Orientador: Mario José Abdalla Saad / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T23:38:58Z (GMT). No. of bitstreams: 1 Caricilli_AndreaMoro_D.pdf: 5503303 bytes, checksum: dd944e8e65716f9ac4355ff0fbc95248 (MD5) Previous issue date: 2012 / Resumo: Fatores ambientais e genéticos do hospedeiro interagem para controlar a microbiota intestinal, que pode ter um papel no desenvolvimento da obesidade e da resistência à insulina. Camundongos deficientes em TLR2, sob condições livres de microorganismos, estão protegidos da resistência à insulina induzida por dieta. Inibição aguda do TLR2 (4 dias de tratamento) com oligonucleotídeo antisense em camundongos alimentados com dieta hiperlipídica leva a um aumento da sensibilidade e da sinalização da insulina em tecido adiposo e muscular. É possível que a presença da microbiota intestinal possa reverter o fenótipo de um animal, induzindo resistência à insulina em um animal geneticamente determinado a ter aumento da sensibilidade à insulina, tal como o camundongo deficiente para TLR2. No presente estudo, nós investigamos a influência da microbiota intestinal nos parâmetros metabólicos, tolerância à glicose, sensibilidade e sinalização da insulina em camundongos deficientes para TLR2. A microbiota intestinal foi investigada (por metagenômica), as características metabólicas e a sinalização da insulina em camundongos deficientes para TLR2 em um biotério convencional. Os resultados mostraram que a perda do TLR2 em camundongos de biotério convencional resulta em fenótipo semelhante ao da síndrome metabólica, caracterizado por diferenças na microbiota intestinal, com um aumento de três vezes na proporção de Firmicutes e um pequeno aumento na de Bacteroidetes, em comparação com os controles. Essas alterações na microbiota foram acompanhadas por um aumento na absorção de LPS, inflamação subclínica, resistência à insulina, intolerância à glicose e posterior obesidade. Essa seqüência de eventos foi reproduzida em camundongos do tipo selvagem por transplante de microbiota intestinal e revertida pelo tratamento com antibióticos. Em nível molecular, o mecanismo demonstrou-se único, com ativação do TLR4, associado com estresse de retículo endoplasmático e ativação da JNK, sem, porém, ativação da via IKK?-I?B-NF?B. Nossos resultados também mostraram que, em camundongos deficientes para TLR2, houve redução de células T regulatórias em tecido adiposo visceral, sugerindo que essa regulação pode contribuir para a resistência à insulina nesses animais. Nesse sentido, nossos resultados enfatizam o papel da microbiota na complexa rede de interações moleculares e celulares que ligam genótipo e fenótipo, e suas potenciais implicações em alterações humanas envolvendo obesidade, diabetes e outras doenças imunológicas / Abstract: Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2 deficient mice, under germ-free conditions are protected from diet-induced insulin resistance. Diet-induced obese mice, acutely treated with TLR2 oligonucleotide antisense during 4 days showed increased insulin sensitivity and signaling in muscle and white adipose tissue. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics and insulin signaling in TLR2 knockout (KO) mice in a non-germ free-facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance and, later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At molecular level the mechanism was unique with activation of TLR4, associated with ER stress and JNK activation, but no activation of the IKK?-I?B-NF?B pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes and even other immunological disorders / Doutorado / Medicina Experimental / Doutor em Ciências

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.

