key: cord-0761178-0zwakfah
authors: Rebuli, Meghan E.; Brocke, Stephanie A.; Jaspers, Ilona
title: Impact of inhaled pollutants on response to viral infection in controlled exposures
date: 2021-07-10
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2021.07.002
sha: fd1c907c75110b593d1150ba1f42e0d91a640863
doc_id: 761178
cord_uid: 0zwakfah

Air pollutants are a major source of increased risk of disease, hospitalization, morbidity, and mortality worldwide. The respiratory tract is a primary target of potential concurrent exposure to both inhaled pollutants and pathogens, including viruses. Although there are various associative studies linking adverse outcomes to co- or subsequent exposures to inhaled pollutants and viruses, knowledge about causal linkages and mechanisms by which pollutant exposure may alter human respiratory responses to viral infection is more limited. In this article, we review what is known about the impact of pollutant exposure on antiviral host defense responses and describe potential mechanisms by which pollutants can alter the viral infection cycle. This review focuses on evidence from human observational and controlled exposure, ex vivo, and in vitro studies. Overall, there are a myriad of points throughout the viral infection cycle that inhaled pollutants can alter to modulate appropriate host defense responses. These alterations may contribute to observed increases in rates of viral infection and associated morbidity and mortality in areas of the world with high ambient pollution levels or in people using tobacco products. Although the understanding of mechanisms of interaction is advancing through controlled in vivo and in vitro exposure models, more studies are needed because emerging infectious pathogens, such as severe acute respiratory syndrome coronavirus 2, present a significant threat to public health.

Air pollutants are a major source of increased risk for disease, hospitalization, morbidity, and mortality worldwide. [1] [2] [3] Because the lungs filter more than 12,000 liters of air per day, they are a primary target of potential concurrent exposure to both inhaled pollutants and pathogens, including viruses. Although there are various associative studies linking adverse outcomes to co-or subsequent exposures to inhaled pollutants and viruses, causal linkages and mechanisms by which pollutant exposure may alter human respiratory response to viral infection are more limited. [4] [5] [6] [7] The limited knowledge on how pollutants may impact response to viral infection has been acutely highlighted with the onset of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and the resulting flurry of review articles that nicely summarize identified associations but do little to capture causal linkages. 8, 9 In this article, we will focus on the impact of pollutant exposure on antiviral host defense responses and describe potential mechanisms by which pollutants can alter the viral infection cycle that have been identified through human observational and controlled exposure studies, as well as mechanistic in vitro studies.

In addition to experimental inoculation with rhinovirus, 10 the Food and Drug Administration-approved live attenuated influenza virus (LAIV) vaccine presents a unique and underused model to mechanistically explore the interaction between pollutants and respiratory viruses in humans in vivo. The live, but attenuated virus replicates only at lower temperatures in the nasal passages, but causes similar innate and adaptive host defense responses, similar to community-acquired infections. 11 Hence, inoculation with LAIV allows the study of active viral infection and immune responses in the nasal passages of human volunteers, without risking the overall safety of the participants. The LAIV model is also ideal for studying the effects of viruses for whom the primary point of entry and activation is likely the nasal passage, such as influenza and SARS-CoV-2. 12 It should be noted that the use of the LAIV model is limited by contraindications in individuals with severe allergic reaction to its ingredients and children and adolescents on asprin therapy. However, the easyto-use LAIV model of viral infection is well suited to determine the effects of ambient air pollutants, such as diesel exhaust and wood smoke, as well as use of tobacco products, including cigarettes and e-cigarettes on host defense responses.

Diesel exhaust contributes a significant percentage of trafficrelated air pollution in many cities, due to increased needs for transportation in urban areas. 13 Acute exposure effects include nose and eye irritation, fatigue, headache, and nausea, whereas chronic exposures increase respiratory symptoms, reduce lung function, and change inflammatory profiles. 13 Diesel exhaust is also known to enhance allergic inflammation and due to its contribution to ambient particulate matter (PM) is likely associated with increased susceptibility to viral infection. To explore this hypothesis, a study was conducted exposing adults who were healthy or had allergic rhinitis to diesel exhaust before inoculation with LAIV. 14 Diesel exhaust exposure was associated with increased LAIV-induced eotaxin-1, eosinophil cationic protein, and influenza RNA in nasal cells, supporting previous literature and animal studies, 15 suggesting that diesel exhaust can act as an allergic adjuvant, promoting inflammation and potentially reducing viral clearance, especially in individuals with underlying allergy.

