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.

Chronic inflammation is an essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration and accumulation of macrophages in damaged tissues. Macrophage motility is highly regulated by adhesive receptors, integrins. Namely, intermediate expression of integrin supports macrophage migration, while a high integrin density inhibits it. Our studies are focused on evaluation of the contribution of related integrins αDβ2 and αMβ2 to macrophage migration and development of chronic inflammation. We found that integrin αDβ2 is upregulated on M1-macrophages in vitro and pro-inflammatory macrophages in atherosclerotic lesions. Interestingly, the expression of ligand-sharing integrin αMβ2 remains unaltered. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that robust adhesion of M1-activated macrophages translates to weak 3D migration, which depends on the high expression of αDβ2, since αD-deficiency decreases M1-macrophage adhesion and improves macrophage migration. In contrast, αD- and αM-knockouts decrease M2-macrophages migration, demonstrating that moderate integrin expression supports cell motility. In model of high fat diet-induced diabetes, αD-deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while αM-deficiency does not affect macrophage accumulation. We detected a new ligand for integrins αMβ2 and αDβ2, 2-(ω-carboxyethyl)pyrrole (CEP). CEP is preferentially generated during inflammation-mediated oxidation and forms adduct with ECM proteins generating novel substrate for αMβ2 and αDβ2. Targeting CEP-dependent macrophage adhesion can be a useful approach to control αDβ2-mediated chronic inflammation. Using specially designed peptide library, protein-protein interaction and adhesion assay, we identified a peptide, called P5, which significantly inhibited αD-CEP binding. P5 peptide regulates macrophage migration in three-dimensional matrix in vitro and reduced macrophage accumulation during thioglycollate-induced peritoneal inflammation. Effect of P5 is completely eliminated in αD-deficient macrophages. Tracking of adoptively-transferred fluorescently-labeled WT and αD-/- monocytes in diabetic mice confirmed that αD-dependent inhibition of macrophage accumulation in adipose tissue is mediated by P5 peptide. Taken together, these results demonstrate the importance of αDβ2 and αDβ2-CEP interaction for the accumulation of infiltrating macrophages during inflammation and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes.

Vitiligo is an autoimmune disease of the skin characterized by epidermal depigmentation that results from CD8+ T cell-mediated destruction of pigment producing melanocytes. Vitiligo affects up to 1% of the population and current treatments are moderately effective at facilitating repigmentation by suppressing cutaneous autoimmune inflammation to promote melanocyte regeneration. In order to cause disease, CD8+ T cells must overwhelm the mechanisms of peripheral tolerance in the skin and if we understand the suppressive mechanisms that are compromised during vitiligo, we can potentially use this information to improve existing treatments or engineer novel interventions. Therefore, my goal is to characterize the regulatory factors in the skin that suppress depigmentation during vitiligo. Our lab has developed a mouse model of vitiligo that accurately reflects human disease and I used this model to demonstrate that regulatory T cells suppress CD8+ T cell-mediated depigmentation and interact with CD8+ T cells in the skin during vitiligo. In this model of disease, I investigated the molecules involved in regulatory T cell function and observed that the chemokine receptors CCR5 and CCR6 play different roles in regulatory T cell suppression. While CCR6 facilitates regulatory T cell migration to the skin, CCR5 is dispensable for migration but required for optimal regulatory T cell function. Additionally, I used our mouse model to demonstrate that Langerhans cells suppress the incidence of disease during vitiligo. Taken together the results from these studies provide novel insights into the mechanisms of suppression during vitiligo.

