Les rétrovirus endogènes humains ont longtemps été considérés comme d'inactifs fossiles de l'ADN humain, mais leur abondance (8%) a récemment été révélée grâce au séquençage du génome humain. Plusieurs études indépendantes ont montré une association au niveau moléculaire entre le rétrovirus endogène humain MSRV de la famille HERV-W et la sclérose en plaques (SEP) ainsi que la schizophrénie (SCZ). Les éléments de la famille HERV-W codent pour une protéine d'enveloppe (Env) très immunopathogène. Cette protéine active une cascade autoimmune et inflammatoire via l'interaction du récepteur TLR4 avec les cellules présentatrices d'antigène, provoque une dérégulation du système immunitaire et induit une cytotoxicité particulière. Grâce à un test ELISA développé pour la détection des protéines HERV-W ex vivo, la présence significative d'une antigénémie MSRV-Env a pu être détectée chez environ 75% des patients SEP, alors que tous les contrôles sains étaient négatifs. Une antigénémie positive d'environ 50% chez des patients SCZ pour les protéines Env et Gag a également été rapportée, montrant une corrélation significative avec un sous-groupe de patients ayant un niveau élevé de protéine C-réactive. De plus, un modèle animal d'encéphalomyélite autoimmune expérimentale a pu être reproduit avec l'utilisation de la protéine Env. Ce modèle a montré des signes d'inflammation et de démyélinisation confirmées par des analyses IRM et histologiques, ainsi qu'une autoimmunité anti-myéline. Un anticorps monoclonal, sélectionné pour ses propriétés inhibitrices par rapport à l'activation du TLR4 par MSRV-Env, a été testé in vivo avec ce nouveau modèle animal et in vitro avec des cellules immunitaires. Une inhibition significative des symptômes cliniques en comparaison avec des contrôles non traités a été obtenue et l'innocuité du traitement a été vérifiée. L'interaction de la protéine Env du rétrovirus MSRV avec des facteurs environnementaux pourrait être à la source de la cascade inflammatoire en association avec la SEP ou la SCZ. Cette protéine représente une nouvelle cible thérapeutique dans le cadre du développement prometteur d'un anticorps thérapeutique monoclonal. L'ensemble des ces travaux a contribué à montrer qu'une partie des rétrovirus endogènes humains ont retenu leur activité, ce qui ouvre de nouvelles perspectives de recherche dans le domaine des maladies complexes humaines / Human endogenous retroviruses (HERVs) have long been considered as inactive fossils in human DNA, but recent sequencing of the human genome revealed that they are very abundant and constitute approximately (8%) of the human genomic sequences. Recent independent molecular studies have also shown an association between the MS-associated retrovirus MSRV of the human endogenous retroviruses type “W” (HERV-W) family and Multiple Sclerosis (MS), as well as Schizophrenia (SCZ). HERV-W elements encode a powerful immunopathogenic envelope protein (Env) that activates a proinflammatory and autoimmune cascade through interaction with Toll-Like receptor 4 (TLR4) on antigenpresenting cells, triggers dysregulation of the immune system and mediates a peculiar cellular toxicity. Using a specific ELISA immunoassay for ex-vivo measurement of HERV-W proteins, a highly significant prevalence of MSRV-Env antigenemia in MS sera (about 75%) was reported, whereas all healthy controls were negative. A positive Env and Gag antigenemia (about 50%) was also found in SCZ patients, and a significant correlation with an elevated level of C-reactive protein was shown in a subgroup of SCZ patients. Moreover, the MSRV-Env protein was shown to reproduce the hallmarks of an Experimental Autoimmune Encephalomyelitis, an animal model for MS, with major inflammatory demyelination confirmed by MRI and histology, as well as anti-myelin autoimmunity. An anti-Env monoclonal antibody, selected for its inhibiting effects on MSRV-Env interaction with TLR4 in human lymphoid cell cultures, was tested in vivo with this new MS model and also in vitro with human immune cells. Significant inhibition and prevention of clinical symptoms compared to untreated controls were observed. These rodent models are also useful for initial investigation of the safety of antibody, since the Env protein is not expressed in animal species except for non-human primates. A relationship between environmental factors and the pro-inflammatory MSRV-Env protein is thus a possible hypothesis regarding initiation of a pathogenic cascade leading to diseases such as MS and SCZ. The MSRV-Env protein now represents a novel target, and the neutralizing antibody being developed by GeNeuro, a promising therapeutical approach for the cure of these severe neurological diseases

