Expression of MATE1, P-gp, OCTN1 and OCTN2, in epithelial and immune cells in the lung of COPD and healthy individuals

Inhaled administration is the main treatment route in chronic obstructive pulmonary disease (COPD), enabling high local drug concentrations, lower systemic exposure and consequently, less risk of side effects. The inhalation device configuration and drug particle size affect the drug deposition pattern in the lung [1, 2]. The mechanisms that govern pulmonary drug disposition after inhalation, such as dissolution, uptake and elimination are however less well understood and are also affected by disease pathology. Knowledge on the parameters affecting drug disposition in the lung is important when predicting tissue retention and the likelihood of reaching sufficient drug concentrations at the pharmacological target.

Membrane transporters have proven important in drug disposition, and consequently in drug efficacy and toxicity [3]. However, drug transporter expression and function in the lung is still an emerging area [4‐6]. Several drug transporter genes are known to be expressed in the lung [7, 8], for example organic cation transporters (e.g. OCT1, OCT3, OCTN1, OCTN2), and organic anion transporting polypeptides (e.g. OATP2A1, OATP2B1, OATP3A1, OATP4A1, OATP4C1) [7, 8]. Similarly, mRNA expression of transporters of the ATP-binding cassette family has been described in lung, including the multidrug resistance proteins (e.g. P-glycoprotein (P-gp)), breast cancer resistance protein (BCRP) and multidrug-resistance associated proteins (e.g. MRP1) [7, 8]. The expression of some of the transporters above was also confirmed on protein level through quantification by mass spectrometry [9].

In the present paper, we have focused on the expression of organic cation transporters OCT1, OCTN1, OCTN2 and MATE1, as well as P-gp. OCT1, OCTN1 and OCTN2 are mainly involved in cellular uptake of compounds, whereas MATE1 secretes organic cations out of cells. P-gp excretes drugs out of cells by an ATP-dependent mechanism. In particular the organic cation transporters, OCT1, OCTN1 and OCTN2 have been found to transport inhaled drugs e.g. muscarinic antagonists and β-adrenergic agonists [10‐13], several of which have poor passive permeability and therefore are dependent on transporters to cross cell membranes [6, 10]. It has been proposed that organic cation transporters are important for these drugs to cross the airway epithelial barrier to reach the smooth muscle cells [11, 14]. Furthermore, organic cation transporters have been demonstrated to impact the disposal of inhaled drugs. As an example, OCT3 facilitates uptake of β-adrenergic agonists into airway smooth muscle cells [15]. MATE1 substrate specificity is similar to OCT1 and OCT2 [16] and we have recently shown that this transporter is expressed in lung [7]. Notably, the muscarinic antagonist ipratropium was recently reported to be a substrate of MATE1 [17]. P-gp is also of interest since it has a broad substrate specificity including basic compounds, a common feature of inhaled drugs. Moreover, P-gp is known to transport some glucocorticosteroids [18, 19], commonly used in the treatment of COPD.

Due to the heterogenic structure of the lung, with highly specific expression patterns [6, 20], it is important to study the spatial distribution and cellular localization of pulmonary drug transporters. High, cell specific expression of a gene/protein, could be masked by low average whole organ expression. In immunohistochemistry (IHC) studies, P-gp has been detected in the apical membrane of bronchial and bronchiolar epithelium and to some degree in alveolar macrophages [21]. Furthermore, OCTN1 and OCTN2 were expressed in respiratory epithelial cells [11], OCTN1 predominantly in the central airways whereas OCTN2 was focused peripherally, e.g.in alveolar epithelia [11], where these transporters may contribute to absorption of cationic bronchodilators [11‐13]. We have recently investigated the mRNA expression of several membrane transporters in pulmonary tissue and detected similar expression levels in the lungs from ex-smoker COPD patients compared to healthy lung tissue [7]. However, little is known about the cell specific expression pattern of membrane transporters, e.g. in inflammatory cells.

COPD is associated with chronic inflammation, tissue destruction, progressive loss of lung function and airway obstruction. Smoking is the main cause of COPD, estimated to be the third leading cause of death in 2020 [22, 23]. Alveolar macrophages are key players in the inflammatory response and their quantity is increased in the lungs of COPD patients and healthy smokers, compared to non-smokers [24‐27]. Alveolar macrophages secrete both pro- and anti-inflammatory cytokines, and components promoting emphysema, fibrosis, and mucohypersecretion [28‐30]. Hence, alveolar macrophages, and other inflammatory cells, are interesting targets for drug development. The expression of drug transporters on the surface of these cells could affect drug concentration at the site of the target. A better understanding of the transporter expression in healthy and diseased lung would facilitate the development of successful drugs for COPD and other lung diseases.

