Le rationnel pour l’utilisation des agonistes des Toll-Like Récepteurs (TLR) dans le traitement du cancer repose sur leurs effets bénéfiques au niveau des cellules du système immunitaire permettant une stimulation de la réponse immunitaire innée et adaptative. Cependant, certains types de cellules tumorales expriment les TLRs qui, une fois activés, peuvent déclencher des effets “délétères” comme la tumorigénèse.Pour mieux disséquer les effets biologiques directs et indirects d’un agoniste de TLR3, l’acide polyadenylique-polyurydilique (poly(A:U)), nous avons utilisé deux modèles tumoraux murins exprimant TLR3 et ne répondant pas à la chimiothérapie, mais capables de produire des grandes quantités de CCL5 et CXCL10 en réponse au poly(A:U) et à l’IFN de type I. In vivo, la combinaison chimiothérapie – poly(A:U) n’a permis d’obtenir qu’une faible activité anti tumorale sauf lorsqu’une vaccination contre les antigènes tumoraux est inclue dans le protocole de traitement et le récepteur CCR5 est bloqué (souris Ccr5-/- ou souris sauvages traitées avec MetRANTES). L’efficacité anti tumorale du traitement combiné est associée à l’induction de cellules T CD8+CXCR3+IFNγ+ et est perdue chez les souris nu/nu, Trif-/- et Cxcr3-/-. La source du ligand de CCR5 est constituée par les cellules tumorales dont la voie TLR3 est activée. L’efficacité du traitement combiné sur ces cellules a été améliorée en inhibant la production de CCL5 à l’aide d’un shARN spécifique pour CCL5. Ces résultats soutiennent la notion que le poly(A:U) peut directement agir sur l’épithélium tumoral pour promouvoir la libération de CXCL10 qui a un effet bénéfique (recrutement des LTCs), mais aussi la sécrétion de CCL5 qui a un rôle délétère (action sur des immunosuppresseurs au niveau de l’hôte). Le découplage des effets opposés des chimiokines et la vaccination anti tumorale préalable peuvent permettre aux LTCs dépendant des ligands de CXCR3 d’infirmer l’action d’immunosuppression CCR5 dépendante et devraient être intégrés dans les essais cliniques à venir utilisant les agonistes de TLR3. / The rationale for the use of Toll –Like Receptor (TLR) agonists in cancer therapy relies upon their “beneficial” effects on immune cells leading to enhanced innate and adaptive immune responses. However, a variety of cancer epithelia express TLRs which, upon triggering, may mediate “deleterious” effects such as tumorigenesis. To further dissect the direct versus indirect biological effects of the TLR3 agonist polyadenylic-polyuridylic acid (poly(A:U)), we took advantage of two murine tumor models expressing TLR3 that failed to respond to chemotherapy but did produce large amounts of CCL5 and CXCL10 in response to the poly(A:U) and type I IFN. In vivo, the combination of chemotherapy and poly(A:U) mediated low tumoricidal activity unless a vaccination against tumor antigens was included in the regimen and the CCR5 receptor was blocked (CCR5 loss-of-function mice or WT animals treated with MetRANTES). The antitumor efficacy of the combination therapy was associated with the elicitation of CD8+CXCR3+IFN+ T cells and abrogated in nu/nu, Trif-/- and Cxcr3-/- mice. The source of CCR5L is the TLR3-activated tumor cells in that stable inhibition of the chemokine production by specific shRNA CCL5 ameliorated the efficacy of the combination therapy. These results support the notion that poly(A:U) can directly act on tumor epithelia to promote the release of beneficial CXCL10 for the recruitment of intratumoral CTLs but also the release of deleterious CCL5 acting on host immunosuppressors. Uncoupling chemokine release and prior vaccination may enable the CXCR3L-dependent CTLs to overrule the CCR5-dependent suppression and may be integrated in future trials using TLR3 agonists.

