Influence of inflammation on drug transporter expression in human airway epithelia: implications for inhaled drug pharmacokinetics

Abstract

The efficacy of inhaled drugs is significantly influenced by airway epithelial transporters that mediate their transport and distribution. Because inhaled drugs are used across diverse populations with inflammatory airway diseases, understanding factors that modulate transporter expression is crucial. Therefore, we investigated how inflammatory states, demographic factors, and airway anatomic location impact drug transporter abundance. Using quantitative targeted absolute proteomics (QTAP), we measured transporter abundance in primary human bronchial epithelia (HBE) from 42 demographically diverse donors. To model distinct inflammatory environments, HBE were exposed to IL-1β (TH1/neutrophilic inflammation) or IL-13 (TH2/eosinophilic inflammation) for four days alongside baseline controls. We also measured transporter protein concentrations in small airway HBE from 5 donors to assess regional influence. In the primary set of HBE, MRP1 (3.96±1.64 pmol/mg protein) and PEPT2 (3.12±0.95 pmol/mg protein) were the most abundant transporters, and their concentrations were responsive to inflammatory signals. Specifically, IL-1β significantly reduced MRP1 (0.80-fold) and PEPT2 (0.65-fold), while IL-13 modestly increased MRP1 (1.1-fold) but reduced PEPT2 (0.73-fold) and OCTN1 (0.44-fold). Several transporters present at lower abundance, including MRP4, MRP5, MRP6, and OCTN1, were also impacted by inflammation. Demographic factors also played a role, with MRP4 higher in females, and age positively correlated with MRP6 but negatively with OCTN1. Analysis of small airway derived HBE did not show a significant impact of anatomic location on transporter abundance. Our findings demonstrate that airway inflammation and donor demographics significantly impact the protein expression of key drug transporters, highlighting dynamic factors crucial for optimizing inhaled drug delivery and efficacy.

1. Introduction

Drug delivery via aerosolization offers an attractive approach to deliver therapeutics for human lung diseases such as asthma, cystic fibrosis (CF), or chronic obstructive pulmonary disease (COPD). Aerosolization delivers drug directly to the epithelia that line the human airway, very often the site of pathophysiology and primary target of the therapeutic. Furthermore, because the total volume of the airway is small relative to systemic circulation¹, aerosolization can enhance therapeutic index by providing high local drug concentrations with small total doses that limit systemic toxicities².

Multiple factors can influence the pharmacokinetic properties of inhaled agents. While the roles of particle deposition and mucociliary clearance are relatively well understood^2–4, the influence of membrane transporters that mediate the influx and/or efflux of drugs within airway epithelia is less clear⁵. Several inhaled agents are reported to interact with membrane transporters⁵. For example, inhaled bronchodilators salbutamol and tiotropium are substrates of apically located organic cation transporters (OCT/OCTN)⁶, and polymyxin class antibiotics such as colistin are substrates and inhibitors of the peptide transporter PEPT2 as well as OCTN2⁷. Efflux transporters such as multidrug resistance proteins (MDRs/MRPs) interact with multiple antibiotics approved for inhalation or commonly utilized off-label.

Previous investigations into airway transporter expression have yielded conflicting results^8–12, with discrepancies potentially arising from methodological limitations such as reliance on RNA expression or analysis of lung homogenates that obscure epithelial-specific signals. Crucially, although most inhaled drugs are used for diseases characterized by airway inflammation, the impact of inflammation on airway epithelial transporter concentrations has not been extensively explored despite the known influence of inflammation on protein expression in other tissues¹³. Furthermore, demographic variations within patient populations are known to impact gene and protein expression^14,15, but few studies have comprehensively addressed these factors in the airway.

To address these gaps, we systematically characterized airway epithelial transporters using quantitative targeted absolute proteomics (QTAP)^16–18 in airway epithelia from a demographically diverse set of human donors. To best mimic in vivo conditions, we utilized primary human bronchial epithelia (HBE) grown at air-liquid interface (ALI), which induces differentiation into cell types most representative of epithelia within the lung^8,19. These findings can provide important insights into the regulation of airway epithelial transporters, providing more personalized modeling strategies to improve inhaled drug delivery strategies.

