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Several studies have reported that administration of cannabinoid receptor agonists in inflammatory/autoimmune and CNS injury models resulted in significant attenuation of clinical disease. The beneficial effects correlated with the observed reduction of inflammatory mediators and peripheral immune cell infiltration into the site of inflammation. Previous studies from our laboratories demonstrated that administration of cannabinoid type 2 receptor agonist attenuated disease score and improved recovery in two murine models of neuroinflammation; spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. The goal of the current investigation was to evaluate the mechanisms through which administration of selective cannabinoid-2 receptor (CB2R) agonists modify inflammatory responses and help to improve function in SCI and EAE. In SCI, an acute neuroinflammatory disorder, administration of CB2R agonist at 1 h and 24 h following contusion injury to the cord resulted in improved recovery of motor function and bladder function (the ability to spontaneously void) compared to control animals. Evaluation of inflammatory mediators at 48h demonstrated a dramatic reduction in the expression of the chemokines CXCL9, 10, 11 and cytokines IL-23 and its receptor in CB2R agonist-treated cords. There was also a reduction in the expression of toll-like receptors (TLR1, TLR4, TLR6, and TLR7), which correlated with a decreased number of immunoreactive microglia. Interestingly, at seven days post injury, CB2R agonist-treated injured cords showed a significant reduction in both hematopoietic and myeloid cell infiltration. In EAE, a chronic neuroinflammatory disorder, our laboratories demonstrated previously that administration of a CB2R agonist led to lower disease scores and improved recovery. In this study, we observed reduced numbers of infiltrating hematopoietic and myeloid cells into the spinal cord and brain of CB2 agonist-treated mice. This reduction was observed at the peak of disease (day 17) and the effect was maintained at the chronic stage of disease (day 30). Evaluation of molecules associated with cell migration showed decreased levels of the adhesion molecule VCAM-1 and matrix metalloproteinases MMP-2 and 9 at peak of EAE in treated mice. The decrease in VCAM-1 correlates with our previous observation of decreased leukocyte rolling and adhesion to brain microvasculature. However, the reduction in MMP-2/9 expression suggests that CB2R agonists may also affect leukocyte transmigration into the perivascular space and further infiltration into the CNS parenchyma. This process requires both chemokine cues and the gelatinases MMP2/9. Animals deficient in these MMPs show leukocyte accumulation in the perivascular space and are resistant to EAE. There are no reports in the literature on possible CB2R agonist effects on gelatinases in myeloid cells. Although both MMP-2 and -9 are produced by antigen-presenting cells and act on similar substrates, MMP-9 appears to play a crucial role in EAE. Therefore, we decided to examine the effects of CB2 signaling on MMP-9 expression in myeloid cells, focusing on myeloid bone marrow-derived dendritic cells (BMDC). Activation of bone marrow-derived macrophages, dendritic cells, and primary microglia with the cytokine cocktail TNFα, IL-1ß, IL-6, containing PGE2, which mimicked an inflammatory milieu, resulted in expression of high levels of MMP-9. Treatment with CB2R agonists reduced MMP-9 in all three cell types. Since migration of DC to various sites is required for their activation and for the initiation of adaptive immune responses, we evaluated the effects of CB2R agonists on migration. The reduced levels of MMP-9 correlated with reduced migration of DC to the draining lymph nodes in vivo, as well as reduced migration vitro in the matrigel migration assay. The effect on MMP-9 expression was mediated through CB2R, resulting in reduction in cAMP levels, subsequent decrease in ERK activation, and reduced binding of c-Fos and c-Jun to the AP-1 site in the MMP-9 promoter. We postulate that, by dampening production of MMP-9 and subsequent MMP-9-dependent DC migration, cannabinoids contribute to resolve acute inflammation and to reestablish homeostasis. Selective CB2R agonists might be valuable future therapeutic agents for the treatment of chronic inflammatory conditions by targeting activated immune cells including DC. / Physiology