L’effet anti-tumoral d'un lipide A, l’OM-174 (partie lipidique des lipopolysaccharides) a été étudié dans un modèle de cancer mammaire chez la souris. In vivo, l’OM-174 augmente la survie de la souris alors qu’in vitro il n'est pas toxique pour les cellules cancéreuses. L’OM-174 se lie au récepteur TLR4 des cellules immunitaires induisant la production de cytokines comme l’IFNγ. In vitro, l’association de cette cytokine au lipide A est cytotoxique. L’objectif de cette thèse est d’en analyser les mécanismes moléculaires. Nous avons montré, aussi bien in vitro qu’in vivo, que la cytotoxicité du lipide A/IFNγ est dépendante du TLR4, du récepteur à l’IFNγ et de l’expression de la NOS II. Nous avons également montré que les espèces radicalaires, NO et anion superoxyde formant le peroxynitrite jouent un rôle crucial dans cette cytotoxicité. L’origine de ces espèces radicalaires se trouve être la NOS II selon un processus de découplage enzymatique. Nous avons également cherché d’autres mécanismes associés pouvant expliquer la cytotoxicité du lipide A/IFNγ. Nous avons ensuite montré que le NO est capable de réagir avec les résidus cystéine de certaines protéines, un processus appelé S-nitrosylation. Une analyse protéomique nous a permis d’identifier au moins une dizaine de protéines qui sont S-nitrosylées dans les cellules cancéreuses mammaires en réponse au lipide A/IFNγ. Nous avons étudié l’impact de cette modification sur la fonction d’une des ces protéines, l’enzyme de conjugaison E2 de l’ubiquitine Ubc13, une protéine impliquée dans la prolifération et la survie cellulaire. Nous avons confirmé la nitrosylation d’Ubc13 et identifié la cystéine 87 comme cible du NO. L’expression d’une forme mutée d’Ubc13 (remplacement de la cystéine 87 par une alanine) inhibe l’auto-ubiquitination de l’enzyme et sa capacité à ubiquitiner une de ses cibles IkBα. Nous avons montré que la S-nitrosylation d’Ubc13 induit sa migration vers le noyau et rend les cellules plus sensibles à l’effet cytotoxique du lipide A/IFNγ. En résumé, nos résultats révèlent un rôle majeur et insoupçonné de la NOS II et du NO dans l’effet antitumoral du lipide A OM-174 dans un modèle de cancer mammaire chez la souris ouvrant la voie pour la conception de nouveaux traitements anticancéreux. / The anti -tumor effect of a lipid A, OM -174 (lipid portion of LPS) was studied in a model of breast cancer in mice. In vivo, OM- 174 increases the survival of mice whereas in vitro it is not toxic to cancer cells. OM -174 binds to TLR4 immune cells inducing the production of cytokines such as IFNγ. In vitro, the combination of IFNγ to lipid A is cytotoxic. The objective of this thesis is to analyze those molecular mechanisms. We have shown both in vitro and in vivo that the cytotoxicity of the lipid A / IFNγ is dependent of TLR4 and of the receptor for IFNγ, and the NOS II expression. We also showed that the radical species, NO and superoxide anion forming peroxynitrite play a crucial role in this cytotoxicity. The origin of these radical species is being NOS II enzyme in a process of decoupling. We also looked for other associated mechanisms that may explain the cytotoxicity of lipid A / IFNγ. We then showed that NO is able to react with the cysteine residues of certain proteins, a process called S- nitrosylation. A proteomic analysis allowed us to identify at least a dozen proteins that are S- nitrosylated in breast cancer cells in response to lipid A / IFNγ. We studied the impact of this change on the basis of one of these proteins, the E2 conjugating enzyme UBC13 ubiquitin, a protein involved in cell proliferation and survival. We confirmed the UBC13 nitrosylation on cysteine 87 and identified as a target of NO. The expression of a mutant of UBC13 (replacement of cysteine 87 with alanine) forms inhibits the auto-ubiquitination of the enzyme and its ability to ubiquitinylated one of its targets IkBα. We have shown that S- nitrosylation of UBC13 induced its translocation to the nucleus and greater sensitivity to the cytotoxic effect of lipid A / IFNγ in cells. In summary, our results reveal an important and unexpected role of NOS II and NO in the antitumor effect of lipid A OM- 174 in a model of breast cancer in mice opening the way for the development of new cancer treatments.

