Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1ß production in air pollution fine particulate matter (PM2.5)-primed macrophages.

Exposure to air pollution fine particulate matter (PM2.5) aggravates respiratory and cardiovascular diseases. It has been proposed that PM2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1ß. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM2.5 surrogates, significant IL-1ß release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1ß release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1ß production in ROFA-exposed NLRP3-deficient (Nlrp3-/-) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1ß release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K+ efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1ß release. Interestingly, specific inhibition of superoxide anion production (O2•-) from mitochondrial respiratory Complex I, but not III, blunted IL-1ß release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM2.5 promotes IL-1ß release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM2.5.

Circulating Autoantibodies Recognizing Immunodominant Epitopes From Human Apolipoprotein B Associate With Cardiometabolic Risk Factors, but Not With Atherosclerotic Disease.

Rationale: Atherosclerosis is a chronic inflammatory disease of large arteries that involves an autoimmune response with autoreactive T cells and auto-antibodies recognizing Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL). Here, we aimed to establish a clinical association between circulating human ApoB auto-antibodies with atherosclerosis and its clinical risk factors using a novel assay to detect auto-antibodies against a pool of highly immunogenic ApoB-peptides. Methods and Results: To detect polyclonal IgM- and IgG-antibodies recognizing ApoB, we developed a chemiluminescent sandwich ELISA with 30 ApoB peptides selected by an in silico assay for a high binding affinity to MHC-II, which cover more than 80% of known MHC-II variants in a Caucasian population. This pre-selection of immunogenic self-peptides accounted for the high variability of human MHC-II, which is fundamental to allow T cell dependent generation of IgG antibodies. We quantified levels of ApoB-autoantibodies in a clinical cohort of 307 patients that underwent coronary angiography. Plasma anti-ApoB IgG and IgM concentrations showed no differences across healthy individuals (n = 67), patients with coronary artery disease (n = 179), and patients with an acute coronary syndrome (n = 61). However, plasma levels of anti-ApoB IgG, which are considered pro-inflammatory, were significantly increased in patients with obesity (p = 0.044) and arterial hypertension (p < 0.0001). In addition, patients diagnosed with the metabolic syndrome showed significantly elevated Anti-ApoB IgG (p = 0.002). Even when normalized for total plasma IgG, anti-ApoB IgG remained highly upregulated in hypertensive patients (p < 0.0001). We observed no association with triglycerides, total cholesterol, VLDL, or LDL plasma levels. However, total and normalized anti-ApoB IgG levels negatively correlated with HDL. In contrast, total and normalized anti-ApoB IgM, that have been suggested as anti-inflammatory, were significantly lower in diabetic patients (p = 0.012) and in patients with the metabolic syndrome (p = 0.005). Conclusion: Using a novel ELISA method to detect auto-antibodies against ApoB in humans, we show that anti-ApoB IgG associate with cardiovascular risk factors but not with the clinical appearance of atherosclerosis, suggesting that humoral immune responses against ApoB are shaped by cardiovascular risk factors but not disease status itself. This novel tool will be helpful to develop immune-based risk stratification for clinical atherosclerosis in the future.

Chronic exposure to polluted urban air aggravates myocardial infarction by impaired cardiac mitochondrial function and dynamics.