2. Experimental Procedures

2.1. HBE culture

Primary HBE were obtained from the University of North Carolina Tissue Procurement and Cell Culture core. Passage two cells were cultured at ALI using standard methods described previously^20,21. Duplicate HBE cultures from each donor were scraped from the culture plates, combined, and stored at −80°C until processed for QTAP. Separate HBE cultures from each donor were exposed to 10 ng/mL IL-1β or 10 ng/mL IL-13 for four days, protocols previously established to reliably induce inflammatory responses^22–25. Duplicate cultures from each inflammatory condition were scraped, combined, and stored as above. Small airway HBE were isolated using previously described methods²⁶ Matched LAE and SAE cells were isolated from the same freshly excised transplant lungs using a bulk enzymatic digestion method, as previously described^27–29. Briefly, LAE and SAE cells were obtained from larger cartilaginous airways (trachea ~3^rd generation bronchi) and peripheral lung tissue, respectively, using Accutase solution supplemented with pronase (10 mg/ml; Roche). At 70–90% confluence, cells were passaged for expansion on inserts coated with human placental type IV collagen. Once confluent, cells were maintained at an air-liquid interface (ALI) by removing apical media and supplying UNC ALI medium to the basal compartment only. Basal media were every other day, and the apical surfaces were washed with PBS. LAE and SAE cells were cultured for 28 days at ALI prior to experimental use.

2.2. QTAP

Detergent-based membrane protein fractionation of cell pellet samples, proteolytic digestion, and isotope dilution LC-MS/MS for QTAP were performed as previously described^16,17. For the membrane protein fractionation a mild detergent, digitonin 0.015 %, first permeabilized cells so that the cytosolic fraction was released and dissolved in the digitonin. After centrifugation and removal of the supernatant the pellet was solubilized in 0.5 % Triton^™ X-100. Further centrifugation removed cell debris and the membrane protein remained in the Triton^™ X-100. For digestion, 20 μg of membrane protein, available for most samples, was digested with trypsin overnight (20 hours). The digestion involved evaporation of membrane protein solution, reconstitution in pH 8 buffer (50 mM ammonium bicarbonate) and 1 % sodium deoxycholate, denaturing with heat, reducing with dithiothreitol and carbamidomethylation of cysteines with iodoacetamide. At this point stable isotope labeled (SIL) peptides (0.5 pmol of each), labeled (¹³C, ¹⁵N) at the c-terminus amino acid and purchased from JPT (Berlin, Germany) (SpikeTides^™_TQL; purified and calibrated) were added as quantifier standards. Trypsin (1 μg) was added at a ratio of at least 1:20 relative to the total protein amount and digestion was performed at 37 °C on a thermal mixer. After stopping the digestion with acid and removing the deoxycholate precipitate the supernatant was cleaned up with solid phase extraction (SPE) (Strata^™-X 33 μ 10 mg/mL cartridges, Phenomenex, Torrance, CA). The SPE eluate was evaporated and reconstituted in modified mobile phase A (2 % acetonitrile rather than 1 % acetonitrile). After centrifuging to remove any particulate matter present, the supernatant was removed for nanoLC-MS/MS analysis on a nanoAcquity (Waters, Milford, MA) interfaced to a QTRAP 5500 (SCIEX, Framingham, MA) mass spectrometer equipped with a NanoSpray III source. A digested protein amount of 0.08 μg (0.4 % of most samples) was injected onto a Symmetry^® C18 Trap Column, 2G-VM, 180 μm × 20 mm, 5 μm particle size (Waters) (part # 186006527) and the peptides were separated on a BEH130 C18 Column, 150 μm × 100 mm, 1.7 μm particle size (Waters) (part # 186003550). The aqueous mobile phase was water containing 1 % acetonitrile and 0.1 % formic acid, and the organic phase was 100 % acetonitrile. The nanoLC gradient injection run time was 35 min. For each peptide, peak areas for two MRMs were summed for unlabeled and two for labeled, and the peak area ratio of unlabeled to labeled was used to calculate peptide concentration based on the amount of SIL peptide added (0.5 pmol of each). The two MRMs for each peptide, both unlabeled and labeled, employed in the analyses, are shown in Supplemental Table 1. NanoLC retention times for unlabeled and SIL peptide MRMs were within ±0.02 min of each other (i.e. unlabeled and SIL peptides co-elute) and equality of response between unlabeled and labeled peptides was assumed. Peaks were integrated using SCIEX MultiQuant 2.0.2 software and the lower limit of quantification (LLOQ) was set at 0.1 pmol/mg protein for all peptides. The lower limit of detection (LLOD) was 0.02 pmol/mg protein.

