“Air That Once Was Breath” Part 1: Wildfire-Smoke-Induced Mechanisms of Airway Inflammation – “Climate Change, Allergy and Immunology” Special IAAI Article Collection: Collegium Internationale Allergologicum Update 2023

Abstract

Background:

Wildfires are a global concern due to their wide-ranging environmental, economic, and public health impacts. Climate change contributes to an increase in the frequency and intensity of wildfires making smoke exposure a more significant and recurring health concern for individuals with airway diseases. Some of the most prominent effects of wildfire smoke exposure are asthma exacerbations and allergic airway sensitization. Likely due to the delayed recognition of its health impacts in comparison with cigarette smoke and industrial or traffic-related air pollution, research on the composition, the mechanisms of toxicity, and the cellular/molecular pathways involved is poor or non-existent.

Summary:

This review discusses potential underlying pathological mechanisms of wildfire-smoke-related allergic airway disease and asthma. We focused on major gaps in understanding the role of wildfire smoke composition in the development of airway disease and the known and potential mechanisms involving cellular and molecular players of oxidative injury at the epithelial barrier in airway inflammation. We examine how PM2.5, VOCs, O₃, endotoxin, microbes, and toxic gases may affect oxidative stress and inflammation in the respiratory mucosal barrier. We discuss the role of AhR in mediating smoke’s effects in alarmin release and IL-17A production and how glucocorticoid responsiveness may be impaired by IL-17A-induced signaling and epigenetic changes leading to steroid-resistant severe airway inflammation.

Key Message:

Effective mitigation of wildfire-smoke-related respiratory health effects would require comprehensive research efforts aimed at a better understanding of the immune regulatory effects of wildfire smoke in respiratory health and disease.

Introduction

Among its recognized health effects, wildfire-smoke-induced asthma exacerbations and allergic airway sensitization count prominently [1-6]. Components of wildfire smoke carry specific pathogens and allergens [7,8] and act as powerful toxic irritants. How wildfire smoke components may damage the respiratory epithelial barrier and sensitize for allergic and inflammatory response is poorly understood. While the immediate consequences (fatalities, emergency room visits, and stress caused by the destruction of homes, livelihoods, and mass evacuations) are readily acknowledged, little attention has been paid to the medium- and long-term respiratory health effects of wildfires. Of great additional concern is the question of how vulnerable populations, children, the elderly, pregnant women, and those from marginalized communities (due to ethnic or socioeconomic status), become at high risk of developing allergic airway disease upon repeated wildfire exposures [8-10]. Here, we aimed to discuss currently available information with a focus on potential cellular and molecular pathways affecting respiratory mucosal barrier function and airway inflammation.

Wildfires generate plums of air pollutants from smoke that quickly spread reducing air quality to hazardous levels across vast areas [11]. The source and composition of inhaled smoke are important as it may elicit differential respiratory effects [4, 9, 12-30]. However, it is not well understood how specific smoke components may induce differential disease outcomes, what are the most toxic components, and how those work. For this reason, workshop reports from the American Thoracic Society in 2021 [31] and National Academies of Sciences in 2022 [32] identified health effect studies on wildfire smoke fuel type, combustion phase (i.e., flaming vs. smoldering), concentration, and exposure duration (i.e., acute vs. repeated episodic) as top priority research areas. We reference here a summary list that was prepared and updated in 2020 by the Office of Environmental Health Hazard Assessment (OEHHA) on inhalational toxicants for their acute, 8-h, and chronic reference exposure level (https://oehha.ca.gov/air/general-info/oehha-acute-8-hour-and-chronic-reference-exposure-level-rel-summary) [33]. The most studied major wildfire smoke components with relevance in eliciting and exacerbating allergic and chronic airway diseases are particulate matter (PM), volatile organic compounds (VOCs), and ozone (O₃). Over the last 3 years in Northern California, the highest ever concurrent O₃ and PM concentrations were recorded, often for weeks at a time.

For review purposes, we defined wildfires as wild and prescribed forest fires, tropical deforestation fires, peat fires, agricultural burning, and grass fires. We compared some findings on wildfire smoke, combustion of indoor household biomass fuels for cooking and heating, and ambient air pollution. We also used information from controlled in vivo and in vitro experimental exposure studies on the effects of PM, VOCs, and O₃ from varying sources. Our search covered the period between 1980 and 2023. We used “PubMed,” “Web of Science,” and Google search engines, and we referenced peer-reviewed original publications, reviews, government publications, and news articles (the latter only to highlight very recent wildfire occurrences).

Particulate Matter

Wildfire smoke contains particles of varying size, but the ones with pathological significance should be smaller than 10 μM in diameter (PM10; coarse particles) as those could pass through the upper respiratory tract and enter the proximal and distal airways and alveoli. Most (>90%) of all particle mass emitted from wildfires are smaller than 2.5 μM in diameter (PM2.5) and are categorized as “fine” or “ultrafine,” if smaller than 0.1 μM in diameter (PM0.1). A review of 45 epidemiological studies across the globe (1986–2015) found that levels of PM10 were 1.2–10 times higher due to wildfire smoke compared to non-fire periods and/or locations. In over 90% of these studies, wildfire smoke was significantly associated with a risk of respiratory morbidity [13]. Studies on the onset of asthma symptoms, oral steroid medication, mean daily symptom count, and mean daily dose of reliever medication showed similar associations with inhaled PM10 and PM2.5 [34] during wildfire events suggesting that coarse and fine PM are similarly responsible for eliciting acute asthma symptoms.

