Air pollution-induced epigenetic changes: disease development and a possible link with hypersensitivity pneumonitis.

Airborne pollutants have adverse effects in the lungs. Recent studies find an association between air pollution exposure and increased incidence of poor lung function and mortality in interstitial lung diseases (ILDs) (Johannson et al. 2015 ). In idiopathic pulmonary fibrosis (IPF), air pollution is associated with increased incidence and harmful effects on health (Harari et al. 2020 ). ILDs comprise an array of heterogeneous parenchymal lung diseases that occur due to fibrosis and inflammation of lung parenchyma. Considerable heterogeneity exists among ILDs, depending on a variety of causative agents, while in some cases the cause is unknown and therefore the disease is classified as idiopathic. Prevalence of ILDs depends largely on geographical locations and incumbent environmental conditions. IPF, sarcoidosis, and HP are the most regularly observed disorders amongst ILDs (Kalchiem-Dekel et al. 2018 ). According to data from Indian registry, HP accounts for 47.3% of all ILD cases and is the most frequent type of ILD in India. HP, also known as extrinsic allergic alveolitis (EAA), is a heterogeneous and immunologically-mediated lung disease. It results from the inhalation of certain organic substances of animal or plant origin and inorganic substances like low molecular weight chemical isocyanates. The most prevalent forms of HP are farmer’s lung disease, developed from bacterial and fungal exposure, and bird fancier’s lung that occur due to the inhalation of avian antigen found in bird droppings. Interestingly, the inhaled offending antigen does not induce the disease in every exposed individual. It is reported that nearly 5–10% of the exposed individuals will develop HP. Prevalence of chemical worker's lung is only 1–4.7% of the isocyanate-exposed workers, while farmer's lung is reported to have developed in 1–19% of the mouldy hay-exposed individual. It is also estimated that only about 6–20% of the avian antigen-exposed people develop bird fancier's lung. Such variations in disease incidence can be traced to the presence of predisposed genetic and epigenetic factors. Individuals who are genetically susceptible (first hit) develop HP when exposed to an inciting antigen (second hit). The second hit—i.e. the exposure to causative antigen—alone cannot induce HP development (Spagnolo et al. 2015 ). A recent study has indicated an association between air pollution level and HP occurrence in India (Singh et al. 2019 ). It is apparent that air pollution could be a risk factor for HP, but how air pollution could influence HP pathogenesis is largely unknown. It has been observed that exposure to specific air pollutants leads to inflammation and altered immune responses. Such exposure is also associated with epigenetic modifications. Therefore, we hypothesize that air pollution-induced epigenetic alterations influence immune responses, thereby making exposed individuals susceptible to the development of HP. The following section outlines the pathophysiology of HP in connection with epigenetic modifications induced by air pollution. HP is an inflammatory lung disease caused by the inhalation of organic or inorganic antigens (summarized in Table 2 ) less than 5 μm in diameter. According to the ‘two-hit’ hypothesis, the coexistence of inducing factors, such as antigen and promoting factors like genetic abnormalities or additional environmental exposures, may lead to an exaggerated immune reaction in the lungs and thereby causes inflammation. HP is characterized by alveolitis, which eventually results in granuloma formation and the development of fibrosis. The development of fibrosis leads to respiratory insufficiency, and finally, to the death of the patient (Selman et al. 2012 ). Table 2 Common HP-inducing antigens Name of the Disease Origin Antigen Source of Antigen 1. Farmer’s lung Bacterial Micropolyspora faeni, Thermoactinomyces vulgaris Moldy hay 2. Summer-type HP Fungal Penicillium frequentans Contaminated old houses 3. Hot tub lung Mycobacterial Mycobacterium avium complex (MAC), composed of M. avium and M. intracellularss Contaminated water 4. Bird fancier’s lung Animal fur protein Avian proteins Feather bloom, avian serum(pigeons, chicken, turkey, goose, avian proteins),and excrement (mainly due to pigeon intestinal mucin) 5. Chemical worker’s lung Low-molecular weight chemicals Diisocyanates, trimellitic anhydride Polyurethane foams, spray paints, dyes, glues Common HP-inducing antigens Micropolyspora faeni, Thermoactinomyces vulgaris