Adenosine 5’ triphosphate (ATP) is a molecule central to life for its role as the cellular energy currency, and a purine nucleotide which serves as a building block of RNA. Thus, on the backdrop of an indispensible intracellular role of ATP, its identification as an extracellular signaling molecule in early 1970s came as a surprise. A novel doctrine, termed as ‘purinergic signaling’, was thus put forth. By definition, purinergic signaling consists of the signaling events triggered by binding of extracellular ATP- a purine nucleotide, and its breakdown products (viz., ADP, AMP, and adenosine) to their cognate receptors, which in turn are termed as ‘purinergic receptors’. Based on their ligand affinity, purinergic receptors are classified into two groups- P1 and P2 receptors. P2 receptors are further subclassified as P2X and P2Y receptors. Till date, four P1 receptors (viz. A1, A2a, A2b, and A3), seven P2X receptors (P2X1-7), and eight P2Y receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and characterized. Conceptually, the first step of purinergic signaling is the release of ATP from an intact cell on encountering a stimulant or a modulator. The main mechanisms of such cellular ATP release include vesicular exocytosis and the release through conductive channels. ATP thus released, binds to its cognate receptors (i.e. P2X receptors, and certain P2Y receptors) and triggers the ‘purinergic signaling’ pathway that modulates the cellular response. In addition to purinergic receptors, cells also express ATP degrading enzymes on their surface, which break ATP down into ADP, AMP, and adenosine. ADP and adenosine, in turn, bind to their cognate receptors (certain P2Y receptors, and P1 receptors respectively) and further contribute to shaping the cellular response to a given cue. Thus, purinergic signaling is a highly dynamic process with pleiotropic downstream effects. First demonstrated in the context of neurotransmission, the phenomenon of purinergic signaling is now widely recognized and has been shown to play a role in regulating functional responses of cells of diverse origins, immune cells being one of them. Purinergic signaling in lymphocytes- an important subset of immune cells- is a common thread for the present research exercise, wherein we have addressed two sets of questions, one of academic curiosity and the other of clinical interest. In the former and the major part, we have examined whether purinergic signaling plays a role in functional aspects of ‘gamma delta (γδ) T cells’, which represent a unique subset of lymphocytes. Whereas, the latter part elaborates on the already identified involvement of purinergic signaling in T cell stimulatory action of ‘hypertonic saline (HS)’, which is used to treat trauma patients. The thesis, thus, is divided into five parts- the ‘Introduction’, ‘Aims and Scope of the study’, ‘Chapter 1’, ‘Chapter 2’, and ‘Summary of the work’. Understanding the questions posed in the present context, strategy designed to answer them, and eventually the experimental results answering these questions invoke basic knowledge of purinergic signaling, which has been attempted to be conferred through the ‘Introduction’ section. The discovery of purinergic signaling, its central theme, and individual molecular players involved in this signaling pathway are highlighted here. From the viewpoint of the present research endeavor, salient findings from the current literatureabout the involvement of purinergic signaling in the functional activities of various subsets of immune cells- are reviewed towards the end of this section. The ‘Introduction’ is followed by definition of the objectives for the present exercise, which are enlisted under ‘Aims and scope of the study’. Here, a brief overview of the background data that led us towards these objectives precedes the actual list of questions which we have approached. Purinergic signaling has been shown to play a role in the activation of ‘conventional αβ T’ cells. So we asked whether a similar purinergic signaling pathway also operates in unconventional γδ T cells. Thus, ‘Chapter 1’ is dedicated to answering the first set of questions about the role of purinergic signaling in γδ T cell activation. The chapter starts off by introducing γδ T cells. The topics such as discovery of γδ T cells, ontology, development, diversity, and distribution of these cells, and most importantly- their antigenic specificity and response are reviewed herein. The details of the experimental procedures employed to answer the defined objectives follow this introduction. We have carried out our experiments on γδ T cells in human