Wildfire PM was shown to cause cell damage and cell death via oxidative stress in mouse macrophages [35, 36]. The coarse PM10 fraction in these studies was more proinflammatory and had several-fold higher concentrations of the oxidation products of phenanthrene and anthracene, phenanthraquinone and anthraquinone, than the fine PM fraction on an equal-dose basis [35]. These data suggested a significant role for atmospheric photochemistry in the formation of secondary pollutants and the possibility of significant additional sources of toxicity depending on the chemical composition of PM. The mechanisms through which oxidative stress, polycyclic aromatic hydrocarbon (PAH) signaling, and proinflammatory cytokines affect the pathogenesis of asthma are discussed below.

The role of endotoxin in wildfire particulate toxicity has also been examined given its presence in wildfire smoke and its ability to cause inflammation via toll-like receptor pathways. To assess the endotoxin’s role in inflammation caused by wildfire particulate, investigators compared the effects of wildfire smoke extract on human [37] and mouse [38] bronchial epithelial cells, with or without polymyxin B pretreatment (used to bind and inactivate endotoxin). Their results suggest that endotoxin played only a minor role in wildfire-mediated macrophage toxicity. Other investigators using similar methods but with endotoxin from North Carolina peat wildfire particulates found that it had a greater portion of toxicity in female CD-1 mice [39]. Dunn et al. [40] demonstrated that mice exposed to smoldering plywood smoke had CXCL-dependent neutrophil influx that was associated with impaired bacterial clearance and greater susceptibility to Pseudomonal pneumonia. These results suggest that wildfire smoke containing endotoxin may modulate immune function in the airways. However, more research is needed to clarify the mechanisms and significance of this effect.

Wildfire smoke has been linked with exposure to plant-derived irritants, pollen and fungal particles potentially evoking immune reaction and allergic sensitization. For example, smoke from fires that burn through poison oak, poison ivy, and poison sumac may contain traces of the irritant urushiol that induces a delayed-type hypersensitivity response upon inhalation. Such exposure can cause severe respiratory distress [41]. Whether wildfire smoke containing allergenic particles could directly induce respiratory allergies is not clear [2]. It has been however reported in a longitudinal observational cohort study design using meteorological data and a patient-level clinical dataset from a local outpatient allergy clinic (n = 842) that there were decreases in respiratory peak flow among allergy clinic patients 1 year after each wildfire event. This study raised the possibility that wildfire smoke itself may elicit a delayed immune response or that it enhances allergic sensitization to other allergens [4].

Kobziar and colleagues [42] used unmanned aircraft systems to analyze the aerosols above high-intensity forest fires in the western USA. They found aerosolization of viable microbes via wildfire smoke and identified both pathogenic and non-pathogenic fungal species using DNA analysis [42]. They also established that wildfire smoke contained about four-fold higher concentrations of microbes with 78% viability, five-fold higher taxon richness, and about three-fold enrichment of ice-nucleating particle concentrations in smoke, implying that wildfire smoke is an important source of diverse bacteria and fungi as well as meteorologically relevant aerosols [43]. These findings are important as they raise the possibility that wildfire smoke inhalation may affect the host’s respiratory microbiome, and local immune milieu, potentially leading to the onset or exacerbation of airway disease.

It is important to understand that as the size of PM decreases, it can travel deeper into the bronchoalveolar spaces. PM2.5 can cross into the circulation, enter the brain and other organs, and cause direct long-term systemic or organ-specific damage. In a recent review by Kunovac et al. [44], the systemic molecular effects of fine and ultrafine particles from a wide range of sources (including wildfire smoke) have been summarized and highlighted. Inhalational exposures to PM2.5 and PM0.1 affect virtually all aspects of cellular functions systemically and in multiple different organs. Human and experimental animal studies showed that PM2.5 and PM0.1 elicited immune, inflammatory, oxidative, and endoplasmic reticulum stress and affected carcinogenic [45], metabolic, and epigenetic pathways [44].

Observational evidence from Southern California suggests that wildfire-specific PM2.5 is up to 10 times more harmful to human health than PM2.5 from other sources [24]. In comparing inflammatory responses between urban and wildfire-derived PM2.5 under in vitro experimental conditions, Nakayama and colleagues at UC Davis found that wildfire PM activated CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1) and CYP1B1 genes to a greater degree than urban (non-combustion-derived) PM [37]. The cytochrome P450 superfamily of enzymes mediates environmental stressors and is involved in numerous biological and pathological processes [46]. Nakayama Wong and colleagues [37] proposed that the differences seen in the effects of wildfire smoke and urban PM were due to differing composition of PAHs that induce oxidative stress via redox cycling. Wildfire particulates also induced the oxidative stress-related gene heme oxygenase 1 (HMOX1, an antioxidant enzyme) that was markedly attenuated after the pretreatment of smoke extracts with deferoxamine, suggesting an important role for iron (Fe⁺⁺) in the generation of reactive oxygen species. By contrast, urban (non-wildfire) PM induced greater levels of the proinflammatory cytokines IL-1α, IL-β, IL-8, CCL20, and GM-CSF than wildfire smoke PM. The authors concluded that wildfire PM as compared to urban PM causes cellular oxidative damage via a transition metal-catalyzed Fenton-like reaction (between Fe⁺⁺ and hydrogen peroxide [H₂O₂]) to produce hydroxyl radicals as well as PAH-mediated redox cycling. Interestingly, other investigators found that wildfire PM may have a PAH composition more prone to cause oxidative stress and chemical components that are more toxic to the lung than equal doses of PM collected from ambient air from the same region [47].