Air pollution exposure positively correlates with increased cardiovascular morbidity and mortality rates, mainly due to myocardial infarction (MI). Herein, we aimed to study the metabolic mechanisms underlying this association, focusing on the evaluation of cardiac mitochondrial function and dynamics, together with its impact over MI progression. An initial time course study was performed in BALB/c mice breathing filtered air (FA) or urban air (UA) in whole-body exposure chambers located in Buenos Aires City downtown for up to 16 weeks (n = 8 per group and time point). After 12 weeks, lung inflammatory cell recruitment was evident in UA-exposed mice. Interestingly, impaired redox metabolism, characterized by decreased lung SOD activity and increased GSSG levels and NOX activity, precede local inflammation in this group. At this selected time point, additional mice were exposed to FA or UA (n = 12 per group) and alveolar macrophage PM uptake and nitric oxide (NO) production was observed in UA-exposed mice, together with increased pro-inflammatory cytokine levels (TNF-α and IL-6) in BAL and plasma. Consequently, impaired heart tissue oxygen metabolism and altered mitochondrial ultrastructure and function were observed in UA-exposed mice after 12 weeks, characterized by decreased active state respiration and ATP production rates, and enhanced mitochondrial H2O2 production. Moreover, disturbed cardiac mitochondrial dynamics was detected in this group. This scenario led to a significant increase in the area of infarcted tissue following myocardial ischemia reperfusion injury in vivo, from 43 ± 3% of the area at risk in mice breathing FA to 66 ± 4% in UA-exposed mice (n = 6 per group, p < 0.01), together with a sustained increase in LVEDP during myocardial reperfusion. Taken together, our data unravel cardiac mitochondrial mechanisms that contribute to the understanding of the adverse health effects of urban air pollution exposure, and ultimately highlight the importance of considering environmental factors in the development of cardiovascular diseases.

Oxidative metabolism in the cardiorespiratory system after an acute exposure to nickel-doped nanoparticles in mice.

There is an increasing concern over the harmful effects that metallic nanoparticles (NP) may produce on human health. Due to their redox properties, nickel (Ni) and Ni-containing NP are particularly relevant. Hence, the aim of this study was to establish the toxicological mechanisms in the cardiorespiratory oxidative metabolism initiated by an acute exposure to Ni-doped-NP. Mice were intranasally instilled with silica NP containing Ni (II) (Ni-NP) (1 mg Ni (II)/kg body weight) or empty NP as control, and 1 h after exposure lung, plasma, and heart samples were obtained to assess the redox metabolism. Results showed that, NP were mainly retained in the lungs triggering a significantly increased tissue O2 consumption rate, leading to Ni-NP-increased reactive oxygen species production by NOX activity, and mitochondrial H2O2 production rate. In addition, an oxidant redox status due to an altered antioxidant system showed by lung GSH/GSSG ratio decreased, and SOD activity increased, resulting in an increased phospholipid oxidation. Activation of circulating polymorphonuclear leukocytes, along with GSH/GSSG ratio decreased, and phospholipid oxidation were found in the Ni-NP-group plasma samples. Consequently, in distant organs such as heart, Ni-NP inhalation alters the tissue redox status. Our results showed that the O2 metabolism analysis is a critical area of study following Ni-NP inhalation. Therefore, this work provides novel data linking the redox metabolisms alterations elicited by exposure to Ni (II) adsorbed to NP and cardiorespiratory toxicity.

Urban air pollution induces alterations in redox metabolism and mitochondrial dysfunction in mice brain cortex.

Previous reports indicate that the central nervous system (CNS) is a target of air pollution, causing tissue damage and functional alterations. Oxidative stress and neuroinflammation have been pointed out as possible mechanisms mediating these effects. The aim of this work was to study the chronic effects of urban air pollution on mice brain cortex, focusing on oxidative stress markers, and mitochondrial function. Male 8-week-old BALB/c mice were exposed to filtered air (FA, control) or urban air (UA) inside whole-body exposure chambers, located in a highly polluted area of Buenos Aires city, for up to 4 weeks. Glutathione levels, assessed as GSH/GSSG ratio, were decreased after 1 and 2 weeks of exposure to UA (45% and 25% respectively vs. FA; p < 0.05). A 38% increase in lipid peroxidation was found after 1 week of UA exposure (p < 0.05). Regarding protein oxidation, carbonyl content was significantly increased at week 2 in UA-exposed mice, compared to FA-group, and an even higher increment was found after 4 weeks of exposure (week 2: 40% p < 0.05, week 4: 54% p < 0.001). NADPH oxidase (NOX) and glutathione peroxidase (GPx) activities were augmented at all the studied time points, while superoxide dismutase (Cu,Zn-SOD cytosolic isoform) and glutathione reductase (GR) activities were increased only after 4 weeks of UA exposure (p < 0.05). The increased NOX activity was accompanied with higher expression levels of NOX2 regulatory subunit p47phox, and NOX4 (p < 0.05). Also, UA mice showed impaired mitochondrial function due to a 50% reduction in O2 consumption in active state respiration (p < 0.05), a 29% decrease in mitochondrial inner membrane potential (p < 0.05), a 65% decrease in ATP production rate (p < 0.01) and a 30% increase in H2O2 production (p < 0.01). Moreover, respiratory complexes I-III and II-III activities were decreased in UA group (30% and 36% respectively vs. FA; p < 0.05). UA exposed mice showed alterations in mitochondrial function, increased oxidant production evidenced by NOX activation, macromolecules damage and the onset of the enzymatic antioxidant system. These data indicate that oxidative stress and impaired mitochondrial function may play a key role in CNS damage mechanisms triggered by air pollution.