Acute Respiratory Distress Syndrome (ARDS) following hemorrhagic shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit” hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with increased activation of the proinflammatory transcription factor NF-κB, and augmented expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which resides in plasma membrane lipid rafts. The objective of this work is to define cellular mechanisms of macrophage priming by oxidative stress following shock resuscitation. The main hypothesis investigated is that altered cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation strategies can diminish these effects. AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface levels of TLR4, which in turn results in augmented NF-κB translocation in response to small doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy iii transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress can be diminished by early exposure to resuscitation regimens with direct or indirect systemic antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline. Hyperosmolarity was found to modulate AM TLR4 gene and protein expression. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling pathway early during shock resuscitation may represent an anti-inflammatory strategy able to ameliorate late morbidity and mortality following S/R.

Orientador: Luciane Alarcão Dias-Melicio / Resumo: A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, prevalente na América Latina, que tem por agentes etiológicos diferentes espécies de Paracoccidioides. Entre eles encontra-se o Paracoccidioides brasiliensis (P. brasiliensis), espécie composta por um complexo de agrupamentos geneticamente isolados, classificados como espécies filogenéticas: S1, PS2, PS3 e PS4. O P. brasiliensis possui distribuído pela parede fúngica e dentro de vesículas citoplasmáticas uma glicoproteína de 43KDa, a gp43. Esta molécula é considerada como o principal componente antigênico produzido e secretado pelo fungo, a qual é encontrada no soro de pacientes acometidos pela PCM e, está associada aos fatores de virulência e escape do patógeno. Sabendo disso, tornou-se extremamente importante a avaliação da interação dessa proteína com as células do sistema imune inato e sua consequente atuação sobre os mecanismos moduladores celulares. Assim, nosso estudo avaliou os neutrófilos polimorfonucleares (PMNs) que estão presentes em abundância desde o início e durante a infecção pelo Paracoccidioides, e que apresentam além de suas ações efetoras diretas contra o fungo, uma importante ação moduladora da resposta imune. Estas ações envolvem o reconhecimento do fungo por Receptores de Reconhecimento de Padrão (PRRs) que culminam em ativação celular com consequente produção e liberação de diferentes mediadores inflamatórios. Dessa maneira, o presente trabalho avaliou o envolvimento dos receptores cel... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, prevalent in Latin America and has as etiological agents Paracoccidioides spp. One of them is Paracoccidioides brasiliensis (P. brasiliensis) composed by a complex of genetically isolated clusters, classified as phylogenetic species: S1, PS2, PS3 and PS4. P. brasiliensis present along the cell wall and inside cytoplasmic vesicles a 43 KDa glycoprotein, known as gp43. This molecule is recognized as the main antigenic component produced and secreted by the fungus into sera of PCM patients and is associated with the escape and virulence factors of the pathogen. Thereby, becomes extremely important to evaluate the interaction mechanisms of this protein with cells from the innate immune system and their consequent action on cellular functions. Polymorphonuclear neutrophils (PMNs) has prominent participation in PCM, once these cells are present in a great amount at the beginning and during Paracoccidioides infection. PMNs perform effector actions directly against the fungi as well as an important immune modulatory function. Such actions involve the recognition of fungal components by pattern recognition receptors (PRRs) that leads to cell activation and consequent production of inflammatory mediators. So, the present study evaluated the involvement of TLR2 and TLR4 on cytokines and eicosanoids release by PMNs from healthy human stimulated with gp43. Cells were initially incubated in the presence or absence of monoclo... (Complete abstract click electronic access below) / Doutor