VOCs and Toxic Gases

Different fire substrates produce disparate effects of combustion by-products in wildfire smoke and affect the ability to predict wildfire smoke toxicity and its linkage with allergy and asthma. Urban fires or wildfires in the wildland-urban interface are particularly challenging for these reasons. Such fires in addition to coarse and fine PM also emit large amounts of toxic gases such as SO₂, nitric oxide (NO), NO₂, CO, CO₂, as well as metals, PAHs, and other VOC (e.g., aldehydes, n-alkanes) [48, 49]. Among the toxic gases, SO₂ is released primarily from the combustion of sulfur-containing coal and oil and it is not considered a major component of wildfire smoke. However, we would mention here that people prone to allergies, especially allergic asthma, can be extremely sensitive to even small amounts of inhaled SO₂ as it is an irritant that penetrates deep into the air spaces where it is converted into bisulfite and interacts with sensory receptors, causing bronchoconstriction (reviewed in [50]).

VOC and toxic agents are released by fire from building construction materials, interior furnishings including plastics, solvents, glues, metals, formaldehyde, and halogens during high-temperature combustion [49]. These components (also referred to as Hazardous Air Pollutants or Toxic Air Contaminants by the California Environmental Protection Agency [CalEPA]) may directly act in concert as respiratory irritants that exacerbate asthma [33, 51, 52]. VOC is particularly hazardous for infants, children, pregnant women and their fetuses, elderly persons, those with existing lung, heart, or liver diseases, and persons engaging in physical activity, such as firefighters. Acetaldehyde, acrolein, formaldehyde, and benzene are of specific interest because of their differential impact on infants and children compared to adults. These contaminants have a cumulative toxic effect and can be present in wildfire smoke in concentrations above the limit determined by the Office of Environmental Health Hazard Assessment (OEHHA) as toxic [33]. Recent systematic reviews on the specific effects of VOC exposure (that were mostly studied in the context of indoor air pollution) uncovered strong associations with asthma [53, 54]. Metanalysis of 32 studies provided robust evidence for the role of VOC by pooled risk ratios for asthma of 1.08 (CI: 1.02–1.14), 1.02 (1.00–1.04), and 1.04 (1.02–1.06) per 1 μg/m³ increase of benzene, toluene, and p-dichlorobenzene, respectively [53, 54]. There was also a pooled risk ratio of 1.12 (1.05–1.19) per 1 μg/m³ increase in benzene exposure of pregnant women for low newborn birth weight. Additionally, a metanalysis of 13 papers demonstrated a pooled odds ratio between formaldehyde exposure and asthma. Each 10 μg/m³ increase in formaldehyde exposure was significantly associated with a 10% increase in the risk of asthma in children (OR =1.10 [CI: 1.00–1.21]) [53]. Formaldehyde exposure was also associated with an increased risk of asthma in adults (1.81 [1.18–2.78]) when separating the high-exposure adult group (FA >22.5 μg/m³) from the rest of the population [54].

VOCs together with nitrogen oxides may also indirectly contribute to the harmful effects of wildfire smoke since through reactions with sunlight, these molecules catalyze the generation of ground-level (tropospheric) O₃. NO and NO² are reactive atmospheric pollutants associated with wildfire smoke and known for their oxidant properties directly or in the form of the more noxious peroxynitrite (ONOO⁻). Exposure to NO₂ by itself was shown to promote allergic airway inflammation and increase asthma susceptibility according to a study by Lu et al. [55]. Using a model of ovalbumin-induced allergic airway inflammation, the authors found that NO² exposure enhanced inflammatory changes, airway hyperreactivity, and remodeling. These effects were associated with increased reactive oxygen species and malone dialdehyde, and decreased glutathione expression, indicating high levels of oxidative stress. De Sousa and colleagues [56] investigated fire-pollutant meteorological variables and their impact on cardio-respiratory mortality in Portugal during wildfire season. The authors studied PM2.5 and PM10, CO, NO₂, O₃, temperature, relative humidity, wind speed, aerosol optical depth, and mortality rates and assessed pollutant-burning interaction and atmospheric-pollutant interaction. Using cluster analysis, they showed that colder and wetter months and higher NO₂ concentration correlated with respiratory symptoms and pneumonia, while warmer and drier months and higher PM10, PM2.5, CO, and O₃ concentrations correlated with respiratory and cardiovascular symptoms and airway disease.

CO and CO₂ are products of incomplete combustion of biomass and fossil fuels. Evidence suggests an association between exposure to CO and moderate or severe asthma exacerbations in adults. In a study conducted in China, a total of 72,430 hospitalized cases showed significant positive exposure-response relationships between ambient CO exposure and respiratory disease [57]. For each 1 mg/m³ increase in CO concentration (lag0-2), hospitalizations for total respiratory diseases, asthma, and chronic obstructive pulmonary disease (COPD) increased by 13.56% (95% CI: 6.76%, 20.79%), 17.74% (95% CI: 1.34%, 36.8%), and 12.45% (95% CI: 2.91%, 22.87%), respectively. Women were more susceptible to ambient CO exposure-associated hospitalizations for asthma and lower respiratory tract infection.