Alterations in oxygen metabolism are associated to lung toxicity triggered by silver nanoparticles exposure.

Along with the AgNP applications development, the concern about their possible toxicity has increasingly gained attention. As the respiratory system is one of the main exposure routes, the aim of this study was to evaluate the harmful effects developed in the lung after an acute AgNP exposure. In vivo studies using Balb/c mice intranasally instilled with 0.1 mg AgNP/kg b.w, were performed. 99mTc-AgNP showed the lung as the main organ of deposition, where, in turn, AgNP may exert barrier injury observed by increased protein content and total cell count in BAL samples. In vivo acute exposure showed altered lung tissue O2 consumption due to increased mitochondrial active respiration and NOX activity. Both O2 consumption processes release ROS triggering the antioxidant system as observed by the increased SOD, catalase and GPx activities and a decreased GSH/GSSG ratio. In addition, increased protein oxidation was observed after AgNP exposure. In A549 cells, exposure to 2.5 µg/mL AgNP during 1 h resulted in augment NOX activity, decreased mitochondrial ATP associated respiration and higher H2O2 production rate. Lung 3D tissue model showed AgNP-initiated barrier alterations as TEER values decreased and morphological alterations. Taken together, these results show that AgNP exposure alters O2 metabolism leading to alterations in oxygen metabolism lung toxicity. AgNP-triggered oxidative damage may be responsible for the impaired lung function observed due to alveolar epithelial injury.

NADPH oxidase and mitochondria are relevant sources of superoxide anion in the oxinflammatory response of macrophages exposed to airborne particulate matter.

Exposure to ambient air particulate matter (PM) is associated with increased cardiorespiratory morbidity and mortality. In this context, alveolar macrophages exhibit proinflammatory and oxidative responses as a result of the clearance of particles, thus contributing to lung injury. However, the mechanisms linking these pathways are not completely clarified. Therefore, the oxinflammation phenomenon was studied in RAW 264.7 macrophages exposed to Residual Oil Fly Ash (ROFA), a PM surrogate rich in transition metals. While cell viability was not compromised under the experimental conditions, a proinflammatory phenotype was observed in cells incubated with ROFA 100 µg/mL, characterized by increased levels of TNF-α and NO production, together with PM uptake. This inflammatory response seems to precede alterations in redox metabolism, characterized by augmented levels of H2O2, diminished GSH/GSSG ratio, and increased SOD activity. This scenario resulted in increased oxidative damage to phospholipids. Moreover, alterations in mitochondrial respiration were observed following ROFA incubation, such as diminished coupling efficiency and spare respiratory capacity, together with augmented proton leak. These findings were accompanied by a decrease in mitochondrial membrane potential. Finally, NADPH oxidase (NOX) and mitochondria were identified as the main sources of superoxide anion () in our model. These results indicate that PM exposure induces direct activation of macrophages, leading to inflammation and increased reactive oxygen species production through NOX and mitochondria, which impairs antioxidant defense and may cause mitochondrial dysfunction.

Role of transition metals present in air particulate matter on lung oxygen metabolism.