過敏性疾病（如過敏性哮喘和過敏性皮炎）發病率在香港及世界均呈上升趨勢。過敏性哮喘是一種慢性反復發作的炎症疾病，而過敏性皮炎是一種慢性皮膚炎症。呼吸道細菌及金黃色葡萄球菌可分別加重過敏性哮喘病人氣道炎症及過敏性皮炎患者的炎症反應。人體對細菌的先天免疫反應主要通過模式識別受體(PRR)介導。NOD樣受體(NLR)和Toll樣受體(TLR)是兩種重要的PRR。 NLR 家族成員NOD2幾乎識別所有細菌中結構保守的胞壁醯二肽(MDP)。而LTR2識別範圍廣泛的病原相關分子模式，如革蘭氏陽性菌中的肽聚糖(PGN)和脂磷壁酸(LTA)，以及人工合成的脂蛋白Pam3CSK4。 / 本研究包括：NOD2配體MDP，哮喘相關的腫瘤壞死因子家族成員LIGHT對共培養的人嗜鹼性粒細胞和人支氣管上皮細胞的活化作用；熱滅活的金黃色葡萄球菌（HKSA），MDP，TLR2配體PGN，LTA，以及Pam3CSK4對共培養的人嗜鹼性粒細胞和人皮膚成纖維細胞的活化作用；在體內NLR配體對卵清蛋白(OVA)致敏的哮喘小鼠的作用。 / 研究發現，在共培養體系中，MDP能顯著增強嗜鹼性粒細胞與支氣管上皮細胞表面粘附因子（細胞間粘附因子ICAM-1 及血管細胞粘附因子VCAM-1）的表達。同時，MDP能顯著促進共培養體系中炎症相關細胞因子IL-6，趨化因子CXCL8及抗菌肽β-防禦素2的釋放。在MDP刺激下，支氣管上皮細胞是共培養體系中釋放IL-6，CXCL8及β-防禦素2的主要細胞。在MDP刺激下，嗜鹼性粒細胞中包括胞核因子-kappaB（NF-κB）在內的幾個核轉錄因子的表達上升。ICAM-1，VCAM-1，IL-6，CXCL8，及β-防禦素2的表達被信號分子化學抑制劑所抑制，結果表明，嗜鹼性粒細胞與支氣管上皮細胞的相互作用受不同的信號通路（NF-κB, p38 MAPK 及 JNK）調節。OVA致敏小鼠實驗表明，NLR配體能增加分泌粘蛋白的杯狀細胞在肺氣管中的數量，使小鼠支氣管下皮結締組織纖維化並增厚。NLR配體進而提高過敏性哮喘小鼠支氣管肺泡灌洗液中CCL5與IL-13 的表達水平。 / 研究表明，在嗜鹼性粒細胞和皮膚成纖維細胞的共培養體系中，HKSA，MDP，PGN，LTA，或Pam3CSK4顯著誘導ICAM-1, IL-6, CXCL8, CCL2 和 CCL5 的表達。而嗜鹼性粒細胞與皮膚成纖維細胞的直接相互作用是釋放IL-6, CXCL8, CCL2 與 CCL5 所必需的。嗜鹼性粒細胞與皮膚成纖維細胞的相互作用並釋放細胞因子與趨化因子受p38 MAPK 及 NF-κB信號通路調控。 / 在嗜鹼性粒細胞與支氣管上皮細胞共培養體系中，LIGHT 可能通過受體HVEM 與 LTβR顯著增強支氣管上皮細胞表面粘附因子的表達，提高細胞因子IL-6, CXCL8 與 MMP-9的釋放。 / 研究結果表明，在過敏炎症中，通過與組織細胞（如支氣管上皮細胞，人皮膚成纖維細胞）相互作用，嗜鹼性粒細胞有利於組織細胞對病原相關的分子模式作出反應。因此，研究結果對細菌介導的先天性免疫應答與過敏炎症的加重之間的聯繫作出了新的解釋。以上結果也增強了我們對LIGHT在氣道重塑中的免疫病理作用及其作為氣道重塑治療靶標的認識。 / The incidences of allergic diseases such as allergic asthma and atopic dermatitis (AD) are increasing in Hong Kong and worldwide. Allergic asthma is a chronically relapsing inflammatory pulmonary disease, while AD is a chronic inflammatory skin disorder. Respiratory bacterial and Staphylococcus aureus (S. aureus) infection can provoke allergen sensitization and subsequently amplify and sustain inflammation in allergic asthma and AD, respectively. The innate immune system recognizes bacterial infection through pattern recognition receptors (PRRs), two important PRRs involving in inflammatory and immune responses are nucleotide-binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs). NOD2 is one member of the NLR family, which senses the conserved structural component muramyl dipeptide (MDP) in almost all bacteria. TLR2 recognizes a wide range of pathogen-associated molecular patterns (PAMPs) including peptidoglycan (PGN) and lipoteichoic acid (LTA) from Gram-positive bacteria and synthetic triacylated lipoprotein N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S] -lysine (Pam3CSK4). / In the present study, we investigated the effect of NOD2 ligand MDP, asthma-related tumor necrosis factor (TNF) family member LIGHT on human basophils co-cultured with human bronchial epithelial cells and the effect of heat-killed S. aureus, MDP, TLR2 ligands PGN, LTA and Pam3CSK4 on basophils co-cultured with human dermal fibroblasts, and the underlying intracellular mechanisms. The in vivo effect of NOD ligands on ovalbumin (OVA)-sensitized allergic asthmatic mice was also studied. / It was found that MDP could significantly enhance the cell surface expression of adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on basophils and primary human bronchial epithelial cells (HBE) in the co-culture system (all p < 0.05). MDP could further enhance the release of inflammatory cytokine interleukin (IL)-6, chemokine CXCL8, and epithelium derived anti-microbial peptide β-defensin 2 in the co-culture. HBE cells were the major source while basophils were the minor source to release IL-6, CXCL8 and β-defensin 2 in the co-culture upon MDP stimulation. The activities of several nuclear transcription factors, including NF-κB, were up-regulated in human basophils upon MDP stimulation. The cell surface expression of ICAM-1 and VCAM-1 and the release of IL-6, CXCL8 and β-defensin 2 were suppressed by the signaling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be differentially regulated by the NF-κB, p38 MAPK and JNK pathways. The animal study showed that iE-DAP and MDP could increase the number of mucin-secreting goblet cells, the thickness and fibrosis of the bronchial subepithelial tissue of airways from the OVA-sensitized mice. The iE-DAP and MDP could further promote the levels of CCL5 and IL-13 (all p < 0.05) in bronchoalveolar lavage fluid (BALF) of allergic asthmatic mice. / It was found that the induction of ICAM-1, IL-6, CXCL8, CCL2 and CCL5 was significantly promoted upon the interaction between human basophils and dermal fibroblasts activated by heat-killed S. aureus, MDP, PGN, LTA or Pam3CSK4. The release of IL-6, CXCL8, CCL2 and CCL5 might depend on the direct interaction of basophils and dermal fibroblasts. The p38 MAPK and NF-κB pathways should be involved in the release of the cytokines and chemokines upon the interaction of basophils and human dermal fibroblasts. / LIGHT could significantly promote the cell surface expression of adhesion molecule, the release of IL-6, CXCL8 and MMP-9 from human bronchial epithelial cells upon the interaction