Pertinent to allergic airway sensitization, CO- and CO₂- level increases in the atmosphere were also linked to prolonged pollen seasons, larger quantity, and allergenicity of pollen produced by plants (reviewed in [58]). Birch pollen extracts from trees grown in warmer temperatures had stronger IgE binding intensity, while ragweed pollen production increased with higher CO₂ levels. Alternaria species growth and allergen production were also increased in an enhanced CO₂ environment [58].

Ozone

The importance of O₃ in airway disease is supported by the fact that in cities with high O₃ levels, people have an over 30% increase in risk of dying from airway disease [59] and children playing outdoor sports have a three times greater chance of developing asthma [8, 60]. O₃, a direct source of oxidative stress (reviewed by Enweasor et al. [61]), can reduce lung function and induce inflammation of the airways, chest pain, coughing, wheezing, and shortness of breath, even in healthy people. Patients with existing airway disease are highly susceptible to the respiratory effects attributed to O₃ exposure that can lead to increased use of medication, school absences, hospital admissions, and emergency room visits for asthma and COPD.

Experimental O₃ exposure was shown to induce significant airway inflammation and hyperreactivity in mouse models of asthma [62-75], asthmatic rhesus macaques [76-79], healthy human subjects [80-83], and patients with asthma and COPD [20, 84-90]. Urban O₃ concentrations steadily declined in the Eastern USA in recent decades because of effective regulations limiting the release of its chemical precursors [91]. O₃-attributable mortality worldwide, however, increased by 46% from 2000 to 2019 [92]. Climate-change-related wildfire activities with heightened VOC and nitrogen oxide emissions may significantly contribute to this trend. PM concentrations in the air are strongly related to O₃ concentrations during wildfires.

The Epithelial Barrier Hypothesis

In a recent targeted proteomic analysis, Aguilera et al. [93] revealed significant changes in molecules critical for immune and barrier function in a study conducted during a 10–12 day-long continuous wildfire smoke exposure period in the San Francisco Bay area. The authors found an association of the zonulin family peptide (zonulin, a molecule responsible for regulating paracellular epithelial barrier tight junctions) with T helper 2 cells, major drivers of allergic sensitization. Thus, wildfire smoke may induce epithelial barrier injury leading to tolerance breakdown [94], increased susceptibility to allergic airway sensitization, and development of asthma and COPD [28] (Fig. 1).

Fig. 1.

Wildfire-smoke-induced oxidative stress leads to epithelial barrier injury and release of alarmins.

In an ex-vivo airway epithelial model [95], wildfire smoke extract increased cellular permeability and blunted autophagy to a greater extent than cigarette smoke. These data suggest that cell fate and epithelial barrier integrity are significantly affected by wildfire smoke. In vitro wildfire smoke exposure also caused a direct and rapid cell death of macrophages [38, 96], a major cell type at the airway epithelial barrier. The level of macrophage toxicity correlated with oxidative stress and NF-kB signaling. These effects were likely due to heat-labile organic compounds and not endotoxin content [38]. Oxidative stress in the airways during wildfire smoke inhalation can be induced exogenously by inhalation of PM and gaseous components, specifically O₃, and endogenously, from reactive oxygen and nitrogen species released from activated inflammatory cells, particularly neutrophils, macrophages, and eosinophils (Fig. 1).

Role of Oxidative Stress

Studies investigating the effects of wildfire smoke components including PM and O₃ often show that the induction of asthmatic airway changes is associated with oxidative stress. When pro- and antioxidant mechanisms are in an imbalance, the resulting oxidative stress causes lipid peroxidation of unsaturated phospholipids, glycolipids, and cholesterol, affecting the function of the pulmonary surfactant phospholipids [97] and cell membranes [98, 99], methionine, and sulfhydryl group oxidation in proteins, depletion of antioxidants, and DNA damage [100-102]. Oxidative stress is characterized by the presence of free radical species such as oxyl radicals, peroxyl radicals, and hydroxyl radicals derived from Fe⁺⁺-mediated reduction of H₂O₂ or non-radical species such as singlet oxygen, O₃, and ONOO⁻ produced by the reaction between NO and superoxide radicals [100-102]. While NO and O₃ cause direct oxidative damage upon inhalation, other reactive chemical species, such as H₂O₂, superoxide anion, hydroxyl and hypohalite radical, are formed endogenously by inflammatory cells through enzymatic action [102].

Indeed, a variety of cells that also participate in the allergic inflammatory response of asthmatic airways, e.g., macrophages, mast cells, neutrophils, eosinophils, and basophils (Fig. 2.), generate superoxide anion, which is rapidly converted to H₂O₂ by the enzyme superoxide dismutase, or hypohalite radical in the presence of Fe⁺⁺ as a secondary reaction [102-104]. Activated neutrophils and macrophages produce myeloperoxidase, while eosinophils specifically release eosinophil peroxidase. While both of these enzymes are members of the mammalian peroxidase family and use halide anions (I⁻, Br⁻, Cl⁻) for activation, myeloperoxidase mainly uses chloride and results in the formation of chlorotyrosine, whereas eosinophil peroxidase selectively uses bromide yielding bromotyrosine as a product. Because of this selectivity, high levels of urine bromotyrosine serve as a biomarker of eosinophil activation in asthma [105].