Erkinis encefalitas (EE) – pati dažniausia ir sunkiausia virusinė nervų sistemos infekcija Lietuvoje, kuria per metus vidutiniškai suserga 400 žmonių. Užsikrėtus EE virusu (EEV), galima besimptomė arba klinikinius požymius sukelianti ligos eiga, turinti platų požymių spektrą – nuo lengvos, meningitinės ligos formos iki sunkaus encefalito. Nors mirštamumas nuo EE yra nedidelis, svarbiausia problema – ilgai trunkantis sveikimo laikotarpis ir ilgalaikiai liekamieji reiškiniai, kurie būdingi 26–46 proc. persirgusiųjų. Neaišku, kodėl užsikrėtę identiško virulentiškumo virusu, vieni žmonės perserga besimptome arba lengva EE forma, o kitiems atsiranda sunkus nervų sistemos pažeidimas. Manoma, kad didelę reikšmę turi žmogaus genetiniai veiksniai. Nesant specifinio priešvirusinio EE gydymo, šios sunkios centrinės nervų sistemos infekcijos patogenezės ir genetinių rizikos veiksnių tyrimai yra ypač svarbūs, siekiant nustatyti galimas veiksmingo gydymo paieškos kryptis ateityje. Šio darbo tikslas – nustatyti CCR5 ir TLR3 genų polimorfizmų paplitimą tarp EE sirgusių asmenų ir šių genų polimorfizmų reikšmę polinkiui sirgti EE bei skirtingoms EE klinikinėms formoms išsivystyti. Šis darbas yra didžiausias iki šiol atliktas genetinių veiksnių reikšmės EEV infekcijos metu tyrimas ir pirmasis tokio pobūdžio vaikų tyrimas. Tyrimo rezultatai patvirtino hipotezę, kad nefunkcionuojantis CCR5 ir funkcionuojantis TLR3 yra reikšmingi simptominės EE formos išsivystymo veiksniai, užsikrėtus EEV. / Tick-borne encephalitis (TBE) is the most common and severe viral infection of the central nervous system in Lithuania, with the average number of 400 cases per year. The clinical spectrum of TBE virus infection varies considerably from asymptomatic to mild meningitis or severe encephalitis. Although the mortality of TBE is relatively low, as many as 26–46% of the patients experience long-lasting sequelae. No specific treatment for TBE exists. A most intriguing question is why certain individuals respond with seve¬re clinical symptoms after infection with TBEV while the majority either remains asymptomatic or develops only mild disease. Studies of host genetic susceptibility to infectious diseases aim to in¬crea¬se our understanding of why some individuals are more susceptable than others. Knowledge of genetic susceptibility may be used in develope¬ment of new therapeutic means and also to recognize individuals who are at increased risk of severe symptoms if infected with a pathogen. The aim of this study – to establish the prevalence of the polymorphisms in CCR5 and TLR3 genes in TBE patients and their role in susceptibility to clinical TBE and disease severity. This study is the largest study on the host genetic risk factors predis¬posing to TBEV infection, and the first study of such kind performed in the pediatric population. The results of this study confirmed that a non¬func¬tional CCR5 protein and a functional TLR3 receptor are associated with the clinical expression... [to full text]

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune inflamatória crônica que possui uma etiopatogênese complexa. Diversos fatores participam da patogênese da doença, dentre eles alterações no balanço de citocinas e quimiocinas. As quimiocinas e seus receptores são fundamentais na regulação da migração de leucócitos durante a inflamação e acredita-se que elas possam ter um papel importante na patogênese de doenças autoimunes, inclusive no LES. Diversos estudos abordaram o papel de quimiocinas e seus receptores no LES, porém, principalmente se tratando dos receptores de quimiocinas CCR5 e CCR2, não existe um consenso. Devido à falta de consenso em relação ao papel dos receptores de quimiocinas na patogênese do LES e considerando a necessidade de mais estudos nesta área, o presente trabalho tem por objetivo investigar o possível papel de polimorfismos na região promotora do CCR5 no desenvolvimento do LES, comparando as frequências dos genótipos e haplótipos entre pacientes e controles, e analisar o possível envolvimento destes polimorfismos nas manifestações clínicas/laboratoriais da doença. O estudo incluiu 382 pacientes com LES (289 Euro-descendentes e 93 Afro-descendentes) e 375 controles (243 Euro-descendentes e 132 Afro-descendentes) genotipados para os polimorfismos CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) e CCR5-59653 C>T (rs1800024) através de PCR-RFLP e