In 1989 the hepatitis C virus was identified as a significant cause of post-transfusion hepatitis. Nearly two decades later there is still no vaccine, inadequate treatment options, and limited understanding of how the virus establishes chronicity in the majority of the people it infects. Recent reports suggest that the interaction of a negative co-stimulatory pathway mediated by PD-1 and PDL-1 is associated with persistent viral infection. The role, if any, that PD-1/PDL-1 has in HCV infection is unknown. In this study we report that PD-1 is upregulated in T-cells from persons with chronic HCV infection when compared to healthy donors. In addition, PD-1 and PDL-1 are upregulated on T-cells from healthy donors when exposed to extracellular HCV core protein (a nucleocapsid protein that is immunosuppressive); upregulation of PD-1 is mediated by core's ability to bind to the complement receptor gC1q. We also report that the observed T-cell function can be restored by blocking the PD-1/PDL-1 interaction. Our results indicate that HCV core can upregulate an important negative T-cell signaling pathway that is associated with viral persistence. This upregulation of PD-1/PDL-1 represents a novel and perhaps shared mechanism that viral pathogens may use to subvert the human immune response. It also represents a potential new treatment option for the millions of people who suffer from chronic hepatitis C infection.

<p>Despite the advanced knowledge of the mechanisms of influenza infection and improved vaccines, Influenza A Virus still causes a life-threatening respiratory disease, especially during pandemics. Past investigations have proposed a synergism between Influenza A virus and a simultaneous or subsequent bacterial superinfection as the predominant cause of death. The recent development of animal models to study these heterologous infections has shed light onto the diverse mechanisms by which Influenza A Virus may increase the susceptibility to contract a secondary bacterial infection. These studies suggested an important role for the innate immune system in mediating such disease. We developed a model of heterologous infection combining Influenza A Virus and <em>Bordetella parapertussis</em> that demonstrated a critical role for MIP-2 to drive pulmonary neutrophilia in the pathology associated with bacterial superinfection of influenza. However, the origin of this increased MIP-2 production and the mechanisms underlying the immunopathology remained to be elucidated. The present studies proposed IL-1β overproduction as the upstream cause of the increased MIP-2 production observed in heterologous infection. This exaggerated IL-1β production was likely related to the increased bacterial burden observed in heterologously infected mice. This study also demonstrated that reduction in IL-1β production by blockade of the inflammasome seemed to provide an improvement in the clinical symptoms and the immunopathology of the disease. Thus, interventions to attenuate the exacerbated bacterial burden and the inflammatory responses derived from the subsequent IL-1β overproduction should be further investigate as possible therapeutic approaches to treat bacterial superinfections.</p> / Master of Science (MSc)

Hyperhomocysteinemia (hHcys) is a widely known pathogenic factor in the progression of end-stage renal disease (ESRD) and it is also associated with an increased risk for injurious cardiovascular pathologies during ESRD. HHcys is linked to the formation and activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, characterized as a critical early mechanism initiating the inflammatory response. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) mediate the activation of the NLRP3 inflammasome in podocytes in response to elevated levels of homocysteine (Hcys) in vitro and in vivo. However, it remains unknown how NLRP3 inflammasome activation is triggered by NOX. The aim of the present study sought to determine the signaling cascade that triggers glomerular injury and sclerosis during hHcys mediated by Vav2, a guanine nucleotide exchange factor (GNEF). Using both genetic and pharmacological interventions of Vav2, we first tested whether this GNEF is involved in hHcys-induced NLRP3 inflammasome activation in podocytes by its role in activation of the Rac-1-NOX complex. Further, we explored whether pharmacological targeting of Vav2 activation may regulate NLRP3 inflammasome signaling pathway during hHcys-induced glomerular injury. We found that mice with hHcys (on the FF diet) or oncoVav2 (a constitutively active form of Vav2) transfection in the kidney exhibited increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1 and elevated IL-1β levels in glomeruli, indicating the formation and activation of the NLRP3 inflammasome. This glomerular NLRP3 inflammasome activation was accompanied by podocyte dysfunction and glomerular injury, even sclerosis. Local transfection of Vav2 shRNA plasmids significantly attenuated hHcys-induced NLRP3 inflammasome activation, podocyte injury, and glomerular sclerosis. In cultured podocytes, Hcys treatment and oncoVav2 transfection increased NLRP3 inflammasome formation and activation. This NLRP3 activation was inhibited by Vav2 shRNA, associated with reduction of Rac-1 activity and ROS production. Administration of NSC23766, a Rac-1 inhibitor substantially attenuated inflammasome formation, desmin expression and decreased podocin expression in glomeruli of hHcys mice. These results suggest that elevated Hcys levels activate Vav2 and thereby increase NOX activity, leading to ROS production. ROS trigger NLRP3 inflammasome activation, podocyte dysfunction and glomerular injury. Therefore, the present study defines a novel mechanism underlying hHcys-induced NLRP3 inflammasome activation and its progression to ESRD.

