Does air pollution explain COVID-19 fatality and mortality rates? A multi-city study in São Paulo state, Brazil.

Since air pollution compromise the respiratory system and COVID-19 disease is caused by a respiratory virus, it is expected that air pollution plays an important role in the current COVID-19 pandemic. Exploratory studies have observed positive associations between air pollution and COVID-19 cases, deaths, fatality, and mortality rate. However, no study focused on Brazil, one of the most affected countries by the pandemic. Thus, this study aimed to understand how long-term exposure to PM10, PM2.5, and NO2 contributed to COVID-19 fatality and mortality rates in São Paulo state in 2020. Air quality data between 2015 and 2019 in 64 monitoring stations within 36 municipalities were considered. The COVID-19 fatality was calculated considering cases and deaths from the government's official data and the mortality rate was calculated considering the 2020 population. Linear regression models were well-fitted for PM2.5 concentration and fatality (R2 = 0.416; p = 0.003), NO2 concentration and fatality (R2 = 0.232; p = 0.005), and NO2 concentration and mortality (R2 = 0.273; p = 0.002). This study corroborates other authors' findings and enriches the discussion for having considered a longer time series to represent long-term exposure to the pollutants and for having considered one of the regions with the highest incidence of COVID-19 in the world. Thus, it reinforces measures to reduce the concentration of air pollutants which are essential for public health and will increase the chance to survive in future respiratory disease epidemics.

Blockade of caspase cascade overcomes malaria-associated acute respiratory distress syndrome in mice.

Malaria is an enormous burden on global health that caused 409,000 deaths in 2019. Severe malaria can manifest in the lungs, an illness known as acute respiratory distress syndrome (ARDS). Not much is known about the development of malaria-associated ARDS (MA-ARDS), especially regarding cell death in the lungs. We had previously established a murine model that mimics various human ARDS aspects, such as pulmonary edema, hemorrhages, pleural effusion, and hypoxemia, using DBA/2 mice infected with Plasmodium berghei ANKA. Here, we explored the mechanisms and the involvement of apoptosis in this syndrome. We found that apoptosis contributes to the pathogenesis of MA-ARDS, primarily as facilitators of the alveolar-capillary barrier breakdown. The protection of pulmonary endothelium by inhibiting caspase activation could be a promising therapeutic strategy to prevent the pathogenicity of MA-ARDS. Therefore, intervention in the programmed death cell mechanism could help patients not to develop severe malaria.

Endothelial Protein C Receptor Could Contribute to Experimental Malaria-Associated Acute Respiratory Distress Syndrome.

The severity of Plasmodium falciparum malaria is associated with parasite cytoadherence, but there is limited knowledge about the effect of parasite cytoadherence in malaria-associated acute respiratory distress syndrome (ARDS). Our objective was to evaluate the cytoadherence of infected red blood cells (iRBCs) in a murine model of ARDS and to appraise a potential function of endothelial protein C receptor (EPCR) in ARDS pathogenesis. DBA/2 mice infected with P. berghei ANKA were classified as ARDS- or hyperparasitemia- (HP-) developing mice according to respiratory parameters and parasitemia. Lungs, blood, and bronchoalveolar lavage were collected for gene expression or protein analyses. Primary cultures of microvascular lung endothelial cells from DBA/2 mice were analyzed for iRBC interactions. Lungs from ARDS-developing mice showed evidence of iRBC accumulation along with an increase in EPCR and TNF concentrations. Furthermore, TNF increased iRBC adherence in vitro. Dexamethasone-treated infected mice showed low levels of TNF and EPCR mRNA expression and, finally, decreased vascular permeability, thus protecting mice from ARDS. In conclusion, we identified that increased iRBC cytoadherence in the lungs underlies malaria-associated ARDS in DBA/2-infected mice and that inflammation increased cytoadherence capacity, suggesting a participation of EPCR and a conceivable target for drug development.

Correction: Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice.

[This corrects the article DOI: 10.1371/journal.ppat.1006054.].