sequenciamento, respectivamente. Dados prévios de nosso grupo em relação ao CCR5delta32 foram incluídos no estudo para a inferência dos haplótipos e como um possível fator de confusão na regressão binária logística. Os resultados obtidos indicam que, em pacientes Euro-descendentes, as frequências reduzidas o polimorfismo CCR5delta32 e o haplótipo HHG*2 observadas em pacientes quando comparados com controles foram associadas com a doença (p=0,001; OR 3,5; 95%CI 1,6-7,5 e 2,0% vs. 7,2%; presidual=2,9E-5; respectivamente). Em pacientes Afrodescendentes, as frequências dos haplótipos HHA/HHB, HHC e HHG*2 foram diferentes em pacientes e controles (10% vs. 20,5%, presidual = 0,003; 29,4% vs. 17,4%; presidual=0,003 e 3,9% vs. 0,8%; presidual=0,023; respectivamente). Em relação às manifestações clínicas da doença, a presença do CCR5delta32 foi confirmada como um fator de susceptibilidade para nefrite classe IV em pacientes Afro-descendentes e no grupo de pacientes como um todo (pcorrigido=0,012; OR 3,0; 95%CI 3,0-333,3 e pcorrigido=0,0006; OR 6,8; 95%CI 1,9-2,48; respectivamente). Em conclusão, o presente estudo indica que polimorfismos na região promotora do CCR5 podem atuar como modificadores no LES. Os resultados observados reforçam o papel do polimorfismo CCR5delta32 como um fator de proteção para o desenvolvimento do LES em Euro-descendentes e como um fator de susceptibilidade à nefrite classe IV em pacientes Afro-descendentes. Além disto, também foram descritos a redução da frequência dos haplótipos HHA/HHB e o aumento da frequência dos haplótipos HHC e HHG*2 em pacientes Afro-descendentes, que possivelmente podem estar associados com uma maior expressão do CCR5 em subtipos específicos celulares e com uma menor expressão deste receptor de maneira geral. / Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, characterized by a complex etiopathogenesis. Many factors are known to participate in the pathogenesis of SLE, including alterations in the cytokines or chemokines balance. Chemokines and their receptors are central players in the regulation of leucocytes chemotaxis in inflammation and they are thought to have an important role in the pathogenesis of autoimmune diseases, including SLE. Several studies have addressed the role of chemokines and their receptors in SLE, however there is no consensus regarding their involvement on the pathogenesis of the disease. Given the lack of consensus considering the role of chemokine receptors in SLE pathogenesis and the need for more studies in this area, the present work aims to investigate a possible role of the CCR5 promoter region polymorphisms in the development of SLE comparing the frequencies of the genotypes and haplotypes with ethnically matched controls and analyze if there is a possible involvement of the polymorphisms in the clinical outcome of the disease. This study included 388 SLE patients (289 classified as Europeanderived and 93 as African-derived) and 375 controls (243 European-derived and 132 African-derived) genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms though PCRRFLP and direct sequencing, respectively. Previous data from CCR5delta32 were included in the study to infer the haplotypes and also as a possible confounding factor in the binary logistic regression. Our results indicated that, in Europeanderived patients, CCR5delta32 and the HHG*2 haplotype reduced frequencies in patients when compared to controls were associated with the disease (p=0.001; OR 3.5; 95%CI 1.6-7.5 and 2.0%, vs. 7.2% residual p= 2.9E-5, respectively). In African-derived patients, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between patients and controls (10% vs. 20.5%, residual p= 0.003; 29.4% vs. 17.4%, residual p=0.003 and 3.9% vs. 0.8%, residual p=0.023; respectively). Considering the clinical manifestations of the disease, CCR5delta32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when patients were considered together (pcorrected=0.012; OR 3.0; 95%CI 3.0-333.3 and pcorrected= 0.0006; OR 6.8; 95%CI 1.9-2.48, respectively). In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces CCR5delta32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also describe a reduced frequency of HHA/HHB and an enhanced frequency of HHC and HHG*2 haplotypes in our African-derived patients, which potentially could reflect in a higher expression of CCR5 in specific cell subsets and in a lower expression of CCR5 overall.