circulation. For in vitro stimulation, we have used anti-CD3 + anti-CD28-coated beads (beads) or isopentenyl pyrophosphate (IPP), a γδ T cell specific stimulant. We observed that, circulating human γδ T cells rapidly release ATP on stimulation with beads or IPP. Pannexin-1 and connexin hemichannels, as well as vesicular exocytosis contribute to the ATP release. Real time RT-PCR data revealed that γδ T cells predominantly express purinergic receptors A2a, P2X1, P2X4, P2X7, and P2Y11. Of these, the inhibition of P2X4 receptors downregulated cytokine expression by γδ T cells post- in vitro stimulation, and also inhibited cytotoxic activity of γδ T cells towards Daudi cells. Selective translocation of P2X4 receptors to the immunological synapse was seen to be the underlying mechanism for these effects. Collectively, these data suggested that autocrine/paracrine purinergic signaling through P2X4 receptors indeed plays an important role in the functional aspects of circulating human γδ T cells. The experimental results are compiled in ‘Chapter 1’; which concludes with the ‘Discussion’ on the mentioned findings, and possible in vivo applications. ‘Chapter 2’ deals with the role of purinergic signaling in HS resuscitation. In addition to restoring the hemodynamic parameters, fluid replacement with small volumes of concentrated NaCl solution (HS) has been reported to reverse the suppression of T cells commonly found in the trauma subjects. Through an in vitro study using Jurkat cells as a model for primary human T cells, it has been shown earlier that, on HS exposure T cells release ATP- which binds to P2X7 receptors and promotes calcium influx. HS treatment also elicits phosphorylation of p38; and put together, Ca2+ influx and phosphorylated p38 synergize with TCR-induced stimulation resulting in the enhancement of transcriptional upregulation of IL-2. However, the mechanism of release of ATP on HS treatment and the possible involvement of P2X1 and P2X4 receptors expressed by T cells had not been addressed in this study. These very questions thus formed the objectives of the second part of present work. Experiments aimed to answer these questions showed that on HS treatment, Jurkat cells release ATP through pannexin-1 hemichannels. The released ATP binds to purinergic receptors P2X1, P2X4, and P2X7. This in turn triggers the downstream signaling cascade leading to phosphorylation of p38 and upregulation of IL-2 transcription, hence augmenting the T cell function. An overview of HS resuscitation, experimental protocols and results, and the discussion on the pathophysiological relevance of these findings comprise ‘Chapter 2’. Hence, we have found the answers to the questions we began with. The results are listed in a point-wise manner under the ‘Summary of the work’. Taken together, our data shows that: (i) Purinergic signaling does play a role in the functional aspects of circulating human γδ T cells. The release of ATP by γδ T cells post-stimulation, and autocrine/paracrine signaling through P2X4 receptors are the main components in this context. (ii) ATP release through pannexin-1 hemichannels, and autocrine/paracrine signaling through P2X1, P2X4, and P2X7 receptors underlie the mechanism of action of HS.

Ecto-nucleotidase triphosphate diphosphohydrolase-2 (NTPDase2) is an ecto-enzyme that is expressed on portal fibroblasts in the liver that modulates P2 receptor signaling by regulating local concentrations of extracellular ATP and ADP. NTPDase2 has protective properties in liver fibrosis and may impact bile duct epithelial turnover. Here, we study the role of NTPDase2 in acute liver injury using an experimental model of acetaminophen (APAP) intoxication in mice with global deletion of NTPDase2. Acute liver toxicity was caused by administration of acetaminophen in wild type (WT) and NTPDase2-deficient (Entpd2 null) mice. The extent of liver injury was compared by histology and serum alanine transaminase (ALT). Markers of inflammation, regeneration and fibrosis were determined by qPCR). We found that Entpd2 expression is significantly upregulated after acetaminophen-induced hepatotoxicity. Entpd2 null mice showed significantly more necrosis and higher serum ALT compared to WT. Hepatic expression of IL-6 and PDGF-B are higher in Entpd2 null mice. Our data suggest inducible and protective roles of portal fibroblast-expressed NTPDase2 in acute necrotizing liver injury. Further studies should investigate the relevance of these purinergic pathways in hepatic periportal and sinusoidal biology as such advances in understanding might provide possible therapeutic targets.

No description available.