Bovine mastitis remains one of the costliest diseases affecting the dairy industry. Individual susceptibility to mastitis and severity of infection varies between animals and can only be partially explained by genetics. As such, understanding how genetic predisposition coordinately interacts with epigenetic modifications and environmental exposures is necessary to bridge the gap in missing heritability. The role of DNA methylation in regulating the response to bacterial lipopolysaccharide (LPS) was first determined by performing reduced representation bisulfite sequencing on fibroblasts isolated from heifers at 5- and 16-months of age that exhibit an age-dependent up-regulation in LPS-responsiveness. More than 14,000 differentially methylated sites were identified between the two sets of cultures with a trend towards decreased methylation with age. Young cultures were also hyper-methylated in gene promoters regulated by NF-κB and exhibited lower expression in genes that regulate the innate immune response, suggesting that methylation contributes to gene regulation in fibroblast innate response. Previously, TLR4 expression was shown to differ in the age-dependent fibroblast model, however, it was not known if variation in TLR4 expression would affect mastitis severity. Therefore, fibroblasts were isolated from sixty lactating, adult Holstein cows and their expression of TLR4, along with LPS-induced production of IL-8 and IL-6, was used to rank the animals from high to low. Six high responders and six low responders were then experimentally infected in one mammary gland with E. coli. Overall, severity of mastitis was quite variable, with a few notable differences between high and low responders. High responding animals had an earlier increase in somatic cell count and febrile response that coincided with more efficient bacterial clearance. However, tissue damage and milk production did not differ between the two groups, indicating that while rapid up-regulation of the innate response addresses bacterial clearance, subsequent down-regulation is required to alleviate damage within the mammary gland. Finally, one-week old bull calves were subjected to treatment with either saline or LPS to determine if neonatal exposure to endotoxin would make calves less responsive to a second LPS challenge at 32-days of age. The initial treatment showed a large effect of LPS as measured by higher plasma IL-6 and TNF-α concentrations in calves treated with LPS over saline. Subsequent treatment of all 10 calves with LPS showed a very similar response between the two treatment groups and significant inter-animal variability in clinical response. Fibroblasts and monocyte-derived-macrophages (MDMs) were also isolated following initial treatment to determine if any changes occurred at the cellular level as a result of LPS exposure. Fibroblasts isolated from calves at 20-days of age had a very low response to LPS that did not differ between the early life treatments. MDMs isolated from calves at 28-days of age were more responsive to LPS, but again no differences were detected between the early life treatments. In summary, our results suggest that DNA methylation likely plays a role in the cellular response to LPS and may partially contribute to differences between animals in severity of E. coli mastitis, however, the appropriate in vitro phenotype to detect susceptible animals still needs to be characterized before epigenetic biomarkers can be identified, and perhaps modified by environmental interventions.

Interleukins, cytokines secreted by leukocytes, are predominant messengers modulating immune responses. Interleukin-27 (IL-27), a key immunomodulatory cytokine, functions to induce both pro- and anti-inflammatory effects in various immune cells. IL-27 is a heterodimeric cytokine, composed of IL-27p28 and Epstein-Bar virus induced gene 3 (EBI3) subunits, and binds to a receptor composed of IL-27Rα (WSX-1) and gp130. Initial studies focused on describing IL-27 functions in skewing T helper cell development to a Th1 response, with few reports on functions in monocytes. Thus, in this thesis, I aimed to characterize novel functions of IL-27 in innate immune responses of monocytes. I initially established that IL-27 induced a pro-inflammatory cytokine profile (IL-6, IP-10, MIP-1α, MIP-1β, and TNF-α) mediated via STAT1/3 and NF-κB signaling pathways. Further investigation led to the discovery that IL-27 could enhance LPS responses via upregulation of TLR4 expression and NF-κB