B-lymphocytes are a subset of immune cells that can be distinguished mainly by carrying clonally diversified membrane-bound immunoglobulin specialized to specific antigen recognition. Together with other immunocytes B-lymphocytes play a central role in adaptive immune system which takes part in defense of the host against wide variety of pathogens. Recently the evidence has supported the emerging concept of different B-cell subpopulations to play a direct or indirect role in a pathogenesis of spectrum of disorders. However, so far the knowledge has been limited mainly in the way of how the specific differentiation stages of B-lymphocytes are involved in pathogenesis of diseases and how course of disease, stage, and eventually different treatment can affect B-cell homeostasis. That is the reason for the thesis to be focused on an analysis of B-cell population profile changes in disease, identification of any association present among specific B-cell subpopulations, as well as association between these subpopulations and clinical parameters. Using polychromatic flow cytometry we analyzed frequencies of 11 B-cell subpopulations including stages of transient B-lymphocytes through effector antibody-producing plasma cells. We examined 81 individuals including 22 healthy controls and 59 patients with...

INTRODUÇÃO: A Leishmaniose Tegumentar Americana (LTA) é uma doença infecciosa endêmica no Brasil, causada por protozoários do gênero Leishmania e possui duas formas clínicas principais, a leishmaniose cutânea (LC) e a leishmaniose mucocutânea (LMC). Os macrófagos têm papel crucial na patogênese da Leishmaniose como hospedeiros do parasita e atuam como células apresentadoras de antígenos, promovendo uma resposta imune de perfil Th1 contra a Leishmania. Os macrófagos polarizados M2, relacionados com resposta anti-inflamatória (Th2), são envolvidos no controle da inflamação e dano tecidual em várias doenças e estão associados à cronicidade nas doenças infecciosas. O objetivo deste trabalho foi verificar a participação dos macrófagos M2 nos sítios de lesão cutânea da LTA MÉTODOS: Sessenta e três biópsias de pele de lesões de LTA, obtidas de 48 doentes com LC e 15 de LMC, foram submetidas ao método imuno-histoquímico com os anticorpos anti-CD163 e anti-CD206, marcadores de macrófagos M2. Dez espécimes foram também submetidos à técnica imuno-histoquímica de dupla marcação com a utilização dos marcadores pSTAT-1 e CD68 para identificação de macrófagos M1 e CMAF e CD163 para identificação de macrófagos M2. RESULTADOS: Os espécimes de pele com reação inflamatória difusa (n = 26) exibiram maior número de células imunomarcadas com CD163, quando comparadas ao grupo com reação granulomatosa bem organizada (n = 37) (p= 0,01). Os macrófagos CD163+ também foram mais numerosos nas lesões de pele com a presença de formas amastigotas de Leishmania (n = 39) ao exame histopatológico (p=0,02), quando comparadas às lesões onde os parasitas não foram observados (n = 24). O mesmo ocorreu quando comparados somente os espécimes do grupo de doentes com LC com a presença de parasitas (n = 31) e ausência dos mesmos (n = 17) (p = 0,04). As lesões de pele de doentes com LMC mostraram maior número de células CD206+ (n = 15) que a de doentes com LC (n = 48) (p=0,008). Houve correlação positiva entre o número de células imunomarcadas com os anticorpos CD163 e CD206 (r de Spearman= 