Several epidemiological studies have shown a positive correlation between daily increases in airborne particulate matter (PM) concentration and the occurrence of respiratory and cardiovascular diseases. Transition metals present in air PM were associated with adverse health effects after PM exposure. The aim of this work was to study lung O2 metabolism after an acute exposure to transition metal-coated nanoparticles (NPs). Female Swiss mice (25g) were intranasally instilled with a suspension of silica NP containing Ni (II), Cd (II), Fe (III), or Cr (VI) at 0, 0.01, 0.05, 0.1, and 1.0mg metal/kg body weight. Lung O2 consumption was found to be significantly increased after the exposure to most doses of Ni-NP and Fe-NP, and the 0.05mg metal/kg body weight dose of Cr-NP, while no changes were observed for Cd-NP. Lucigenin chemiluminescence (as an indicator of NADPH oxidase (NOX) activity) was evaluated in lung homogenates. Only Ni-NP and Fe-NP have shown the ability to induce a significant increase in lucigenin chemiluminescence. In order to establish the possible occurrence of pulmonary oxidative stress, TBARS levels and the GSH/GSSG ratio were determined. The higher doses of Ni-NP and Fe-NP were able to induce an oxidative stress condition, as shown by changes in both TBARS levels and the GSH/GSSG ratio. Taken together, the present results show differential effects for all the metals tested. These findings emphasize the importance of transition metals present air PM in PM adverse health effects, and contribute to the understanding of the pathological mechanisms triggered by the exposure to environmental PM.

Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning.

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30min of ischemia and 120min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6±2.4 vs. 39.2±2.1%, p<0.05), but this protection was abolished in DN-Trx1-PostC group (49.7±1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9±2.1 and 38.4±1.3%, p<0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3ß phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3ß survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.

Ischemic postconditioning confers cardioprotection and prevents reduction of Trx-1 in young mice, but not in middle-aged and old mice.

Thioredoxin-1 (Trx-1) is part of an antioxidant system that maintains the cell redox homeostasis but their role on ischemic postconditioning (PostC) is unknown. The aim of this work was to determine whether Trx-1 participates in the cardioprotective mechanism of PostC in young, middle-aged, and old mice. Male FVB young (Y: 3 month-old), middle-aged (MA: 12 month-old), and old (O: 20 month-old) mice were used. Langendorff-perfused hearts were subjected to 30 min of ischemia and 120 min of reperfusion (I/R group). After ischemia, we performed 6 cycles of R/I (10 s each) followed by 120 min of reperfusion (PostC group). We measured the infarct size (triphenyltetrazolium); Trx-1, total and phosphorylated Akt, and GSK3ß expression (Western blot); and the GSH/GSSG ratio (HPLC). PostC reduced the infarct size in young mice (I/R-Y: 52.3 ± 2.4 vs. PostC-Y: 40.0 ± 1.9, p < 0.05), but this protection was abolished in the middle-aged and old mice groups. Trx-1 expression decreased after I/R, and the PostC prevented the protein degradation in young animals (I/R-Y: 1.05 ± 0.1 vs. PostC-Y: 0.52 ± .0.07, p < 0.05). These changes were accompanied by an improvement in the GSH/GSSG ratio (I/R-Y: 1.25 ± 0.30 vs. PostC-Y: 7.10 ± 2.10, p < 0.05). However, no changes were observed in the middle-aged and old groups. Cytosolic Akt and GSK3ß phosphorylation increased in the PostC compared with the I/R group only in young animals. Our results suggest that PostC prevents Trx-1 degradation, decreasing oxidative stress and allowing the activation of Akt and GSK3ß to exert its cardioprotective effect. This protection mechanism is not activated in middle-aged and old animals.

Selective TNF-α targeting with infliximab attenuates impaired oxygen metabolism and contractile function induced by an acute exposure to air particulate matter.