signaling. Together, these studies described novel signaling mechanisms (NF-κB and JAK/STAT crosstalk) and gene targets (cytokines and TLR4) of IL-27 that drive inflammatory responses. In continuing the quest for novel IL-27 functions in innate immunity, I reported IL-27 can upregulate expression of the IFN-responsive, antiviral protein called BST-2. My results showing IL-27-induced expression of BST-2 mRNA and cell surface protein were supported by previous studies reporting IL-27-induced expression of other antiviral molecules. Furthermore, previous studies showed IL-27 could inhibit HIV replication via antiviral gene induction, pointing to potential for IL-27 immunotherapies. In light of the posited role for IL-27 in therapeutics, it became inherently critical to describe how IL-27 functions in the setting of HIV infection. Thus, in my final thesis chapters, I described the effect of HIV infection on IL-27 expression and functions, addressing a substantial void in literature. Interestingly, a trend of decreased IL-27 expression and significant impairment of IL-27-induced gene expression was observed in HIV infection. Therefore, decreased circulating IL-27 and decreased IL-27 responsiveness may collectively dysregulate IL-27 function in HIV. This thesis describes novel, IL-27-driven, proinflammatory responses, and highlights impairment of IL-27 function in HIV infection. This work bridged a gap in knowledge of IL-27 functions in monocytes and highlighted multifaceted mechanisms underlying immunoregulation by IL-27. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2012-04-12 13:07:50.588

Acute Respiratory Distress Syndrome (ARDS) following hemorrhagic shock/resuscitation (S/R) is an important contributor to late morbidity and mortality in trauma patients. S/R promotes ARDS by inducing oxidative stress that primes cells of the innate immune system for excessive responsiveness to small inflammatory stimuli, termed the “twohit” hypothesis. Activated alveolar macrophages (AM) play a central role and when recovered from S/R animals exhibit an exaggerated responsiveness to lipopolysaccharide (LPS) with increased activation of the proinflammatory transcription factor NF-κB, and augmented expression of cytokines. LPS triggers AM signalling through Toll like receptor 4 (TLR4), which resides in plasma membrane lipid rafts. The objective of this work is to define cellular mechanisms of macrophage priming by oxidative stress following shock resuscitation. The main hypothesis investigated is that altered cellular distribution of TLR4 can lead to macrophage priming and antioxidant resuscitation strategies can diminish these effects. AM of rodents, exposed in vivo to oxidant stress following S/R, increase their surface levels of TLR4, which in turn results in augmented NF-κB translocation in response to small doses of LPS. Furthermore, in vitro H2O2 treatment of RAW 264.7 macrophages results in similar TLR4 surface translocation. Depletion of intracellular calcium, disruption of the cytoskeleton or inhibition of the Src kinases prevents the H2O2-induced TLR4 translocation, suggesting the involvement of receptor exocytosis. Further, fluorescent resonance energy iii transfer between TLR4 and lipid rafts as well as biochemical raft analysis demonstrated that oxidative stress redistributes TLR4 to surface lipid rafts. Preventing the oxidant-induced movement of TLR4 to lipid rafts using methyl-ß-cyclodextrin precluded the increased responsiveness of cells to LPS after H2O2 treatment. Further, AM priming by oxidative stress can be diminished by early exposure to resuscitation regimens with direct or indirect systemic antioxidant effects, such as 25% albumin, N-acetylcysteine and hypertonic saline. Hyperosmolarity was found to modulate AM TLR4 gene and protein expression. Collectively, these studies suggest a novel mechanism whereby oxidative stress might prime the responsiveness of cells of the innate immune system. Targeting the TLR4 signalling pathway early during shock resuscitation may represent an anti-inflammatory strategy able to ameliorate late morbidity and mortality following S/R.

Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.