This study aimed at investigating the pulmonary cellular expression patterns of the human membrane transporters OCT1, OCTN1, OCTN2, MATE1 and P-gp. IHC analyses with specific anti-bodies were performed on lung biopsies from ex-smokers with severe stage of COPD and healthy non-smokers. Furthermore, drug transporter expression in bronchoalveolar lavage as well as in a model of macrophages was investigated.

In the current study, we present for the first time the localization of MATE1 protein, an important drug transporter, in the human lung e.g. on the apical side of bronchial and bronchiolar epithelial cells, and in particular in alveolar macrophages and other immune cells. In addition, we demonstrate that P-gp, OCTN1 and OCTN2 proteins are expressed in epithelial and inflammatory cells in the lung including in alveolar macrophages, and confirmed mRNA expression of all these transporters, including MATE1 as well as OCT1, in BAL cells obtained from healthy subjects. Notably, mRNA encoding for P-gp (ABCB1) and MATE1 (SLC47A1) were down-regulated in BAL cells from smokers as compared to non-smokers whereas a difference was not observed in non-smoking COPD patients when compared to healthy volunteers. These findings are important to consider when developing drugs towards inflammatory lung disease. As a result of variability in transporter expression, drug disposition at the site of target, e.g. intracellularly in alveolar macrophages, could be distinct in various study populations, e.g. smokers compared to non-smokers or a healthy lung compared to an inflamed lung. Early phase clinical studies are often conducted in young healthy males and it is important to keep in mind when analyzing early biomarkers that drug disposition in this population might not completely reflect the situation in patients.

We recently showed that MATE1 mRNA (SLC47A1) was expressed in lung tissue from COPD patients and healthy subjects [7]. In the present study, we demonstrate MATE1 protein expression on the apical membrane of epithelial cells in bronchial and bronchiolar epithelium. Previously, MATE1 protein has mainly been studied in kidney and liver where it is located on the apical membrane of proximal tubule cells and hepatocytes, respectively [35].

In accordance with previous literature, OCTN1, OCTN2 and P-gp proteins were also expressed in epithelial cells in the bronchi [5, 6, 11, 21]. Several inhaled drugs such as β-adrenergic agonists and muscarinic antagonists are good substrates of organic cation transporters, and are dependent on these transporters to cross cell membranes, due to their polarity [6, 10]. It has been suggested that transport of β-adrenergic agonists and muscarinic antagonists by OCT1, OCTN1 and OCTN2, over the apical membrane of epithelial cells in the bronchi serves as the first step for these compounds to be transported across the epithelial cell barrier to their target receptors in the smooth muscle cells [11, 14]. However, the transporter that secretes these cationic compounds on the basolateral side of the airway epithelial cells has not yet been identified. In the renal proximal tubule cells, the basolateral expression of OCT2 works in concert with the apically expressed MATE1 to transport cationic drugs like cimetidine from blood to urine. Thus, MATE1 would be a plausible candidate to transport cationic inhaled drugs out of airway epithelial cells on the basolateral side. However, we here demonstrate that MATE1 is located on the apical side of bronchial and bronchiolar epithelial cells, thus making this role for MATE1 unlikely. Interestingly, in an in vitro model of bronchial epithelium using Calu-3 cells, it was recently demonstrated that ipratropium, a muscarinic antagonist that is a substrate for MATE1 [17], was not transported trans-vectorially through the cell layer but was instead both taken up and excreted on the apical side of the cells [36]. Although it is not known if MATE1 is expressed in Calu-3 cells, our finding of MATE1 on the apical side of the bronchial epithelium in the human lung indicates that is the case. Therefore, MATE1 may be responsible for the efflux of ipratropium on the apical side of Calu-3 cells. Thus, the mechanism of muscarinic antagonist and β-agonist absorption through the bronchial epithelial cell layer to the smooth muscle cells, in particular the efflux on the basolateral side, still remains to be clarified. Interestingly, expression of MATE1 protein, as well as the other investigated transporters OCTN1, OCTN2 and P-gp, was also found in immune cells, with particularly strong expression in alveolar macrophages. Both epithelial cells and macrophages form the surface that is exposed to a large variety of environmental factors, such as microorganisms, inhaled drugs, cigarette smoke and other pollutants, and function as a defense system. In addition, these cell types are key components of the pulmonary inflammatory response in COPD and other diseases. Expression of membrane transporters in immune cells, in the lung or other tissues, has not been as extensively studied. However, it is known that transporters are expressed in several types of immune cells, including T-cells, monocytes, macrophages, and dendritic cells [37]. Transporters are believed to be involved in active secretion of endogenous compounds in these cell types, for example inflammatory mediators such as prostaglandins, leukotrienes, sulfatides and cyclic nucleotides [37]. Additionally, cholesterol [38], cortisol, platelet activating factor and sphingosine 1-phosphate are substrates for P-gp, which is expressed in several immune cells [39‐41] . Although not fully elucidated, there are thus several indications that membrane transporters play an active role in the lung immune response and in inflammatory diseases in the lung through their uptake and efflux of immunomodulating agents. Transporters may also be important for the disposal of drugs by inflammatory cells and may thus play a role in targeting cells involved in disease progression.