The pathogenesis of HP is complex and involves both Type III (immune complex-mediated) and Type IV (T cell-mediated) hypersensitivity reaction . Several studies have suggested that the alveolitis, (inflammation in alveoli branches) initiates with almost an exclusive predominance of neutrophils, eventually involving lymphocytes and becoming solely lymphocytic at later stages. The human lung contains approximately 10 11 alveolar macrophages which play a major role in granuloma and fibrosis formation in HP. In the susceptible individual with repeated exposures, the disease-causing antigen forms an immune complex with the pre-existing antigen-specific antibody (IgG). This event triggers the activation of the complement cascade and results in the recruitment of macrophages, neutrophils, and lymphocytes at the site infection to induce the formation of granuloma (Patel et al. 2001 ). These immune cells also secrete several molecular mediators that induce the migration and stimulation of fibroblast to induce fibrosis. It is reported that chronic forms of HP are regulated by the T cells. Analyses of the bronchoalveolar lavage fluid (BALF) of chronic HP patients have suggested an involvement of Th2 T cells with a higher CD4+ / CD8+ ratio and a concomitant reduction in the number of γδT cells in disease development (Caillaud et al. 2012 ; Simonian et al. 2009 ). It is likely that the CD4 to CD8 ratio varies according to the type of the inhaled antigen. Activation of alveolar macrophages in the acute and sub-acute phases also promotes CD8+ T cells accumulation, granuloma formation, and subsequent development of fibrosis. Natural killer cells are also reported to be involved in HP pathogenesis (Korosec et al. 2007 ). Various reports have demonstrated that Th1 cytokine plays an essential role in disease pathogenesis (Barrera et al. 2008 ). The induction and perpetuation of the inflammatory responses in HP, including the recruitment of the different inflammatory cells, are mediated by a myriad of cytokines and chemokines. The pathophysiology of HP is not dependent on any one component of cellular immunity, but it is developed due to a multimodal interaction between these components, each of which may play a crucial role in disease progression. Neutrophils are the body’s first line of defence against foreign bodies and plays a crucial role in inflammatory diseases like HP. After inhalation of the causative antigen, neutrophils accumulate in the alveoli and small airways of the lungs. Upon stimulation, neutrophils secrete various cytokines that are implicated in the pathogenesis of HP (Pardo et al. 2000 ). The secretion of IFN β, IL-10, and IL-6 by neutrophil is regulated by epigenetic modifications. The binding of two transcription factors, PU.1 and C/EBPβ, favours activation of the transcription of these three cytokines (Ostuni et al. 2016 ). Additionally, the matrix metalloproteinases (MMPs), collagenase-2 (MMP 8), and gelatinase B (MMP 2) produced by neutrophils are also reported to play a key role in the disease progression (Pardo et al. 2000 ). An epigenetic study on MMP2 reveals that its expression is dominated by DNA and histone modifications (Santiago-Ruiz et al. 2019 ). Patients with chronic HP have an increase of neutrophils loaded with MMP8 and MMP9, and many studies show that the expressions of MMPs are regulated by histone and DNA methylation (Campos et al. 2016 ). Macrophages are predominantly involved in granuloma formation in HP (Suga et al. 1997 ). The lncRNA MEG3-4 has been identified as a tissue-specific regulator of inflammatory responses in alveolar macrophages during bacterial infection through the transcriptional regulation of immune response genes. It has been confirmed that the lncRNA MEG3-4 binds to the microRNA miR-138 in a competitive manner, with mRNA encoding the proinflammatory cytokine IL-1β, thereby increasing the abundance of IL-1β and enhancing the inflammatory response to bacterial infection in alveolar macrophages (Li et al. 2018b ). A recent study on epigenome analysis has revealed that DNA methylation is involved during macrophage differentiation by changing the binding sites of transcription factors (Dekkers et al. 2019 ). T cells are also considered to be important regulators of HP development (Simonian et al. 2009 ). Epigenome maps combined with mechanistic studies, have demonstrated that T cells undergo extensive epigenome remodelling in response to signals, which has a strong effect on phenotypic stability and function of the lymphocytes (Schmidl et al. 2018 ). Moreover, it was shown that the second-hand smoke and ambient air