Avaliação do citoesqueleto e da barreira endotelial pulmonar na malária experimental / Evaluation of the cytoskeleton and pulmonary endothelial barrier in experimental malaria

Infecções por Plasmodium sp. podem levar a um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda e síndrome do desconforto respiratório agudo (LPA/SDRA). Inflamação aguda, lesão do endotélio alveolar e do parênquima pulmonar, disfunção e aumento da permeabilidade da barreira alvéolo-capilar e, consequente, formação de edema, caracterizam esta síndrome. O modelo experimental, que utiliza o parasita murino Plasmodium berghei ANKA e camundongos da linhagem DBA/2, é empregado no estudo de mediadores imunológicos e fatores que propiciam o estabelecimento das lesões pulmonares associados à LPA/SDRA. Diversos estímulos podem atuar diretamente no aumento da permeabilidade endotelial por meio da desestabilização dos microtúbulos, rearranjo dos microfilamentos de actina e contração das células endoteliais, via sinalização de Rho-GTPases, causando disfunção da barreira endotelial. Desta forma, este trabalho tem como objetivo avaliar as alterações do citoesqueleto em células endoteliais primárias pulmonares de camundongos DBA/2 (CEPP-DBA/2), as vias de sinalização das principais Rho-GTPases e o estresse oxidativo, causados pela presença de eritrócitos parasitados com esquizontes de P. berghei ANKA (EP-PbA). As CEPP-DBA/2 foram estimuladas com TNF, VEGF ou IFNγ, em diferentes tempos de exposição, seguido da incubação com EP-PbA. Assim, foram realizados ensaios de imunofluorescência para análise do rearranjo de microfilamentos de actina e da desestabilização de microtúbulos. As vias de sinalização das Rho-GTPases foram avaliadas por Western blot, para as expressões proteicas de RhoA, Cdc42 e MLC. Além disso, ensaio fluorométrico foi realizado para detectar a produção de espécies reativas de oxigênio, resultantes do estímulo com eritrócitos parasitados. CEPP-DBA/2 estimuladas por EP-PbA, VEGF, TNF ou IFNγ, em associação ou não, apresentaram alterações morfológicas nos microfilamentos de actina e aumento dos espaços interendoteliais. Imagens de imunofluorescência também mostram desestabilização de microtúbulos e desfosforilação de FAK, causadas por EP-PbA. Os ensaios de permeabilidade validam que os eritrócitos parasitados com formas maduras de P. berghei induziram aumento da permeabilidade microvascular nas CEPP-DBA/2. Além disso, estas células, estimuladas com EP-PbA, demonstraram elevada produção de espécies reativas de oxigênio (EROs), o que pode estar contribuindo com o desenvolvimento de estresse oxidativo e com a injúria endotelial, assim como, com o aumento da permeabilidade vascular. O mais interessante é que estas alterações endoteliais podem estar relacionadas ao aumento da razão RhoA/Cdc42, da expressão proteica de MLC fosforilada e do sinal de ativação de RhoA. Em conjunto, estes resultados mostram envolvimento dos eritrócitos parasitados com esquizontes de Plasmodium berghei ANKA na desorganização do citoesqueleto e na disfunção da barreira alvéolo-capilar, via RhoA/Rho-kinase, o que pode estar contribuindo com a patogênese da LPA/SDRA associada à malária

Infections by Plasmodium sp. can lead to a serious respiratory condition with pulmonary complications, named acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Acute inflammation, alveolar endothelium and lung parenchyma injuries, dysfunction and increased permeability of the pulmonary alveolar-capillary barrier and consequent formation of edema characterize this syndrome. Several stimuli can directly increase endothelial permeability through actin microfilaments rearrangement, via Rho- GTPases signaling, leading to endothelial barrier dysfunction. DBA/2 mice infected with Plasmodium berghei ANKA develop ALI/ARDS similar to that observed in humans. The purpose of this research was to assess cytoskeletal changes in DBA/2 mice primary microvascular lung endothelial cells (PMLEC), verify the signaling pathways of the Rho- GTPases and analyze the oxidative stress on these cells in the presence of P. berghei ANKA-infected red blood cells (PbA-iRBC). PMLEC were stimulated by TNF, VEGF or IFNγ followed by incubation with PbA-iRBC. Immunofluorescence assays were performed to analyze actin microfilaments rearrangement and microtubules destabilization. Western blot for RhoA, Cdc42 and MLC proteins were conducted to assess alterations in signaling pathways of Rho-GTPases. In addition, a fluorimetric assay was performed to detect the production of reactive oxygen species resulting from PbA-iRBC stimulus. P. berghei ANKA, VEGF, TNF and IFNγ stimuli, in association or not, caused morphological disturbances in actin microfilaments of PMLEC and an increase of intercellular spaces. Moreover, immunofluorescence images showed microtubules destabilization and FAK dephosphorylation in these cells, caused by PbA-iRBC. The permeability assay showed that PbA-iRBC induced an increase of microvascular permeability in PMLEC. In addition, PMLEC stimulated by PbA-iRBC, showed elevated production of ROS, which may be contributing to oxidative stress and increasing the damage of endothelial cells, as well as an increase of vascular permeability. Interestingly, these endothelial changes may be related to the increased RhoA/Cdc42 protein expressions ratio, augmented protein expression of phosphorylated MLC and RhoA activation signal. Taken together, these data demonstrate the involvement of P. berghei ANKA-infected red blood cells in cytoskeleton disorganization and alveolar-capillary barrier dysfunction, through of RhoA / Rho-kinase signaling pathway, which may contribute to ALI/ARDS pathogenesis

Association of Heme Oxygenase 1 with Lung Protection in Malaria-Associated ALI/ARDS.