0,369). Os macrófagos +pSTAT1/CD68+ (M1) mostraram-se mais numerosos que os macrófagos CMAF+/CD163+ (M2) nos sítios de lesão cutânea da LTA. CONCLUSÕES: Os resultados obtidos sugerem que os macrófagos M2 têm um papel na resposta imune \"in situ\" da LTA. Essas células devem atuar como células alvo, facilitando a entrada dos parasitas, a progressão da doença e parecem estar envolvidos nos mecanismos de cronicidade e nos processos de remodelamento tecidual dessa doença. As moléculas CD163 e CD206 podem ser consideradas bons marcadores teciduais de macrófagos M2, uma vez que mostraram correlação positiva em sua expressão. A técnica de dupla marcação confirmou a presença dos macrófagos M2 nas lesões cutâneas da LTA. Embora ocorra a presença de macrófagos M2, os macrófagos M1 são a população macrofágica predominante da resposta imune \"in situ\" da LTA / INTRODUCTION: American tegumentary leishmaniasis (ATL) is an endemic infectious disease in Brazil, caused by Leishmania parasites and it has two main clinical forms, cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). Macrophages play a key role in leishmaniasis pathogenesis. They are definite parasite host and act as antigen presenting cells, eliciting a Th1 pattern of immune response (M1) against Leishmania. M2 polarized macrophages, related to an anti-inflammatory response (Th2), are involved in controlling inflammation and tissue damage in several diseases and are related to chronicity in infectious diseases. The aim of this study was verifying M2 macrophages \"in situ\" participation in ATL. METHODS: Sixty-three skin biopsies from LTA lesions, obtained from patients with CL (n=48) and MCL (n=15), were subjected to imunohistochemical technique with M2 macrophages markers CD163 and CD206 antibodies. Ten samples were selected for double staining technique with pSTAT1 and CD68 markers for M1 identification, and CMAF and CD163 markers for M2 macrophages identification. RESULTS: Skin samples displaying diffuse inflammatory reaction (n = 26) exhibited higher number of CD163 immune stained macrophages when compared to the well-organized granulomatous reaction group (n = 37) (p=0.01). CD163+ macrophages were higher in samples displaying Leishmania parasites (n = 39) at histopathology exam than the samples without parasites (n = 24) (p=0.02). Same result was observed when comparing the lesions of CL lesions with (n = 31) and without (n = 17) parasites (p = 0.04). MCL skin lesions (n = 15) showed higher number of CD206+ cells (p = 0.008) in comparison with skin lesions taken from CL patients (n = 48). Spearman test demonstrated a positive correlation between the number of CD163+ and CD206+ immune stained cells (Spearman r = 0,369). M1 macrophages (pSTAT1+/CD68+) were present in higher number when compared to M2 macrophages (CMAF+/CD163+) in the samples studied. CONCLUSIONS: The results suggest that M2 macrophages play a role in the in situ immune response of ATL. M2 polarized macrophages may act as host target cells facilitating parasites entry and promoting disease progression, but also seem to be involved with chronicity and tissue remodeling of ATL lesions. CD163 and CD206 molecules may be considered as good markers for M2 macrophages. Double staining technique confirmed the presence of M2 macrophages, although M1 macrophages are the predominant macrophagic component in skin lesions of ATL