Modulation of the endocannabinoid system by the administration of exogenous agonists and selective antagonists has been shown to have potential to attenuate the contribution of inflammation to secondary injury in the CNS. The two most well-defined receptors are the CB1 and CB2 receptors. CB2, the cannabinoid receptor expressed primarily on hematopoietic cells and activated microglia, mediates the immunoregulatory functions of cannabinoids. The involvement of CB2 in central nervous inflammation has been demonstrated by using both endogenous and exogenous ligands. We showed previously that CB2 selective agonists inhibited leukocyte rolling and adhesion to CNS microvasculature and ameliorate clinical symptom in both chronic and remitting-relapsing EAE models; and our previous studies also demonstrated therapeutic potential of CB2 agonist improving recovery following spinal cord injury in the mouse. The goal of the current investigation was to evaluate the mechanisms through which administration of a selective cannabinoid-2 (CB2) agonist modifies inflammatory responses and helps to improve function following the injury in central nervous system. In the EAE project, we showed that Gp1a, a highly selective CB2 agonist with a four log higher affinity for CB2 than CB1, reduced clinical scores and facilitated recovery in EAE in conjunction with long term reduction in demyelination and axonal loss. We also established that Gp1a affected EAE through at least two different mechanisms, i.e. an early effect on Th1/Th17 differentiation in peripheral immune organs, and a later effect on the accumulation of pathogenic immune cells in the CNS, associated with reductions in the expression of CNS and T cell chemokine receptors, chemokines and adhesion molecules. This is the first report on the in vivo CB2-mediated Gp1a inhibition of Th17/Th1 differentiation. We also confirmed the Gp1a-induced inhibition of Th17/Th1 differentiation in vitro, both in non-polarizing and polarizing conditions. The CB2-induced inhibition of Th17 differentiation is highly relevant in view of recent studies emphasizing the importance of pathogenic self-reactive Th17 cells in EAE/MS. In spinal cord injury project, we showed that spinal cord injury mice CB2 agonist O-1966 (with affinities to the CB1 and CB2 receptors of 5055±984 and 23±2.1 nM, respectively) had improved motor function, autonomic function. They also had significant reductions in CXCL-9, CXCL-11, dramatic reductions in IL-23p19 expression and its receptor IL-23r, and reduction in the number of immunoreactive microglia. The results reported in this thesis, demonstrated that the combined effect on Th17 differentiation and immune cell accumulation into the CNS, may contribute to the usefulness of CB2 selective ligands as potential therapeutic agents in neuroinflammation. / Physiology

Chemokines, immune proteins that induce chemotaxis and adhesion, and their G-protein coupled receptors distribute throughout the central nervous system (CNS), regulate neuronal patterning, and mediate neuropathology. These chemo-attractant molecules may provide a neuro-immune "link" by regulating CNS systems. The purpose of this study was to investigate the interactions of specific chemokines, stromal cell-derived factor (SDF)-1a/CXCL12, and fractalkine/CX3CL1, and their receptors, CXCR4 and CX3CR1, with the serotonin (5-hydroxytryptamine; 5-HT) and opioid systems using anatomical and electrophysiological techniques in the rat brain. In the serotonin dense midbrain raphe nuclei (RN), SDF-1a, CXCR4, fractalkine and CX3CR1 co-localize over 70% with 5-HT neurons. CX3CR1 also localizes to microglia in the RN and hippocampus. Functionally, SDF-1a (10 nM) increases spontaneous inhibitory postsynaptic current (sIPSC) frequency and evoked IPSC (eIPSC) amplitude, while decreasing paired-pulse ratio (PPR) selectively in 5-HT neurons, thus stimulating presynaptic GABA release at these neurons. Alternatively, fractalkine (10 nM) increases sIPSC and eIPSC amplitude without changing PPR selectively in 5-HT neurons, thereby elevating the postsynaptic GABA receptor number or sensitivity. These results are dose-dependent and receptor-mediated. Chemokine interactions with serotonin, a neurotransmitter regulating mood, may lead to therapies for depression comorbid with immune diseases. Additional immunohistochemical analysis in the brain shows CXCR4 and CX3CR1 neuronal co-localization with the mu-opioid receptor (MOR) in the hippocampus, cingulate cortex, periaqueductal grey (PAG), nucleus accumbens, ventral tegmental area, globus pallidus, but not in the striatum or habenular nuclei, suggesting region specific receptor