In contrast to mammals, adult zebrafish undergo successful neural regeneration following spinal cord injury (SCI). Radial glia (RG) lining the zebrafish central canal undergo injury-induced proliferation and subsequent neuronal differentiation to replace damaged cells and restore motor function. However, the molecular mechanisms that underlie these processes remain elusive. Here, we demonstrate that signaling through the evolutionarily conserved purinergic P2X7 receptor is involved. Within the zebrafish spinal cord, P2X7 receptors have widespread distribution with specific localization to neurons and radial glia. At the protein level, the predominant P2X7 receptor isoforms in zebrafish did not include the full-length variant expressed throughout the murine central nervous system, but two truncated splice variants. In response to SCI, protein expression of the 50 kDa isoform became downregulated at 7 dpi and returned to basal levels of expression at 14 and 21 dpi when compared to naïve controls. Meanwhile, expression of the 37 kDa isoform did not change following injury. Pharmacological activation of P2X7 following SCI resulted in a greater number of proliferating cells around the central canal by 7 dpi, while P2X7 inhibition appeared to have no effect. At 14 dpi, these treatments did not have a significant effect on the number of neurons within the injured spinal cord. This data indicates that P2X7 receptor activation is sufficient to induce cellular proliferation, but not a necessary mediator of either proliferation or neurogenesis following SCI in adult zebrafish. Our findings suggest that unlike in humans, P2X7 signaling may not play a maladaptive role following SCI in adult zebrafish. / Thesis / Master of Science (MSc) / Spinal cord injury in mammals causes widespread neuronal cell death and paralysis. In comparison, zebrafish regenerate damaged neurons and restore motor function. Radial glial cells within the zebrafish spinal cord maintain stem-cell properties. Following injury, these cells divide and replace motor neurons. Since mammals have similar cell-types within the spinal cord, understanding the molecular cues driving this adaptive response is of great interest. Here, we examined the evolutionarily conserved purinergic signaling system and found that the expression of the P2X7 receptor varies significantly from mammals and promotes radial glia division following injury.

No description available.

Trichomonas vaginalis é o agente etiológico da doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos de tricomonose a cada ano. O estabelecimento da infecção se deve principalmente à capacidade de adesão do parasito às células epiteliais vaginais, cervicais ou de próstata, seguida pela intensa reação inflamatória, resultado da infiltração de neutrófilos no sítio da infecção. Nucleotídeos e nucleosídeos, especialmente ATP e adenosina, são liberados para o espaço extracelular por células em situações de estresse ou injúria tecidual e desenvolvem seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, as ectonucleotidases, NTPDase e ecto-5’-nucleotidase, são capazes de hidrolisar os nucleotídeos gerando adenosina e finalmente, a enzima adenosina deaminase (ADA) é responsável pela conversão de adenosina em inosina. A expressão gênica de cinco NTPDases putativas presentes no genoma de T. vaginalis foram investigadas, assim como o envolvimento da sinalização purinérgica na relação parasito-hospedeiro. Nossos resultados mostraram que diferentes isolados de T. vaginalis expressam os genes TvNTPDase1, 2, 3, 4 e 5, sendo observado o maior número de transcritos para TvNTPDase1, 2 e 4. A sequência preditiva de aminoácidos revelou a presença das cinco regiões conservadas da apirase, domínios transmembrana, sítios de fosforilação, peptídeos sinais e os prováveis sítios ativos da enzima. A análise filogenética demonstrou maior similaridade das TvNTPDases com as formas intracelulares da enzima, como as NTPDases 4 e 7 humanas e a de Saccharomyces cerevisiae. Além disso, a restrição de soro promoveu aumento significativo da atividade da NTPDase de T. vaginalis, no entanto sem corresponder com o aumento de expressão gênica de determinada(s) sequência(s). Quanto à participação da sinalização purinérgica na resposta inflamatória de células do hospedeiro frente ao parasito, foram utilizados como modelos celulares as células epiteliais vaginais (HMVII), cervicais (HeLa) e neutrófilos humanos. As linhagens HMVII e HeLa mostraram expressar todos os subtipos de receptores P1, P2X e P2Y e XI os diferentes isolados de T. vaginalis, que foram cocultivados com as células, mostraram hidrolisar eficientemente os nucleotídeos ATP, ADP e AMP. Ainda, o isolado clínico fresco TV-LACM6 foi o único a apresentar elevada citotoxicidade frente