Adenosine A2A receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage.

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells-glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host-pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron-glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae-Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. Enzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR analysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzyme-linked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

Adenosine A2a receptor as a potential regulator of Mycobacterium leprae survival mechanisms: new insights into leprosy neural damage

Background: Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can lead to a disabling neurodegenerative condition. M. leprae preferentially infects skin macrophages and Schwann cells­glial cells of the peripheral nervous system. The infection modifies the host cell lipid metabolism, subverting it in favor of the formation of cholesterol-rich lipid droplets (LD) that are essential for bacterial survival. Although researchers have made progress in understanding leprosy pathogenesis, many aspects of the molecular and cellular mechanisms of host­pathogen interaction still require clarification. The purinergic system utilizes extracellular ATP and adenosine as critical signaling molecules and plays several roles in pathophysiological processes. Furthermore, nucleoside surface receptors such as the adenosine receptor A2AR involved in neuroimmune response, lipid metabolism, and neuron­glia interaction are targets for the treatment of different diseases. Despite the importance of this system, nothing has been described about its role in leprosy, particularly adenosinergic signaling (AdoS) during M. leprae­Schwann cell interaction. Methods: M. leprae was purified from the hind footpad of athymic nu/nu mice. ST88-14 human cells were infected with M. leprae in the presence or absence of specific agonists or antagonists of AdoS. nzymatic activity assays, fluorescence microscopy, Western blotting, and RT-qPCR nalysis were performed. M. leprae viability was investigated by RT-qPCR, and cytokines were evaluated by enzymelinked immunosorbent assay. Results: We demonstrated that M. leprae-infected Schwann cells upregulated CD73 and ADA and downregulated A2AR expression and the phosphorylation of the transcription factor CREB (p-CREB). On the other hand, activation of A2AR with its selective agonist, CGS21680, resulted in: 1) reduced lipid droplets accumulation and pro-lipogenic gene expression; 2) reduced production of IL-6 and IL-8; 3) reduced intracellular M. leprae viability; 4) increased levels of p-CREB. Conclusion: These findings suggest the involvement of the AdoS in leprosy neuropathogenesis and support the idea that M. leprae, by downmodulating the expression and activity of A2AR in Schwann cells, decreases A2AR downstream signaling, contributing to the maintenance of LD accumulation and intracellular viability of the bacillus.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14 C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial-Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial­Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy. (AU)

Reduction of host cell mitochondrial activity as Mycobacterium leprae's strategy to evade host innate immunity.

Leprosy is a much-feared incapacitating infectious disease caused by Mycobacterium leprae or M lepromatosis, annually affecting roughly 200,000 people worldwide. During host-pathogen interaction, M leprae subverts the immune response, leading to development of disease. Throughout the last few decades, the impact of energy metabolism on the control of intracellular pathogens and leukocytic differentiation has become more evident. Mitochondria play a key role in regulating newly-discovered immune signaling pathways by controlling redox metabolism and the flow of energy besides activating inflammasome, xenophagy, and apoptosis. Likewise, this organelle, whose origin is probably an alphaproteobacterium, directly controls the intracellular pathogens attempting to invade its niche, a feature conquered at the expense of billions of years of coevolution. In the present review, we discuss the role of reduced host cell mitochondrial activity during M leprae infection and the consequential fates of M leprae and host innate immunity. Conceivably, inhibition of mitochondrial energy metabolism emerges as an overlooked and novel mechanism developed by M leprae to evade xenophagy and the host immune response.

Envolvimento dos receptores P2X7 e A2a na patogênese causada pelo Mycobacterium leprae / Involvement of P2X7 and A2a receptors in the pathogenesis caused by Mycobacterium leprae

A hanseníase, causada pelo Mycobacterium leprae, um bacilo intracelular obrigatório com tropismo por macrófagos e células de Schwann, é uma doença infecciosa que acomete a pele e os nervos periféricos e pode acarretar danos irreversíveis se não tratada. O Brasil ocupa o segundo lugar no ranking mundial entre os países com maior número de casos doença, o que torna essa enfermidade um importante problema de saúde pública. ATP e adenosina (ADO) extracelulares, assim como um conjunto de ecto-enzimas e receptores constituem os principais mediadores do sistema purinérgico. Este sistema vem sendo amplamente descrito regulando respostas imunes em diferentes modelos de doenças, inclusive infecciosas como a tuberculose. Entretanto, nada foi estudado até os dias atuais sobre o papel deste complexo sistema com relação a patogênese da hanseníase. Assim, o presente estudo buscou avaliar a participação deste sistema na interação de monócitos humanos com o M. leprae. Nossos dados, obtidos a partir de experimentos in vitro, usando as técnicas de citometria de fluxo e western blotting mostram que o M. leprae aumenta a expressão das ecto-enzimas CD39 e CD73 e adenosina desaminase (ADA) que hidrolisam ATP/ADP em AMP, AMP em ADO e ADO em inosina (INO) respectivamente, após 24h de infecção, entretanto, após 48h de infecção a expressão de CD73 e ADA parecem ser diminuídas frente a infecção.

Estes dados sugerem que a regulação dos níveis extracelulares destes mediadores purinérgicos parece ser relevante na interação monócito-M. leprae. A infecção também aumentou a expressão de panexina 1, uma proteína envolvida na secreção de ATP, mas diminui a expressão do receptor P2X7, que ativa uma resposta pró-inflamatória, e aumenta a expressão do receptor A2A, um potencial receptor anti- inflamatório. Estes dados em conjunto suportam a hipótese de que a infecção deve estar reduzindo os níveis extracelulares de ATP, em contrapartida, deve aumentar os níveis de ADO/INO no meio extracelular após 24h, entretanto, após 48h, os níveis de ADO/INO estariam reduzidos. Dados de LC-MS/MS mostram que a infecção após 48h reduz os níveis de INO e de seus metabólitos, hipoxantina, xantina e ácido úrico, quando comparada às células não infectadas, sugerindo que a produção de ácido úrico não seria benéfica para o estabelecimento da infecção. Observamos, ainda por ensaios de ELISA usando agonistas e antagonistas do receptor A2a, que este parece regular positivamente a produção de IL-10 e IL-1beta e da quimiocina MCP1, entretanto, sua ativação reduz a produção de IL8. A produção de IL-1beta também foi aumentada com o tratamento com ATP. M. leprae subverte o metabolismo lipídico do hospedeiro em seu benefício e a literatura mostra que o receptor P2X7 e receptor A2a estão envolvidos na homeostase lipídica. (AU)