key: cord-0998195-rbh5sh02 authors: Frontera, Antonio; Cianfanelli, Lorenzo; Vlachos, Kostantinos; Landoni, Giovanni; Cremona, George title: Severe air pollution links to higher mortality in COVID-19 patients: the “double-hit” hypothesis. date: 2020-05-21 journal: J Infect DOI: 10.1016/j.jinf.2020.05.031 sha: b8f8aa80224023f51e1c5dc0d5328a3df27c41ec doc_id: 998195 cord_uid: rbh5sh02 OBJECTIVES: In areas of SARS-CoV-2 outbreak worldwide mean air pollutants concentrations vastly exceed the maximum limits. Chronic exposure to air pollutants have been associated with lung ACE-2 over-expression which is known to be the main receptor for SARS-coV2. The aim of this study was to analyse the relationship between air pollutants concentration (PM 2.5 and NO2) and COVID-19 outbreak, in terms of transmission, number of patients, severity of presentation and number of deaths. METHODS: COVID-19 cases, ICU admissions and mortality rate were correlated with severity of air pollution in the Italian regions. RESULTS: The highest number of COVID-19 cases were recorded in the most polluted regions with patients presenting with more severe forms of the disease requiring ICU admission. In these regions, mortality was two-fold higher than the other regions. CONCLUSIONS: From the data available we propose a “double-hit hypothesis”: chronic exposure to PM 2.5 causes alveolar ACE-2 receptor overexpression. This may increase viral load in patients exposed to pollutants in turn depleting ACE-2 receptors and impairing host defences. High atmospheric NO2 may provide a second hit causing a severe form of SARS-CoV-19 in ACE-2 depleted lungs resulting in a worse outcome. From the data available we propose a "double-hit hypothesis": chronic exposure to PM 2.5 causes alveolar ACE-2 receptor overexpression. This may increase viral load in patients exposed to pollutants in turn depleting ACE-2 receptors and impairing host defences. High atmospheric NO2 may provide a second hit causing a severe form of SARS-CoV-19 in ACE-2 depleted lungs resulting in a worse outcome. The potential correlation between air pollution and outbreaks of COVID-19 have been described in the recent literature [1] [2] [3] [4] . While SARS-CoV-2 diffusion has been documented in every country and a pandemic has been declared [5] , there is still debate about the death rate and the severity of pneumonia encountered in specific countries, like Italy and China. In Italy the majority of severe cases and deaths occurred in the Po Valley, accounting for three quarters of the entire Italian caseload, pointing to a specific characteristic of the territory that may favour spread and severity of SARS-CoV-2 virus. The worst COVID-19 outbreaks have been reported in the Po valley, in the cities of Lodi, Cremona, Bergamo and Brescia which are known to be the four Italian cities with highest pollution levels [6] . Here we propose a hypothesis linking PM 2.5 and NO2 concentrations and the severity of SARS-CoV-2 which could have important implications in the prevention and management of the pandemic. We collected population data provided by the Italian Civil Protection Agency and data concerning the emissions of air pollutants registered in every Italian region (air pollution data have been collected from Air-matters App and websitehttps://www.air-matter.com). Pearson correlation analysis was used to examine relationship between COVID- 19 A clear association is apparent between PM 2.5 levels and COVID-19 outbreak distribution (see Table 1 and Table 2 ). Our data show a significant relationship between the mean PM 2.5 concentration during February 2020, one month before the beginning of the outbreak, and the number of COVID-19 cases per region (updated to March 31 st ), confirming how more polluted areas are the ones where the contagion is more widespread. More significantly patients in polluted areas present with more severe forms of the disease requiring ICU. Mortality is two-fold higher than the other regions despite similar rates of ICU admission (crude death rate 14 vs 7 %) (see Figure 1 ). One explanation for the geographical disparity in the number of cases, is that the high level of pollutants may favour the transmission of viral illnesses and increase their persistence in the community. A recent study indicates that the virus may remain viable in aerosol for some hours [7] . The high pollutant levels and specific climate in the Po Valley may enhance aerosol stability of the virus, explaining the on-going outbreaks and the associated high rate of contagion. However, this does not explain the high fatality rate in the aforementioned areas. Atmospheric pollution may have a