Resumo: Os objetivos deste trabalho foram a padronização de metodologia alternativa de genotipagem do SNP AY_731081:g.1900T>C do gene CD14 equino por PCR-RFLP, bem como a caracterização em equinos da raça Mangalarga deste e de outros dois polimorfismos, o AY_005808: c.1530A>G do TLR4 e o AX_463789: g.133T>C do Cε, a fim de promover o embasamento necessário para futuras pesquisas visando associação entre marcadores de DNA e características relacionadas à fisiologia do sistema imune na raça. Para tanto, foram utilizados 151 animais Mangalarga, de ambos os sexos e de idades variadas, representativos da população do estado de São Paulo. O método de PCR-RFLP mostrou-se adequado para a genotipagem do SNP AY_731081: g.1900T>C do gene CD14 equino. Entretanto, tal polimorfismo provavelmente não ocorre em equinos Mangalarga, impossibilitando estudos de associação com o marcador na raça. Os parâmetros genético-populacionais obtidos para os polimorfismos AY_005808:c.1530A>G do gene TLR4 e o AX_463789:g.133T>C do gene Cε demonstraram a possibilidade de realização de pesquisas visando a associação entre os marcadores e características relacionadas ao sistema imune na raça Mangalarga / Abstract: The objective of this work was to contribute to the molecular characterization of the equine Mangalarga, aiming at future studies on the association of DNA polymorphisms and traits associated with the immune system physiology of this equine breed. An alternative PCR-RFLP genotyping method was developed for the SNP AY_731081:g.1900T>C, in the gene CD14. Furthermore, this SNP plus the AY_005808: c.1530A>G, in the gene TLR4, and the AX_463789: g.133T>C, in the gene Cε, were used to analyze 151 Mangalarga individuals. The analyzed horses are representative of the São Paulo State population and consisted of male and female animals of several ages. The PCR-RLP method has been demonstrated to be suitable for equine genotyping using the SNP AY_731081: g.1900T>C of the gene CD14. However, this polymorphism is apparently absent in the Mangalarga breed. The population genetic data obtained for the polymorphisms AY_005808:c.1530A>G, of the gene TLR4, and the AX_463789:g.133T>C, in the gene Cε, indicated the feasibility of these SNPs for further studies aiming at the association of molecular markers with traits related to the immune system of the Mangalarga horse breed / Orientador: Marcílio Dias Silveira da Mota / Coorientador: Rogério Abdallah Curi / Banca: Humberto Tonhati / Banca: Lenira El Faro Zadra / Mestre

Submitted by Erika Demachki (erikademachki@gmail.com) on 2016-08-17T20:24:56Z No. of bitstreams: 2 Tese - Hélio Galdino Júnior - 2013.pdf: 3450540 bytes, checksum: 01adc52cca765449b5eed357351fb5ee (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-18T12:50:37Z (GMT) No. of bitstreams: 2 Tese - Hélio Galdino Júnior - 2013.pdf: 3450540 bytes, checksum: 01adc52cca765449b5eed357351fb5ee (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-18T12:50:37Z (GMT). No. of bitstreams: 2 Tese - Hélio Galdino Júnior - 2013.pdf: 3450540 bytes, checksum: 01adc52cca765449b5eed357351fb5ee (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-07-26 / American Tegumentary Leishmaniasis (ATL) is a disease caused by protozoa Leishmania. In Brazil, the most prevalent species is L. (Viannia) braziliensis. Several cytokines and receptors are involved in immunopathogenesis of ATL, however, the role of interleukin 32 (IL-32) was not investigated in this disease. Besides, toll-like receptors (TLR) were poorly evaluated in Leishmania infection, especially when it is caused by L. (V.) braziliensis. The aim of the present study was to evaluate IL-32, TNF and IL-10 expression in ATL lesions; the induction of IL-32 by L. (V.) braziliensis amastigotes in human peripheral blood mononuclear cells (PBMC) cultures as well as the involvement of TLR4 in monocyte/macrophage response to L. (V.) brazilienis amastigotes. Biopsies fragments from cutaneous and mucosal lesion and healthy tissues were used to investigate the subgenus of the parasites by PCR-RFLP assay; expression of IL-32, TNF and IL-10 was assayed by immunohistochemistry and expression of IL-32 isoforms     , TNF and IL-10 was analysed by qRT-PCR. The PBMC were cultured with L. (V.) braziliensis amastigotes in the absence or presence of IFN and IL-32 induction was assayed by qRT-PCR; and TNF and IL-10, by ELISA. TLR4 was neutralized by monoclonal antibodies and lipopolysaccharide (LPS) was used as TLR4 agonist. The expression of TLR4 in monocyte/macrophages was evaluated by flow cytometry. Thirty five patients were evaluated, 23 with cutaneous leishmaniasis (CL) and 12 with mucosal leihsmaniasis (ML). All parasite positive samples contained L. (Viannia) sp. The expression of IL-32 (protein and mRNA) was