interactions. Electrophysiological recordings following morphine, SDF-1?? or fractalkine in vitro treatment reveal morphine (10 ?M)-mediated hyperpolarization of the membrane potential and reduction of the input resistance of PAG neurons, however, SDF-1??and fractalkine at 10 nM do not impact either parameter. In combination, SDF-1? inhibits morphine's actions in all PAG neurons tested, and fractalkine blocks morphine-mediated changes in 60% of PAG neurons examined. Thus, CXCR4 as well as CX3CR1, although less consistently, both appear to desensitize MOR at the neuronal level. Chemokine-opioid receptor interactions may mediate novel mechanisms to treat neuro-inflammatory pain and opiate abuse. The combined anatomical and electrophysiological results support chemokines as neuromodulatory proteins that may provide communication between the nervous and immune systems. / Anatomy

L'échinococcose alvéolaire (EA) est une maladie parasitaire en relation non seulement avec la destruction hépatique quiaccompagne le développement du stade larvaire (métacestode) d'Echinococcus multilocularis, mais aussi avecl'importante réponse immunitaire granulomateuse qui l'entoure. La plupart des travaux antérieurs visant à caractériser le;relations hôte-parasite ont porté sur les cellules de l'immunité dans des sites éloignés de l'habituelle localisationparasitaire, dans le foie. Ce travail de thèse rapporte les mécanismes impliqués dans les modifications de l'homéostasiehépatique aux différents stades de l'infection, mais aussi l'analyse détaillée des profils de cytokines et chimiokinesprésents dans l'infiltrât cellulaire périparasitaire hépatique, de la présence du transforming growth factor-beta et desautres acteurs de sa voie d'activation, et de l'implication possible du Fibrinogen-like protein-2 (FGL2), une moléculeeffectrice des lymphocytes T-régulateurs récemment identifiée. Les résultats indiquent que la réaction inflammatoire quientoure le métacestode dans le foie contribue significativement à la sécrétion de cytokines et de chimiokines et auxmécanismes fonctionnels immunitaires de l'interaction hôte-parasite ; ils révèlent aussi pour la première foisl'intervention cruciale de FGL2 dans la tolérance vis-à-vis d'£. multilocularis. Ces résultats contribuent à identifier denouvelles cibles pour une thérapeutique immunologique qui permettrait de pallier les conséquences pathologiques del'infection par E. multilocularis et de complémenter l'action seulement parasitostatique des benzimidazoles, seuls agentsthérapeutiques connus actuellement. / Alveolar echinococcosis (AE) is a parasitic disease predominantly caused not only by thé direct hepatic damage whichfollows thé continuous tumor-like prolifération of thé larval stage (métacestode) of Echinococcus multilocularis, but alsoindirectly by thé intense local granulomatous immune response which surrounds thé parasitic tissue. Most of previousstudies which aimed at characterizing thé host-parasite relationship hâve been performed on cells of thé immune responsiin peripheral sites, far from thé usual location of E. multilocularis larvae, i.e. thé liver. This PhD thesis reports on thémechanisms involved in thé changes in hepatic homeostatis at thé various stages of infection, and also on a detailedanalysis of thé cytokine and chemokine profiles in thé hepatic periparasitic cell infiltrate, of thé présence of transforminggrowth factor-beta and other actors of its metabolic pathway, and of thé possible involvement of Fibrinogen-like protein-:(FGL2), a recently identified effector molécule of T-regulatory lymphocytes. Results indicate that thé inflammatoryreaction which surrounds thé métacestode in thé liver significantly contributes to cytokine and chemokine sécrétion and tthé functional immune mechanisms of thé host-parasite interactions; they also reveal for thé first time thé crucialintervention of FGL2 in thé tolérance towards E. multilocularis. Thèse results contribute to identify new targets fortherapeutic immune modulation in order to alleviate thé pathological conséquences of E. multilocularis and tocomplément thé parasitostatic-only action of benzimidazoles, thé currently available chemotherapy of thé disease