Wood smoke is a major and continually growing source of PM in the United States and globally as wildfire events occur with increasing frequency. 16 Furthermore, a substantial percentage of the world population uses wood and biomass for heating or cooking, resulting in up to 30% of ambient fine PM in some areas of the United States during the winter months, and greater percentages in developing countries. [17] [18] [19] Exposure to wood smoke and biomass is associated with increased morbidity, including reduced lung function, upper respiratory tract symptoms, and inflammation, and mortality due to respiratory infection in many populations. [20] [21] [22] [23] [24] [25] The effects of wood smoke on LAIV were examined in healthy study participants exposed to either 500 mg/m 3 of wood smoke particulate or filtered air for 2 hours. 26 LAIV-induced IFN-g-induced protein 10 levels, as measured in nasal lavage, were suppressed in the nasal mucosa of all participants. Furthermore, an Exposure by Sex interaction was observed, with males showing greater inflammation-related gene expression, whereas in females, host defense-related gene expression was mildly decreased in nasal lavage fluid cells. These results support sex-specific responses to viral infection, which can be augmented with the additional interaction of pollutant exposure, in this case wood smoke.

Epidemiological evidence repetitively links tobacco smoke exposure to increased risk of viral infection. 7, 27 To better understand the mechanisms of this association between tobacco product use and respiratory infection, various studies in users and nonusers have been conducted using the LAIV model. An observational cohort study was first conducted to understand differential responses in active cigarette smokers, nonsmokers, and those exposed to secondhand smoke after LAIV inoculation. 28 In this initial study, nasal lavage fluid IL-6 response was significantly suppressed in active smokers, along with decreased median IFN-g-induced protein 10, and IFN-g, whereas viral RNA levels were significantly increased in smokers as compared with nonsmokers. Individuals exposed to secondhand smoke generated responses that were intermediate between active smokers and nonsmokers, suggesting mechanisms for increased susceptibility to infection in tobacco product users and those exposed secondhand.

Most recently, the LAIV model has been used to investigate potential mechanisms by which electronic cigarettes (e-cigarettes) may affect susceptibility to viral infection. 29 Because e-cigarettes were deemed a tobacco product 30 and, similar to cigarettes, contain nicotine and other additives, it was hypothesized that e-cigarettes may also increase susceptibility to respiratory viral infection. Thus, a cohort of cigarette smokers, e-cigarette users, and nonsmokers was inoculated with LAIV and monitored for effects on immune gene expression and antibody response. 29 Overall, there was substantial downregulation of critical immune genes in nasal biopsy samples, especially in e-cigarette users compared with nonsmokers. In particular, altered host defense mediators, IFN-g, IL-6, and IL12p40, were found in cigarette smokers and e-cigarette users when compared with nonsmokers. It was also found that nasal mucosal anti-LAIV IgA levels were significantly lower in cigarette smokers and e-cigarette users than in nonsmokers, demonstrating that e-cigarette use can alter response to viral infection, affecting host defense mediators and antibody production.

Although the LAIV model has provided significant insight into the effects of inhaled pollutants on viral infection in humans in vivo, there are still many aspects of the effects of inhaled pollutant exposures on response to viral infection that are poorly understood. For example, many of these studies focus on acute exposures, whereas most human population is exposed chronically to inhaled pollutants, which may impact response to viral infection. Furthermore, only a limited number of model pollutants have been investigated and more research is needed on pollutants such as PM, gaseous pollutants such as NO 2 and ozone, and pollutant mixtures. The field would also benefit from longer follow-up periods in LAIV-based studies to understand the effect of pollutant exposure on adaptive immunity and studies that include the exploration of potential targeted interventions to prevent pollution-induced effects (eg, therapeutics, dietary, and use of personal and household filtration devices). 31,32