with basophils, probably through the receptors HVEM and LTβR. / The results suggest that, through the interaction with tissue-resident cells such as bronchial epithelial cells and dermal fibroblasts, basophils may facilitate the activation of tissue-resident cells in response to the PAMPs in allergic inflammation. The results therefore provide a new insight of the crucial link between the bacterial-mediated innate immune response and the exacerbation of allergic inflammation. The above results also enhance our understanding on the immunopathological roles of LIGHT in airway remodeling, and the potential therapeutic target for airway remodeling. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qiu, Huaina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 165-196). / Abstract also in Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 摘要 --- p.ix / Publications --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Asthma and atopic dermatitis (AD) --- p.1 / Chapter 1.2 --- Human basophils in allergic inflammation --- p.3 / Chapter 1.2.1 --- Development and morphology of basophils --- p.3 / Chapter 1.2.2 --- Receptors and products of basophils --- p.4 / Chapter 1.2.3 --- Cell surface markers on basophils --- p.7 / Chapter 1.2.4 --- Basophils in allergic inflammation --- p.7 / Chapter 1.3 --- Human bronchial epithelial cells in airway inflammation --- p.10 / Chapter 1.4 --- Human fibroblasts in AD --- p.11 / Chapter 1.5 --- Staphylococcus aureus (S. aureus) in AD --- p.12 / Chapter 1.6 --- NOD2 and TLR2 in allergic inflammation --- p.14 / Chapter 1.7 --- IL-33 in allergic inflammation --- p.18 / Chapter 1.8 --- IL-6 in allergic inflammation --- p.18 / Chapter 1.9 --- CXCL8 in allergic inflammation --- p.20 / Chapter 1.10 --- CCL2 in allergic inflammation --- p.21 / Chapter 1.11 --- CCL5 in allergic inflammation --- p.22 / Chapter 1.12 --- β-defensin 2 (HBD-2) in allergic inflammation --- p.23 / Chapter 1.13 --- ICAM-1 and VCAM-1 in allergic inflammation --- p.25 / Chapter 1.14 --- LIGHT and airway remodeling in allergic asthma --- p.25 / Chapter 1.15 --- Signal transduction pathways in allergic inflammation and pharmacological inhibitors --- p.26 / Chapter 1.15.1 --- Signal transduction pathways in allergic inflammation --- p.26 / Chapter 1.15.2 --- Signaling molecule inhibitors as new drugs for inflammatory diseases --- p.31 / Chapter 1.16 --- Aims and scope of the study --- p.32 / Chapter Chapter 2: --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Reagents and buffers for the purification of human basophils --- p.35 / Chapter 2.1.2 --- Primary cells and cell lines --- p.36 / Chapter 2.1.3 --- Heat-killed Staphyloccocus aureus (HKSA) --- p.38 / Chapter 2.1.4 --- Ligands for NLR and TLR2 --- p.39 / Chapter 2.1.5 --- Recombinant human cytokines --- p.39 / Chapter 2.1.6 --- Reagents and buffer solutions for flow cytometry --- p.40 / Chapter 2.1.7 --- RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and real-time quantitative PCR (qPCR) --- p.45 / Chapter 2.1.8 --- Cytometric Bead Array (CBA) Kits --- p.48 / Chapter 2.1.9 --- MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel Kit --- p.49 / Chapter 2.1.10 --- Enzyme-linked immunosorbent assay (ELISA) kits --- p.49 / Chapter 2.1.11 --- Procarta Transcription Factor Assay kit --- p.50 / Chapter 2.1.12 --- Signal transduction inhibitors --- p.50 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Purification of primary human basophils and basophil culture --- p.50 / Chapter 2.2.2 --- Culture of KU812 cells --- p.51 / Chapter 2.2.3 --- Culture of primary human bronchial epithelial cells --- p.51 / Chapter 2.2.4 --- Culture of BEAS-2B cells --- p.52 / Chapter 2.2.5 --- Culture of human dermal fibroblasts --- p.52 / Chapter 2.2.6 --- Co-culture of primary human bronchial epithelial cells/human bronchial epithelial cell line (BEAS-2B) cells and basophils/KU812 cells --- p.52 / Chapter 2.2.7 --- Co-culture of human dermal fibroblasts and basophils/KU812 cells --- p.52 / Chapter 2.2.8 --- Co-culture of fixed primary human bronchial epithelial cells and basophils --- p.53 / Chapter 2.2.9 --- Co-culture of human dermal fibroblasts and basophils in the presence of transwell inserts --- p.53 / Chapter 2.2.10 --- CBA assay --- p.53 / Chapter 2.2.11 --- ELISA --- p.54 / Chapter 2.2.12 --- Human Transcription