In addition to host defense, alternative functions of immune cells are emerging. Immune cells are crucial during healing of injured tissue, in formation of new blood vessels, angiogenesis, and also in maintaining the balance in inflammation having immune regulating functions. Over the last decade a higher degree of heterogeneity and plasticity of immune cells have been reported and immune cells develop different characteristics in different situations in vivo. This thesis investigates roles for immune cells in situations of muscle hypoxia and reduced blood perfusion, wound healing in skin and at sites of transplantation of allogeneic islets of Langerhans and on top of this, ways to steer immune cell function for future therapeutic purposes. A specific neutrophil subset (CD49d+VEGFR1+CXCR4high) was found to be recruited to VEGF-A released at hypoxia and these neutrophils were crucial for functional angiogenesis. In muscle with restricted blood flow macrophages were detected in perivascular positions and started to express aSMA and PDGFR1b and were found to directly assist in blood flow regulation by iNOS-dependent NO production. This essential function in muscle regain of function could be boosted by plasmid overexpression of CXCL12 where the effect of these macrophages chaperoning the vasculature was amplified improving limb blood flow regulation. The effect on macrophages accelerating tissue regeneration being amplified by CXCL12 was tested in a model of cutaneous wound healing where the administration of CXCL12 was optimized for high bioavailability. In the skin, CXCL12-treatment induced accumulation of TGFb-expressing macrophages close to the wound driving the healing process, and subsequently the wounds healed with an efficiency never reported before. In the last study means to circumvent systemic immune suppressive therapy required in allogeneic transplantation was investigated. Allogeneic islets of Langerhans transplanted to muscle were immediately destroyed by the host immune system. Co-transplanting islets and CCL22-encoding plasmids we could curb this fast rejection for 10 days by accumulating CD4+CD25+FoxP3+ regulatory T lymphocytes at the site for transplantation preventing islet grafts from being attacked by the host cytotoxic T lymphocytes. In summary this thesis outlines distinct immune cell subsets being essential for regain of tissue function in hypoxia, ischemia and post injury and ways to amplify specific immune cell functions in these situations that are feasible for clinical use.

Chronic inflammation in the brain results in the development of several CNS diseases, including Alzheimer’s and Parkinson’s diseases, multiple sclerosis, and tumors. IL-1, a pro-inflammatory cytokine released by activated microglia and astrocytes, instigates the expression of factors promoting the progression of these CNS disorders, including cytokines, chemokines, and components of matrix remodeling systems, such as the plasminogen activator system. IL-1 also increases the mRNA expression and activity of SphK, the enzyme that phosphorylates Sph to form S1P, a bio-active sphingolipid. This thesis demonstrates that IL-1 and S1P enhance the mRNA and protein expression of PAI-1 and uPAR, two key components of the plasminogen activator system, in glioblastoma cells. The S1P-induced mRNA expression of PAI-1 and uPAR is mediated by the S1P2 receptor, and requires Rho-kinase and MEK1. However, IL-1 regulation of PAI-1 and uPAR mRNA expression is independent of SphK, and thus S1P. IL-1- and S1P-induced mRNA expression of PAI-1 and uPAR results in the increased in vitro invasion of glioblastoma cells. Since significant amounts of IL-1 are secreted from gliomas, and it increases the production of S1P via inciting the activity and mRNA expression of SphK, we propose a mechanism by which S1P and IL-1 influence the invasion of glioblastoma cells by increasing the mRNA and protein expression of uPAR and PAI-1. IL-1 and S1P also influence the mRNA expression of chemokines implicated in the development and progression of multiple sclerosis, namely IP-10 and RANTES, in primary human astrocytes. IP-10 and RANTES attract T cells, which are the major pathological cause of multiple sclerosis. This thesis demonstrates a novel mechanism by which S1P significantly inhibits the IL-1-induced mRNA expression of these chemokines. The mechanism by which S1P reduces IL-1-induced IP-10 and RANTES mRNA expression involves the prolonged hyperphosphorylation of TAK1, as well as the inhibition of IL-1-stimulated IFN beta production and the phosphorylation of STAT1 and STAT2. In summary, this dissertation describes the mechanisms by which S1P and IL-1 control the mRNA expression of two chemokines associated with multiple sclerosis, and the components of the plasminogen activator system, which are critical for the invasion of glioblastoma cells; thus, indicating future therapeutic targets for destructive CNS disorders.