pollution, which deteriorate respiratory health, were associated with DNA hypermethylation and a decreased expression of IFN-γ and Foxp3 in T cells population (Runyon et al. 2012 ). Recently, Singh et al. have reported that ambient air pollution is a promoting factor in the development of HP. The study demonstrates that the odds of developing HP is 7% greater for every 10 μg/m 3 increase in air pollution (PM2.5 level). The authors hypothesize that fine-particulate air pollutants lead to airway inflammation, reduce mucociliary clearance in the alveoli, and lead to an immune-mediated response that ultimately leads to the development of HP (Singh et al. 2019 ). It is apparent that more rigorous studies are necessary to establish this hypothesis. It is evident from the ‘Table 1 ’ that exposures to PM2.5, PM10, NO2, SO2, CO, and O3 were found to be associated with epigenetic changes like alteration in LINE-1 methylation, methylation on CpG sites of many immunoregulatory genes, histone modifications, and miRNA regulations. These epigenetic changes were again associated with inflammation and poor respiratory health. Therefore, exposure to those pollutants may alter immune responses, which could influence HP development. Moreover, HP pathogenesis involves many components of our immune systems that are under epigenetic regulations (Fernández-Morera et al. 2010 ). Wu et al. have reported methylation of EBF3 gene in peripheral blood samples of a Turkic ethnic group (Uygurs) with bird fancier’s lung. The authors hypothesized that EBF3 hypomethylation might be associated with IL 10 dysregulation in HP subjects (Wu et al. 2018 ). In another study on Chinese Uygur population by the same group, methylation of the gene Smad3 is reported. Smad 3 protein is one of the key players in the formation of fibrosis; it is likely that Smad3 gene hypomethylation promotes pulmonary fibrosis by increasing Smad3 mRNA expression (Wu et al. 2017a ). Diagnosis and effective management of HP often pose to be a challenge to the pulmonologists. HP is curable if diagnosed correctly in its early stages, i.e. before occurrence of irreversible lung damage. The prognosis of HP is not dependent only on the dose and duration of exposure to the causative agents; the genetic and environmental factors also play a major role in disease pathogenesis. Recent studies have shown that HP development is associated with ambient air pollution, which is known to be linked with inflammation and other lung diseases. An understanding of epigenetic alterations associated with air pollution has emerged in recent times and in many cases, such alterations are associated with diverse diseases including ILDs (Fig. 2 ). Fig. 2 Mechanism showing HP development by increasing host susceptibility through air pollution exposure. a) Emission of industrial and traffic-related air pollution. Pollutants reach the alveolar spaces in the lungs and induce epigenetic changes in the lungs’ microenvironment, thereby influencing host susceptibility to disease development. b) The interaction between the causative antigen deposited in alveolar spaces and the circulating IgG antibody forms precipitating antigen-antibody immune complexes in interstitial spaces. Immune complexes trigger activation of both complement and pulmonary macrophages. Activated macrophages secrete the cytokines IL-1 and TNF-a that promote adhesion of leukocytes to activated endothelium. Complement activation enhances vascular permeability and attracts inflammatory cells. As a consequence of recruitment and activation, inflammatory cells release toxic mediators that promote acute lung injury, which eventually leads to HP development Mechanism showing HP development by increasing host susceptibility through air pollution exposure. a) Emission of industrial and traffic-related air pollution. Pollutants reach the alveolar spaces in the lungs and induce epigenetic changes in the lungs’ microenvironment, thereby influencing host susceptibility to disease development. b) The interaction between the causative antigen deposited in alveolar spaces and the circulating IgG antibody forms precipitating antigen-antibody immune complexes in interstitial spaces. Immune complexes trigger activation of both complement and pulmonary macrophages. Activated macrophages secrete the cytokines IL-1 and TNF-a that promote adhesion of leukocytes to activated endothelium. Complement activation enhances vascular permeability and attracts inflammatory cells. As a consequence of recruitment and activation, inflammatory cells release toxic mediators that promote acute lung injury, which eventually leads to HP development