Malaria is a serious disease, caused by the parasite of the genus Plasmodium, which was responsible for 440,000 deaths in 2015. Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one of the main clinical complications in severe malaria. The murine model DBA/2 reproduces the clinical signs of ALI/ARDS in humans, when infected with Plasmodium berghei ANKA. High levels of HO-1 were reported in cases of severe malaria. Our data indicated that the HO-1 mRNA and protein expression are increased in mice that develop malaria-associated ALI/ARDS (MA-ALI/ARDS). Additionally, the hemin, a HO-1 inducing drug, prevented mice from developing MA-ALI/ARDS when administered prior to the development of MA-ALI/ARDS in this model. Also, hemin treatment showed an amelioration of respiratory parameters in mice, high VEGF levels in the sera, and a decrease in vascular permeability in the lung, which are signs of ALI/ARDS. Therefore, the induction of HO-1 before the development of MA-ALI/ARDS could be protective. However, the increased expression of HO-1 on the onset of MA-ALI/ARDS development may represent an effort to revert the phenotype of this syndrome by the host. We therefore confirm that HO-1 inducing drugs could be used for prevention of MA-ALI/ARDS in humans.

Targeting Neutrophils to Prevent Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome in Mice.

Malaria remains one of the greatest burdens to global health, causing nearly 500,000 deaths in 2014. When manifesting in the lungs, severe malaria causes acute lung injury/acute respiratory distress syndrome (ALI/ARDS). We have previously shown that a proportion of DBA/2 mice infected with Plasmodium berghei ANKA (PbA) develop ALI/ARDS and that these mice recapitulate various aspects of the human syndrome, such as pulmonary edema, hemorrhaging, pleural effusion and hypoxemia. Herein, we investigated the role of neutrophils in the pathogenesis of malaria-associated ALI/ARDS. Mice developing ALI/ARDS showed greater neutrophil accumulation in the lungs compared with mice that did not develop pulmonary complications. In addition, mice with ALI/ARDS produced more neutrophil-attracting chemokines, myeloperoxidase and reactive oxygen species. We also observed that the parasites Plasmodium falciparum and PbA induced the formation of neutrophil extracellular traps (NETs) ex vivo, which were associated with inflammation and tissue injury. The depletion of neutrophils, treatment with AMD3100 (a CXCR4 antagonist), Pulmozyme (human recombinant DNase) or Sivelestat (inhibitor of neutrophil elastase) decreased the development of malaria-associated ALI/ARDS and significantly increased mouse survival. This study implicates neutrophils and NETs in the genesis of experimentally induced malaria-associated ALI/ARDS and proposes a new therapeutic approach to improve the prognosis of severe malaria.

Predictive criteria to study the pathogenesis of malaria-associated ALI/ARDS in mice.

Malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS) often results in morbidity and mortality. Murine models to study malaria-associated ALI/ARDS have been described; we still lack a method of distinguishing which mice will develop ALI/ARDS before death. This work aimed to characterize malaria-associated ALI/ARDS in a murine model and to demonstrate the first method to predict whether mice are suffering from ALI/ARDS before death. DBA/2 mice infected with Plasmodium berghei ANKA developing ALI/ARDS or hyperparasitemia (HP) were compared using histopathology, PaO2 measurement, pulmonary X-ray, breathing capacity, lung permeability, and serum vascular endothelial growth factor (VEGF) levels according to either the day of death or the suggested predictive criteria. We proposed a model to predict malaria-associated ALI/ARDS using breathing patterns (enhanced pause and frequency respiration) and parasitemia as predictive criteria from mice whose cause of death was known to retrospectively diagnose the sacrificed mice as likely to die of ALI/ARDS as early as 7 days after infection. Using this method, we showed increased VEGF levels and increased lung permeability in mice predicted to die of ALI/ARDS. This proposed method for accurately identifying mice suffering from ALI/ARDS before death will enable the use of this model to study the pathogenesis of this disease.