more fundamental role by increasing the susceptibility to the infection and mining pulmonary defence mechanisms favouring the establishment of more severe forms of the disease. Pulmonary RAS consists of two axes participating in local inflammatory responses with opposing functions [9] : ACE/AngII/AT1R axis is involved in release of pro-inflammatory cytokines (such as IL-6 and TNF-alfa); ACE-2/Ang1-7/Mas axis culminates in Mas activation that represses STAT3 and ERK exerting an anti-inflammatory effect. ACE-2 cleaves Ang II into the cardioprotective Ang 1-7, which acts through Mas receptors to counterbalance the detrimental effects of Ang II signalling. Therefore, ACE2 protects against RAS-induced injuries through two processes: 1) degrading Ang I and Ang II to limit substrate availability in the adverse ACE/Ang II/AT1 receptor axis; 2) generating Ang 1-7 to increase substrate availability in the protective ACE2/Ang 1-7/Mas receptor axis [9] . ACE-2 knock-out mice are more prone to develop lung injury after exposure to PM 2.5 and pulmonary repair in this context is attenuated relative to controls. This suggests a crucial role for ACE-2 in lung protection from air pollutants [10] . Chronic exposure to PM 2.5 in mice causes upregulation of pulmonary ACE expression and activity [10, 11] which may be a protective response to a chronic deleterious insult. Despite having normal lung structure and function, ACE2 knockout mice exhibit very severe pathology of acute respiratory distress syndrome (ARDS) compared with wild-type control mice [10, 12] . ACE-2 has been shown to be a co-receptor for viral entry of SARS-CoV-2, through interaction with viral spike proteins (VPS) [13] . ACE-2 is expressed mainly at alveolar level, explaining the viral tropism for lower airways. Binding and entry of both SARS-CoV and SARS-CoV-2 into human cells is facilitated by the interaction between receptor-binding domain (RBD) of the S1 subunit on viral spike glycoproteins with the ectodomain of ACE-2 [14] . SARS-CoV infection and challenge with recombinant SARS-Spike protein trigger a marked downregulation of ACE-2 expression in lungs and in cell culture causing more severe acute lung injury [15] . Viral depletion of ACE-2 appears to be crucial in mediating lung injury [16, 17] . We postulate that patients chronically exposed to high levels of PM 2.5 overexpress ACE-2 facilitating viral penetration and subsequent depletion of ACE-2 leads to more severe forms of the disease. This may explain the low incidence of severe pneumonia in children most of whom are asymptomatic. The limited exposure to PM 2.5 due to their young age may exempt them from pulmonary ACE-2 receptor overexpression. In China out of 72.314 infected patients <1% of the cases were children younger than 10 years old [18] and developed milder disease [19] . Similarly, chronic upregulation of ACE-2 in a PM 2.5 concentration-dependent manner could explain the high variability in clinical presentation ranging from asymptomatic patients to patients presenting with mild, moderate or severe form of the disease [20, 21] . Early in March 2020, the Italian village of Vo' Euganeo near Padua, an area with relatively low atmospheric levels of pollution (yearly means PM 2.5 and NO2 respectively 19 and 14μ g/m 3 [22] ) experienced an exceptionally high number of cases of SARS-CoV-2. Swabs were performed in all the 3.003 inhabitants. It was found that 50-75% of positive SARS-CoV2 patients were asymptomatic [23] . Mean viral loads of severe cases of SARS-CoV2 have been reported to be 60 times higher than mild cases [24] . In this case patients who had low exposure to PM 2.5 would have low pulmonary expression of ACE-2 with subsequent low viral load and mild symptoms. Numerous sources of NO2 exist, making it one of the most common and widespread air pollutants [25] with a geographical distribution overlapping that of PM 2.5. Acute inhalation of high concentrations of NO2 occurring in conditions such as Silo filler's disease causes increased permeability pulmonary oedema [26] . Long-term low-to moderatelevel air pollutant exposure, including NO2, is associated with a greater risk of developing ARDS after severe trauma [27] . Being a free radical, NO2 acts as a potent oxidant depleting anti-oxidant stores leading to impaired tissue defences, increased inflammatory response and cellular damage. Two days after exposure to moderate levels of NO2 in an ice arena, exposed individuals experienced dry cough (97%), headache (45%), haemoptysis (35%) and dyspnoea (65%) [28] . The acute intoxication was secondary to the malfunctioning of ice resurfacer engine associated with poor