Factor Plex Assay --- p.54 / Chapter 2.2.13 --- Milliplex Human Cytokine / Chemokine Magnetic Panel assay --- p.54 / Chapter 2.2.14 --- Bio-Plex mouse cytokine assay --- p.55 / Chapter 2.2.15 --- Flow cytometric analysis of cell surface expression of target molecules --- p.55 / Chapter 2.2.16 --- Flow cytometric analysis of intracellular expression of target molecules --- p.55 / Chapter 2.2.17 --- Allergic asthmatic mice model --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3: --- Muramyl Dipeptide Mediated Activation of Human Bronchial Epithelial Cells Interacting with Basophils: A Novel Mechanism of Airway Inflammation --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Results --- p.61 / Chapter 3.2.1 --- Cell surface expression of CD203c on basophils --- p.61 / Chapter 3.2.2 --- Intracellular expression of NOD2 protein --- p.63 / Chapter 3.2.3 --- Cell surface expression of adhesion molecules on basophils and primary human bronchial epithelial cells activated by MDP --- p.67 / Chapter 3.2.4 --- Induction of cytokine, chemokine and β-defensin 2 upon the interaction of basophils and bronchial epithelial cells stimulated by MDP --- p.71 / Chapter 3.2.5 --- Bronchial epithelial cells were the main source for the release of IL-6, CXCL8 and β-defensin 2 in co-culture --- p.74 / Chapter 3.2.6 --- Effects of signaling inhibitors on MDP-induced cytokines and adhesion molecules --- p.77 / Chapter 3.2.7 --- Differential activation of intracellular signaling pathways involved in the interaction of KU812 and BEAS-2B upon MDP stimulation --- p.84 / Chapter 3.2.8 --- In vivo effect of NOD1,2 ligands on IgE and chemokine production in serum and BALF in allergic asthmatic mice --- p.89 / Chapter 3.3 --- Discussion --- p.93 / Chapter Chapter 4: --- NOD2 and TLR2 Ligands Mediated Activation of Basophils Interacting with Human Dermal Fibroblasts in Atopic Dermatitis --- p.100 / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Results --- p.102 / Chapter 4.2.1 --- Cell surface expression of adhesion molecules ICAM-1 on human dermal fibroblasts activated by heat-killed Staphyloccocus aureus (HKSA) --- p.102 / Chapter 4.2.2 --- Induction of chemokines upon the interaction of basophils and human dermal fibroblasts stimulated by HKSA --- p.104 / Chapter 4.2.3 --- Expression of NOD2 and TLR2 protein --- p.107 / Chapter 4.2.4 --- Cell surface expression of adhesion molecule ICAM-1 on human dermal fibroblasts activated by MDP, PGN, LTA or Pam3CSK4 --- p.110 / Chapter 4.2.5 --- Induction of cytokine and chemokines upon the interaction of basophils (with or without IL-33 priming) and human dermal fibroblasts stimulated by MDP, PGN, LTA or Pam3CSK4 --- p.112 / Chapter 4.2.6 --- Direct interaction between human dermal fibroblasts and basophils was required for the release of IL-6, CXCL8, CCL2 and CCL5 upon the stimulation of MDP, PGN, LTA and Pam3CSK4 --- p.118 / Chapter 4.2.7 --- Effect of signaling molecular inhibitors on the expression of adhesion molecule ICAM-1 --- p.121 / Chapter 4.2.8 --- Effect of signaling molecule inhibitors on the release of cytokine and chemokines upon the stimulation by NOD2 and TLR2 ligands --- p.123 / Chapter 4.2.9 --- Differential activation of intracellular signaling pathways involved in the interaction of human dermal fibroblasts and basophilic KU812 upon stimulation of NOD2 and TLR2 ligands --- p.127 / Chapter 4.3 --- Discussion --- p.131 / Chapter Chapter 5: --- Effect of Tumor Necrosis Factor Family Member LIGHT on the Activation of Basophils Interacting with Bronchial Epithelial Cells: Potential Therapeutic Target for Airway Remodeling --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Cell surface expression of HVEM and LTβR --- p.139 / Chapter 5.2.2 --- Effect of LIGHT on the expression of ICAM-1 on basophil or BEAS-2B alone or co-culture --- p.141 / Chapter 5.2.3 --- Induction of cytokine and chemokine upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.144 / Chapter 5.2.4 --- Induction of MMP-9 upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.147 / Chapter 5.2.5 --- Effect of LIGHT on the release of TGFβ-1, histamine and periostin upon the interaction of basophils and BEAS-2B cells --- p.149 / Chapter 5.3 --- Discussion --- p.152 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.156 / Chapter 6.1 --- General conclusions --- p.156 / Chapter 6.2 --- Future perspectives --- p.160 / Appendix --- p.163 / References --- p.165