Fig. 2.

Wildfire-smoke-induced oxidative stress leads to activation of residential immune and inflammatory cells and to neutrophilic and eosinophilic airway inflammation. This diagram depicts the pluripotent effects of type-2 polarizing alarmins on the immune and inflammatory cells that reside in the airway submucosal tissue.

Like O₃, NO is a highly reactive atmospheric pollutant associated with wildfire smoke, with oxidant properties directly or in the form of the more noxious ONOO⁻. NO, however, can also be produced endogenously, particularly in the lung, through the enzymatic action of NO synthase. NO functions as a vasodilator, bronchodilator, neuro-transmitter, and inflammatory mediator (reviewed in [106]). Patients with asthma have high levels of NO in their exhaled breath and high levels of inducible NO synthase enzyme expression in the epithelial cells of their airways. Exhaled NO therefore is also an indirect marker for upregulation of airway inflammation and is used to diagnose eosinophilic, type 2 asthma.

Inflammatory Mechanisms

Oxidative stress in the airways leads to the release of alarmins including cytokines (IL-1β, IL-6, IL-23, IL-25, IL-33, TNF-α, and TSLP) and other inflammatory mediators such as arachidonic acid derivatives and damage-associated molecular patterns triggering a cascade of proinflammatory changes in structural and immune cells in the respiratory mucosal tissue [61, 68, 71, 78,107-114]. Such changes are also hallmarks of severe asthma exacerbations [111] (Fig. 2.).

In the healthy lung, the primary inflammatory cells recruited to the airways following oxidative injury (studied in response to O₃ [81, 82] or endotoxin-containing PM [39]) are the neutrophils. These cells appear in the airways within minutes and accumulate in significant numbers as early as 1–2 h after exposure [64, 68, 76]. In wildfire smoke-exposed infant monkeys [115] and O₃-exposed healthy human subjects [81, 83, 89], such inflammatory changes were associated with a decrease in lung function. Importantly, when allergen-sensitized mice, asthmatic rhesus macaques, or allergic human subjects were exposed to O₃, a marked influx of both eosinophilic and neutrophilic granulocytes was observed [68, 78, 116, 117] with greatly amplified lung function impairment, indicating the pathological significance of O₃ exposure in asthma.

Among the many mediators released in response to oxidative damage of the airway epithelium IL-25, IL-33 and TSLP play an important role in priming type 2 airway inflammation and causing severe asthma exacerbation [68, 72, 108-113] (Fig. 2). Specifically, our laboratory and others previously found that IL-33 transcription as well as release was upregulated by oxidative damage in the lung in a time-dependent manner [68, 110]. Interestingly, Michaudel and colleagues [110] also showed that the absence of IL-33 or neutralization of the IL-33 receptor (ST2) although reduced airway responsiveness led to enhanced epithelial cell injury with protein leak and increased neutrophilia that was reversed by replacement of IL-33. These results suggested that like many components of the oxidative stress/inflammation cascade, IL-33 may have both pro- and anti-inflammatory roles.

Destruction of Anti-Inflammatory and Antioxidant Mechanisms

Oxidative stress not only causes lipid peroxidation, methionine, and sulfhydryl group oxidation in proteins, and DNA damage, but also depletes antioxidants, thereby generating an oxidant imbalance [100, 101]. In addition to the nutritionally obtained ascorbic acid, α-tocopherol, lycopene, and β-carotene, the major endogenously produced antioxidants that fight against the effects of oxidative stress are the antioxidant enzymes such as superoxide dismutase, catalase, and glutathione. While there are no studies investigating the effects of wildfire smoke on antioxidant expression, Sahiner and colleagues [102] found that antioxidants were expressed at significantly lower levels in asthmatic children compared with healthy controls (reviewed in [102]).

Against inflammatory injury, the lung mounts immuno-protective mechanisms such as production of the epithelial-cell-derived lung collectins, surfactant protein A and D. We and others showed that SP-D plays an important anti-inflammatory function in allergic airway inflammation [71, 116, 118, 119]. Oxidative stress however was shown to destroy the biologically active quaternary and tertiary structure of this molecule [70, 120, 121]. Indeed, a de-oligomerized form of SP-D can readily enter the circulation from the lung during lung injury. We have recently found that wildfire smoke exposure of healthy volunteers caused a significant increase in the levels of circulating SP-D (unpublished information) indicating wildfire-smoke-induced oxidative lung injury and the biomarker potential of this molecule.

The mainstay of treatment in asthma and other chronic inflammatory diseases is formed by glucocorticoid therapy. However, studies on mice [69], dogs [122], rhesus macaques [123], and asthma patients [84, 124] showed diminished effectiveness of glucocorticoids in inhibiting exacerbation of asthmatic airway inflammation caused by exposure to air pollutants. Because oxidative stress causes neutrophil influx in asthma and because neutrophils are poorly responsive to glucocorticoids [125], wildfire-smoke-induced asthma exacerbations could potentially be resistant to glucocorticoid treatment, generating an important clinical problem (reviewed in [61]).