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune inflamatória crônica que possui uma etiopatogênese complexa. Diversos fatores participam da patogênese da doença, dentre eles alterações no balanço de citocinas e quimiocinas. As quimiocinas e seus receptores são fundamentais na regulação da migração de leucócitos durante a inflamação e acredita-se que elas possam ter um papel importante na patogênese de doenças autoimunes, inclusive no LES. Diversos estudos abordaram o papel de quimiocinas e seus receptores no LES, porém, principalmente se tratando dos receptores de quimiocinas CCR5 e CCR2, não existe um consenso. Devido à falta de consenso em relação ao papel dos receptores de quimiocinas na patogênese do LES e considerando a necessidade de mais estudos nesta área, o presente trabalho tem por objetivo investigar o possível papel de polimorfismos na região promotora do CCR5 no desenvolvimento do LES, comparando as frequências dos genótipos e haplótipos entre pacientes e controles, e analisar o possível envolvimento destes polimorfismos nas manifestações clínicas/laboratoriais da doença. O estudo incluiu 382 pacientes com LES (289 Euro-descendentes e 93 Afro-descendentes) e 375 controles (243 Euro-descendentes e 132 Afro-descendentes) genotipados para os polimorfismos CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) e CCR5-59653 C>T (rs1800024) através de PCR-RFLP e sequenciamento, respectivamente. Dados prévios de nosso grupo em relação ao CCR5delta32 foram incluídos no estudo para a inferência dos haplótipos e como um possível fator de confusão na regressão binária logística. Os resultados obtidos indicam que, em pacientes Euro-descendentes, as frequências reduzidas o polimorfismo CCR5delta32 e o haplótipo HHG*2 observadas em pacientes quando comparados com controles foram associadas com a doença (p=0,001; OR 3,5; 95%CI 1,6-7,5 e 2,0% vs. 7,2%; presidual=2,9E-5; respectivamente). Em pacientes Afrodescendentes, as frequências dos haplótipos HHA/HHB, HHC e HHG*2 foram diferentes em pacientes e controles (10% vs. 20,5%, presidual = 0,003; 29,4% vs. 17,4%; presidual=0,003 e 3,9% vs. 0,8%; presidual=0,023; respectivamente). Em relação às manifestações clínicas da doença, a presença do CCR5delta32 foi confirmada como um fator de susceptibilidade para nefrite classe IV em pacientes Afro-descendentes e no grupo de pacientes como um todo (pcorrigido=0,012; OR 3,0; 95%CI 3,0-333,3 e pcorrigido=0,0006; OR 6,8; 95%CI 1,9-2,48; respectivamente). Em conclusão, o presente estudo indica que polimorfismos na região promotora do CCR5 podem atuar como modificadores no LES. Os resultados observados reforçam o papel do polimorfismo CCR5delta32 como um fator de proteção para o desenvolvimento do LES em Euro-descendentes e como um fator de susceptibilidade à nefrite classe IV em pacientes Afro-descendentes. Além disto, também foram descritos a redução da frequência dos haplótipos HHA/HHB e o aumento da frequência dos haplótipos HHC e HHG*2 em pacientes Afro-descendentes, que possivelmente podem estar associados com uma maior expressão do CCR5 em subtipos específicos celulares e com uma menor expressão deste receptor de maneira geral. / Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, characterized by a complex etiopathogenesis. Many factors are known to participate in the pathogenesis of SLE, including alterations in the cytokines or chemokines balance. Chemokines and their receptors are central players in the regulation of leucocytes chemotaxis in inflammation and they are thought to have an important role in the pathogenesis of autoimmune diseases, including SLE. Several studies have addressed the role of chemokines and their receptors in SLE, however there is no consensus regarding their involvement on the pathogenesis of the disease. Given the lack of consensus considering the role of chemokine receptors in SLE pathogenesis and the need for more studies in this area, the present work aims to investigate a possible role of the CCR5 promoter region polymorphisms in the development of SLE comparing the frequencies of the genotypes and haplotypes with ethnically matched controls and analyze if there is a possible involvement of the polymorphisms in the clinical outcome of the disease. This study included 388 SLE patients (289 classified as Europeanderived and 93 as African-derived) and 375 controls (243 European-derived and 132 African-derived) genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms though PCRRFLP and direct sequencing, respectively. Previous data from CCR5delta32 were included in the study to infer the haplotypes and also as a possible confounding factor in the binary logistic regression. Our results indicated that, in Europeanderived patients, CCR5delta32 and the HHG*2 haplotype reduced frequencies in patients when compared to controls were associated with the disease (p=0.001; OR 3.5; 95%CI 1.6-7.5 and 2.0%, vs. 7.2% residual p= 2.9E-5, respectively). In African-derived patients, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between patients and controls (10% vs. 20.5%, residual p= 0.003; 29.4% vs. 17.4%, residual p=0.003 and 3.9% vs. 0.8%, residual p=0.023; respectively). Considering the clinical manifestations of the disease, CCR5delta32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when patients were considered together (pcorrected=0.012; OR 3.0; 95%CI 3.0-333.3 and pcorrected= 0.0006; OR 6.8; 95%CI 1.9-2.48, respectively). In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces CCR5delta32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also describe a reduced frequency of HHA/HHB and an enhanced frequency of HHC and HHG*2 haplotypes in our African-derived patients, which potentially could reflect in a higher expression of CCR5 in specific cell subsets and in a lower expression of CCR5 overall.