Many viral pathogens, including influenza, parainfluenza, and coronaviruses, including SARS-CoV-2, depend in part on proteolytic activation of the virus, regulating the ability of the virus to enter the host cell 33, 34 (Fig 1, step 1 ). For example, attachment and subsequent entry of SARS-CoV-2 into the host cell occurs via binding of the virus spike (S)-protein to angiotensinconverting enzyme 2 (ACE2) receptors expressed on many different cell types. The S protein has 2 functional domains: the S1 domain, which binds to the ACE2 receptor, and the S2 domain, which mediates the fusion between the virus and the host cell membrane. Proteolytic cleavage of the S protein is required to enable the S2 domain to become active. 35 This can be accomplished by a number of proteases, such as transmembrane protease, serine 2 (TMPRSS2), furin, cathepsins, neutrophil elastase (reviewed in El-Shimy et al 35 and Meyer and Jaspers 36 ), and potentially other proteases that prime and regulate viral entry of SARS-CoV-2 into the host cell. Human nasal and bronchial mucosa abundantly express ACE2 and are rich sources of proteases, such as TMPRSS2, furin, cathepsins, and other proteases. 33, [37] [38] [39] [40] Inhibition of protease activity in the respiratory mucosa has been explored as a therapeutic target, but can also be regulated endogenously by antiproteases, such as alpha 1 antitrypsin and secretory leukocyte protease inhibitor. Pollutant exposure has been shown to dysregulate the protease/antiprotease balance in the respiratory mucosa. For example, exposure to ozone increases secreted levels of TMPRSS2 and decreases levels of secretory leukocyte protease inhibitor, which was linked to increased viral entry of influenza virus. 41 Similarly, analysis of lung tissue from mice chronically exposed to ozone showed elevated expression of Tmprss2. 42 Many pollutants also enhance neutrophil elastase levels in the respiratory tract.

The effects of inhaling cigarette smoke on protease/antiprotease balance in the respiratory mucosa are well established and causally linked to smoking-related lung diseases. [43] [44] [45] [46] Some groups have shown that expression of ACE2 and TMPRSS2 is similar in bronchial epithelial cells from current and never smokers, 47 whereas others have observed significant increases in either ACE2 and/or TMPRSS2 in cells from smokers. [48] [49] [50] Expression and activity of antiproteases, including secretory leukocyte protease inhibitor and alpha 1 antitrypsin, are also modified by smoking. 51, 52 Controlled exposures to model particulate air pollutants, such as diesel exhaust, 53 demonstrate increased expression of ACE2 and TMPRSS2 in human pluripotent stem cell-derived alveolar epithelial cells and alveolar organoids. Expression of ACE2 and TMPRSS2 might be regulated by Randomized, placebocontrolled study 500 mg/m 3 wood smoke particles or filtered air and LAIV inoculation

Healthy adults IP-10 suppression in nasal mucosa in all exposed participants. Increased inflammatory-related gene expression in male exposed subjects and decreased host defense-related gene expression in female exposed subjects, compared with controls several consensus motifs for binding of the aryl hydrocarbon receptor, a common pathway activated by ambient air pollutants. 54, 55 The pulmonary surfactant, which lines the alveoli, serves as another line of defense against pathogens. Surfactant proteins (SPs) SP-A and SP-D assist in the clearance of bacteria and virus from the lung by directly opsonizing pathogens and enhancing their uptake by phagocytic immune cells. 56 Air pollutants such as PM and ozone as well as cigarette smoke have all been shown to interfere with the pulmonary surfactant. Specifically, ozone A1AT, Alpha 1 antitrypsin; AhR, aryl hydrocarbon receptor; BAL, bronchoalveolar lavage; HA, hemaggluttinin; HAT, human airway trypsin-like protease; IL-12p40, IL-12 subunit p40; IP-10, IFN-g-induced protein 10; NF-kB, nuclear factor kappa-light-chain-enhancer; NK, natural killer; ROR, receptor tyrosine kinase-like orphan receptor; RSV, respiratory syncytial virus; SLPI, secretory leukocyte protease inhibitor; STAT1, signal transducer and activator of transcription 1; TLR, Toll-like receptor. exposure decreases the phagocytic index of SP-A. 57 Diesel exhaust exposures dampened production of SP-D in allergensensitized individuals 58 and decreased SP-A and SP-D levels in mice, which was associated with increased influenza infections. 59 Finally, smokers, who are generally more susceptible to viral infections, have been reported to have less SP-A and SP-D present in bronchoalveolar lavage fluid compared with nonsmokers. 60