2.3. RNA sequencing data

Bulk RNA sequencing data were obtained from Gene Expression Omnibus that included results from a published study on the impact of hypoxia on gene expression in HBE³⁰ (GEO accession: GSE227502). This database was chosen because the methods utilized to generate this RNA sequencing data were performed by investigators at the same institution using identical culture techniques as the current study. Data from the six HBE cultures grown under normoxic conditions as the control group were utilized for comparison.

2.4. Data analysis and statistics

Differences between groups were analyzed by T-tests or ANOVA and correlations using linear regression. Paired statistics were utilized to compare protein concentrations in baseline HBE to those exposed to inflammatory stimuli. Analysis of demographic factors influence on protein concentrations were performed by multiple linear regression. Sex, Black race, and Hispanic ethnicity were coded as 0 or 1 with year of age as a continuous variable. All analyses utilized GraphPad Prism v10.4 (Boston, MA).

3. Results

3.1. Quantification of transporters in HBE.

HBE were cultured using established methods²⁰ from 42, donors, 20 male and 22 female, average age 34.8±18.6 years (range 12–73) (Table 1). Donors were from a range of races/ethnicities, with 21 reported as White, 9 as Black, 11 as Hispanic, and one as Pacific Islander.

Table 1:

Donor Demographics

	Large Airway	Paired Large/Small Airway
N =	42	5
Sex, male (%)	20 (48%)	4 (80%)
Age in years, (mean ± SD)	34.8 ± 18.6	39.8 ± 17.0
Age range in years (min – max)	12–73	20–60
Race/ethnicity
White (%)	21 (50%)	4 (80%)
Black (%)	9 (21%)	1 (20%)
Hispanic (%)	11 (26%)	0 (0%)
Pacific Islander (%)	1 (2%)	0 (0%)

QTAP was utilized to quantity abundance of 30 transporters in cells evaluated under baseline, untreated conditions as well as after exposure to either IL-1β or IL-13 using standardized protocols^22–25. Among the transporters evaluated, 9 were present in HBE, defined as membrane abundance above the lower limit of quantification of 0.1 pmol/mg protein in at least 20% of untreated (baseline) samples. Multidrug resistance protein (MRP)1 and peptide transporter (PEPT)2 were present at the highest abundance, and transporters detected routinely at lower concentrations included MRP3, MRP4, MRP5, MRP6, the organic anion transporter (OAT)4C1, the organic cation/carnitine transporter (OCTN)1, and the equilibrative nucleoside transporter (ENT)1 (Table 2). Of note, MDR1/P-gp was below quantification limits in all samples though was present in a whole lung homogenate utilized as a quality control (not shown).

Table 2:

Transporter Protein Concentrations and RNA expression (transcripts/million) at Baseline in HBE^*