Inflammation plays a central role in the onset and progression of cardiovascular diseases associated with the exposure to air pollution particulate matter (PM). The aim of this work was to analyze the cardioprotective effect of selective TNF-α targeting with a blocking anti-TNF-α antibody (infliximab) in an in vivo mice model of acute exposure to residual oil fly ash (ROFA). Female Swiss mice received an intraperitoneal injection of infliximab (10 mg/kg body wt) or saline solution, and were intranasally instilled with a ROFA suspension (1 mg/kg body wt). Control animals were instilled with saline solution and handled in parallel. After 3 h, heart O2 consumption was assessed by high-resolution respirometry in left ventricle tissue cubes and isolated mitochondria, and ventricular contractile reserve and lusitropic reserve were evaluated according to the Langendorff technique. ROFA instillation induced a significant decrease in tissue O2 consumption and active mitochondrial respiration by 32 and 31%, respectively, compared with the control group. While ventricular contractile state and isovolumic relaxation were not altered in ROFA-exposed mice, impaired contractile reserve and lusitropic reserve were observed in this group. Infliximab pretreatment significantly attenuated the decrease in heart O2 consumption and prevented the decrease in ventricular contractile and lusitropic reserve in ROFA-exposed mice. Moreover, infliximab-pretreated ROFA-exposed mice showed conserved left ventricular developed pressure and cardiac O2 consumption in response to a ß-adrenergic stimulus with isoproterenol. These results provides direct evidence linking systemic inflammation and altered cardiac function following an acute exposure to PM and contribute to the understanding of PM-associated cardiovascular morbidity and mortality.

[Gastric tricobezoar: a rare cause of tumoral syndrome and pyloric obstruction]. / Tricobezoar gástrico: una causa poco frecuente de síndrome tumoral y de obstrucción pilórica.

UNLABELLED: A Trichobezoar is a rare tumoral mass of the gastrointestinal tract, formed mainly from the ingestion of hair. It contains also mucus and foods debris. CASE PRESENTATION: A 22 years old female with a 10 years history of surgery secondary to gastric foreign body (trichobezoar), presents with abdominal pain, swelling, nausea and vomiting. Physical examination of the abdomen revealed a palpable mass in the epigastric and left upper quadrant regions. It was also noted areas of alopecia of the scalp. DISCUSSION: Ninety percent of the trichobezoars present in females with a high frequency between 10-19 years. The treatment of bezoars (unless small in size) is mainly surgical. A psychiatric evaluation and follow up is important after surgery hence in most cases there is a psychological disorder that lead to the ingestion of hair.

Tricobezoar gástrico: una causa poco frecuente de síndrome tumoral y de obstrucción pilórica / Gastric Tricobezoar: a rare cause of tumoral syndrome and pyloric obstruction

El tricobezoar es una rara formación tumoral gástrica causada por un cuerpo extraño compuesto fundamentalmente por pelo; también contiene moco y fragmentos de alimentos. Presentación del caso: Paciente de 22 años que hace 10 años fue intervenida por cuerpo extraño gástrico (tricobezoar), ahora refiere cuadro de dolor abdominal, distensión, náuseas y vómitos, al examen físico presencia de masa abdominal palpable en epigástrico y cuadrante superior izquierdo. En la cabeza se observaron zonas alopécicas. Discusión: El 90% de los tricobezoares se presentan en el sexo femenino. Su máxima frecuencia es entre 10 y 19 años. El tratamiento de los bezoares, excepto los más pequeños, es quirúrgico. El control psiquiátrico postoperatorio es fundamental ya que en casi todos los casos existe en el fondo un conflicto afectivo que perpetúa el hábito de ingerir cabello.

A Trichobezoar is a rare tumoral mass of the gastrointestinal tract, formed mainly from the ingestion of hair. It contains also mucus and foods debris. Case presentation: A 22 years old female with a 10 years history of surgery secondary to gastric foreign body (trichobezoar), presents with abdominal pain, swelling, nausea and vomiting. Physical examination of the abdomen revealed a palpable mass in the epigastric and left upper quadrant regions. It was also noted areas of alopecia of the scalp. Discussion: Ninety percent of the trichobezoars present in females with a high frequency between 10 - 19 years. The treatment of bezoars (unless small in size) is mainly surgical. A psychiatric evaluation and follow up is important after surgery hence in most cases there is a psychological disorder that lead to the ingestion of hair.