às células epiteliais vaginais e cervicais, no entanto não foi detectado aumento da liberação de ATP pelas células após o cocultivo, provavelmente devido à alta atividade da enzima NTPDase observada nesse isolado. Os trofozoítos de T. vaginalis não foram capazes de aumentar a produção de IL-8 e IL-6 pelas linhagens HMVII e HeLa, e apenas os isolados ATCC30236 e TV-LACM6 causaram aumento na secreção da citocina MIP-3α pelas células epiteliais cervicais. Finalmente, o nucleotídeo ATP e o nucleosídeo adenosina não modularam a produção dos mediadores inflamatórios investigados. Em relação aos neutrófilos, estes mostraram aumentar a produção de espécies reativas de oxigênio (ERO) e IL-8 após incubação com os trofozoítos de T. vaginalis. Os nucleotídeos e nucleosídeos da adenina e guanina não produziram efeito na produção de ERO e IL-8; no entanto, quando o nucleosídeo adenosina foi incubado junto com o inibidor da enzima ADA (EHNA) observou-se uma redução significativa da produção de ERO e IL-8 pelos neutrófilos, devido à inibição da ADA e consequentemente, ao aumento da concentração de adenosina disponível no meio extracelular. Os nossos resultados indicaram a ativação do receptor A1 dos neutrófilos nessa condição. O conjunto de dados aqui obtidos contribuiu para uma melhor caracterização da família de enzimas NTPDases de T. vaginalis assim como para um maior conhecimento acerca da influência da sinalização purinérgica na relação parasito-hospedeiro. / Trichomonas vaginalis is the agent of the most common non-viral sexually transmitted disease worldwide, causing 276.4 million new cases a year. The establishment of the infection is closely related to the parasite ability to adhere to vaginal, cervical and prostate epithelial cells, followed by an intense inflammatory response as result of neutrophil infiltration. Nucleotides and nucleosides, mainly ATP and adenosine, are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, NTPDase and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing adenosine and finally, the adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine. We investigated the gene expression of five putative NTPDases found in T. vaginalis genome as well as the involvement of purinergic signaling on the host-parasite relationship. Our results showed that different T. vaginalis isolates are able to express TvNTPDase1, 2, 3, 4 and 5 and that TvNTPDase1, 2 and 4 are the most expressed genes. Predictive amino acid sequence revealed the presence of the five apyrase conserved regions, transmembrane domains, phosphorylation sites, signal peptides and the active sites. Phylogenetic analysis showed that TvNTPDases share more similarity with the intracellular enzymes, such as human NTPDase 4 and 7 and Saccharomyces cerevisiae NTPDase. In addition, the serum limitation caused a significant increase in NTPDase activity, but without association with the gene expression of a specific TvNTPDase sequence. Regarding the participation of purinergic signaling on the inflammatory responses against the parasite, the vaginal (HMVII) and cervical (HeLa) epithelial cells and the human neutrophils were used as cellular models. HMVII and HeLa cell lines showed to express all subtypes of P1, P2X and P2Y receptors and the different T. vaginalis isolates, which were co-cultured with the cells, showed to hydrolyze efficiently ATP, ADP and AMP. Furthermore, only the fresh clinical isolate, TV-LACM6, caused a profound cytotoxicity against the vaginal and cervical epithelial cells. Interestingly, it was not detected an increase in ATP release by the cells after cocultivation, probably due to the high NTPDase activity dislplayed by TV-LACM6 isolate. The T. vaginalis trophozoites were not able to increase the production of IL-8 and IL-6 by HMVII and HeLa cells and only ATCC30236 and TV-LACM6 isolates enhanced MIP-3α secretion by the cervical epithelial cells. Finally, neither ATP nor adenosine has modulated the production of the inflammatory mediators here investigated. Considering the neutrophils, T. vaginalis stimulated the production of reactive oxygen species (ROS) and IL-8 by these immune cells and both adenine as guanine nucleotides and nucleosides did not cause any effect on ROS and IL-8 levels. However, when adenosine was incubated with an ADA inhibitor (EHNA) we observed a significant reduction of ROS and IL-8 production by neutrophils, due to inhibition of ADA with a subsequent increase of adenosine concentration in the extracellular milieu. . Our results suggested the participation of A1 receptor in this condition. The data set obtained in this study contributed to the characterization of T. vaginalis NTPDases family as well as to a better understanding of the influence of purinergic signaling on host-parasite relationship.