Gene	Protein	# Detected (>0.1 pmol/mg protein)	QTAP (pmol/mg protein)	RNA (transcripts/million)
ABCB1	MDR1	1	0.001±0.004	0.00±0.01
ABCG2	BCRP	8	0.044±0.082	0.18±0.15
	BCRP*2	1	0.018±0.033
ABCC1	MRP1	42	3.955±1.638	34.3±7.5
ABCC2	MRP2	1	0.013±0.032	0.78±0.30
ABCC3	MRP3	10	0.056±0.103	4.99±1.11
ABCC4	MRP4	42	0.385±0.160	7.40±2.81
ABCC5	MRP5	20	0.239±0.378	35.6±4.0
ABCC6	MRP6	13	0.174±0.307	15.5±5.4
ABCC12	MRP9	1	0.013±0.031	0.00±0.00
SLC15A2	PEPT2	42	3.124±0.948	74.3±18.6
SLC22A7	OAT2	1	0.011±0.030	0.00±0.00
SLC22A8	OAT3	0	0.005±0.017	0.00±0.00
SLC22A1	OCT1	0	0.004±0.012	0.13±0.09
SLC22A2	OCT2	0	0.002±0.009	0.00±0.00
SLC22A3	OCT3	6	0.049±0.109	0.47±0.59
SLC22A4	OCTN1	41	0.707±0.338	31.2±13.2
SLC22A5	OCTN2	2	0.004±0.018	15.0±1.5
SLC28A1	CNT1	4	0.038±0.133	0.18±0.19
SLC28A3	CNT3	1	0.084±0.537	0.67±0.16
SLC29A1	ENT1	11	0.646±1.357	6.53±1.27
SLC29A2	ENT2	2	0.027±0.124	8.45±1.12
SLC29A4	ENT4	0	0.001±0.004	0.31±0.15
SLC47A1	MATE1	4	0.015±0.042	0.91±0.36
SLC47A2	MATE2	2	0.008±0.026	2.98±0.77
	MATE2-K	3	0.030±0.121
SLCO1A2	OATP1A2	1	0.004±0.025	0.04±0.04
SLCO2A1	OATP2A1	1	0.008±0.023	2.53±3.21
SLCO1B1	OATP1B1	0	0.003±0.012	0.02±0.03
SLCO1B3	OATP1B3	1	0.006±0.027	4.79±2.52
SLCO2B1	OATP2B1	1	0.003±0.016	0.00±0.00
SLCO4C1	OATP4C1	14	0.093±0.139	16.1±3.2

^*Transporter protein concentrations are from this study and are reported as average concentrations of recorded values across all samples, including those below LLOQ. Transporter mRNA concentrations were obtained from an online database. See Methods for details.

3.2. Comparison of protein to mRNA levels.

To explore our proteomic findings relative to mRNA expression, we compared our data to RNA sequencing data available from a previously published study of HBE obtained from the same cell culture core, at the same institution as this study, and cultured using similar methods³⁰. There was a statistically significant correlation between protein abundance measured by QTAP and mRNA expression (Fig. 1). All 9 proteins we defined as present by QTAP had RNA levels of >5 transcripts per million, whereas the majority of proteins not detected by QTAP had RNA levels of <1 transcript per million (15 of 21). A small number of transporters had discrepant QTAP and RNA findings, with MATE2, OATP1B3, OATP2A1, OCTN2, and ENT2 not detected by QTAP despite having mRNA levels >1 transcript/million.

Figure 1.

Relationship between mean abundance (pmol/mg membrane protein) of transport proteins in HBE as measured by QTAP to RNA expression (transcripts/million) of the corresponding genes measured by RNA sequencing in a prior study. Linear regression R² = 0.41, p<0.003.

3.3. Impact of inflammation and demographics.

To assess the impact of inflammation, we measured transporter abundance by QTAP in HBE exposed to either IL-1β or IL-13, focusing on those transporters routinely detected in HBE. IL-1β exposure resulted in statistically significant reductions in the protein concentrations of MRP1 (0.78-fold) and PEPT2 (0.66-fold). In contrast, abundance of MRP4 was increased 1.4-fold (Fig. 2). IL-13 exposure resulted in a modest increase in MRP1 concentrations (1.1-fold) but more substantial decreases in MRP4 (0.45-fold), PEPT2 (0.69-fold), and OCTN1 (0.42-fold).

Figure 2.

Protein abundance (pmol/mg membrane protein) of transporters expressed in HBE under baseline conditions (baseline, blue bars) and after 4-day exposure to 10 ng/mL IL-1β (red bars) or IL-13 (green bars). Abundance of the highly expressed MRP1 and PEPT2 transporters was altered by IL-1β and IL-13 exposure, as were several other transporters. Data are shown as mean and standard deviation (n=42, *=p<0.05, ****=p<0.001).

To assess the impact of demographic factors, we performed a multivariate analysis for each transporter using sex, Black race, Hispanic ethnicity, and age as predictive variables. In this analysis, MRP4 was significantly impacted by sex (higher in females) and MRP5 and OCTN1 were significantly impacted by age (MRP5 higher and OCTN1 lower in older individuals) (Table 3). P-values for MRP1 by age and for MRP4 in Black donors were <0.1 but these did not reach the conventional threshold to be considered statistically significant.