Role of the Aryl Hydrocarbon Receptor

The aryl hydrocarbon receptor (AhR) mediates the effects of wildfire smoke components while regulating asthma-related genes. AhR is expressed intracellularly in myeloid, lymphoid, and structural cells and functions as a transcription factor (TF) activated by environmental chemicals (such as aromatic hydrocarbons) or endogenous indole derivatives (such as kynurenine) [126]. AhR is critical for a wide range of immune functions including the maintenance of innate and adaptive cell populations at mucosal barrier sites, and the control of inflammation at steady-state or during ongoing inflammatory responses in asthma is involved in cell differentiation, cell adhesion, mucus and cytokine production [72, 127, 128]. Upon ligand binding, the AhR complex translocates into the nucleus and heterodimerizes with AhR nuclear translocator to induce gene transcription. AhR is a main activator of the genes encoding cytochrome P450 and the cytokines IL-17A and IL-22 (Fig. 3). The effects of AhR on immune cell differentiation (including Th17 or Treg polarization) are influenced by the nature of the ligand and the local cytokine milieu, and it could be either protective or pro-asthmatic (reviewed in detail by Poulain-Godefroy et al. [126]).

Fig. 3.

Wildfire smoke induces mixed eosinophilic and neutrophilic airway inflammation by stimulating AhR signaling on immune cells. Intracellular AhR in epithelial, myeloid, and lymphoid cells mediates the effects of PAHs and other ligands by transcriptional regulation. Upon ligand binding, the AhR complex translocates into the nucleus and heterodimerizes with AhR nuclear translocator (ARNT) to induce transcription of cytokines such as IL-17A through RORγt. The type 2 immune profile (characterized by IL-5 and IL-13 production) in the asthmatic airways may be skewed toward IL-17A production by activating the AhR-ARNT-RORγt signaling.

AhR interferes with the action of nuclear factor-kappa B (NF-κB), a proinflammatory TF in different ways. For example, NF-κB induces AhR expression, but AhR then regulates NF-κΒ signaling [128]. By interacting with the function of other TFs, AhR promotes IL-22 (RORγt), IL-10, and IL-21 (cMaf) as well as Aiolos and its own expression (STAT3). Through Aiolos, AhR inhibits expression of the T-cell growth factor, IL-2 [128]. Thus, on the one hand AhR promotes Th17 cell differentiation; on the other, it induces Th17 cell plasticity into IL-10 producing protective Tr1 cells. While both IL-17A and IL-22 can elicit airway neutrophilia, IL-22 can also play a protective role when produced during epithelial or tissue damage. Chronic oxidative lung injury was reported to be associated with increased tryptophan and lipoxin A4 (activators of AhR), and recruitment of IL-17A and IL-22-expressing cells. T-cell-specific AhR depletion enhanced lung inflammation, indicating that oxidative stress activates AhR, which controls airway inflammation by reduction of IL-22 expression [72].

IL-17A and Glucocorticoid Responsiveness

IL-17A was implicated in wildfire-smoke-induced airway inflammation as one of the main players [129, 130]. The IL-17A and IL-22 genes are activated by the RORγt proinflammatory signaling pathway [131, 132]. Combustion-derived PM containing environmentally persistent free radicals induced IL-17-related airway inflammation as well as activation of Cyp1a1 and Cyp1b1 gene expression in airway epithelial cells indicating the involvement of AhR. Using single-cell RNA sequencing analysis, combustion-derived PM exposure of the mouse lung showed that epithelial cells acquire a transcriptomic profile indicative of increased IL-17 signaling, AhR activation, EGFR signaling, and T-cell receptor and costimulatory signaling pathways. AhR activation was brought on by Ahr/AhR nuclear translocator and activation of tyrosine kinase c-Src, EGFR, and subsequently Erk1/2 pathways [130]. Thus, PM initiates mixed neutrophilic and eosinophilic asthma through epithelial, dendritic, and T-cell AhR activation involving IL-17A release (Fig. 3.).

While Th17 cells are identified as the main producers of IL-17A, in our in vivo studies, O₃-induced asthma exacerbation in mice did not show T-cell activation or migration of T cells into the lung prior to the O₃-prompted neutrophil influx [68]. These results implied that Th17 cells do not participate in IL-17A release in the early phases of the O₃ response. Mathews et al. [132] proposed that the source of IL-17A in response to acute O₃ exposure is the γδ T cell. In addition, innate lymphoid cells were shown to be essential and sufficient to elicit the development of O₃-induced neutrophilia [68] and the ensuing airway hyperresponsiveness in mice. These studies suggest the importance of innate immune players in O₃-induced IL-17A pathways. Taken together, in addition to Th cells, both IL-17A and IL-22 can be produced by ILC3, γδ T, and NK cells, after stimulation with IL-1β, TGF-β, IL-6 or IL-23, and the TF RORγt [72]. These mechanisms are important because IL-17A-mediated neutrophilia in response to oxidative stress feeds back to a vicious cycle by releasing additional free radicals into the airways. The clinical relevance of these experimental findings is supported by the fact that increased IL-17A expression in the lung was associated with steroid-insensitive neutrophilic asthma in patients [133]. While glucocorticoid responsiveness varies among individuals depending on genetic, environmental, and immune factors, wildfire-smoke-induced oxidative stress, inflammatory mediators, and epigenetic changes [61, 104, 134, 135] can all reduce immune cell glucocorticoid responsiveness leading to persistent inflammation. IL-17 is particularly interesting in this mechanism for several reasons [136].