La plus grande famille de récepteurs membranaires est constituée par des récepteurs à sept domaines transmembranaires couplés aux protéines G (RCPG). Ces récepteurs sont impliqués dans un grand nombre de réponses cellulaires physiologiques et pathologiques et représentent la ciblé de une grande majorité des produits thérapeutiques. La fonction d’un récepteur est déterminée par la quantité de récepteur fonctionnel à la surface cellulaire, qui dépend de différents paramètres comme le niveau de biosynthèse, l’export vers la surface cellulaire à partir de stocks intracellulaires, l’endocytose et les modifications post-transcriptionelles (ex. phosphorylation). Le nouveau concept d’export régulé pour les RCPG présent l’importance physiologique de la rétention de récepteurs, leur relargage, leur interaction avec les partenaires chaperonnes et les escortes. Les études présentées ici concernent les mécanismes d’export régulé de deux RCPG, le récepteur métabotropique de l’acide γ-amino butyrique (GABAB) et le récepteur de chimiokines CC 5 (CCR5). GABAB est un récepteur constitué de deux sous-unités GB1 et GB2 et CCR5 est probablement un homo-dimer. GB1 ainsi que CCR sont retenus dans des compartiments intracellulaire (RE et appareil Golgi) d’où ils sont relâchés en réponse à un signal extern (CCR5) ou/et en interagissant avec protéines d’escorte (comme CD4 pour CCR5 et GB2 pour GB1). L’objectif de ces études était de comprendre le mécanisme de rétention de ces récepteurs et leur régulation. Dans ce contexte, nous avons déterminé en utilisant des approches biophysiques et biochimiques que ces récepteurs interagissent de façon spécifique avec les membres de Prenylated Rab Acceptors Family (PRAF). Ces protéines sont résidentes dans le RE (PRAF2 et PRAF3) et dans le appareil Golgi (PRAF1) où elles fonctionnent comme de gatekeepers pour les récepteurs. Nous avons pu démontrer que PRAF2 interagie de manière spécifique avec des motifs de rétention connus pour leur implication dans la rétention de récepteurs. Cette interaction détermine une rétention au niveau de RE donc régule de façon négatif l’export vers la membrane cellulaire. Dans le cas de récepteur GABAB, l’interaction de GB2 avec GB1 permet la libération de GB1 de sa rétention par PRAF2 par simple compétition. La modification de l’équilibre stoichiométrique entre les gatekeepers PRAF et les protéines d’escorte pour les récepteurs induit des modifications de la fonction du récepteur in vitro et in vivo. Les PRAFs sont ubiquitaires et peuvent interagir avec plusieurs RCPG représentant dans ce cas des régulateurs majors de la fonction de RCPG dans des conditions physiologiques et pathologiques. / The largest family of membrane receptors is constituted by conserved seven-membrane domain spanning receptors, the G-protein coupled receptors (GPCRs). They are involved in numerous cell responses and diseases thus being a major drug target. Receptor function is determined by the amount of active receptors at the cell surface, which depends on various parameters, such as the biosynthetic rate, the export to the cell surface from internal stores, the endocytosis and post-transcriptional modifications (i.e. phosphorylation). Only recently, the importance of the regulated export has emerged, shedding new light on the physiological role of receptor retention, release, chaperoning and escorting. This work concerns the regulated export mechanisms of two members of the GPCRs family, the chemokine receptor 5 (CCR5) and the metabotropic receptor of the g amino butyric acid (GABAB). Whereas CCR5 is likely a homo-dimer of 2 identical protomers, GABAB is an obligatory hetero-dimer of 2 distinct subunit known as GB1 and GB2. Both CCR5 and GB1 are retained in intracellular compartments (the ER and the Golgi) from which they are released in response to external signals (CCR5) and/or interaction with “private escort proteins” (CD4 for CCR5 and GB2 for GB1). The main goal of our work was to understand the mechanism of retention of these receptors and its regulation. In this context, we determined using biochemical and biophysical approaches that these GPCRs specifically interact with the members of the Prenylated Rab Acceptor Family (PRAF). These proteins are resident either in the ER (PRAF2 and PRAF3) or in the Golgi apparatus (PRAF1) where they function as receptor gatekeepers. Indeed, we could document for PRAF2 that this protein likely interacts directly with previously identified receptor retention motifs and inhibits receptor egress from the ER and subsequent trafficking to the plasma membrane. In the context of the GABAB receptor, PRAF2-dependent retention of GB1 can be overridden by GB2 via simple competition. Perturbing the stoichiometry of PRAF gatekeepers respective to that of receptors significantly perturbs receptor function both in vitro and in vivo. Because PRAFs are ubiquitous and seem to interact with many other GPCRs, they might represent major regulators of receptor function both in physiological and pathological conditions.