Once the virus enters the host cell, it is met with an organized antiviral host defense response, aimed at limiting the replication and release of new virions. 61 Infection triggers a rapid antiviral signaling pathway, beginning by activation of Toll-like receptors and resulting in the secretion of type I and type III interferons (IFNs) by the infected cell (Fig 1, steps 2-4) . Type I IFNs act in either an autocrine fashion or a paracrine fashion to stimulate the second wave of antiviral responses by activating the Janus kinase/signal transducer and activator of transcription signaling pathway and resulting in the expression of numerous IFNstimulated genes, whose role is to shut off or limit the replication of the virus in the host cell. 61 Similarly, type III IFNs, which are massively induced in respiratory epithelium on viral infection, activate Janus kinase/signal transducer and activator of transcription pathways to upregulate expression of immunomodulatory genes. 62 The effects of cigarette smoke exposure on antiviral host defense responses have been well studied, including recent studies demonstrating that exposure to cigarette smoke increases SARS-CoV-2 infection. 63 Effects of smoking include inhibition of Toll-like receptor activation, inactivation of nuclear factor kappa-light-chain-enhancer response, 64 epigenetic modulation of IFN response genes, 65 decreased expression of antiviral host defense genes, 66, 67 and decreased activation of IFN signaling pathways. 68, 69 Much less is known about ambient air pollutant effects on antiviral host defense responses. In the context of model air pollutant exposures, such as diesel exhaust and ozone, type I IFN responses were not affected by the exposures and increase with the level of infection. 7, 41, 70 In contrast, exposure to ambient PM suppresses virus-induced IFN-b expression in macrophages and epithelial cells. 41, 71, 72 Hence, whether and to what extent inhalation of ambient air pollutants modulates the ability to mount an effective antiviral host defense response needs to be further explored.

Control of viral replication and pathogenesis depends on the ability of infected epithelial cells to limit spread of virus to neighboring cells while also increasing the production of cytokines and chemokines that recruit virus-specific immune effector cells to the infection site (Fig 1, step 5) . However, increases in pollutant-induced oxidative stress, which has been observed after diesel exhaust, PM, and ozone exposures, 73 can result in epithelial cell damage, limiting their capacity to function. 7, 9, 74, 75 Release of cytokines and chemokines, which recruit and activate immune cells, is also critical for coordinated innate and adaptive immune responses. 76 These include IFN-inducible cytokines, such as C-X-C motif chemokine ligand (CXCL)9/ monokine induced by IFN-g, CXCL10/IFN-g-induced protein 10, and CXCL12/stromal cell-derived factor 1 alpha, as well as neutrophil chemokines (CXCL8/IL-8 and CXCL1/GROa), lymphocyte chemoattractants (C-C motif chemokine ligand 5/ RANTES and CXCL16), and monocyte chemoattractants (C-C motif chemokine ligand 2/monocyte chemoattractant protein 1). The production of many of these inducible cytokines has been shown to be altered by pollutant exposure including tobacco products and wood smoke, indicating potentially impaired coordinated immune responses to infection. 26, 29 Along with reduced production of important immune signaling molecules, the functions of innate immune cells recruited by these signals are also impaired by inhaled pollutant exposure (Fig 1, step 6 ). For example, neutrophil phagocytosis and neutrophil extracellular trap formation, macrophage phagocytosis, and cytotoxic natural killer cell responses were all altered ex vivo with e-cigarette exposure. 77 Furthermore, gaseous pollutants such as ozone have been shown to alter macrophage function 7,74 while diesel exhaust particle exposure induces apoptosis of human macrophages at levels that cause minimal cytotoxicity to bronchial epithelial cells. 78 In addition, critical to a coordinated response to future viral infection is the production of virus-specific antibodies, which have recently been shown to be impaired with exposure to e-cigarettes in an LAIV inoculation model. 29 

Overall, there are a myriad of ways throughout the viral infection cycle that inhaled pollutants can alter appropriate host defense responses, which are summarized in Table I . These alterations may contribute to observed increases in rates of viral infection and associated morbidity and mortality in areas of the world with high ambient pollution levels or in people using tobacco products. There are limitations to using human controlled exposure models, such as the limitation of LAIV only permitting viral replication in the nose and limiting our ability to study effects that may differ in the lower airway. [79] [80] [81] Despite these limitations, this experimental design does allow for the examination of the effects of pollutants and viral infection in a model with immediate public health relevance. Controlled in vivo and in vitro exposure models have allowed us to begin to explore these mechanisms, yet as new viruses and pollutants continue to emerge, further study is needed. The current coronavirus disease 2019 pandemic has illustrated that better understanding of potential interactions between pollutants or toxicant exposures and effects on antiviral host defense responses is needed.

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