Células T reguladoras (Treg) são cruciais na tolerância periférica e no controle da inflamação. Nós usamos dois modelos bem estabelecidos de tolerância de mucosas para a asma alérgica e a tolerância inalatória local induzida pela exposição crônica a OVA para estudar o aparecimento e função das Treg. Nós mostramos que a tolerância nasal distinguiu da tolerância oral pela produção sistêmica de IgG1 e desenvolvimento da inflamação alérgica na cavidade peritoneal ou pela indução da inflamação das vias aéreas de camundongos RAG-/- reconstituídos com células T CD4+ após desafios com OVA. Observamos também que Treg Foxp3+ migraram para o pulmão alérgico e expressaram fenótipo de ativação e memória que distinguiu essas células das Treg presentes nos linfonodos drenantes. Células T CD4+CD25+ do pulmão dos animais alérgicos suprimiram a proliferação das células T CD4+CD25-, mas não a produção de citocinas Th2. Finalmente, a exposição crônica a OVA levou ao aumento da apoptose de eosinófilos que infiltraram o pulmão resultando na resolução da inflamação alérgica pulmonar. / Regulatory T cells (Treg) are critical for peripheral tolerance and control of inflammation. We used two well established models of mucosal tolerance to allergic airway disease and the local inhalational tolerance induced by chronic OVA exposure to study the appearance and function of Treg cells. We found that nasal tolerance distinguished from oral tolerance by systemic IgG1 antibody production and development of allergic inflammation in the peritoneal cavity or by induction of airway inflammation in RAG-/- mice reconstituted with CD4+ T cells after OVA challenge. We also found that Foxp3+ T cells migrated to allergic lung and expressed an effector/memory phenotype that distinguished them from Treg cells present in lung draining lymph nodes. Lung infiltrating CD4+CD25+ T cells from allergic mice suppressed CD4+CD25- T cell proliferation but not Th2 cytokines production by these cells. Finally, chronic OVA exposure leaded to increased apoptosis of infiltrating lung eosinophils resulting in the resolution of allergic lung inflammation.

Results of the above studies thus suggested that immune dysregulation in SLE result in derangement of a spectrum of inflammatory mediators leading to possible multiple organs auto-inflammatory damages. However, the exact etio-pathogenic mechanism could not simply be explained by these phenomena. Infection has been invoked as an underlying etiology or trigger for the induction of autoimmune disease. Epstein-Barr virus (EBV) possesses multiple features that characterise its involvement in initiating or perpetuating SLE disease. Several research groups demonstrated that the peripheral blood EBV DNA load is significantly higher in SLE patients, yet cell-free viral DNA was also reported in other EBV-associated diseases such as nasopharyngeal carcinoma (NPC) and certain lymphomas, suggesting that relatively little is known about its biology and dynamic distribution in the blood circulation. In the second part of our study, we examined the cell-free and cell-associated distribution profile of EBV DNA load in SLE. Our data showed that the distribution of EBV DNA in the cell-free and cell-associated compartments exhibited a heterogeneous pattern in SLE patients. Contrary to the exclusive presence of circulating cell-free EBV DNA in NPC patients, both cell-free and cell-associated EBV DNA were detected in some SLE patients, while in others, no EBV DNA was measurable in either blood compartments. The level of cell-associated EBV viral load was significantly higher in SLE patients with active disease than those who presented with milder disease activity. This phenomenon indicated a possible association of EBV viral infection with the level of immune competence in SLE patients. It has been reported that EBV encodes proteins which shares significantly homology sequence with human IL-6, IL-8, IL-10, IL-12 and colony-stimulating factor (CSF)-1. This proposition brought our attention to the immune perturbation by EBV on the cytokine balance, possibly constitute in part, to the immune dysregulation and Th1 and Th2 dichotomy in SLE exacerbation. (Abstract shortened by UMI.) / The first section of this research study aimed to explore the messengers that influence Th1/Th2 cells differentiation, development, effector functions and hence their plausible contribution in SLE immunopathogenesis. We focused on studying the expression of cytokine and chemokine milieu that directs the traffic of T lymphocytes; co-stimulatory molecules in the activation of T lymphocytes; transcription factors T-bet and GATA-3 in regulating the differentiation of Th1 and Th2 cell lineage. We also investigated the involvement of the lymphocyte subpopulation, Th17 in the auto-inflammatory axis of SLE exacerbation. / Lit, Choi Wan. / Advisers: Christopher W.K. Lam; Y.M. Dennis Lo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3358. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 203-235). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.