At birth, the immune system is immature and the gut microbiota influences immune maturation. Staphylococcus aureus (S. aureus) and lactobacilli are part of the neonatal gut microbiota and have seemingly opposite effects on the immune system. S. aureus is a potent immune activator and early-life colonization associates with higher immune responsiveness later in life. Lactobacilli-colonization associates with reduced allergy-risk and lower immune responsiveness. Further, lactobacilli modulate immune-activation and have probiotic features. Here, we investigated S. aureus-induced activation of human lymphocytes, including T regulatory cells (Tregs), conventional T-cells (CD4+ and CD8+), unconventional T-cells (γδ T-cells and MAIT-cells) and NK-cells from children and adults, together with the modulatory effect of lactobacilli on immune-activation. Further, early-life colonization with these bacteria was related to lymphocyte-maturation, plasma cytokine- and chemokine-levels and allergy.  S. aureus cell free supernatant (CFS) and staphylococcal enterotoxin (SE) A induced an increased percentage of FOXP3+ Tregs and of CD161+, IL-10+, IFN-γ+ and IL-17A+ Tregs (Paper I). The same pattern was observed in children with a lower degree of activation, possibly due to lower CD161-expression and poor activation of naive T-cells (Paper II). S. aureus-CFS induced IFN-γ-expression, proliferation and cytotoxic capacity in conventional and unconventional T-cells, and NK-cells. SEA, but not SEH, induced activation of unconventional T-cells and NK-cells by unknown mechanism(s) (Paper III, extended data). Lactobacilli-CFS reduced S. aureus-induced lymphocyte activation without the involvement of IL-10, Tregs or monocytes, but possibly involving lactate (Paper III). Early-life colonization with S. aureus associated with increased percentages of CD161+ and IL-10+ Tregs while lactobacilli-colonization negatively correlated with the percentage of IL-10+ Tregs later in life (Paper II). Allergic disease in childhood associated with double allergic heredity, being born wintertime and with higher plasma levels of TH2-, TH17- and TFH-related chemokines early in life. Lactobacilli-colonization associated with lower prevalence of allergy, reduced chemokine-levels and increased levels of IFN-γ in plasma (Paper IV).    This thesis provides novel insights into S. aureus- and SE-mediated activation of Tregs, unconventional T-cells and NK-cells and suggests an overall impairment of immune-responsiveness towards this bacterium in children. Further, S. aureus-colonization may influence the maturation of peripheral Tregs. Our data show that lactobacilli potently dampen lymphocyte-activation in vitro and that colonization associates with Treg-responsiveness, altered plasma cytokine- and chemokine-levels and with remaining non-allergic, thereby supporting the idea of lactobacilli as important immune-modulators. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Le but de cette thèse était d’identifier de nouveaux substrats au complexe FANC Core,déficient dans l’Anémie de Fanconi, une pathologie génétique rare. Cette maladie estcaractérisée par un phénotype hétérogène associant une pancytopénie à des malformationscongénitales et une prédisposition accrues aux leucémies myéloïdes aigues.L’anémie de Fanconi est causée par la mutation biallélique dans un des seize gènesFANC. Les protéines produites par ces gènes participent à une même voie moléculaireimpliquée dans la signalisation des dommages de l’ADN. Huit de ces protéines forment lecomplexe FANC Core, une E3 ubiquitine ligase, dont les seuls substrats à ce jour sontFANCD2 et FANCI.Dans le but d’identifier de nouveaux substrats du complexe FANC Core, j’ai réalisé uneanalyse protéomique après immunoprécipitation des peptides modifiés par l’ubiquitine ou parles ubiquitin-like NEDD8 et ISG15. Cette expérience a été faite dans des cellules déficientespour la voie FANC, mutées sur les gène FANCA ou FANCC et comparée à des cellulescorrigées par l’expression de ces gènes.Cette analyse révèle que FANCD2 et FANCI sont les seules cibles du complexe FANCCore en réponse à des dommages de l’ADN.Néanmoins, je montre l’existence d’autres protéines qui sont modifiées d’une manièreFANCA dépendante. Ces protéines sont pour la grande majorité des protéines membranairesou associées aux membranes cytoplasmiques. Parmi celles-ci, j’ai pu déterminer que lerécepteur aux chimiokines, CXCR5, était modifié d’une manière FANCA dépendante parl’ubiquitin-like NEDD8. Cette modification