Table 3:

Multivariate analysis

	MRP1		MRP4		MRP5		OCTN1
	β ± SE	p-value	β ± SE	p-value	β ± SE	p-value	β ± SE	p-value
Black	0.15 ± 0.66	0.820	0.11 ± 0.06	0.080	0.10 ± 0.14	0.505	−0.06 ± 0.13	0.647
Hispanic	−0.04 ± 0.61	0.950	0.03 ± 0.06	0.611	−0.02 ± 0.13	0.872	0.10 ± 0.12	0.389
Sex	0.24 ± 0.51	0.650	0.11 ± 0.05	0.027	0.07 ± 0.11	0.504	0.11 ± 0.10	0.262
Age	0.025 ± 0.014	0.081	0.001 ± 0.001	0.393	0.008 ± 0.003	0.010	−0.007 ± 0.003	0.016

β = regression coefficient. SE = standard error

3.4. Small airway epithelia.

Standard methods for isolating HBE obtain cells from larger airways, but small airways are often a primary site of disease pathophysiology and are increasingly targeted by inhaled agents³¹. To assess whether airway region affected transporter abundance, we employed alternative methods²⁶ to isolate small airway epithelia (SAE) from five human donors in parallel with large airway epithelia (LAE) isolated using established standard protocols (demographics in Table 1). Transporter protein concentrations measured by QTAP were similar in SAE and LAE (Fig. 3), and no significant differences were observed comparing paired SAE and LAE or comparing SAE to the larger dataset of 42 donors.

Figure 3.

Protein abundance (pmol/mg membrane protein) of transporters expressed in HBE isolated from paired large airway (LAE) and small airway (SAE) culture from the same donor. Data are shown as mean and standard deviation. No significant differences were noted between LAE and SAE (n=5).

4. Discussion

Our findings demonstrate that HBE express several transporters relevant to inhaled drugs, and that concentrations of these transporters are altered by demographic factors and inflammatory processes common in airway diseases. In general, our baseline results are comparable to prior studies of human bronchial epithelia. Consistent with findings from Sakamoto et al.^8,9, we observed that the basolaterol efflux pump MRP1 had the highest level of protein expression and that the apical influx transporters OCTN1 and PEPT2 were also present in relatively high concentrations (Fig. 4). These findings suggest that the predominant transporter activity in HBE is primarily directional from the apical to basolateral compartments, with some apical efflux via MRP4 expressed at much lower concentrations. In contrast, we did not observe significant expression at the protein or mRNA level of the apical efflux pump MRP2 or the apical influx organic anion (OATs, most OATPs) or cation influx transporters (OCTs). This observation contrasts with Sokomato⁹, who observed modest levels of MRP2, OCT1, OCT2, OAT3, and OATP2B1 in cultured HBE. A few proteins did not meet our thresholds for protein detection but did have meaningful mRNA expression. These discrepancies highlight the fact that protein levels are influenced by translation efficiency as well as factors such as protein half-life and turnover, which are regulated by post-translational modifications like phosphorylation, glycosylation, or ubiquitination³². Therefore, techniques such as QTAP provide a more reliable prediction of protein function, and caution is warranted when relying solely on mRNA expression results to predict protein levels. Further analysis of tissue and cellular distribution could be achieved by cutting-edge spatial proteomics technologies^33,34, which have the potential to deepen our insight into transport protein location.

Figure 4.

Schematic showing the previously identified localization of transporters⁵ measured in this study within differentiated airway epithelia. Transporters detected in our study are on the left. For reference, transporters previously reported in airway epithelia⁹ but not detected in our study are shown on the right. Created in BioRender. Esther, C. (2026) https://BioRender.com/3w3c34m.

Notably, we did not detect expression of MDR1/P-gp in HBE at the protein or mRNA level, which is consistent with some studies^9,35 but differs from other investigations^11,12. Most studies that report MDR1 expression have relied on whole lung tissue rather than airway epithelia, and MDR1 expression could arise from other tissue types within the lung. In fact, we did detect MDR1 in a lung homogenate run for quality assurance purposes. Similarly, our failure to detect meaningful concentrations of BCRP protein or mRNA expression was surprising since this transporter is widely reported to be expressed in airway epithelia^35–37, although in at least one prior study the BCRP mRNA levels were much lower than those of other transporters³⁵. Overall our findings suggest that neither MRD1/P-gp nor BCRP are expressed at meaningful concentrations in airway epithelia, at least in our system, suggesting they may not play a significant role in PK of inhaled drugs on airway surfaces.