Epigenetic signatures reflect changes in the cellular environment and can also be found in the human circulatory system in case of several diseases (Ladd-Acosta 2015 ; Bhargava et al. 2018a ). These signatures seem promising and can be explored to identify robust biomarkers to monitor clinical outcomes as well as early detection of diseases. An ideal biomarker should show a high degree of specificity and sensitivity and ease of measurement; it should also be capable of detecting a disease in its early stage (Byrnes and Weigl 2018 ). Although the plasticity of the human epigenome makes it difficult to establish true epigenetic marks for a particular condition, diverse diseases show alterations in their epigenetic signatures. A recent study reports that epigenetic alterations take place even on seasonality and weather influence (Ricceri et al. 2014 ; Xu et al. 2020 ). A significant number of studies have been conducted to understand the epigenome during cancer. The cancer-specific hypermethylation of CpG islands shows clinical importance (Jin et al. 2011 ; Wilhelm-Benartzi et al. 2011 ). Epigenetic marks, which are established as an effect of air pollution, can also be used as biomarkers for risk assessments for diseases associated with air pollution. Methylation in DNA sequences has several advantages. DNA is a relatively stable molecule. It can be collected from a large number of sources and stored for a long time after being collected from patients. Moreover, DNA methylation is a widespread change throughout the cancer genome and hence many points in the genome can be used for the assay to generate highly predictive models (Huang et al. 2018a , 2018b ; Tommasi et al. 2012 ). Sophisticated techniques such as methylation-specific PCR (MSP), mass spectrometry-based methylation detection (EpiTYPER), MethyLight, and pyrosequencingetc, allow determination of the methylation state of a specific DNA location even from a minimum sample volume (Olkhov-Mitsel and Bapat 2012 ). This makes it feasible to evaluate the diagnostic and prognostic potential of the methylation state of a large number of DNA sequences for many cancer types. The Cancer Genome Atlas project (TCGA) provides a platform for information regarding DNA methylation changes in cancers (Lee 2016 ). A case control study in 2008 utilized a series of methylated genes to predict disease recurrence after surgery in Stage I lung cancer patients. The promoter hypermethylation of just four genes—CDH13, RASSF1A, APC, and CDKN2A (p16) was reported to predict tumor recurrence, thus establishing a link between gene methylation and tumor recurrence in lung cancer patients (Brock et al. 2008 ). This study established that the epigenetic status of tumors plays a critical role in clinical outcomes as well as in determining the aggressiveness of tumor development. MGMT (O6-methylguanine-DNA methyltransferase)-promoter methylation has been used to determine the treatment response among patients with glioblastoma (Butler et al. 2020 ). MGMT is a DNA-repair enzyme, and the efficiency of DNA repair in cancer cells plays an important role in therapeutic resistance. Thus, MGMT inactivation can sensitize such cells to therapeutic drugs (Verbeek et al. 2008 ). Many other epigenetic changes have started to emerge as predictive markers for the response to chemotherapy. BRCA1 hypermethylation is common in breast and ovarian cancers (Esteller et al. 2000 ). It has been found that BRCA1 methylation status can predict chemosensitivity and treatment response to cisplatin (Stefansson et al. 2012 ). The methylation of GSTP1 and MLH1 also predicts therapeutic outcomes in many cancer types (Shivapurkar and Gazdar 2010 ). Another oncogene, KRAS is itself capable of mediating epigenetic alterations, which are essential for cell transformation. KRAS has been found to promote DNA methylation by facilitating the recruitment of DNMT1 in the promoter regions of the specific genes (Serra et al. 2014 ). In H-Ras-transformed rat fibroblasts, MMP2 and clusterin gene expressions are suppressed by promoter DNA hypermethylation (Lund et al. 2006 ). Aberrant histone modifications can also be utilized as predictive biomarkers to evaluate the progression and treatment outcomes in cancers. A significant number of proteins that regulate histone modifications are themselves mutated in tumors. The polycomb group protein Enhancer of Zeste 2 (EZH2) acts as a critical regulator in prostate cancer development. EZH2 is involved in chromatin silencing by methylating histone3 on lysine27 (H3K27me3). EZH2 has been found to be overexpressed in