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

Orientador: Lício Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T06:28:45Z (GMT). No. of bitstreams: 1 Santos_AndressaCoopedos_D.pdf: 9572929 bytes, checksum: ce4c4fb2095c6ffe99868ec77f19d5b3 (MD5) Previous issue date: 2011 / Resumo: A ativação da sinalização através do toll-like receptor-4 (TLR4) e a indução de estresse de retículo endoplasmático (ERE) são importantes mediadores da resistência à insulina na obesidade e em outras situações nas quais há um excesso de ácidos graxos saturados. Em um estudo recente, demonstrou-se que sinalização através de TLR4 pode, per se, induzir a ativação de ERE, sugerindo que a ativação do TLR4 é o evento inicial para a indução do estresse celular que contribui para o aumento da expressão de genes de resposta inflamatória. No entanto, os mecanismos que conectam essas duas vias distintas são desconhecidos. As chaperonas GRP78 e GRP94 exercem uma função importante no processamento das moléculas recém-traduzidas do TLR4. Além disso, a chaperona GRP94 é responsável por sua translocação e conteúdo na superfície celular. Durante uma ativação prolongada da via de sinalização do TLR4, a demanda por novas moléculas sintetizadas aumenta, e consequentemente, a demanda por novas chaperonas. Por esta razão, nós aventamos a hipótese de que sob uma ativação extrema da via do TLR4, a síntese de proteínas sobrepujaria a expressão de chaperonas, dessa forma induzindo ERE. Para testar essa hipótese, monócitos da linhagem THP-1 foram incubados com LPS e foi avaliada a expressão e ativação de proteínas responsivas ao ERE por real-time PCR, citometria de fluxo, imunoprecipitado e western blot. Em alguns experimentos, as células foram privadas de glicose ou tratadas com siRNA para aumentar ou diminuir, respectivamente, a expressão das chaperonas. Experimentos de time-course revelaram que o LPS induz um aumento de 2,5 vezes na expressão do TLR4, iniciando após 8 h, com um pico após as 24 h e permanecendo significantemente aumentado após 48 h. A expressão de GRP78 foi aumentada em três vezes com um aumento acentuado após 24 h sem aumento às 8 h, enquanto o GRP94 aumenta apenas 1,5 vezes com um pico após 2 h que retorna aos valores basais após 8 h do estímulo. Não houve aumento da expressão protéica das chaperonas após 48 h. A indução de ERE por LPS foi detectada antes de 4 h do estímulo observado pela avaliação da via da PERK/eIF2a, IRE1 e ATF6 e se mantém ativado após 48 h. Adicionalmente, a privação de glicose em células THP-1 aumenta a expressão de GRP94 e GRP78 em 2,5 e 11 vezes, respectivamente. Na ausência de glicose, o tratamento com LPS não induz ERE. A inibição da expressão das chaperonas por siRNA anula o efeito da privação de glicose em proteger as células do desenvolvimento de ERE induzido por LPS. Portanto, a hiperexpressão das chaperonas GRP78 e GRP94 protegem as células do ERE induzido por LPS. Assim, defeito na expressão das chaperonas induzido por TLR4 é um mecanismo envolvido na integração da sinalização do TLR4 e ERE / Abstract: TLR4 activation and the induction of endoplasmic reticulum stress (ERS) are two of the most important mechanisms connecting excessive fat with insulin resistance. Recently, it was shown that activation of TLR4 can, per se, induce ERS, suggesting that TLR4 is a primary event in the induction of the cellular stress that contributes to increased inflammatory gene expression. However, the mechanisms linking these molecular events are unknown. The chaperones GRP78 and GRP94 play an important role during the assembly of newly translated TLR4 molecules. In addition, the chaperone GRP94 escorts the protein to the cell membrane. Under prolonged activation, the demand for newly synthesized TLR4 molecules increases, and thus, the demand for new chaperones. Therefore, we hypothesized that under increased activation of TLR4, the synthesis of the protein would not be matched by the expression of chaperones, thus, triggering ERS. To test this hypothesis, the monocyte cell line THP-1 was incubated with LPS and the expression/activation of proteins involved in ERS was determined by real-time PCR, flow-cytometry, immunoprecipitation and immunoblot. In some experiments, cells