Importantly, we observed that inflammation had a significant impact on airway epithelial transporter expression. Our findings that IL-1β generally resulted in a reduction in transporter abundance are comparable to those form other tissues³⁸. However, they contrast somewhat with Aguiar et al., who noted smoking and COPD mediated increases in gene expression of several ABC transporters³⁹, and those of Berg et al who reported no significant gene expression differences in lung transporters in those with COPD^11,40. Of note, those studies utilized lung homogenates and were not epithelial specific signals. Notably, the impact of TH2/eosinophilic inflammation modeled by IL-13 exposure was distinct from that of TH1/neutrophilic inflammation modeled by IL-1β exposure, suggesting different transporter expression profiles in diseases such as CF or COPD characterized by neutrophilic inflammation versus atopic asthma characterized by eosinophilic inflammation.

Anatomic location and demographic diversity did not appear to play a major role in airway epithelial transporter concentrations, though we did observe some influence of age and sex. MRP4 was higher in HBE from female donors, and MRP6 increased significantly with donor age (and a similar trend for MRP4) whereas OCTN1 was negatively correlated with age. Differential expression of MRPs by sex¹⁴ and age^41,42 have been reported in other models, though not previously in airway epithelia. Although our study is one of the largest in terms of number of donors examined, we cannot rule out the possibility that further investigation with a larger set of donors would reveal other meaningful differences by these factors.

The findings presented here have significant implications on the pharmacokinetic properties of several transporter substrate drugs administered via the inhaled route for airways disease and ultimately on therapeutic outcomes⁵. For example, the inhaled antibiotic colistin utilized for treatment of resistant airway infections in CF is transported by PEPT2⁷. CF is characterized by increased airway IL-1β²², and reduced influx from the ~35% decrease in the apical PEPT2 transporter might increase the concentration and/or duration of colistin within the airway. However, inflammation could also have adverse consequences. The bronchodilator albuterol must cross through airway epithelia to reach its target of smooth muscle cells, and studies have suggested that albuterol transport is mediated by apical organic cation influx transporters such as OCTN1⁴³. The >50% reduction in this transporter with IL-13, which is elevated in the airways of those with allergic asthma⁴⁴, could reduce the efficacy of albuterol, particularly in those with poor control⁴⁵. Interestingly, decreased systemic albuterol concentration have been reported in asthma, and while this has been attributed to lower deposition within constricted asthmatic airways⁴⁶, decreased transport across airway epithelia could also play a role.

There are several limitations to this study. While cultured primary HBE offer an optimal approach towards isolating the impact of epithelial transporters and are more representative than cell lines, it is possible that cultured cells differ from epithelia present in situ. While examining proteins from freshly isolated lungs could address this limitation, it is not readily feasible to isolate airway epithelia from the lung without using methods such as protease digestion that alter cell surface proteins. Furthermore, while our study evaluated protein concentrations, we did not directly assess localization or activity, and we are relying on prior histological studies to allow us to predict the localization and impact of these transporters.

Another limitation is that we examined SAE from a relatively small number of donors. Different expression patterns of SAE and LAE have been described for other proteins²⁶, and it is possible that evaluation of a larger set of donors would reveal differences in transporter concentrations. However, we feel our data are sufficient to rule out large differences by anatomic location, and we note that this sample size is consistent with multiple previous proteomic studies of HBE^9,47.

In conclusion, our evaluation defines the complement of transporters present in HBE and demonstrates that inflammation impacts transporter concentration in a manner that could influence the PK of inhaled agents. Evaluations of inhalational drugs should account for the presence of these epithelial transporters and the effects of airway inflammation on their expression.

Significance:

Human airway epithelia express multiple transporters, with MRP1 and PEPT2 found at the highest levels. Transporter abundance is influenced by inflammation, age, and biological sex though not by airway anatomic location. This information can guide modeling of inhaled drug pharmacokinetics.

Data Availability Statement:

The authors declare that all other data supporting the findings of this study are available within the paper and its Supplemental Data.