The P2X7 receptor mediates Toxoplasma gondii Control in Macrophages through canonical NLRP3 inflammasome activation and reactive oxygen species production

Toxoplasma gondii (T. gondii) is the protozoan parasite that causes toxoplasmosis, a potentially fatal disease to immunocompromised patients, and which affects approximately 30% of the world\u27s population. Previously, we showed that purinergic signaling via the P2X7 receptor contributes to T. gondii elimination in macrophages, through reactive oxygen species (ROS) production and lysosome fusion with the parasitophorous vacuole. Moreover, we demonstrated that P2X7 receptor activation promotes the production of anti-parasitic pro-inflammatory cytokines during early T. gondii infection in vivo. However, the cascade of signaling events that leads to parasite elimination via P2X7 receptor activation remained to be elucidated. Here, we investigated the cellular pathways involved in T. gondii elimination triggered by P2X7 receptor signaling, during early infection in macrophages. We focused on the potential role of the inflammasome, a protein complex that can be co-activated by the P2X7 receptor, and which is involved in the host immune defense against T. gondii infection. Using peritoneal and bone marrow-derived macrophages from knockout mice deficient for inflammasome components (NLRP3-/-, Caspase-1/11-/-, Caspase-11-/-), we show that the control of T. gondii infection via P2X7 receptor activation by extracellular ATP (eATP) depends on the canonical inflammasome effector caspase-1, but not on caspase-11 (a non-canonical inflammasome effector). Parasite elimination via P2X7 receptor and inflammasome activation was also dependent on ROS generation and pannexin-1 channel. Treatment with eATP increased IL-1β secretion from infected macrophages, and this effect was dependent on the canonical NLRP3 inflammasome. Finally, treatment with recombinant IL-1β promoted parasite elimination via mitochondrial ROS generation (as assessed using Mito-TEMPO). Together, our results support a model where P2X7 receptor activation by eATP inhibits T. gondii growth in macrophages by triggering NADPH-oxidase-dependent ROS production, and also by activating a canonical NLRP3 inflammasome, which increases IL-1β production (via caspase-1 activity), leading to mitochondrial ROS generation

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

The P2X7 Receptor Mediates Toxoplasma gondii Control in Macrophages through Canonical NLRP3 Inflammasome Activation and Reactive Oxygen Species Production

Toxoplasma gondii (T. gondii) is the protozoan parasite that causes toxoplasmosis, a potentially fatal disease to immunocompromised patients, and which affects approximately 30% of the world’s population. Previously, we showed that purinergic signaling via the P2X7 receptor contributes to T. gondii elimination in macrophages, through reactive oxygen species (ROS) production and lysosome fusion with the parasitophorous vacuole. Moreover, we demonstrated that P2X7 receptor activation promotes the production of anti-parasitic pro-inflammatory cytokines during early T. gondii infection in vivo. However, the cascade of signaling events that leads to parasite elimination via P2X7 receptor activation remained to be elucidated. Here, we investigated the cellular pathways involved in T. gondii elimination triggered by P2X7 receptor signaling, during early infection in macrophages. We focused on the potential role of the inflammasome, a protein complex that can be co-activated by the P2X7 receptor, and which is involved in the host immune defense against T. gondii infection. Using peritoneal and bone marrow-derived macrophages from knockout mice deficient for inflammasome components (NLRP3−/−, Caspase-1/11−/−, Caspase-11−/−), we show that the control of T. gondii infection via P2X7 receptor activation by extracellular ATP (eATP) depends on the canonical inflammasome effector caspase-1, but not on caspase-11 (a non-canonical inflammasome effector). Parasite elimination via P2X7 receptor and inflammasome activation was also dependent on ROS generation and pannexin-1 channel. Treatment with eATP increased IL-1β secretion from infected macrophages, and this effect was dependent on the canonical NLRP3 inflammasome. Finally, treatment with recombinant IL-1β promoted parasite elimination via mitochondrial ROS generation (as assessed using Mito-TEMPO). Together, our results support a model where P2X7 receptor activation by eATP inhibits T. gondii growth in macrophages by triggering NADPH-oxidase-dependent ROS production, and also by activating a canonical NLRP3 inflammasome, which increases IL-1β production (via caspase-1 activity), leading to mitochondrial ROS generation

Pyrimidinergic Receptor Activation Controls <i>Toxoplasma gondii</i> Infection in Macrophages

<div><p>Infection by the protozoan parasite <i>Toxoplasma gondii</i> is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. <i>T</i>. <i>gondii</i> is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with <i>T</i>. <i>gondii</i> tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca²⁺-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y₂ and P2Y₄ and P2Y₆ showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls <i>T</i>. <i>gondii</i> infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against <i>T</i>. <i>gondii</i> infection.</p></div

Non-canonical NLRP3 inflammasome activation and IL-1β signaling are necessary to L. amazonensis control mediated by P2X7 receptor and leukotriene B4.