ventilation. Interestingly, anosmia is frequently present also in the context of NO2 intoxication [29] . Environmental concentrations of NO2 increase susceptibility to pneumonia by Klebsiella pneumoniae [30] and Streptococcus pneumoniae [31] in experimental murine models. The increased susceptibility seems to be linked to an impairment of pulmonary defence mechanisms, especially phagocytic activity at progressively higher concentrations [32, 33] and is associated with high mortality rates [34] . Rose et al. reported that mice exposed to NO2 prior to cytomegalovirus infection required a viral load 100-fold lower than in control mice and re-infection from viral sources was more common [35, 36] . In this model, mice exposed to 5 ppm of NO2 were readily developing viral From this initial observation, we formulated our working hypothesis according to which a linkage exists between the air pollution levels and COVID-19 outbreak, in terms of transmission, number of patients, severity of presentation and number of deaths. Air pollutants, (such as PM 2.5 and NO2) plus SARS-CoV-2 give a "double-hit" to the lungs leading to acute lung injury by attenuating tissue remodelling and influencing local inflammatory response. In Italy, the areas with the highest incidence of cases and deaths are the ones with levels of PM 2.5 and NO2 that are chronically high or with recent increases in the 2 months prior to the outbreak. Chronic exposure of the lungs to high levels of PM 2.5 causes upregulation of its protective mechanisms, such as ACE-2. SARS-CoV-2 has shown a specific affinity for ACE-2 receptors and overexpression in patients subjected to chronic pollutants could represent a trojan horse for viral infection. Moreover, the SARS-CoV-2 binding to ACE-2 may induce deficient anti-inflammatory action leading to acute lung injury, attenuating local tissue repair. It is therefore likely that patients who present overexpression are the ones more readily infected and the ones with more severe presentations. Chronic lung exposure to NO2 may favour viral injury due to local damage induced by oxidative stress and due to local reduction of macrophage function and adaptive immune responses. In addition, a putative role of NO2 in worsening pulmonary damage can be hypothesized in these patients. Many of the symptoms and signs of COVID-19 infection resemble that of moderate NO2 poisoning, including anosmia. It is possible that ACE-2 depletion following COVID-19 infection increases tissue vulnerability to NO2 toxicity that eventually contributes to the acute lung injury observed in patients with pneumonia-induced ARDS. (Figure 2) . Table 1 . Distribution of SARS-CoV-2 cases on March 31 th 2020 (total number of cases, ICU admitted patients, deaths) according to each Italian region and the mean value of PM 2.5 registered in each region during February 2020, the month before the beginning of the outbreak in Italy. (Air pollution data have been collected from Air-matters app which include daily measurements from air quality measurement stations all over Italian territory. Data from P.A. Bolzano, Sardegna, Valle d'Aosta, Molise and Basilicata were unavailable at the time of writing). Air Pollution and the Novel Covid-19 Disease: a Putative Disease Risk Factor COVID-19 as a factor influencing air pollution? 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WHO Guidelines for Indoor Air Quality: Selected Pollutants Silo-filler's disease Low to Moderate Air Pollutant Exposure and Acute Respiratory Distress Syndrome after Severe Trauma An outbreak of nitrogen dioxide-induced respiratory illness among ice hockey players Nitrogen dioxide pollution exposure is associated with olfactory dysfunction in older U Chronic Toxicity of Nitrogen Dioxide Health effects of short-term inhalation of nitrogen dioxide and ozone mixtures Modulation of Pulmonary Defense Mechanisms by Acute Exposures to Nitrogen Dioxide Short-term Exposure to Nitrogen Dioxide Enhances Susceptibility to Murine Respiratory Mycoplasmosis and Decreases Intrapulmonary Killing of Mycoplasma pulmonis Role of time as a factor in the toxicity of chemical compounds in intermittent and continuous exposures. part I. effects of continuous exposure The Pathophysiology of Enhanced Susceptibility to Murine Cytomegalovirus Respiratory Infection during Short-term Exposure to 5 ppm Nitrogen Dioxide Altered susceptibility to viral respiratory infection during short-term exposure to nitrogen dioxide We wish to thank all the people who assisted with this research, in particular: Rosalba Lembo (Msc, Anaesthesia and Intensive Care, San Raffaele Scientific Institute, Milan, Italy) for the graph and statistics;