studied the functional effects of matrix on cell behaviour in vitro using recombinant laminins and a line of spontaneously immortalised mouse HPCs. Compared to other laminin chains, Lama5 selectively promotes HPC adhesion and spreading. These effects are partially blocked by antibodies against beta-1 integrin. Lama5 also significantly enhances HPC migration, resulting in an increase in cell migration. Furthermore, only Lama5 enhances HPC survival in serum-free medium, with an increase in cell viability. Culturing HPCs on HPCs maintained in culture on plastic synthesise Lama5 chain. Knock-down of endogenous Lama5 production using siRNA results in reduced proliferation and increased hepatocytic differentiation, with increased albumin production. I then studied the effects in vivo using transgenic Cre-lox mouse strains that allow conditional knock-out of either laminin alpha 5 or beta-1 integrin in HPCs. The effects of gene deletion were examined in healthy mice and two dietary models of HPC activation: the DDC diet and a choline-deficient, ethionine-supplemented (CDE) diet. Although these experiments were limited by a low number of experimental animals and low recombination rates, there was a suggestion of impaired HPC expansion associated with loss of laminin alpha 5. There was also a significant increase in hepatocellular injury and fibrosis in response to the DDC diet seen with loss of laminin alpha 5 expression. Laminin alpha 5-containing matrix is deposited around HPCs during liver regeneration and supports progenitor cell attachment, migration and maintenance of an undifferentiated phenotype. This work identifies a novel target for enhancing liver regeneration.

Large efforts have been made to target integrin αVβ3 of endothelial cells. We have successfully developed a new class of protein (Ref to as ProAgio) by rational protein design using a stable host protein, domain 1 of cell adhesion protein CD2. ProAgio is designed to target integrin αVβ3 at a novel site and induces angiogenic endothelial cell apoptosis by recruiting and activating caspase 8 to the cytoplasmic domain of the targeted integrins. Tests with tumor xenograft models show that ProAgio strongly inhibits tumor growth. Histology analyses indicate that tumor vessels are reduced, while the established vasculatures are not affected. Toxicity analyses demonstrate that ProAgio is not toxic to mouse. Our study develops an effective anti-angiogenesis agent and provides a new platform for development of therapeutics by targeting integrins. We have successfully developed an anti-angiogenesis protein targeting integrin αVβ3 at a novel site by rational protein design. The developed agent is not toxic to non-cancerous blood vessels and other tissue/organs, providing an excellent candidate for future potential clinical development. Our developed protein is one of the very few examples that do not act through targeting VEGF/VEGFR or any other RTK pathways. The βA groove is present in almost all other β integrins. This approach may be applicable to develop agents targeting the similar βA groove of other integrin pairs, which can address wide array of pathological conditions such as AMD, Rheumatoid Arthritis, Osteoporosis etc.

Intestinal inflammatory disorders such as Crohn’s disease contribute significantly to human mortality and morbidity. Although the cells and molecules involved in suppression of intestinal inflammation have been extensively documented in mouse models, a full understanding of how these work together in the healthy and diseased gut remains elusive. It is known, however, that tight regulation over TH17 cells and regulatory T cells (Treg) is required to maintain immune homeostasis in the intestine. Activation of the cytokine transforming growth factor-β (TGFβ), which is secreted by immune cells as an inactive complex, plays a crucial role in the induction of both Treg and TH17 cells. Recent work has shown that the αvβ8 integrin is required for activation of TGFβ by murine dendritic cells (DC). Murine integrin αvβ8 is thus of fundamental importance for Treg and TH17 induction and, subsequently, for intestinal immune homeostasis. However, little is known about the signals controlling the expression of integrin αvβ8 on intestinal DC. Furthermore, whether this system is also important for regulation of the human system is entirely unknown. Here, expression of integrin αvβ8 is shown on human intestinal CD1c+CD103+SIRPα+ DC and CD1c+CD103-SIRPα+ DC, but not on CD141+CD103+SIRPα- DC. Expression of integrin αvβ8 is increased on DC from Crohn’s disease patients, and on DC from non-IBD donors treated with LPS. Both LPS and a number of probiotic bacteria were also able to induce integrin αvβ8 expression on human monocyte-derived DC (moDC), which suggested that the microbiota may play a role in the regulation of human integrin αvβ8. Importantly, we have also shown that TGFβ is activated by integrin αvβ8 on human moDC, and that integrin αvβ8 promotes expression of forkhead box P3 (FOXP3) in naïve human T cells in vitro. Together, these data suggest that integrin αvβ8-mediated activation of TGFβ by DC may play an important role in the regulation of human T cell responses in the human intestine, and that this pathway may be perturbed during intestinal inflammation.