Leishmaniasis is a neglected tropical disease affecting millions of individuals worldwide. P2X7 receptor has been linked to the elimination of Leishmania amazonensis. Biological responses evoked by P2X7 receptor activation have been well-documented, including apoptosis, phagocytosis, cytokine release, such as IL-1β. It was demonstrated that NLRP3 inflammasome activation and IL-1β signaling participated in resistance against L. amazonensis. Furthermore, our group has shown that L. amazonensis elimination through P2X7 receptor activation depended on leukotriene B4 (LTB4) production and release. Therefore, we investigated whether L. amazonensis elimination by P2X7 receptor and LTB4 involved NLRP3 inflammasome activation and IL-1β signaling. We showed that macrophages from NLRP3-/-, ASC-/-, Casp-1/11-/-, gp91phox-/- , and IL-1R-/- mice treated with ATP or LTB4 did not decrease parasitic load as was observed in WT mice. When ASC-/- macrophages were treated with exogenous IL-1β, parasite killing was noted, however, we did not see parasitic load reduction in IL-1R-/- macrophages. Similarly, macrophages from P2X7 receptor-deficient mice treated with IL-1β also showed decreased parasitic load. In addition, when we infected Casp-11-/- macrophages, neither ATP nor LTB4 were able to reduce parasitic load, and Casp-11-/- mice were more susceptible to L. amazonensis infection than were WT mice. Furthermore, P2X7-/- L. amazonensis-infected mice locally treated with exogenous LTB4 showed resistance to infection, characterized by lower parasite load and smaller lesions compared to untreated P2X7-/- mice. A similar observation was noted when infected P2X7-/- mice were treated with IL-1β, i.e., lower parasite load and smaller lesions compared to P2X7-/- mice. These data suggested that L. amazonensis elimination mediated by P2X7 receptor and LTB4 was dependent on non-canonical NLRP3 inflammasome activation, ROS production, and IL-1β signaling

The activation of different P2Y receptor subtypes reduce <i>T</i>. <i>gondii</i> infection in peritoneal macrophages.

<p>Mouse peritoneal macrophages were infected with <i>T</i>. <i>gondii</i> tachyzoites at a ratio of 5:1 for 2 h and then treated with specific P2Y receptor agonists and antagonists for 30 min, prior to infection index determination. (A) Treatment 2 Thio-UTP at concentrations of 0.05 or 0.1 μM (to activate P2Y₄) and 0.5 or 1 μM of 2 Thio-UTP (to activate P2Y₂) led to similar reductions in the infection index relative to untreated controls. (B) The infection index was also reduced after treatment with MRS 2693, a specific P2Y₆ agonist, and MRS 2693 effect was totally blocked by pre-treatment with the specific P2Y₆ antagonist MRS 2578. The 20, 50, 100 represent on the x-axis concentration of the agonist/antagonist. * statistically significant relative to untreated. # statistically significant relative to reduction of infection index. Data represent mean and SEM of three independent experiments. * p < 0,05; **,^## p < 0.001; ***, ^### p < 0.0001.</p

<i>T</i>. <i>gondii</i> tachyzoites that egress prematurely from nucleotide-treated macrophages have reduced infectivity.

<p>Mouse peritoneal macrophages infected with tachyzoites at a 5:1 ratio were treated with 100 μM UTP or UDP for 30 min. Immediately after nucleotide treatment, prematurely egressed parasites were recovered from culture supernatants and allowed to interact with cultures of freshly harvested peritoneal macrophages for 24 h. Cells were then processed for light microscopy analysis of the % of infected cells (A) and the infection index (B). Untreated control parasites represented those that had not invaded the untreated cells 2 h post-interaction. Cytoplasmic vacuole acidification was analyzed by fluorescence microscopy using the acidic compartment probe Lysotracker red (C). Arrows indicate parasites inside host cells. Phagolysosomal fusion inhibition (with lack of Lysotracker red staining) was observed in cells infected with control parasites obtained from infected mice (Fresh). In contrast, in cultures infected with parasites rescued from nucleotide-treated cells (UTP or UDP), tachyzoites were found inside acidic (Lysotracker red-positive) parasitophorous vacuoles, indicating that phagolysosomal fusion occurred during infection. (A, B) Data represent mean and SEM of three independent experiments. * p < 0.05; ** p < 0.001; *** p < 0.0001, relative to untreated controls.</p