similar in CL and ML lesions but was higher than in health tissues. Only IL-32 was detected. The proteins TNF and IL-10 were detected in similar levels in CL and ML lesions, but TNF mRNA was present in higher levels in ML (4.069x) than in CL lesions (141 x, p < 0.05). L. (V.) braziliensis amastigotes induced IL-32, TNF and IL-10 in IFN pre-treated PBMC. The production of TNF and IL-10 was TLR4 dependent and treatment of PBMC with LPS further increased the production of TNF induced by amastigotes (p < 0.05). However, LPS did not altere the IL-10 production. Treatment with IFN enhanced the percentage of TLR4+ monocyte/macrophage (p < 0.05), which was decreased following incubation with amastigotes (p = 0.06). The results showed that IL-32 is produced during L. (Viannia) infection and TLR4 mediates L. (V.) braziliensis amastigote-induced TNF and IL-10 production in human PBMC. Moreover, the data suggest that amastigotes can lead to TLR4 internalization what can allow parasite to evade of innate immune response. This study indicates that IL-32 and TLR4 are important players in human infection caused by L. (Viannia), especially L. (V.) braziliensis. Whether TLR4 is also important to IL-32 production by human monocytes/macrophages deserves further investigation. / A leishmaniose Tegumentar Americana (LTA) é uma doença infecto-parasitária, causada, no Brasil, principalmente pela espécie Leishmania (Viannia) braziliensis. Na imunopatogenia da LTA participam várias citocinas e receptores. A interleucina 32 (IL-32) é uma citocina pro-inflamatória, cuja participação na LTA ainda não foi investigada. O papel dos receptores similares a Toll (TLR) na LTA tem sido pouco investigado, especialmente considerando infecção por L. (V.) braziliensis. O objetivo deste estudo foi avaliar a expressão da IL-32, do TNF e da IL-10 nas lesões de pacientes com LTA, a indução de IL-32 por formas amastigotas de L. (V.) braziliensis em células mononucleares (CMN) humanas, bem como avaliar a participação do TLR4 na infecção de monócitos/macrófagos humanos com formas amastigotas de L. (V.) brazilienis. Fragmentos de lesões cutâneas ou mucosas de pacientes com LTA e tecidos sadios foram obtidos, sendo usados para a determinação do subgênero de Leishmania, usando a PCR-RFLP; para análise de IL-32, TNF e IL-10 por imunoistoquímica; e para análise da expressão das isoformas     da IL-32, do TNF e da IL-10, por qRT-PCR. As CMN foram cultivadas com amastigotas de L. (V.) braziliensis, na ausência ou presença de IFN e a indução de IL-32 foi avaliada por qRT-PCR e TNF e IL-10, por ELISA. Para neutralização de TLR4 foram usados anticorpos monoclonais e lipopolissacarídeo (LPS), como agonista de TLR4. A expressão de TLR4 nos monócitos/macrófagos presentes nas CMN foi avaliada por citometria de fluxo. Foram analisados 35 pacientes sendo 23 leishmaniose cutânea (LC) e 12 com leishmaniose mucosa (LM). Todas as amostras PCR positivas continham parasitos pertencentes ao subgênero Viannia. Nas lesões de pacientes com LC e LM foi detectada uma maior expressão tanto da proteína quanto do mRNA da IL-32 do que nos tecidos controles, porém níveis similares foram encontrados nas duas formas clínicas. Somente a isoforma IL-32 foi detectada. As proteínas TNF e a IL-10 foram detectadas nas lesões, em níveis similares na LC e na LM, porém, o mRNA do TNF estava em níveis mais elevados nas lesões de LM (4.069 vezes) do que nas lesões de LC (141 vezes, p < 0,05). As formas amastigotas de L. (V.) braziliensis indiziram a produção de IL-32 TNF e de IL-10 nas CMN pré-tratadas com IFN. A produção de TNF e IL-10 foi dependente de TLR4 e o tratamento das CMN com LPS aumentou a produção do TNF induzido pelas amastigotas (p < 0,05), mas não alterou a produção da IL-10. A incubação com IFN aumentou significantemente a porcentagem de monócitos/macrófagos TLR4+ (p < 0,05), a qual foi diminuída pelas formas amastigotas (p = 0,06). Os resultados mostram que a IL-32 é produzida durante a infecção humana causada por L. (Viannia) e evidenciam a participação de TLR4 na indução de TNF e IL-10 em CMN humanas por formas amastigotas de L. (V.) braziliensis. Ainda, os dados sugerem que as formas amastigotas causam a internalização dos TLR4, o que pode ser um mecanismo de evasão da resposta imune. Portanto, a IL-32 e o TLR4 parecem importantes componentes na infecção humana causada por L. (Viannia), especialmente L. (V.) braziliensis, podendo contribuir para a imunopatogenia ou para o controle da infecção.