Trichomonas vaginalis é o agente etiológico da doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos de tricomonose a cada ano. O estabelecimento da infecção se deve principalmente à capacidade de adesão do parasito às células epiteliais vaginais, cervicais ou de próstata, seguida pela intensa reação inflamatória, resultado da infiltração de neutrófilos no sítio da infecção. Nucleotídeos e nucleosídeos, especialmente ATP e adenosina, são liberados para o espaço extracelular por células em situações de estresse ou injúria tecidual e desenvolvem seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, as ectonucleotidases, NTPDase e ecto-5’-nucleotidase, são capazes de hidrolisar os nucleotídeos gerando adenosina e finalmente, a enzima adenosina deaminase (ADA) é responsável pela conversão de adenosina em inosina. A expressão gênica de cinco NTPDases putativas presentes no genoma de T. vaginalis foram investigadas, assim como o envolvimento da sinalização purinérgica na relação parasito-hospedeiro. Nossos resultados mostraram que diferentes isolados de T. vaginalis expressam os genes TvNTPDase1, 2, 3, 4 e 5, sendo observado o maior número de transcritos para TvNTPDase1, 2 e 4. A sequência preditiva de aminoácidos revelou a presença das cinco regiões conservadas da apirase, domínios transmembrana, sítios de fosforilação, peptídeos sinais e os prováveis sítios ativos da enzima. A análise filogenética demonstrou maior similaridade das TvNTPDases com as formas intracelulares da enzima, como as NTPDases 4 e 7 humanas e a de Saccharomyces cerevisiae. Além disso, a restrição de soro promoveu aumento significativo da atividade da NTPDase de T. vaginalis, no entanto sem corresponder com o aumento de expressão gênica de determinada(s) sequência(s). Quanto à participação da sinalização purinérgica na resposta inflamatória de células do hospedeiro frente ao parasito, foram utilizados como modelos celulares as células epiteliais vaginais (HMVII), cervicais (HeLa) e neutrófilos humanos. As linhagens HMVII e HeLa mostraram expressar todos os subtipos de receptores P1, P2X e P2Y e XI os diferentes isolados de T. vaginalis, que foram cocultivados com as células, mostraram hidrolisar eficientemente os nucleotídeos ATP, ADP e AMP. Ainda, o isolado clínico fresco TV-LACM6 foi o único a apresentar elevada citotoxicidade frente às células epiteliais vaginais e cervicais, no entanto não foi detectado aumento da liberação de ATP pelas células após o cocultivo, provavelmente devido à alta atividade da enzima NTPDase observada nesse isolado. Os trofozoítos de T. vaginalis não foram capazes de aumentar a produção de IL-8 e IL-6 pelas linhagens HMVII e HeLa, e apenas os isolados ATCC30236 e TV-LACM6 causaram aumento na secreção da citocina MIP-3α pelas células epiteliais cervicais. Finalmente, o nucleotídeo ATP e o nucleosídeo adenosina não modularam a produção dos mediadores inflamatórios investigados. Em relação aos neutrófilos, estes mostraram aumentar a produção de espécies reativas de oxigênio (ERO) e IL-8 após incubação com os trofozoítos de T. vaginalis. Os nucleotídeos e nucleosídeos da adenina e guanina não produziram efeito na produção de ERO e IL-8; no entanto, quando o nucleosídeo adenosina foi incubado junto com o inibidor da enzima ADA (EHNA) observou-se uma redução significativa da produção de ERO e IL-8 pelos neutrófilos, devido à inibição da ADA e consequentemente, ao aumento da concentração de adenosina disponível no meio extracelular. Os nossos resultados indicaram a ativação do receptor A1 dos neutrófilos nessa condição. O conjunto de dados aqui obtidos contribuiu para uma melhor caracterização da família de enzimas NTPDases de T. vaginalis assim como para um maior conhecimento acerca da influência da sinalização purinérgica na relação parasito-hospedeiro. / Trichomonas vaginalis is the agent of the most common non-viral sexually transmitted disease worldwide, causing 276.4 million new cases a year. The establishment of the infection is closely related to the parasite ability to adhere to vaginal, cervical and prostate epithelial cells, followed by an intense inflammatory response as result of neutrophil infiltration. Nucleotides and nucleosides, mainly ATP and adenosine, are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, NTPDase