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune inflamatória crônica que possui uma etiopatogênese complexa. Diversos fatores participam da patogênese da doença, dentre eles alterações no balanço de citocinas e quimiocinas. As quimiocinas e seus receptores são fundamentais na regulação da migração de leucócitos durante a inflamação e acredita-se que elas possam ter um papel importante na patogênese de doenças autoimunes, inclusive no LES. Diversos estudos abordaram o papel de quimiocinas e seus receptores no LES, porém, principalmente se tratando dos receptores de quimiocinas CCR5 e CCR2, não existe um consenso. Devido à falta de consenso em relação ao papel dos receptores de quimiocinas na patogênese do LES e considerando a necessidade de mais estudos nesta área, o presente trabalho tem por objetivo investigar o possível papel de polimorfismos na região promotora do CCR5 no desenvolvimento do LES, comparando as frequências dos genótipos e haplótipos entre pacientes e controles, e analisar o possível envolvimento destes polimorfismos nas manifestações clínicas/laboratoriais da doença. O estudo incluiu 382 pacientes com LES (289 Euro-descendentes e 93 Afro-descendentes) e 375 controles (243 Euro-descendentes e 132 Afro-descendentes) genotipados para os polimorfismos CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) e CCR5-59653 C>T (rs1800024) através de PCR-RFLP e sequenciamento, respectivamente. Dados prévios de nosso grupo em relação ao CCR5delta32 foram incluídos no estudo para a inferência dos haplótipos e como um possível fator de confusão na regressão binária logística. Os resultados obtidos indicam que, em pacientes Euro-descendentes, as frequências reduzidas o polimorfismo CCR5delta32 e o haplótipo HHG*2 observadas em pacientes quando comparados com controles foram associadas com a doença (p=0,001; OR 3,5; 95%CI 1,6-7,5 e 2,0% vs. 7,2%; presidual=2,9E-5; respectivamente). Em pacientes Afrodescendentes, as frequências dos haplótipos HHA/HHB, HHC e HHG*2 foram diferentes em pacientes e controles (10% vs. 20,5%, presidual = 0,003; 29,4% vs. 17,4%; presidual=0,003 e 3,9% vs. 0,8%; presidual=0,023; respectivamente). Em relação às manifestações clínicas da doença, a presença do CCR5delta32 foi confirmada como um fator de susceptibilidade para nefrite classe IV em pacientes Afro-descendentes e no grupo de pacientes como um todo (pcorrigido=0,012; OR 3,0; 95%CI 3,0-333,3 e pcorrigido=0,0006; OR 6,8; 95%CI 1,9-2,48; respectivamente). Em conclusão, o presente estudo indica que polimorfismos na região promotora do CCR5 podem atuar como modificadores no LES. Os resultados observados reforçam o papel do polimorfismo CCR5delta32 como um fator de proteção para o desenvolvimento do LES em Euro-descendentes e como um fator de susceptibilidade à nefrite classe IV em pacientes Afro-descendentes. Além disto, também foram descritos a redução da frequência dos haplótipos HHA/HHB e o aumento da frequência dos haplótipos HHC e HHG*2 em pacientes Afro-descendentes, que possivelmente podem estar associados com uma maior expressão do CCR5 em subtipos específicos celulares e com uma menor expressão deste receptor de maneira geral. / Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, characterized by a complex etiopathogenesis. Many factors are known to participate in the pathogenesis of SLE, including alterations in the cytokines or chemokines balance. Chemokines and their receptors are central players in the regulation of leucocytes chemotaxis in inflammation and they are thought to have an important role in the pathogenesis of autoimmune diseases, including SLE. Several studies have addressed the role of chemokines and their receptors in SLE, however there is no consensus regarding their involvement on the pathogenesis of the disease. Given the lack of consensus considering the role of chemokine receptors in SLE pathogenesis and the need for more studies in this area, the present work aims to investigate a possible role of the CCR5 promoter region polymorphisms in the development of SLE comparing the frequencies of the genotypes and haplotypes with ethnically matched controls and analyze if there is a possible involvement of the polymorphisms in the clinical outcome of the disease. This study included 388 SLE patients (289 classified as Europeanderived and 93 as African-derived) and 375 controls (243 European-derived and 132 African-derived) genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms though PCRRFLP and direct sequencing, respectively. Previous data from CCR5delta32 were included in the study to infer the haplotypes