impacte sur la localisation de la protéine à lamembrane et à des conséquences sur la migration des cellules.J’ai aussi montré que FANCA participe d’une manière similaire à la régulation de lalocalisation membranaire d’autres protéines comme APLP2.Ainsi, il est proposé par ce travail un rôle de la protéine FANCA en dehors du complexeFANC Core et en dehors de la réparation des dommages à l’ADN. Comment la protéineFANCA participe à la régulation du trafic des protéines membranaires reste un point nonrésolu à ce jour. / The aim of this thesis was to find new substrates of the E3-ubiquitin ligase activity of theFANC Core complex, mutated in the rare genetic disorder Fanconi Anemia. This disease ischaracterized by bone marrow failure, developmental abnormalities and predisposition tocancer. Eight of the 16 known FANC proteins participate in the FANCcore nuclear complex,which has E3 ubiquitin-ligase activity and monoubiquitinates FANCD2 and FANCI inresponse to replication stress.In this thesis, I used mass spectrometry to compare cellular extracts from FANC Corecomplex deficient FA-A and FA-C cells to their ectopically corrected counterparts after agenotoxic stress.FANCD2 and FANCI appear to be the only true direct targets of the FANCcore complex.However, I also identified other proteins that undergo post-translational modifications throughFANCA- or FANCC-specific direct or indirect mechanisms that are independent of theFANCcore complex. The majority of these potential FANCA or FANCC target proteinslocalize to the cell membrane.Finally, I demonstrated that (a) the chemokine receptor CXCR5 is a neddylated protein; (b)FANCA, surprisingly, appears to modulate CXCR5 neddylation through a currently unknownmechanism; (c) CXCR5 neddylation is involved in the targeting of this receptor to the cellmembrane; and (d) CXCR5 neddylation stimulates cell migration/motility.I also confirmed that the role of FANCA in neddylation is not restrict to CXCR5 but also to,at least, one other protein, APLP2.My work has uncovered a new signaling pathway that is potentially involved in the rarehuman syndrome Fanconi Anemia and in cell motility and has highlighted a potential newfunction for the FANCA protein independant of the FANC Core complex and of a genotoxicstress.

A Cardiomiopatia Chagásica Crônica (CCC) é uma cardiomiopatia de natureza inflamatória, que ocorre em cerca de 30% dos indivíduos infectados pelo protozoário Trypanosoma cruzi 5-30 anos após infecção. Na doença de Chagas crônica e na CCC, há importante produção de citocinas próinflamatórias do padrão Th1 e quimiocinas, mesmo na ausência de disfunção ventricular. Foi demonstrado que células mononucleares que infiltram o tecido cardíaco de pacientes CCC produzem algumas dessas citocinas inflamatórias. Entretanto, os fatores que determinam a composição do infiltrado inflamatório e contribuem para a migração e acúmulo das células inflamatórias dentro do tecido cardíaco na CCC são ainda desconhecidos. Sabendo-se que a CCC apresenta pior prognóstico que as cardiomiopatias dilatadas de natureza não inflamatória, é possível hipotetizar que diversos mediadores inflamatórios produzidos localmente estejam envolvidos no pior prognóstico. Dentro deste contexto, nosso objetivo no presente trabalho foi avaliar a expressão gênica de citocinas do padrão próinflamatório/Th1, quimiocinas envolvidas na migração de células T de memória e seus receptores e quimiocinas envolvidas na migração diferencial de linfócitos Th1/Th2 e seus receptores em amostras de miocárdio de pacientes com CCC e outras cardiomiopatias. Para isso, utilizamos a técnica de qRT-PCR e imunofluorescência com microscopia confocal para esses mediadores/receptores em amostras de miocárdio (ventrículo esquerdo) de pacientes CCC, portadores de cardiomiopatia não inflamatória (CNI) e doadores saudáveis, obtidos durante o procedimento de transplante. Observamos a expressão gênica aumentada da citocina pró-inflamatória IL-18, das quimiocinas CCL3/MIP-1, CCL4/MIP-1, CCL5/RANTES, CXCL9/Mig, CXCL10/IP-10, CCL17/TARC e CCL19/ELC e dos receptores CXCR3, CCR5 e CCR4 em amostras de miocárdio de pacientes com CCC quando comparadas com amostras de miocárdio de pacientes com CNI ou tecido cardíaco controle. Entretanto, observamos a expressão diminuída ou ausente de genes como TGF-, Foxp3, IL-4 e IL-13 , sugerindo a ausência de células T regulatórias ou células Th2 funcionais. Adicionalmente, a presença de células mononucleares CXCR3+, CCR5+ e CCR4+ foi observada em amostras de miocárdio