and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing adenosine and finally, the adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine. We investigated the gene expression of five putative NTPDases found in T. vaginalis genome as well as the involvement of purinergic signaling on the host-parasite relationship. Our results showed that different T. vaginalis isolates are able to express TvNTPDase1, 2, 3, 4 and 5 and that TvNTPDase1, 2 and 4 are the most expressed genes. Predictive amino acid sequence revealed the presence of the five apyrase conserved regions, transmembrane domains, phosphorylation sites, signal peptides and the active sites. Phylogenetic analysis showed that TvNTPDases share more similarity with the intracellular enzymes, such as human NTPDase 4 and 7 and Saccharomyces cerevisiae NTPDase. In addition, the serum limitation caused a significant increase in NTPDase activity, but without association with the gene expression of a specific TvNTPDase sequence. Regarding the participation of purinergic signaling on the inflammatory responses against the parasite, the vaginal (HMVII) and cervical (HeLa) epithelial cells and the human neutrophils were used as cellular models. HMVII and HeLa cell lines showed to express all subtypes of P1, P2X and P2Y receptors and the different T. vaginalis isolates, which were co-cultured with the cells, showed to hydrolyze efficiently ATP, ADP and AMP. Furthermore, only the fresh clinical isolate, TV-LACM6, caused a profound cytotoxicity against the vaginal and cervical epithelial cells. Interestingly, it was not detected an increase in ATP release by the cells after cocultivation, probably due to the high NTPDase activity dislplayed by TV-LACM6 isolate. The T. vaginalis trophozoites were not able to increase the production of IL-8 and IL-6 by HMVII and HeLa cells and only ATCC30236 and TV-LACM6 isolates enhanced MIP-3α secretion by the cervical epithelial cells. Finally, neither ATP nor adenosine has modulated the production of the inflammatory mediators here investigated. Considering the neutrophils, T. vaginalis stimulated the production of reactive oxygen species (ROS) and IL-8 by these immune cells and both adenine as guanine nucleotides and nucleosides did not cause any effect on ROS and IL-8 levels. However, when adenosine was incubated with an ADA inhibitor (EHNA) we observed a significant reduction of ROS and IL-8 production by neutrophils, due to inhibition of ADA with a subsequent increase of adenosine concentration in the extracellular milieu. . Our results suggested the participation of A1 receptor in this condition. The data set obtained in this study contributed to the characterization of T. vaginalis NTPDases family as well as to a better understanding of the influence of purinergic signaling on host-parasite relationship.

Trichomonas vaginalis é o agente etiológico da doença sexualmente transmissível não viral mais comum no mundo, sendo registrados aproximadamente 276 milhões de novos casos de tricomonose a cada ano. O estabelecimento da infecção se deve principalmente à capacidade de adesão do parasito às células epiteliais vaginais, cervicais ou de próstata, seguida pela intensa reação inflamatória, resultado da infiltração de neutrófilos no sítio da infecção. Nucleotídeos e nucleosídeos, especialmente ATP e adenosina, são liberados para o espaço extracelular por células em situações de estresse ou injúria tecidual e desenvolvem seus efeitos sinalizadores através da ativação de purinoceptores. Ainda, as ectonucleotidases, NTPDase e ecto-5’-nucleotidase, são capazes de hidrolisar os nucleotídeos gerando adenosina e finalmente, a enzima adenosina deaminase (ADA) é responsável pela conversão de adenosina em inosina. A expressão gênica de cinco NTPDases putativas presentes no genoma de T. vaginalis foram investigadas, assim como o envolvimento da sinalização purinérgica na relação parasito-hospedeiro. Nossos resultados mostraram que diferentes isolados de T. vaginalis expressam os genes TvNTPDase1, 2, 3, 4 e 5, sendo observado o maior número de transcritos para TvNTPDase1, 2 e 4. A sequência preditiva de aminoácidos revelou a presença das cinco regiões conservadas da apirase, domínios transmembrana, sítios de fosforilação, peptídeos sinais e os prováveis sítios ativos da enzima. A análise filogenética demonstrou maior