The mRNA expression of ABCB1 (P-gp) and SLC47A1 (MATE1) was significantly lower in BAL cells isolated from smokers as compared to never smokers. Notably, the decrease in P-gp and MATE1 expression correlated to the extent of smoking i.e. pack years or number of cigarettes/day. The regulation of drug transporters is often specific to each cell and tissue, leading to a differential expression pattern of transporters across tissues as well as a tissue specific response to endogenous and exogenous factors. Drugs and other xenobiotics have the capability to both induce and repress expression of transporter mRNA, by binding to nuclear hormone receptors [42], and P-gp is known to be upregulated via the nuclear receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) [42]. However, there is very limited knowledge about nuclear hormone receptor dependent regulation of MATE1 expression although hepatocyte nuclear factor 4α (Hnf4α) is likely essential for its expression in liver [43].

The reduced expression of ABCB1 (P-gp) and SLC47A1 (MATE1) in BAL cells from smokers as compared to non-smokers may be a result of different stimuli. Cigarette smoke contains a wide range of chemical substances known to directly interact with nuclear hormone receptors. This includes polyaromatic hydrocarbons, smoke components with the ability to activate the arylhydrocarbon receptor (AhR), resulting in strong induction of drug disposition genes e.g. CYP1B1 in the lung [44]. Downregulation of MATE1, and other transporters, by cigarette smoke extract and the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has previously been seen in HepRG cells, a hepatocyte cell line [45]. However, regulation of P-gp by AhR has to our knowledge not been described in the literature, and no change in P-gp expression was observed in lung tissue from smokers or COPD patients when compared to healthy tissue [7, 46]. It is therefore probable that another mechanism regulates the observed downregulation of ABCB1 (P-gp) expression, observed in this study. In addition, cytokines released during inflammation are known to down-regulate several CYP enzymes [47]. Interestingly, pro-inflammatory cytokines have been shown to downregulate MATE1 expression in synovial fibroblasts from human subjects with rheumatoid arthritis [48]. Similarly, P-gp was downregulated in an acute inflammation model using human peripheral blood mononuclear cells [49]. The mechanism of downregulation of P-gp and MATE1 mRNA that was observed in BAL cells from smokers needs to be further investigated. It is important to point out that expression regulation in disease is complex and may be affected by different factors, in the case of COPD by e.g. inflammatory mediators and smoke components.

The human monocyte cell line THP-1 is widely used as a model system for studying various mechanisms in monocytes and macrophages, and the cells adopt several key features of tissue macrophages after differentiation with PMA [50]. The PMA treatment induces the differentiation into proinflammatory macrophage-like cells that however retain the capability to be further stimulated by LPS [51] and other Toll Like Receptor ligands (data not shown). The THP-1 cell line has also been used for in vitro stimulations using cigarette smoke extract, and these studies have shown that smoke extract exposure induce a range of transcriptional changes, related to inflammation, oxidative stress and several other cellular processes [52], and can potentiate the LPS-induced NF-kB signaling [53]. The THP-1 cell line has also been used to study the expression and function of membrane transporters. Shimizu et al. studied SLC22A4 (OCTN1) in the context of colitis, and described an increase in gene expression after PMA differentiation of THP-1 cells [54]. Perdomo and coworkers [55] detected P-gp protein in differentiated THP-1 cells, and found that benznidazole can further upregulate the protein levels of this protein. We analyzed the expression of several genes encoding for membrane transporter and found significant differences in SLC22A1 (OCT1) and ABCB1 (P-gp) mRNA levels, in differentiated versus undifferentiated cells. This indicates that the macrophage-like THP-1 cells, with several characteristics resembling tissue macrophages, may be more adapted to actively transport a variety of small compounds, compared to monocytes. We further stimulated the differentiated cells with LPS or CSE, though this did not significantly change the expression of the analyzed genes. While this model has some limitations, this suggests that short-term exposure to smoke or inflammatory stimuli is not sufficient to induce the downregulation of gene expression, that is seen for ABCB1 (P-gp) and SLC47A1 (MATE1) in the human primary BAL cells from smokers. Thus, additional signaling mechanisms, related to the long-term smoke exposure in the lungs of habitual smokers, are involved in this transcriptional regulation.

We have demonstrated the expression of key drug transporters in pulmonary tissue and inflammatory cells that are targets for treatment of lung diseases such as COPD. We also observed a difference in transporter expression in BAL cells from smokers and non-smokers. Transporter proteins may affect drug disposition in lung and consequently the ability of drugs to reach their intracellular targets. Moreover, transporter proteins are involved in the uptake and release of endogenous mediators from inflammatory cells. Thus, understanding drug transporter expression and regulation is important in the development of successful new inhaled drug therapies.