metastatic prostate cancer, and tumors that express a higher level of EZH2 are associated with poorer prognosis (Gan et al. 2018 ; Liu et al. 2019 ). In addition, EZH2 overexpression has shown poor clinical outcomes in other cancers such as esophageal cancer, breast cancer, head and neck cancer, and glioblastoma (He et al. 2010 ). Lysine-specific demethylase 1 (LSD1) is another histone-modifying protein, which is linked with cancer development. LSD-1 removes the mono- and di-methyl groups from H3K4 and functions as a transcriptional repressor (Majello et al. 2019 ). The role of LSD1 in breast cancer has not been clearly established. A study has shown that inactivation of LSD1 by both genetic and pharmacological approaches leads to aggressiveness in luminal breast cancer, while another study shows that LDS1 mediates chemoresistance in the disease (Verigos et al. 2019 ; Hu et al. 2019 ). Bioinformatics, as a new emerging discipline, combines mathematics, information science, and biology and helps answer biological questions. There are several bioinformatics tools for DNA methylation analyses, but only a few platforms can correlate DNA methylation and gene expression for customized analyses. Several bioinformatics tools like COHCAP (Warden et al. 2013 ), PiiL tool (Moghadam et al. 2017 ), ViewBS (Huang et al. 2018a , 2018b ), correlate DNA methylation with gene expression. MethHC databases can also be utilized to visualize DNA methylation and gene expression on a web platform (Huang et al. 2015 ). MethGET is another web-based bioinformatics platform that correlates genome-wide DNA methylation and gene expression (Teng et al. 2020 ). The Gene-Ontology (GO) database provides a useful tool to analyse the functions of all dysregulated genes (Harris et al. 2004 ). Database for Annotation, Visualization, and Integrated Discovery (DAVID) is another online bioinformatics tool that provide biological meaning behind gene profile (Dennis Jr et al. 2003 ). In addition to that, Reactome also provides intuitive bioinformatics tools for visualisation, interpretation and analysis of pathways associated with genes (Fabregat et al. 2017 ). We anticipate that this review will provoke further investigation on the potential association between HP and air pollution, considering epigenetic modifications as a potential threat by which air pollution exerts its effect on disease development. It would be useful to identify a set of predictive biomarkers to assess the risk factors for the development and progression of the diseases associated with air pollution. However, in this review article we did not explore whether air pollution directly affects our genome by generating novel mutation, deletion, DNA breakage or chromosomal aberrations. We also did not discuss the genetic and immunological basis of HP development, although it could be crucial to understand the impact of air pollution in disease pathogenesis and may lead to the development of novel biomarkers to assess the risk of HP development due to air pollution.

The adverse effect of air pollution on our health is a matter of increasing concern. PM and other chemicals associated with air pollution pose various disease risks through both genetic and epigenetic alterations. Although the link between environmental exposure and disease risk due to epigenetic modifications is clearly established, little is known about the signaling pathways that connect the pollutants to the epigenome. Here we summarize the air pollutants that are associated with epigenetic modifications linked with increasing risk factors for several diseases. This discussion prompts the question whether these epigenetic changes could be utilized as risk assessment biomarkers for specific diseases associated with air pollution. Further research may identify more robust epigenetics marks that are associated a particular disease induced by specific pollutants. Moreover, such modifications may pass through several generations and predispose an individual to disease risks even without any direct exposure. These epigenetic alterations may serve as biomarkers for disease susceptibility for the next generation. Recently, it has been indicated that HP is associated with ambient air pollution. Pollution-mediated epigenetic changes in HP is yet to be explored. Such changes may sensitize the immune system, thereby making individuals susceptible to developing HP when exposed to the causative agents. Future research may use the findings of this paper to more comprehensively understand HP pathogenesis as well as to develop biomarkers for early detection and disease prognosis.