and also as a possible confounding factor in the binary logistic regression. Our results indicated that, in Europeanderived patients, CCR5delta32 and the HHG*2 haplotype reduced frequencies in patients when compared to controls were associated with the disease (p=0.001; OR 3.5; 95%CI 1.6-7.5 and 2.0%, vs. 7.2% residual p= 2.9E-5, respectively). In African-derived patients, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between patients and controls (10% vs. 20.5%, residual p= 0.003; 29.4% vs. 17.4%, residual p=0.003 and 3.9% vs. 0.8%, residual p=0.023; respectively). Considering the clinical manifestations of the disease, CCR5delta32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when patients were considered together (pcorrected=0.012; OR 3.0; 95%CI 3.0-333.3 and pcorrected= 0.0006; OR 6.8; 95%CI 1.9-2.48, respectively). In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces CCR5delta32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also describe a reduced frequency of HHA/HHB and an enhanced frequency of HHC and HHG*2 haplotypes in our African-derived patients, which potentially could reflect in a higher expression of CCR5 in specific cell subsets and in a lower expression of CCR5 overall.

HIV-1 primarily utilizes the CCR5 receptor as a co-receptor, but over time, viruses can evolve to use the CXCR4 protein. Changes in the viral envelope V3 loop mediate this switch. The emergence of CXCR4-utilizing viruses has been presumed to occur as a consequence of decreased humoral immunity. We show that exclusively CXCR4-using (X4) viruses contain a 2 to 3 amino acid insertion in the V3 loop. Structural modeling revealed that this insertion caused a protrusion in the V3 loop, which impacts CCR5 receptor interaction. These genotypic and structural motifs affected neutralization susceptibility because X4, as compared to co-circulating CCR5-utilizing (R5) viruses, were less neutralization sensitive to autologous contemporaneous and heterologous plasma. Individuals with co-circulating X4 and R5, as compared to those with only R5, viruses had similar neutralization breadth and potency indicating that the emergence of X4 viruses is not associated with decreased humoral immunity. These results suggest that X4 viruses are neutralization escape variants and arise due to humoral selective pressure. This work has implications for future antibody-based therapeutics. Along with providing a framework for developing an HIV-1 vaccine, broadly neutralizing antibodies (bnAbs) are also being investigated as a potential therapeutic. BnAbs target a limited number of conserved HIV-1 envelope structures, including glycans in and around the V1/V2 and V3 domains. Along with the V3 loop, changes in V1/V2 are also known to impact co-receptor usage. We show that viruses that exclusively use the CXCR4 co-receptor, as compared to variants that only utilize CCR5, were less neutralization sensitive to V1/V2 and V3 directed bnAbs. In contrast, R5 and X4 viruses did not demonstrate neutralization differences to bnAbs that target non-V1/V2 and V3 envelope regions, such as the CD4 binding site and the membrane proximal external region. Structural modeling revealed that the predicted orientation of the V1/V2 loop among diverse HIV-1 variants predicts susceptibility to V3 loop directed bnAbs. In aggregate, our results suggest that viruses with different co-receptor usage have differing bnAb susceptibility. Furthermore, structural modeling may be used as a tool to predict neutralization susceptibility to bnAbs against regions associated with co-receptor usage. / 2020-06-12T00:00:00Z

The chemokine receptor CCR5 has been implicated in the pathogenesis of prostate cancer. A novel natural product, anibamine, was isolated and found to be a micromolar inhibitor of the receptor. Anibamine was used as a new anti-prostate cancer lead compound. To discover the pharmacophore, analogs of anibamine were designed using the “deconstruction-reconstruction-elaboration” approach and synthesized. The establishment of a stereoselective route to only one isomer was explored, to increase yield and eliminate elaborate purification procedures. Analogs were found to have anti-prostate cancer activity at levels higher than the parent compound. The molecular modeling studies of the deconstructed analogs indicate that due to the psuedo-symmetry of the parent compound, the binding conformation of the deconstructed analogs may not be very different from each other. All this information together may help identify a next generation lead compound for anti-prostate cancer treatment.