similaridade das TvNTPDases com as formas intracelulares da enzima, como as NTPDases 4 e 7 humanas e a de Saccharomyces cerevisiae. Além disso, a restrição de soro promoveu aumento significativo da atividade da NTPDase de T. vaginalis, no entanto sem corresponder com o aumento de expressão gênica de determinada(s) sequência(s). Quanto à participação da sinalização purinérgica na resposta inflamatória de células do hospedeiro frente ao parasito, foram utilizados como modelos celulares as células epiteliais vaginais (HMVII), cervicais (HeLa) e neutrófilos humanos. As linhagens HMVII e HeLa mostraram expressar todos os subtipos de receptores P1, P2X e P2Y e XI os diferentes isolados de T. vaginalis, que foram cocultivados com as células, mostraram hidrolisar eficientemente os nucleotídeos ATP, ADP e AMP. Ainda, o isolado clínico fresco TV-LACM6 foi o único a apresentar elevada citotoxicidade frente às células epiteliais vaginais e cervicais, no entanto não foi detectado aumento da liberação de ATP pelas células após o cocultivo, provavelmente devido à alta atividade da enzima NTPDase observada nesse isolado. Os trofozoítos de T. vaginalis não foram capazes de aumentar a produção de IL-8 e IL-6 pelas linhagens HMVII e HeLa, e apenas os isolados ATCC30236 e TV-LACM6 causaram aumento na secreção da citocina MIP-3α pelas células epiteliais cervicais. Finalmente, o nucleotídeo ATP e o nucleosídeo adenosina não modularam a produção dos mediadores inflamatórios investigados. Em relação aos neutrófilos, estes mostraram aumentar a produção de espécies reativas de oxigênio (ERO) e IL-8 após incubação com os trofozoítos de T. vaginalis. Os nucleotídeos e nucleosídeos da adenina e guanina não produziram efeito na produção de ERO e IL-8; no entanto, quando o nucleosídeo adenosina foi incubado junto com o inibidor da enzima ADA (EHNA) observou-se uma redução significativa da produção de ERO e IL-8 pelos neutrófilos, devido à inibição da ADA e consequentemente, ao aumento da concentração de adenosina disponível no meio extracelular. Os nossos resultados indicaram a ativação do receptor A1 dos neutrófilos nessa condição. O conjunto de dados aqui obtidos contribuiu para uma melhor caracterização da família de enzimas NTPDases de T. vaginalis assim como para um maior conhecimento acerca da influência da sinalização purinérgica na relação parasito-hospedeiro. / Trichomonas vaginalis is the agent of the most common non-viral sexually transmitted disease worldwide, causing 276.4 million new cases a year. The establishment of the infection is closely related to the parasite ability to adhere to vaginal, cervical and prostate epithelial cells, followed by an intense inflammatory response as result of neutrophil infiltration. Nucleotides and nucleosides, mainly ATP and adenosine, are released into the extracellular space by cells under stress or injury and they exert their signaling effects through activation of the purinoceptors. Moreover, the ectonucleotidases, NTPDase and ecto-5'-nucleotidase, are capable of hydrolyzing the nucleotides producing adenosine and finally, the adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine. We investigated the gene expression of five putative NTPDases found in T. vaginalis genome as well as the involvement of purinergic signaling on the host-parasite relationship. Our results showed that different T. vaginalis isolates are able to express TvNTPDase1, 2, 3, 4 and 5 and that TvNTPDase1, 2 and 4 are the most expressed genes. Predictive amino acid sequence revealed the presence of the five apyrase conserved regions, transmembrane domains, phosphorylation sites, signal peptides and the active sites. Phylogenetic analysis showed that TvNTPDases share more similarity with the intracellular enzymes, such as human NTPDase 4 and 7 and Saccharomyces cerevisiae NTPDase. In addition, the serum limitation caused a significant increase in NTPDase activity, but without association with the gene expression of a specific TvNTPDase sequence. Regarding the participation of purinergic signaling on the inflammatory responses against the parasite, the vaginal (HMVII) and cervical (HeLa) epithelial cells and the human neutrophils were used as cellular models. HMVII and HeLa cell lines showed to express all subtypes of P1, P2X and P2Y receptors and the different