were deprived of glucose or treated with siRNA to increase or decrease, respectively, the expression of the chaperones. Time-course experiments revealed that LPS led to a 2.5-fold increase of TLR4 expression starting as early as 8h, peaking after 24h and remaining significantly increased after 48h. The expression of GRP78 underwent a 3-fold increase with a sharp rise at 24h (no increase at 8h), while GRP94 increased by only 1.5-fold with a peak at 2h and an early return to base-line levels. None of the chaperones were increased after 48h. LPS-induced ERS was detected as early as 4h after stimulus as detected by the evaluation of PERK/eIF2?, IRE1 and ATF6 pathways. Strong signals of ERS were still present after 48h. The pre-incubation of THP-1 in glucose-deprived medium produced 2.5 and 11-fold increases of GRP94 and GRP78, respectively. Upon glucosedeprivation, LPS could no longer induce ERS. Inhibition of chaperone expression by siRNA completely abrogated the effect of glucose deprivation to protect cells from LPS-induced ERS. Thus, the hyperexpression of GRP78 and GRP94 protect cells from LPS-induced ERS. Defective TLR4-induced chaperone expression is a mechanism involved in the integration of TLR4 signaling and ERS / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Abstract Intrauterine infections apparently cause about 85% of preterm deliveries at less than 28 weeks of gestation. Infection and inflammation play an important role in morbidity and mortality during perinatal period. The inflammatory mechanisms and pathophysiological consequences of intrauterine infection are poorly understood. According to current evidence from animal studies, the phenotypes of fetal inflammatory response are variable, ranging from spontaneous preterm birth to fetal death. The fetus is rather well protected against infectious agents by both structural and functional barriers. Toll-like receptors (TLR) are a part of innate immune system. Binding of bacterial or viral component to TLR induces inflammatory response in the host. The present study addressed the hypothesis that the effects of bacterial lipopolysaccharide (LPS) on the fetus, depends on the route by which it reaches the fetus. In this experimental study we hypothesized that an acute inflammation within the amniotic cavity may cause an innate immune response in the fetus consequently leading to cardiovascular distress. Secondly the role of the placenta in protecting the fetus from acute infectious challenges was evaluated. Additionally, the role of gestational age in the fetal immune response was studied. In the present study, LPS from Gram-negative bacteria caused an acute intrauterine inflammation in mice as indicated by the elevated levels of inflammatory mediators (e.g. cytokines) in amniotic fluid. The fetal heart revealed mRNA and protein expression of TLR4, which recognizes LPS. Moreover, the data showed a cytokine response in the fetal heart and severe cardiac dysfunction. In contrast, intraperitoneal LPS administered to the mother did not cause an acute cytokine response in the fetus. After intraperitoneal LPS the placenta showed severe blood congestion and a cytokine response. The data suggest that TLRs play roles in regulating the acute inflammatory responses in the placenta. Despite the absence of a fetal immune response, the placental lesions caused a fetal cardiovascular compromise. In addition, the present data indicate that the fetal expression of TLR4 is tissue specific and developmentally regulated. Present study provides new insights into the acute inflammatory mechanisms in maternal and fetal compartments and the pathophysiological changes in fetal cardiovascular hemodynamics. / Tiivistelmä Hyvin ennenaikaisen, alle 28. raskausviikolla tapahtuneen synnytyksen taustalta löytyy usein kohdunsisäinen infektio (IUI). Kohdunsisäisen infektion aiheuttama synnytys on suurimpia sikiön vammautumiseen tai kuolemaan johtavia syitä. Tulehdukselliset mekanismit, jotka johtavat synnytykseen tai sikiön vammautumiseen ovat huonosti tunnettuja. Tollin kaltaiset reseptorit (TLR) ovat osa synnynnäisen immuniteetin puolustusjärjestelmää. Niiden tarkoitus on tunnistaa nopeasti elimistölle vieraat, usein bakteeri- tai virusperäiset rakenteet ja käynnistää tulehdusvaste tuottamalla tulehdusvälittäjäaineita. Nämä välittäjäaineet