IL-17A through IL-17RA [137-140] can directly activate signaling pathways such as the NF-κB p38-MAPK and PI3K pathway that antagonize GR function [134, 141]. IL-17A-induced NF-κB activity was shown to consume and compete for transcription coactivators, essential for both transactivation of anti-inflammatory genes and transrepression of proinflammatory ones by the GR. NF-κB can also interfere with GR nuclear translocation and DNA access (at glucocorticoid response elements) and can promote post-translational modifications (such as phosphorylation) and degradation of the GR. IL-17A was also shown to activate a dominant negative isoform (GRβ) leading to reduced anti-inflammatory effects. Th17 cell differentiation was shown to be in fact upregulated by dexamethasone treatment in vitro and in mice in vivo [142], along with other factors like increased Bcl-2 expression, heightened RORγt activity, amplified Stat3 phosphorylation, and unique metabolic traits such as higher level of glucose and glutaminolysis [143]. Low levels of glucocorticoidinduced leucine zipper protein and histone deacetylase 2, and elevated IL-6, IL-23, IL-22, and STAT3 activation were described as main players in glucocorticoid resistance. Taken together, IL-17A potentially contributes in major ways to glucocorticoid resistance in wildfire-smoke-exposed populations especially those suffering from chronic severe inflammatory airway disease, involving intricate molecular pathways and various immune and inflammatory cell types.

Epigenetic Modifications of Asthma-Related Gene Expression

Accumulating evidence obtained from studies on exposure to wildfire smoke, air pollution, and other toxic sources suggests that inflammation and oxidative stress are linked with epigenetic changes (including DNA methylation, histone modification, and microRNAs) [144-149]. Airborne PM has been shown to induce redox-sensitive signaling mechanisms and irreversible epigenetic changes that may persist through generations [150].

The role of extracellular vesicles in wildfire smoke response in the airways has recently been investigated by Carberry et al. [149]. Using in vitro and animal models of exposures to smoke condensate from peat and red oak, the authors found increases in miR-150, miR-183, miR-223-3p, miR-30b, and miR-378a in extracellular vesicles. These miRNAs were identified to regulate hypoxia and cell stress-related gene transcription. Wildfire smoke might contribute to the transmission of epimutations from gametes to zygotes by involving mitochondrial DNA, parental allele imprinting, histone withholding, and non-coding RNAs. Larger prospective studies using innovative, integrated epigenome-wide strategy are highly warranted to further investigate epigenetic inheritance and the associated effects of wildfire smoke on allergic airway disease [150].

DNA methylation was also associated with immune and inflammatory changes aggravating models of allergic airway inflammation [151-153]. The dynamic DNA methylation landscape can be rapidly altered (within hours) in peripheral leukocytes following PM2.5 exposure, linking systemic inflammation and oxidative stress [154, 155]. Indeed, DNA methylation changes were identified in inflammatory and oxidative stress response genes [156]. In rats, traffic-related PM exposure induced DNA methylation alterations in both blood and lung tissues that were dependent on exposure dose and duration, as well as the sex of the rat [157]. Most evidence in humans is based on experiments utilizing a heterogeneous mixture of leukocytes [154, 155, 158, 159], but specific loss of methylation in inflammatory genes and subsequent inflammatory responses, specifically in circulating T helper cells, was found in vivo after environmental challenge [160, 161]. A human intervention study of 2-h exposure to median 234.0 μg/m³ PM2.5 substantially modified DNA methylation in CD4⁺ T helper cells, with changes in genes involved in mitochondrial oxidative energy metabolism [162] suggesting these epigenetic changes as potential mediators of adverse health outcomes [163].

PM2.5 from different sources also induces significant DNA methylation changes in cultured bronchial epithelial cells at different doses and time points [164, 165]. Current and early life exposure to PM2.5 from outdoor and indoor environments has been associated with DNA methylation changes in nasal epithelial cells [164, 166, 167]. Brown and colleagues at UC Davis studied long-term baseline epigenetic changes associated with early-life exposure to wildfire smoke in rhesus macaques. Using whole genome bisulfite sequencing of nasal epithelial cells, they identified 3,370 differentially methylated regions annotated to genes significantly enriched for synaptogenesis signaling, protein kinase A signaling, and a variety of immune processes. Some DMRs significantly correlated with gene expression differences, suggesting that early-life exposure to wildfire smoke leads to long-term changes in the methylome over genes impacting both the nervous and immune systems [148].

PM-sensitive CpG sites were also mapped to diseaserelated genes, including pulmonary disease, in peripheral blood leukocytes of 8,397 healthy subjects [168]. PM exposure was further linked with methylation in the CFTR cystic fibrosis gene. Long-term PM exposure was associated with altered DNA methylation in individuals with COPD [169]. Other studies similarly revealed abnormal DNA methylation in patients with asthma [166, 170-173] and COPD [174, 175]. Importantly, DNA methylation changes linked to wildfire PM2.5 in pregnant women and their relevance to respiratory function remain unstudied.