T. vaginalis isolates, which were co-cultured with the cells, showed to hydrolyze efficiently ATP, ADP and AMP. Furthermore, only the fresh clinical isolate, TV-LACM6, caused a profound cytotoxicity against the vaginal and cervical epithelial cells. Interestingly, it was not detected an increase in ATP release by the cells after cocultivation, probably due to the high NTPDase activity dislplayed by TV-LACM6 isolate. The T. vaginalis trophozoites were not able to increase the production of IL-8 and IL-6 by HMVII and HeLa cells and only ATCC30236 and TV-LACM6 isolates enhanced MIP-3α secretion by the cervical epithelial cells. Finally, neither ATP nor adenosine has modulated the production of the inflammatory mediators here investigated. Considering the neutrophils, T. vaginalis stimulated the production of reactive oxygen species (ROS) and IL-8 by these immune cells and both adenine as guanine nucleotides and nucleosides did not cause any effect on ROS and IL-8 levels. However, when adenosine was incubated with an ADA inhibitor (EHNA) we observed a significant reduction of ROS and IL-8 production by neutrophils, due to inhibition of ADA with a subsequent increase of adenosine concentration in the extracellular milieu. . Our results suggested the participation of A1 receptor in this condition. The data set obtained in this study contributed to the characterization of T. vaginalis NTPDases family as well as to a better understanding of the influence of purinergic signaling on host-parasite relationship.

Les récepteurs purinergiques P2X sont des canaux ioniques activés par l’ATP. Ils sont exprimés très largement dans l’organisme, et possèdent de nombreux rôles physiologiques et pathologiques. Les récepteurs P2X4 en particulier ont été impliqués dans les processus de douleur chronique. Suite à une lésion nerveuse, l’expression des récepteurs P2X4 est induite de novo dans la microglie spinale activée, où ils sont responsables de l’hypersensibilité mécanique caractéristique des douleurs neuropathiques.Notre étude montre que les récepteurs P2X4 neuronaux sont également des acteurs centraux dans plusieurs processus pathologiques, et notamment dans la douleur inflammatoire périphérique chronique. Les récepteurs P2X4 sont exprimés par les neurones sensoriels des ganglions rachidiens et semblent impliqués dans la libération du BDNF dans la corne dorsale de la moëlle épinière. Cette libération conduit à l’activation des voies de signalisation de la voie BDNF/TrkB, et en particulier à la diminution de l’expression de KCC2. Ce processus est en partie responsable de l’allodynie tactile et de l’hyperalgésie mécanique observée en cas de douleur inflammatoire chronique. Notre étude a également permis d’étendre cette hypothèse à un modèle d’excitotoxicité in vitro dans l’hippocampe, mimant une activité épileptiforme. Nos résultats indiquent que les récepteurs P2X4 neuronaux pourraient être des acteurs importants de la libération de BDNF dans l’hippocampe lors d’un évènement excitotoxique. / Purinergic receptors P2X are ATP-gated ion channels widely expressed in the organism and involved in many physiological and pathological states. Particularly, P2X4 receptors have been involved in chronic pain. Following nerve injury, their expression is induced de novo in activated spinal cord microglia where they are responsible for the BDNF release leading to tactile allodynia, a characteristic of neuropathic pain. Our study shows that neuronal P2X4 receptors are crucial actors of other pathological processes, including inflammatory pain. We show that P2X4R are expressed in sensory neurons in dorsal root ganglions and seem involved in BDNF release in the spinal cord. This release leads to activation of BDNF/TrkB signalization pathways, and particularly to the downregulation of KCC2. This process underlies the spinal hyperexcitability in chronic inflammatory pain states. These results have been extended to model of excitotoxicity in the hippocampus mimicking the lesions caused by an epileptic activity. Our preliminary results suggest that neuronal P2X4 receptors are likely major actors in the BDNF release in the hippocampus following an excitotoxicitic insult.

Editorial on the Research Topic. Purinergic Signaling 2020: The State-of-The-Art Commented by the Members of the Italian Purine Club.