Air pollution has become a global threat to human health as it predisposes the risks of various diseases including cancers. In today’s world, a whole range of industries such as petrochemical firms, chemical factories, fertilizer units, and metallurgical setups contribute to air pollution. Apart from the industrial sector, the main emissions from certain types of power stations, cars, railways, airways, combustion engines also cause air pollution. Certain field-cultivation techniques release ammonia in the air from heavily fertilized fields and consequently pollute the environment. Even natural disasters like forest fire, volcanic erosion, and dust storms pollute the air. Other than these, biomass fuel uses, agricultural burning and utilization of adulterated fuels are some of the sources of air pollution (Manisalidis et al. 2020 ). The use of wood and charcoal for cooking and the operation of computers, printers, and photocopy machines, among others, contribute to indoor air pollution. Rapid urbanization also contributes to air pollution as it involves huge construction works. Moreover, some people are exposed to air pollution due to their professions—for instance, mining workers, traffic police and drivers are exposed to large amounts of air pollutants (Shukla et al. 2019 ). Air pollution is linked with a range of diseases like asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), cardiovascular disease, metabolic disorders, various reproductive conditions, dysregulation in immune response, neurodegenerative disorders, and cancer (Breton and Marutani 2014 ; Kurt et al. 2016 ). In 2013, World Health Organization (WHO) declared outdoor air pollution as a leading environmental cause of death associated with cancer (WHO 2013 ). A new study has indicated that air pollution may increase the risk of death due to COVID-19, an infectious disease that emerged in 2019 and is caused by novel coronavirus SARS-CoV-2. It has been suggested that 15% deaths across the world due to covid-19 could be linked with long-term exposure to air pollution (Pozzer et al. 2020 ). Outdoor air is contaminated with various chemicals such as engine exhausts, solvents, metals, dusts, and particulate matter (PM). Although air pollution is linked with numerous diseases, the impact of pollutants at both cellular and molecular levels has not been explored in detail. At the cellular level, exposure to different air pollutants or other toxic elements may lead to apoptosis by targeting mitochondria either directly or indirectly. Airborne particles have been found to induce apoptosis associated with ROS production, cellular stress, and DNA fragmentation (Andreau et al. 2012 ). Air pollution also induces inflammation in tissues by influencing specific cytokines and may also impact our endocrine system and metabolism processes (Prada et al. 2020 ). Moreover, recent studies indicate that epigenetic alterations could be an important pathway through which environmental factors exert their effects (Alfano et al. 2018 ). Epigenetic is the alteration at the gene expression level without changing the underlying DNA sequence such as DNA methylation, histone modification, miRNA, and non-coding RNA expression (Weinhold 2006 ; Jin et al. 2011 ). Cancers and many other diseases are often associated with epigenetic modifications (Weinhold 2006 ). Exposure to environmental stimuli may result in epigenetic changes, which, in turn, can impact gene expression and predisposition to disease risk (Breton and Marutani 2014 ). Little is known about how specific air pollutants generate specific epigenetic marks associated with a particular disease. Understanding epigenetic alterations due to specific pollutants may lead to the development of biomarkers to assess the disease risk due to air pollution. This narrative review provides an overview of the association between air pollution-induced epigenetic modification and disease development. It also explores whether such modification plays a role in HP pathogenesis. We have searched the PubMed and MEDLINE databases using the keywords ‘air pollution’, ‘air pollutant’, ‘air pollution-associated diseases’, ‘epigenetics’, and ‘hypersensitivity pneumonitis’. We have included all the original research articles published on the topic in the last 20 years. Overall, this review paper summarizes the findings of 235 articles, including 145 original research papers, 76 review articles, 6 systematic review and meta-analyses, 6 clinical trials, and 2 comparative studies. We have only included the studies where disease development is associated with air pollutants as well as with epigenetic changes. We have not included studies in which air pollution induces mutations or other alterations at the genetic level or causes other physiological changes associated with disease pathogenesis. The objective of this review is to discuss the association between certain air pollutants, epigenetic modifications, and diseases linked with those pollutants. These epigenetic alterations could be the mechanisms underlying the diseases associated with air pollution. Such knowledge may guide the prevention and treatment of the diseases. We also discuss HP pathogenesis because certain pollutants could influence HP development, and epigenetic modifications could be a mechanism involved in this process. Moreover, this review will help researchers to develop epigenetic biomarkers for early detection and monitoring prognosis of diseases associated with air pollution. Other mechanisms involved in air pollution-associated disease development have not been discussed in this review article.