Two enzyme systems, namely, DNA methyltransferase and ten-eleven translocation (TET) proteins, methylate and demethylate DNA from C to 5-methylcytosine and back to C to establish, genome-wide, a methylation pattern required for gene regulation and cell fate decisions [176] (Fig. 4a). The potential role of gene-specific epigenetic pathways in mediating the effects of wildfire smoke is highlighted by the Tet1 gene [166, 177]. Methylation of this gene promoter itself has been associated with childhood asthma and traffic-related air pollution (reviewed by Poulain-Godefroy et al. [126]).

Fig. 4.

Tet1 proposed as a potential master epigenetic regulator of genes involved in mediating wildfire smoke effects in asthma. a DNA methylation, histone modification, chromatin accessibility, and transcription factor (TF) binding influence each other and coordinately regulate gene expression. It is proposed that Tet proteins regulate all these mechanisms. 5mC, 5-methylcytosine; 5hmC: 5-hydroxmethylcytosine. b Tet1 regulates AhR expression through histone modifications and chromatin accessibility. Tet1 is known to form a complex with SIN3A (a scaffold protein) and hMOF (a histone acetyltransferase), which prevents auto-acetylation of hMOF and facilitates it to add H4K16ac and activate gene expression. H3K4me3 (added by MLL) and H3K27ac (added by p300/CBP) are also markers for active transcription. Tet1 can form a complex with p300/CBP. c Tet proteins may regulate cytokine production of ILC2 through promoter demethylation, which allows the binding of lineage-specific TFs (e.g., GATA3). In response to PM2.5 exposure, Tet1 may induce expression of IL17A through promoting AhR binding in ILC2 and result in ILC2 plasticity. d Tet may regulate promoter methylation of NR3C1 gene and promote the expression of glucocorticoid receptor through demethylation. Reducing the recruitment of Tet proteins to NR3C1 promoter downregulates the expression of the receptor and reduces the responsiveness to steroid.

TET family proteins may work as transcriptional activator or repressor through their enzymatic and non-enzymatic activity in multiple cellular processes. Our recent study on house dust mite-challenged Tet1−/− mice (compared to Tet1+/+ mice) showed an increased allergic airway inflammation associated with a decrease in AhR-dependent protective Cyplal and Aldhlal gene expression and an increase in interferon signaling. In human bronchial epithelial cells, activation of TET1 differentially regulated interferon and AhR signaling pathways, with predicted binding of transcriptional factors with relevant functions in their promoters, and the presence of histone marks generated by histone enzymes. TET1 forms a complex with hMOF (a histone acetyltransferase) and SIN3A (a scaffold protein), promotes acetylation of H4K16ac, and regulates genes in DNA repair. Thus, TET1 may play a protective role in allergic airway inflammation through activation of the AhR signaling pathways in airway epithelial cells, probably through epigenetic modifications of the Ahr gene (Fig. 4b) [178].

Depending on the cell type and which TFs and histone-modifying enzymes TET proteins interact with, they can either activate or repress gene expression. For example, TET1 regulates hypoxia-induced epithelial-mesenchymal transition by acting as a co-activator [179]. In embryonic stem cells with catalytically inactive Tet1 (Tet1^m/m) and Tet1 knockout (Tet1^−/−), loss of Tet1, rather than its catalytic activity, led to abnormal upregulation ofbivalent developmental genes, involving chromatin modifiers like SIN3A and reducing H3K27 trimethylation and deacetylation without changes in DNA methylation. Tet1^−/− embryos expressed elevated levels of Gata6 and experienced developmental delays, highlighting the crucial non-catalytic roles of TET1 in regulating H3K27 modifications in gene silencing [180]. Especially relevant to wildfire smoke exposure, AhR and TET proteins have a complex regulatory relationship in hematopoietic and lymphoid cells, in particular, in modifying cell differentiation and lineage specificity during inflammation [181]. For example, AhR directly upregulates Tet2 expression (reviewed in [182]). TET2 is involved in promoting plasticity rather than differentiation of hematopoietic cells leading to malignancies and chronic inflammatory conditions (reviewed in [183]). This is important as during wildfire smoke exposure TET2 can enable AhR activation of genes such as Il17a leading to plasticity (Fig. 4c) [177,184]. The role of TET proteins and their interactions with AhR in wildfire smoke and other toxic exposures warrant further clarifications.

Conclusions

We highlighted the mechanisms of how the harmful components PM2.5, VOCs, O₃, endotoxin, microbes, and toxic gases in wildfire smoke may contribute to oxidative stress and inflammation in the airways by disrupting the respiratory tract’s immune and barrier functions increasing susceptibility to allergies, asthma, and COPD. Oxidative stress, both from inhaled PM and endogenously from inflammatory cells, triggers alarmin release, damages DNA, proteins, and cell membranes, and inactivates pulmonary antioxidant and immune protective molecules like SP-A and SP-D. The AhR expressed on immune and structural cells plays a central role in directly and indirectly mediating the smoke’s effects, affecting cell differentiation, and IL-17A production, implicated in neutrophilic airway inflammation. For these reasons, glucocorticoids are less effective against smoke-induced exacerbations of chronic airway diseases. The underlying mechanisms strongly point to the role of epigenetic regulation, including DNA methylation and histone modifications. Understanding these mechanisms is crucial for developing effective prevention and treatment strategies during wildfire events, highlighting the significance of basic, mechanistic research in this area.