In the central nervous system (CNS), oligodendrocytes (OLs) generate myelin to facilitate the rapid propagation of neuronal impulses. In multiple sclerosis (MS), chronic demyelination leads to irreversible neurodegeneration that eventually impairs physical and cognitive function. Much effort is directed at elucidating the mechanisms underlying OL development in hope to unveil therapeutic targets for promoting remyelination in MS. Many aspects of OL biology are regulated by the integrins, a large family of transmembrane extracellular matrix (ECM) receptors. ECM components such as laminin and fibronectin bind to OL integrin receptors and initiate downstream signaling cascades involved in survival, proliferation, differentiation/myelination and migration. Integrin-linked kinase (ILK), an adaptor protein that binds to integrin cytosolic tails, works to stabilize the ECM-integrin connection by indirectly targeting the actin cytoskeleton to ECM adhesion sites. We hypothesized that ILK played an important role in OL migration, differentiation and capacity to myelinate neuronal projections. To address this hypothesis, we developed three cell culture techniques to assess these cellular phenomena in vitro. Conditional knockout of Ilk compromised both the morphological and molecular differentiation of primary mouse OLs in vitro, and reduced their capacity to produce myelin-like membrane. ILK was required for proper OL ensheathment of neuronal extensions when co-cultured with primary neurons. Conditional ablation of Ilk in vivo produced a transient amyelination defect that was endogenously compensated for at later time points. Loss of ILK in primary OLs was associated with upregulated RhoA signaling, and pharmacological inhibition of the RhoA axis restored the morphology of a distinct subset of NG2+ OPCs. ILK depletion in OL precursor cells (OPCs) resulted in a substrate-dependent defect in migration velocity and migration initiation. Inhibition of the RhoA signaling pathway enhanced the migratory velocity of wild-type OPCs, an effect that was dependent on ILK expression. In sum, we established three primary mouse OL cell culture techniques, with which we defined roles for ILK in OL biology. Our work highlights the importance of integrin signaling in OLs and provides new experimental methods useful in MS research.

abstract: Integrins are a family of αβ heterodimeric transmembrane receptors. As an important class of adhesion receptors, integrins mediate cell adhesion, migration, and transformation through bidirectional signaling across the plasma membrane. Among the 24 different types of integrins, which are notorious for their capacity to recognize multiple ligands, the leukocyte integrin αMβ2 (Mac-1) is the most promiscuous member. In contrast to other integrins, Mac1 is unique with respect to its preference for cationic ligands. In this thesis, a new Mac-1 cationic ligand named pleiotrophin (PTN) is uncovered. PTN is an important cytokine and growth factor. Its activities in mitogenesis and angiogenesis have been extensively researched, but its function on immune cells was not widely explored. In this research, the cell biology and biochemical evidences show that PTN can regulate various Mac-1-expressing cells functions through the activation of the extracellular signal regulated kinases. Direct interactions between PTN and the αM I-domain, the major ligand-binding domain of Mac-1, has been shown using biolayer interferometry analyses and confirmed by solution NMR spectroscopy. The binding epitopes and the binding mechanism of PTN and αM I-domain interaction were further revealed by peptide array analysis and microscale thermophoresis. The data suggested that PTN’s thrombospondin type-1 repeat (TSR) domains and αM I-domain metal-ion-dependent adhesion site (MIDAS) are the major binding sites. In addition, this interaction followed a novel metal-ion independent binding mechanism which has not been found in other integrins. After a series of characterizations of αM I-domain using both experimental and computational methods, it showed that activated αM I-domain is significantly more dynamic than inactive αM I-domain, and the dynamics seem to modulate the effect of Mg2+ on its interactions with cationic ligands. To further explore the PTN induced Mac-1 structure rearrangement, intact Mac-1 was studied by negative stain electron microscopy. The results showed that the Mac-1 exhibited a very heterogeneous conformation distribution in detergents. In contrast, the Mac-1 adopted predominantly the bent conformation in phospholipid nanodisc condition. This Mac-1 nanodisc model provides a new platform for studying intact Mac-1 activation mechanism in a more physiologically relevant manner in the future. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22354号 / 医博第4595号 / 新制||医||1042(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 松田 道行, 教授 武藤 学, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13386号 / 論医博第2218号 / 新制||医||1048(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 椛島 健治, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM