University of Groningen Air pollution exposure of lung models Cattani Pinto Cavalieri, Isabella

University of Groningen

Air pollution exposure of lung models

Cattani Pinto Cavalieri, Isabella

10.33612/diss.172080794

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Cattani Pinto Cavalieri, I. (2021). Air pollution exposure of lung models: focus on inflammation, oxidative stress and cyclic AMP signaling. University of Groningen. https://doi.org/10.33612/diss.172080794

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Chapter 1

10 Air pollution

Air pollution is a modern world problem with high impact on human health as it increasingly contributes to overall morbidity and mortality. Health problems associated with air pollution are responsible for the leading cause of death in 58% of patients dying from ischemic heart disease and stroke, 18% in those with COPD and acute lower respiratory infections, and 6% in subjects with lung cancer (WHO , Mills, Donaldson et al. 2009, Fischer, Marra et al. 2015, Wolf, Stafoggia et al. 2015, Raaschou-Nielsen, Beelen et al. 2016, Consonni, Carugno et al. 2018, Miller and Newby 2020). Additionally, an association between air pollution and increased mortality and hospitalization of patients with COPD, including underlying (patho)physiological mechanisms, has been reported (Atkinson, Kang et al. 2014, Li, Sun et al. 2016, DeVries, Kriebel et al. 2017, Steiner, Bisig et al. 2016, Carlsten, Blomberg et al. 2016).

Air pollution primarily affects the cardiovascular and respiratory systems, with the latter generally being affected first because of the direct and frequent contact with air pollutants. Air pollution problems occur mainly in urban areas, where the presence of air pollution sources is relatively high and usually exceeds WHO air quality guideline levels (WHO 2006). In addition, populations of low- and middle-income countries are more susceptible to the impact of air pollution on health (WHO 2006). The main sources related to an increase of air pollution are industry, transport vehicles, the burning of fossil fuels, and burning from natural processes. Among the pollutants released during these processes, the most common are particulate matter (PM), nitrogen dioxide, ozone, sulfur oxides, carbon monoxide and hydrocarbons (Chen and Kan 2008).

PM may vary in size, chemical composition, and source; because of these heterogenous characteristics it constitutes one of the main problems of air pollution. The aerodynamic diameter is used to classify PM; coarse particles are known as PM10 and have a diameter <10 µm, PM2.5 are fine particles with a diameter < 2.5 µm, and PM0.1 are considered ultrafine particles with a diameter < 0.1 µm (EPA 2010). In urban centers, the main source of air pollution is diesel fuel combustion. In addition to the release of the aforementioned pollutants, diesel exhaust particles (DEP) are produced as well (Steiner, Bisig et al. 2016). DEP contain metals (cadmium, chromium, copper and iron) and organic compounds, such as polycyclic aromatic hydrocarbons (PAH) (benzo [a] anthracene, benzo [a] pyrene and benzo [b] fluoranthene), absorbed in their surface, whereas the center core consists of elemental carbon (Wichmann 2007). As a result of their composition and small size (diameter <2.5 µm), DEP are associated with high toxicity to health (Lucking, Lundback et al. 2011, Xu, Barregard et al. 2013, Carlsten,

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Blomberg et al. 2016, Zhang, Zhang et al. 2017, Vilcassim, Thurston et al. 2019, Cattani-Cavalieri, Valenca et al. 2020, Coleman, Ezzati et al. 2021). Because of the deleterious effects of burning fossil fuels, biodiesel has been considered as a potential alternative in order to reduce harmful emissions. Biodiesel is mainly produced from vegetable oil or animal fats, and its use showed a reduced emission of particulate matter, carbon monoxide and total hydrocarbons as compared to conventional diesel (Schmidt 2007). The most common biodiesel blend with a composition of 20% biodiesel and 80% diesel exhibits a reduced emission of particulate matter of 10.1 % (EPA 2002, Christian Rodriguez Coronado 2009). Therefore, studies are needed to understand the molecular mechanisms underlying the effects of different sources of air pollutants, and, more specifically, identify their potential impact on lung (patho)physiology (Fig. 1).

Figure 1. Schematic overview of in vivo and in vitro models for the study of the potential impact of air pollution exposure on the lung. Diesel-biodiesel particulate matter (DBPM) and/or diesel exhaust particles (DEP) induce inflammation and oxidative stress, mitochondrial dysfunction, and changes in cAMP, ultimately promoting lung injury. These models will reveal if DEP and DB differentially impact these parameters. For further details, see main text.

12 Oxidative stress and Nrf2 pathway

Reactive oxygen species (ROS) are produced under physiological conditions as a result of normal cellular metabolism, including mitochondrial respiration. ROS consist of radical and non-radical oxygen species generated by the partial reduction of oxygen (Ray, Huang et al. 2012). The free radicals are highly reactive and unstable because of at least one unpaired electron in the outer shell of the oxygen atom. Some examples for free radicals include superoxide anion (O∙−2), oxygen radical (O∙∙2), and hydroxyl radical (OH∙), while the most commonly known non-radical is hydrogen peroxide (H2O2) (Auten and Davis 2009).

ROS production can also occur in response to exogenous triggers/irritants, such as air pollutants and cigarette smoke (Cloonan, Kim et al. 2020). Under physiological conditions and in relatively low concentrations, ROS production is essential for the regulation of various cellular processes (Ray, Huang et al. 2012). On the other hand, high concentrations and aberrant production of ROS can promote oxidative stress and oxidative damage to (mitochondrial) lipids, DNA, and proteins (Ray, Huang et al. 2012). The imbalance between oxidants and antioxidants is further accelerated under conditions of inflammation, hyperoxia, ischemia, and reperfusion. Such pathological conditions seem to contribute to the development and progression of several diseases, including COPD, acute respiratory syndrome, lung cancer and asthma (Cloonan, Kim et al. 2020). The antioxidant system acts as a first line defense system in order to reduce the effects of ROS overproduction and oxidative stress (Ighodaro and Akinloye 2018). The main components of the enzymatic antioxidants system are superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (Gpx) (Ighodaro and Akinloye 2018). Other antioxidants acting as part of the antioxidant system include glutathione, thioredoxin, and heme oxygenase-1 (HO-1)(Lu and Holmgren 2014, Ighodaro and Akinloye 2018).

Exposure of BALB/C mice to DEP by intratracheal instillation for 24 h has been shown to promote oxidative stress, as measured by elevated ROS levels in lung tissue (Nemmar, Al-Salam et al. 2015). In addition, exposure of 3- and 15-month-old (equivalent to humans aged 20-30 and 50-69 years, respectively) C57BL/6 male mice to diesel exhaust for 30 days elevated SOD-1 (CuZn) activity in lung tissue (Ribeiro Junior, de Souza Xavier Costa et al. 2019). Conversely, exposure of outbred mice to DEP by intratracheal instillation for 24 h reduced SOD activity in bronchoalveolar lavage (Nemmar, Subramaniyan et al. 2012). This is in accordance with reports demonstrating

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that mouse strains are differentially susceptible to the exogenous trigger cigarette smoke (Santos-Silva, Pires et al. 2012, Pouwels, Faiz et al. 2017).

Nuclear factor erythroid-derived-like 2 (Nrf2) is a crucial transcriptional factor involved in the defense against oxidative stress (Fig. 2) (Niture, Kaspar et al. 2010). Under conditions of oxidative stress, Nrf2 dissociates from its repressor, kelch-like ECH-associated protein 1 (Keap-1) (Nguyen, Nioi et al. 2009). Nrf2 can then translocate to the nucleus where it (together with the antioxidant response element (ARE)) promotes transcriptional activity, subsequently allowing the transcription and release of phase II antioxidant as well as cytoprotective genes (Niture, Kaspar et al. 2010). Some of the enzymes involved in the Nrf2 pathway that participate in the cellular detoxification are: HO-1, thioredoxin, NAD(P)H quinone oxidoreductase 1 (NQO1), thioredoxins, and γ-glutamyl cysteinyl synthetase (γ-GCS) (composed of a catalytic (GCLC) and a modifier (GCLM) subunit) (Niture, Kaspar et al. 2010).

Eight-hour exposure of human bronchial epithelial cells (BEAS-2B) to primary ultrafine particles (generated from diesel) upregulated the heme oxygenase-1 (HMOX1) gene (Grilli, Bengalli et al. 2018). Furthermore, exposure of BEAS-2B cells to DEP (collected from a diesel engine from a 2004 model Volkswagen bus with a 6-cylinder MWM x-10 engine) for 2 h upregulated Nrf2, HO-1, and NQO1 genes (Frias, Gomes et al. 2020), whereas primary bronchial epithelial cells subjected to DEP (collected from a tractor engine) for 24 h exhibited elevated HMOX1 and Gpx gene expression (Ji, Upadhyay et al. 2018). These findings demonstrate that specific gene transcription associated with the antioxidant response is effectively induced by DEP exposure in human bronchial epithelial cells.

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Figure 2. Schematic representation of the nuclear factor erythroid- derived-like 2 (Nrf2) pathway.

Under basal conditions, Nrf2 is bound to kelch-like ECH-associated protein 1 (Keap-1) and sequestered in the cytosol. Oxidative stress causes Nrf2 and Keap-1 to dissociate, allowing nuclear translocation of Nrf2. In the nucleus, Nrf2 binds to the antioxidant response element (ARE) in the promoter regions of various detoxification and antioxidant genes, and induces the transcription of cytoprotective enzymes such as heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), catalase (CAT), thioredoxin, and glutamine-cysteine ligase (GCL) subunits. Reactive oxygen species (ROS). For further details, see text.

Mitochondrial function

Mitochondria are no longer simply considered a powerhouse of the cell but have also been recognized to play important regulatory roles in various cellular events, including cell cycle, metabolism, and survival (Chandel 2015). The mitochondrial electron transport chain is composed of 4 transmembrane protein complexes (i.e., complexes I-IV): complex I (NADH-ubiquinone oxidoreductase) has been identified as the largest enzyme complex in the electron transport chain (ETC) (Efremov and Sazanov 2011); complex II (succinate dehydrogenase) belongs to both the Krebs cycle and the ECT, as such representing a link between metabolism and oxidative phosphorylation (Cecchini 2003); complex III (ubiquinol-cytochrome c oxidoreductase) transfers the electrons across the intermembrane space to cytochrome c (Chandel 2010); and lastly, complex IV (cytochrome c oxidase) transfers electrons from cytochrome c to the terminal electron

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acceptor, thereby reducing oxygen to water. It is well established that mitochondria are the main source of cellular ROS. Part of the electrons can leak out of the respiratory chain to form the free radical superoxide. Mitochondrial ROS are not only generated under pathological conditions and play an important role physiologically as well. Indeed, ROS have been implicated as second messengers in several cellular signaling pathways, such as Nrf2-, mitogen-activated protein kinase- (MAPK), and redox-factor 1-associated signaling (De Giusti, Caldiz et al. 2013, Brand 2016).

Oxidative stress can alter cellular function in several ways, including the promotion of mitochondrial dysfunction. Mitochondrial dysfunction affects various processes such as electron transport and ATP synthesis leading to an increase in ROS production and subsequent oxidative stress, inflammation, DNA damage, and cell death (Turrens 2003, Cloonan and Choi 2016, Cloonan, Kim et al. 2020). A relationship between mitochondrial dysfunction as an underlying pathological mechanism and several chronic diseases, including cardiovascular diseases, COPD, and asthma, has been reported (Cloonan and Choi 2016).

Inflammation and the NF-κB pathway

Nuclear factor κB (NF-κB)-mediated signaling is a well-recognized pathway responsible for the regulation of several genes involved in different types of cellular processes, including immune and inflammatory responses. To date, five members belonging to the NF-κB transcription factor family of proteins have been identified in mammals: NF-κB1 (or p50), NF-κB2 (or p100/52), RelA (or p65), RelB and c-Rel (Liu, Zhang et al. 2017). Under physiological conditions, NF-κB is sequestered in the cytoplasm and its activity is inhibited by IκB proteins. Activation of NF-κB can be induced by a wide range of stimuli, including oxidative stress and inflammation. Upon activation, NF-κB is translocated to the nucleus, where it promotes the expression of a variety of genes, including those encoding cytokines (TNF-α, IL-1β and IL-6), chemokines (IL-8), growth factors, and cell adhesion molecules (Lawrence 2009).

Long-term exposure (12 weeks) of rats to diesel engine exhaust elevated IL-8, IL-6 and TNF-α protein levels and mRNA expression in bronchoalveolar lavage and lung tissue, respectively (Wang, Li et al. 2019). Another study performed in mice exposed to DEP (collected after 1 day of routine operation of a 210-hp engine bus from São Paulo's metropolitan fleet) for 30 days revealed an increased number of IL-6-, TNF-α-, and p65-NF-κB positive cells in the alveolar septa of lung slices (Santana, Pinheiro et al. 2019). In accordance with these findings, mice intratracheally instilled with DEP for 24 h

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exhibited elevated levels of NF-κB in lung tissue (Nemmar, Al-Salam et al. 2018). Some mechanistic insight came from an earlier study in human epithelial cells, in which exposure to particulate matter (PM2.5) for 6 h induced nuclear translocation of NF-κB (Dagher, Garcon et al. 2007).

Epithelial-to-mesenchymal transition

Epithelial to mesenchymal transition (EMT) is a cellular process during which epithelial cells assume a more mesenchymal cell-like phenotype; transforming growth factor-β1 (TGF-β1) is among the best-known factors that can drive EMT (Zuo, Cattani-Cavalieri et al. 2019). It has been reported that EMT can also be induced by ROS, and details are provided in Chapter 2. EMT involves the loss of cell–cell junctions, disrupted cell interactions with the basal membrane, and reduced apicobasal polarity. EMT converts the cells from cube- to spindle-shaped (fibroblastoid), which enhances the cellular ability to effectively migrate and invade (Thiery, Acloque et al. 2009, Zuo, Cattani-Cavalieri et al. 2019). Several epithelial and mesenchymal cell markers have been identified to closely monitor the progression of the EMT process. In chronic inflammatory lung diseases, such as COPD, a wide spectrum of EMT states have been discussed (Sohal, Reid et al. 2010, Milara, Peiro et al. 2013, Sohal and Walters 2013, Wang, Wang et al. 2013, Jolly, Ward et al. 2018, Zuo, Trombetta-Lima et al. 2020). Thus, EMT is typically characterized by a loss of the epithelial markers E-cadherin and ZO-1 concomitant with an increase in the mesenchymal cell phenotype markers collagen, β-catenin, vimentin, and -smooth muscle actin (-SMA) (Thiery, Acloque et al. 2009, Jolly, Ward et al. 2018).

It has been shown that exposure of human bronchial epithelial cells to DEP reduces E-cadherin and increases vimentin protein expression (Rynning, Neca et al. 2018), indicating the induction of EMT in these cells in response to DEP.

Cyclic adenosine monophosphate (cAMP) signaling

Cyclic adenosine monophosphate (cAMP) is one of the most important second messengers known to control several physiological cellular processes, including, but not limited to, cell adhesion, migration, and proliferation (Oldenburger, Maarsingh et al. 2012, Schmidt, Dekker et al. 2013, Zuo, Cattani-Cavalieri et al. 2019). cAMP signaling (Fig. 3) is initiated through the activation of Gs-protein-coupled receptors, such as the β2-adrenoceptor (β2-AR) and distinct prostanoid E receptors (EP), subsequently resulting in

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the stimulation of adenylyl cyclase (AC) and conversion of adenosine triphosphate (ATP) into cAMP (Oldenburger, Maarsingh et al. 2012). Interestingly, cAMP-generating G-protein-coupled receptors have been reported to be functionally repressed in diseased lungs (Oldenburger, Maarsingh et al. 2012, Musheshe, Schmidt et al. 2018, Haak, Kostallari et al. 2019, Haak, Ducharme et al. 2020). The intracellular level of cAMP is tightly controlled by phosphodiesterases (PDEs), and more specifically PDE1, PDE3 and PDE4, which degrade cAMP in 5'-AMP (Zuo 2019). Although not discussed in detail here, it is worth mentioning that dual-specific PDEs such as PDE1 and PDE3 also degrade cGMP (Zuo, Han et al. 2018, Zuo 2019, Zuo, Faiz et al. 2020). In addition to cAMP-producing receptors, expression and function of PDEs (primarily PDE4 and PDE3) are altered in lung pathologies (Zuo, Han et al. 2018, Zuo, Faiz et al. 2020).

Cyclic-AMP signaling is coordinated by several members of the A-kinase anchoring protein (AKAP) family, known to associate with mitochondria (Poppinga, Munoz-Llancao et al. 2014) (Fig. 3). Regulation of cellular functions by cAMP seems to occur through activation of distinct intracellular effectors, including protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), of which 2 subtypes (Epac1 and Epac2) have been identified (Schmidt, Dekker et al. 2013, Raker, Becker et al. 2016). Additionally, PKA and Epacs can both associate with certain AKAP family members, which tether these effectors to the mitochondria where they affect mitochondrial function and cellular homeostasis. Previously, our group reported that β2-ARs, EPs, Epacs and AKAPs are differentially linked to EMT, inflammation and oxidative stress (Oldenburger, Maarsingh et al. 2012, Oldenburger, Roscioni et al. 2012, Oldenburger, Timens et al. 2014, Oldenburger, van Basten et al. 2014, Jansen, Poppinga et al. 2016). For example, we demonstrated that exposure of airway smooth muscle cells to cigarette smoke extract reduced the protein expression of Epac1 but not Epac2 (Oldenburger, van Basten et al. 2014). Furthermore, we showed that exposure of mice to cigarette smoke promoted different functional responses of Epac1 and 2; thus, Epac1 was able to inhibit remodeling linked to EMT, whereas Epac2 promoted inflammation (Oldenburger, Timens et al. 2014). We also demonstrated that Epac1 links β-catenin to the EMT process in alveolar epithelial adenocarcinoma cells (Jansen, Poppinga et al. 2016). Taken together the exogeneous trigger cigarette smoke altered cAMP signaling and induced EMT, inflammation and oxidative stress processes associated with the development and progression of COPD (Oldenburger, Timens et al. 2014, Zuo, Trombetta-Lima et al. 2020). However, our current knowledge about a potential association between cAMP signaling and air pollution exposure is rather limited, particularly considering that different air pollutants seem to promote a distinct damage pattern in the lung (Nemmar, Al-Salam

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et al. 2018, Cattani-Cavalieri, Valenca et al. 2019, Cattani-Cavalieri, da Maia Valenca et al. 2020).

Figure 3. Schematic illustration of cAMP signaling in the lung. Stimulation of Gs-protein-coupled

receptors (e.g., β2-AR and EP4) leads to activation of adenylyl cyclase (AC) and subsequent generation of cAMP. The cellular level of cAMP is tightly controlled by the activity of phosphodiesterases (PDEs). The intracellular cAMP effectors protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) are activated by cAMP and associate with A-kinase anchoring protein (AKAP) family members. Further details, see text.

Potential drugs to diminish lung damage by air pollutants: dimethyl fumarate The increasing health risk associated with lung damage caused by air pollution exposure (Prüss-Üstün 2016, Choi, Oh et al. 2018) emphasizes the importance of implementing therapeutic strategies aimed at reducing air pollutant-related lung injury. For example, it has been shown that emodin - a drug known for its anticancer and antioxidant activities – diminished inflammation and oxidative stress in BALB/C mice exposed to DEP (Nemmar, Al-Salam et al. 2015). In addition, our group has previously demonstrated that eugenol administered to BALB/C mice 1 h after exposure to DEP limited inflammation and alveolar collapse (Zin, Silva et al. 2012).