säätelevät tulehdusprosessia, toisaalta niiden liiallinen tuotto voi aikuisilla johtaa elinvaurioihin kuten sydämen toimintahäiriöön. On esitetty että tulehdusvälittäjäaineet käynnistäisivät myös ennenaikaisen synnytyksen IUI:ssa. TLR-reseptorien merkityksestä sikiön tulehdusvasteessa tai synnytyksen käynnistymisessä on vain vähän tietoa. Väitöskirjatyössä tutkittiin kokeellisen hiirimallin avulla bakteeriperäisen lipopolysakkaridin (LPS) kykyä aiheuttaa sikiön tulehdusvaste, sekä muutoksia sikiön sydämen ja verenkierron toiminnassa, riippuen LPS:n reitistä sikiöön. Toisaalta kokeellisella mallilla tutkittiin istukan kykyä suojata sikiötä voimakkaalta tulehdusreaktiolta. Tarkoituksena oli tutkia myös sikiön kehitysasteen vaikutusta LPS:n aiheuttamaan tulehdusvasteeseen. Tutkimuksessa havaittiin LPS:n aiheuttavan kohdunsisäisen tulehduksen kun sitä annettiin suoraan lapsiveteen. Sikiön sydämessä todettiin TLR4 lähetti-RNA:n ja proteiinin ilmentyminen sekä nopea sytokiinien tuotannon lisääntyminen kuvastaen äkillistä tulehdusprosessia. Samanaikaisesti ultraäänitutkimuksessa todettiin sikiön sydämen vaikea toimintahäiriö. Toisaalta, jos LPS annettiin kantavalle emolle vatsaonteloon, tulehdusvälittäjäaineiden tuotannon lisääntymistä sikiössä ei havaittu. Sen sijaan istukassa todettiin akuutti verentungos ja tulehdusvaste. Edellä kuvattu istukan toimintahäiriö johti myös sikiön sydämen ja verenkierron toiminnan vaikeutumiseen huolimatta siitä, ettei sikiön sydämessä havaittu tulehduksellista vastetta. Lisäksi sikiöstä ja istukasta saadut tulokset viittaavat siihen, että TLR-reseptorit ovat mukana LPS:n tunnistamisessa ja äkillisen tulehdusvasteen käynnistymisessä. Tutkimus antaa uutta tietoa raskauden aikaisista tulehduksellisista mekanismeista sekä sikiön sydämen toiminnan äkillisistä muutoksista voimakkaan tulehduksen aikana.

A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but their tendency to display low immunogenicity in humans has hindered their usage, despite excellent tolerability and safety profiles. Various approaches have been used to improve the immunogenicity of DNA vaccines. Recent human study data re-established the value of DNA vaccines, especially in priming high-level antigen-specific antibody responses. Data suggests that innate immune responses to the DNA vaccine plasmid itself contribute to the immunogenicity of DNA vaccines, however the underlying mechanisms responsible remain unclear. In this dissertation, we investigate the role of innate immunity in shaping antigen-specific adaptive immune responses following DNA vaccination. The current belief is that the cytosolic DNA sensing pathways govern DNA vaccine immunogenicity. To date, only the type I interferon inducing STING/TBK1 regulatory pathway has been identified as required for DNA vaccine immunogenicity. Surprisingly, neither the upstream receptor nor the downstream signaling molecules in this pathway have been characterized. I therefore investigated a candidate cytosolic DNA receptor, as well as the downstream transcription factors required for generation of antigen-specific immune responses. Additionally, the effects of pro-inflammatory signaling on DNA vaccine immunogenicity have yet to be comprehensively studied. Previous studies have only provided indirect evidence for the role of inflammatory v signaling in DNA vaccination. As such, I also investigated the role of the DNA sensing AIM2 inflammasome in DNA vaccination. My data indicates that AIM2 is a key modulator in DNA vaccination via a previously unrecognized connection to type I interferon. Importantly, this marks the first time a DNA vaccine sensor has been identified. Of note, this dissertation represents a departure from many published works in the field. Whereas previous studies have mostly utilized model antigens and only focused on the adaptive immune responses generated, I analyzed the effects on innate immunity as well. Using various innate gene knockout murine models, I quantified antigen-specific humoral and T cell responses, as well as serum cytokine and chemokines following immunization with a clinically relevant DNA vaccine. Overall, this data provides a basis for understanding the mechanisms of DNA vaccination, allowing for the design of more effective vaccines.

Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.

T cell development in the thymus produces multiple lineages of cells, including conventional naïve CD4+ and CD8+ T cells, regulatory T cells, and innate T cells. Innate T cells encompass γδ T cells, invariant natural killer (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and H2-M3-restricted cells (Berg, 2007). Although they are a minor subset of all thymocytes, innate T cells develop in the thymus and share characteristics of the innate and adaptive immune systems (Berg, 2007). These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation (Berg, 2007). However, in several strains of mice harboring mutations in T cell signaling proteins or transcriptional regulators, conventional CD8+ T cells develop as innate cells that share characteristics with memory T cells (Atherly et al., 2006b; Broussard et al., 2006; Fukuyama et al., 2009; Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). One of these signaling proteins, inducible T cell kinase (Itk) is a nonreceptor protein tyrosine kinase that signals downstream of the T cell receptor (TCR) (Berg et al., 2005). Upon TCR activation, Itk is activated and recruited to the TCR signaling complex, where Itk interacts with Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), linker for activation of T cells (LAT), and phospholipase C γ1 (PLCγ1) (Berg et al., 2005). Thus, in Itk-deficient mice, TCR signaling is disrupted, which results in mature CD4- CD8+ (CD8SP) thymocytes that are CD44high, CD62Lhigh, CD122+, and CXCR3+ and that express high levels of the transcription factor, Eomesodermin (Eomes) (Atherly et al., 2006b; Broussard et al., 2006; Weinreich et al., 2010). Recently, it was determined that the development of these innate CD8SP thymocytes in itk-/- mice is dependent on IL-4 produced in the thymic environment by a poorly characterized subset of CD3+ thymocytes expressing the transcriptional regulator, promyelocytic leukemia zinc finger (PLZF) (Gordon et al., 2011; Verykokakis et al., 2010b; Weinreich et al., 2010). Here we show that a sizeable proportion of mature CD4+ CD8- (CD4SP) thymocytes in itk-/- mice also develop as Eomesodermin+ innate T cells. These Eomes+ innate CD4+ T cells are CD44high, CD62Lhigh, CD122+, and CXCR3+ (Atherly et al., 2006b; Broussard et al., 2006; Dubois et al., 2006; Weinreich et al., 2010). Surprisingly, neither CD4SP nor CD8SP innate thymocytes in itk-/- mice are dependent on γδ T cells for their development as was previously hypothesized (Alonzo and Sant'Angelo, 2011). Instead, both subsets of innate itk-/- T cells require the presence of a novel PLZF-expressing, SAP-dependent thymocyte population that is essential for the conversion of conventional CD4+ and CD8+ T cells into Eomesodermin-expressing innate T cells with a memory phenotype. This novel subset of PLZF-expressing SAP-dependent innate T cells preferentially home to the spleen and mesenteric lymph nodes and have a restricted TCR repertoire. Thus, we have christened this subset as CD4+ PLZF + MAIT-like cells. We have characterized multiple subsets of innate T cells that expand in the absence of Itk. Therefore, we were interested in how innate T cells respond to infection. Although Itk KO mice have defects in cytolytic function and cytokine production during an acute infection, these mice are able to clear viral infections (Atherly et al., 2006a; Bachmann et al., 1997). Hence, we hypothesized that Itk-deficient memory CD8+ T cells would be able to provide protection upon a challenge infection. Conversely, we found this not to be true although Itk-deficient memory CD8+ T cells were present in similar frequencies and cell numbers as WT memory CD8+ T cells at 42 days post-infection. Furthermore, Itk-deficient memory CD8+ T cells were able to produce IFNγ and exert cytolytic function upon stimulation. Although the function of Itk-deficient memory CD8+ T cells appeared to be intact, we found that these cells were unable to expand in response to a challenge infection. Remarkably, conventional memory CD8+ T cells lacking Itk were able to expand and form protective memory responses upon challenge. Thus, the inability of Eomes+ innate CD8+ T cells to form protective memory responses does not appear to be intrinsic to cells deficient in Itk. This thesis is divided into six major chapters. The first chapter will provide an introduction to T cell development and the role of Itk in T cell development. Additionally, it will introduce a variety of innate T cell subsets that will be discussed throughout this thesis and will provide an overview of CD4+ and CD8 + T cell differentiation during infection. This section will explain the role of Itk in CD4+ helper T cell differentiation and describe how Itk-deficient CD8+ T cells respond to acute infection. The introduction will also discuss the generation of conventional memory CD8+ T cells. The second chapter will provide the details of the experimental procedures used in this thesis. The third chapter will describe the characterization and development of Eomes+ innate CD4+ T cells that develop in the absence of Itk. Additionally, this chapter will address the subset of PLZF+ innate T cells that induce the expression of Eomes in innate T cells. The fourth chapter will further characterize and explore the development of itk-/- CD4+ PLZF+ MAIT-like T cells. The fifth chapter will examine the role of Eomes + innate CD8+ T cells in protective memory responses. Chapters three through five will display work that is in preparation to be submitted to a peer-reviewed journal. The sixth chapter will discuss the results of this thesis and their implications.

Innate sensing of nucleic acids lies at the heart of antiviral host defense. However, aberrant activation of innate sensors by host nucleic acids can also lead to the development of autoimmune diseases. Such host nucleic acids can also be released from stressed, damaged or dying cells into the tissue microenvironment. It however remains unclear how the extracellular nucleic acids impacts the quality of the host immune responses against viral infections. Using a mouse model of influenza A virus (IAV) infection, we uncovered an important immune-regulatory pathway that tempers the intensity of the host-response to infection. We found that host-derived DNA from necrotic cells accumulates in the lung microenvironment during IAV infection, and is sensed by the DNA receptor Absent in Melanoma 2 (AIM2). AIM2-deficiency resulted in severe immune pathology highlighted by enhanced recruitments of immune cells, and excessive systemic inflammation after IAV challenge, which led to increased morbidity and lethality in IAV-infected mice. Interestingly, these effects of AIM2 were largely independent of its ability to mediate IL-1β maturation through inflammasome complexes. Finally, ablation of accumulated DNA in the lung by transgenic expression of DNaseI in vivo had similar effects. Collectively, our results identify a novel mechanism of cross talk between PRR pathways, where sensing of hostderived nucleic acids limits immune mediated damage to virus infected tissues.