Human immunodeficiency virus type 1 (HIV-1) persists in certain CNS cell populations, despite peripheral control of the infection with modern antiretroviral therapy. Infected and/or activated cells release viral proteins, such as trans-activator of transcription (Tat) and various pro-inflammatory factors such as CCL5, creating a positive loop of neuro-inflammation. This serves as the basis for the resulting sublethal and lethal neuropathology that manifests as a spectrum of HIV-mediated CNS impairments, known as HIV-associated neurocognitive disorders (HAND). Opiates, which exist as an interlinked epidemic with HIV-1 infections, exacerbate these neurological effects through direct and indirect mechanisms that disrupt both glial and neuronal function. We hypothesize this is due to converging actions on the CCL5-CCR5 signaling axis by HIV-1 Tat and morphine co-exposure, primarily mediated at the level of the glia, whose consequent activation leads to neuronal damage. We performed repeated measure studies on mixed glia and neuron co-cultures obtained from C57Bl/6J and/or CCR5 knockout mice, treated with Tat and/or morphine for 72 hours. As established in prior studies, morphine worsened Tat-induced neurotoxicity in wild-type co-cultures; substitution of CCR5-null glia eliminated the interactive effects of Tat and morphine, but substitution of CCR5-null neurons did not. Overall, these results suggest that glial CCR5, but not neuronal CCR5, is a convergence point for the interactive effects of Tat and morphine that result in neuron loss. Additional experiments involving treatments with naloxone, a MOR antagonist, or the CCR5 antagonist maraviroc, confirmed each receptor’s role in mediating Tat + morphine toxicity. Quite surprisingly, in co-cultures of wild-type neurons and CCR5-null glia, morphine entirely protected neurons from the neurotoxic effects of Tat. We hypothesize that this effect may reflect an imbalance of neurotrophic factors, particularly BDNF and its neurotoxic precursor proBDNF, whose levels are altered in HIV+ and illicit drug-using patients and may contribute to changes in neuronal signaling and survival exhibited in HAND. Related behavioral tests of anxiety, motor and cognitive function – three areas of neurologic decline seen in HAND – were performed in inducible Tat-transgenic mice that were treated with maraviroc via oral gavage. Tat-mediated impairment was observed in the Barnes Maze, a measure of spatial memory, and was ameliorated by maraviroc. Finally, we assessed the role of CCR5 in mediating Tat and/or morphine effects on psychomotor sensitization and dendritic morphology. With both in vitro and in vivo studies, our findings support the hypothesis that CCR5 plays a central role in driving HIV-1 Tat and/or morphine-mediated neuronal damage.

Little is known about the outward trafficking of receptor dimers from the endoplasmic reticulum to the plasma membrane, or the role that trafficking plays in assembly, targeting and specificity of receptor signalling. Bimolecular fluorescence complementation was used to follow prescribed receptor homo/heterodimers in Jurkat cells and clarify the trafficking itineraries those receptors follow to reach the plasma membrane. Chemokine receptors CXCR4 and CCR5 were chosen due to their implication in numerous pathologies including, HIV and cancer, and their ability to form homo and hetero-oligomers. This study demonstrates that although the individual receptors composing heterodimeric complexes are the same as in homodimeric complexes, the heterodimer traffics and signals independently of its constituent homodimers. The presence of CD4 affects the trafficking of CCR5 containing dimers but not the CXCR4 homodimer. These observations demonstrate the importance of considering receptor heterodimers as distinct signalling entities that should be more carefully and individually characterized.