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Dimethyl fumarate (DMF) (Fig. 4) is a fumaric acid ester which has been used in the treatment of psoriasis and relapsing forms of multiple sclerosis. In experimental models of multiple sclerosis and traumatic brain injury, DMF represented the most pharmacologically effective fumaric ester acid based on its oxidative, anti-inflammatory and immune-modulating modes of action (Mrowietz and Asadullah 2005). The pharmacokinetics of fumaric acid esters are not well documented. However, it has been suggested that orally administered DMF is rapidly converted to monomethylfumarate (MMF) in the small intestine (Dubey, Kieseier et al. 2015). The main mechanisms of action of DMF include the activation of Nrf2, which importantly regulates cellular antioxidant responses (as earlier described and depicted in Fig. 2), and inhibition of the NF-κB pathway linked to immunomodulation. DMF interferes with the binding between Nrf2 and Keap-1, allowing Nrf2 to translocate into the nucleus and subsequently induce transcription of cytoprotective enzymes, such as HO-1 and SOD (Kourakis, Timpani et al. 2020).

Figure 4. Molecular structure of dimethyl fumarate (DMF).

DMF inhibited proinflammatory cytokine secretion through inhibition of NF-kB in airway smooth muscle cells (Seidel, Merfort et al. 2009). Further, rats subjected to sepsis and treated with DMF showed a decrease in the oxidative stress marker malondialdehyde, and a reduction of neutrophil infiltration and nitric oxide metabolism followed by an increase of SOD and CAT activities in the lung (Giustina, Bonfante et al. 2018). An in vivo study in a mouse model of hepatic injury revealed that DMF treatment reduced NF-κB and elevated Nrf2 and HO-1 protein levels in the liver (Abdelrahman and Abdel-Rahman 2019). Despite its promising pharmacological profile, DMF has not been studied in experimental models of air pollution exposure. Despite these promising observations and an apparent association between DMF treatment and induction of the antioxidant response in several organs, including the lung, the effects of DMF have not been studied in experimental models of air pollution exposure.

20 Scope of the thesis

In this thesis, we investigated mechanisms of air pollution-induced inflammation and oxidative stress, and, more specifically, the impact on cAMP signaling and mitochondrial function. To this aim, we utilized different in vitro and in vivo models of air pollution exposure and studied the effects of diesel-biodiesel particulate matter (DBPM) and diesel exhaust particles (DEP) on these parameters (Fig.1).

In Chapter 2, we provide an overview on how cAMP scaffolds and distinct signalosomes in different subcellular compartments may contribute to COPD. In addition, the epithelial-to-mesenchymal transition (EMT) process, including the involvement of cAMP compartmentalization, and its potential role in the development and progression of COPD are described.

Chapter 3 is a comprehensive review that focuses on the regulation of several PDEs and the therapeutic potential of using (combinations of) selective PDE inhibitors in the treatment of COPD and asthma.

In Chapter 4, we review how targeting the mitochondrial cAMP nanodomain may have therapeutic potential in reducing the air pollutant-induced decrease in cardiopulmonary function. We suggest that mitochondrial dysfunction induced by exposure to air pollutants — particularly DEP — is key to cardiopulmonary impairments. Furthermore, we propose that specifically targeting the mitochondrial cAMP nanodomain may effectively diminish the DEP-induced decline in cardiopulmonary function.

In Chapter 5, we explore the effects of acute exposure to DBPM on Nrf2 and NF-kB signaling pathways in the lungs using an in vivo mouse model.

In Chapter 6, we investigate the impact of DEP on cAMP signaling and mitochondrial function in human bronchial epithelial (BEAS-2B) cells. The effects of DEP exposure on cAMP levels, parameters of mitochondrial function, markers of oxidative stress, inflammation and EMT as well as expression of Gs-protein-coupled receptors and members of the AC superfamily will be discussed.

In Chapter 7, we evaluate the therapeutic potential of DMF to ameliorate air pollution-induced inflammation and oxidative stress in the lungs using a mouse model of chronic DEP exposure.

Chapter 8 provides a summary and general discussion of our results. In addition, several perspectives for future research are outlined.

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