de pacientes com CCC utilizando imunofluorescência confocal. As quimiocinas CCL5/RANTES e CXCL9/Mig foram detectadas em células mononucleares do infiltrado inflamatório de tecido cardíaco de pacientes com CCC. A expressão diferencial dos genes aqui estudados permitiu obter um quadro panorâmico dos mediadores inflamatórios produzidos no miocárdio de pacientes com CCC. A expressão gênica aumentada de IL-18 e de quimiocinas e seus receptores no miocárdio de pacientes com CCC contribuem para a migração e acúmulo de células de CCR5+, CXCR3+ de perfil Th1 e as correlações observadas entre esses mediadores e receptores sugerem um feedback positivo atuando na manutenção e amplificação do processo inflamatório, possivelmente em associação com outros mediadores expressos no miocárdio. A resposta inflamatória intensa e predominantemente Th1 com a expressão aumentada de diversos mediadores inflamatórios no miocárdio de pacientes com CCC pode ocorrer pela ausência de células T regulatórias Foxp3+ ou TGF-+, e a expressão de alguns mediadores como IL-18 e CCL21/SLC pode estar associada ao desenvolvimento de hipertrofia e fibrose, sugerindo um papel fisiopatológico adicional para a expressão desses mediadores no grupo de pacientes com CCC. / Chronic Chagas disease Cardiomyopathy (CCC) is an inflammatory cardiomyopathy that affects around 30% of individuals infected by the protozoan Trypanosoma cruzi and happens 5-30 years after the infection. In Chronic Chagas disease and CCC, there is a significant production of proinflammatory Th1 cytokines and chemokines even in the absence of ventricular dysfunction. Mononuclear cells inflitrating the heart tissue of CCC patients produce some of these inflammatory cytokines. However, the factors that determine the composition of the inflammatory infiltrate and contribute to the migration, accumulation and distribution of inflammatory cells inside heart tissue in the CCC are still unknown. Considering that CCC has worse prognosis than dilated cardiomyopathy of non-inflammatory etiology, we hypothesized that the production of several inflammatory mediators in situ could be involved in the worse prognosis of CCC. Taking this into consideration, our aim in the present study was to analyze the gene expression of pro-inflammatory/Th1 cytokines, chemokines involved in cell T memory migration and its receptors and chemokines involved in Th1/Th2 lymphocyte migration and its receptors. qRT-PCR and immunofluorescence with confocal microscopy were employed to detect the expression these mediators/receptors in left ventricular free wall samples from end-stage CCC patients, patients with non-inflammatory cardiomyopathy (NIC) and healthy donors, obtained upon transplantation. We observed a significant increase in the expression of pro-inflammatory cytokine IL-18, chemokines CCL3/MIP- 1, CCL4/MIP-1, CCL5/RANTES, CXCL9/Mig, CXCL10/IP-10, CCL17/TARC and CCL19/ELC and its receptors CXCR3, CCR5 and CCR4 in the samples of CCC patients compared to NIC patients and control heart samples. On the other hand, we observed absence of expression or downregulation or of TGF-, Foxp3, IL-4 and IL-13, suggesting the absence of regulatory T cells and functional Th2 cells. In addition, the presence of mononuclear CXCR3+, CCR5+ and CCR4+ cells was observed in myocardium of CCC patients using immunofluorescence with confocal microscopy. The chemokines CCL5/RANTES and CXCL9/Mig were detected in mononuclear cells of inflammatory infiltrates of heart tissue CCC patientes. The differential gene expression observed in this study allowed us to elaborate a global profile of inflammatory mediator production in the myocardium CCC patients. The up-regulated gene expression of IL-18 and chemokines and its receptors in the myocardium CCC patients contribute to the migration and accumulation of CCR5+, CXCR3+ Th1 cells and the correlation observed between these mediators and their receptors suggest a positive feedback contributing to the maintenance and amplification of inflammatory process, possibly in association with another mediators expressed in the myocardium. The intense Th1 inflammatory response with the up-regulated expression of various inflammatory mediators in the myocardium of CCC patients could be enhanced by the absence of Foxp3+ or TGF-+, regulatory T cells and the expression of mediators as IL-18 and CCL21/SLC could play a role in the development of hypertrophy and fibrosis suggesting an additional pathophysiologic role of expression of these mediators in CCC patients.