Current smoking alters phospholipid- and surfactant protein A levels in small airway lining fluid: An explorative study on exhaled breath.

Small airways are difficult to access. Exhaled droplets, also referred to as particles, provide a sample of small airway lining fluid and may reflect inflammatory responses. We aimed to explore the effect of smoking on the composition and number of exhaled particles in a smoker-enriched study population. We collected and chemically analyzed exhaled particles from 102 subjects (29 never smokers, 36 former smokers and 37 current smokers) aged 39 to 83 years (median 63). A breathing maneuver maximized the number exhaled particles, which were quantified with a particle counter. The contents of surfactant protein A and albumin in exhaled particles was quantified with immunoassays and the contents of the phospholipids dipalmitoyl- and palmitoyl-oleoyl- phosphatidylcholine with mass spectrometry. Subjects also performed spirometry and nitrogen single breath washout. Associations between smoking status and the distribution of contents in exhaled particles and particle number concentration were tested with quantile regression, after adjusting for potential confounders. Current smokers, compared to never smokers, had higher number exhaled particles and more surfactant protein A in the particles. The magnitude of the effects of current smoking varied along the distribution of each PEx-variable. Among subjects with normal lung function, phospholipid levels were elevated in current smokers, in comparison to no effect of smoking on these lipids at abnormal lung function. Smoking increased exhaled number of particles and the contents of lipids and surfactant protein A in the particles. These findings might reflect early inflammatory responses to smoking in small airway lining fluid, also when lung function is within normal limits.

Introduction

The patency of small airways (<2 mm internal diameter) is important for gas exchange. Pathological changes in this peripheral region give rise to few symptoms, thus, they are typically detected only in the later stages of lung disease. Small airways are largely inaccessible, and there is no easy method for retrieving a biological sample for the identification of biomarkers to use, for example, in screening. Biomarkers that reflect pathological changes in small airways may contribute to the early detection of adverse effects of airborne exposures, and possibly, to more effective treatment of lung diseases that affect small airways, such as chronic obstructive pulmonary disease (COPD). With this in mind, a method was developed for collection of particles in exhaled air (PEx).

Human breath aerosol contains endogenously generated droplets, also referred to as particles, of a very broad size distribution [1, 2]. PEx refers to exhaled particles in the size range of 0.5–5 μm that is being counted and collected, non-invasively, with the PExA method (i.e. the collection method) [3, 4]. The PEx content can be determined with various analytical assays [5-7].

In general, it is thought that a portion of small airways close upon expiration, and re-open upon inspiration. When small airways re-open during inspiration, the fluid lining the airways bursts, which generates particles that are small enough to be carried in the breath aerosol during the subsequent exhalation [4, 8]. The PExA method involves a specific breathing maneuver to maximize small airway closure, followed by small airway re-opening [4].

Airway closure increase with age and in lung diseases, like COPD [9]. The PEx number concentration, collected with the PExA method, is suggested to reflect the degree of airway closure followed by airway re-opening [4]. Particles larger than those collected with the PExA method are also exhaled, but these particles are possibly mainly generated in the upper airways [10].

Pulmonary surfactant, the major constituent of small airway lining fluid, is a complex mixture of phospholipids and proteins that prevents alveolar collapse by modulating surface tension. Moreover, the lining fluid is important in the host immune defense of the lungs; both as a barrier but also by specific proteins and lipids binding to inhaled material to enhance its elimination [11-13]. The two major phospholipids found in lung surfactant are the saturated di-palmitoyl-phosphatidyl-choline (DPPC), produced by alveolar type II cells, and the unsaturated palmitoyl-oleoyl-phosphatidylcholine (POPC).

Tobacco smoking is a well-known risk factor for COPD [14]; the smoke-associated inflammatory response is known to affect surfactant homeostasis by disturbing both lipid degradation and transport [15, 16]. Reduced DPPC and POPC contents have been found in the broncho-alveolar lavage (BAL) fluid collected from patients with COPD [17]. Taken together, these findings have raised interest in the interaction between tobacco exposure and changes in the lining fluid of small airways, especially at early stage, as there still is a lack of methods for early detection of disease, before damage to small airways is permanent.

Surfactant protein/lipid interactions are essential for maintaining surfactant homeostasis. The most abundant lung-specific protein in surfactant is surfactant protein A (SP-A), which has several important immunological functions. SP-A is mainly produced by the alveolar type II cells. SP-A promotes the re-uptake of oxidized surfactant lipids by alveolar type II cells [13] and is also an important opsonin of inhaled material [18]. Previous studies have suggested that SP-A might serve as a potential biomarker for early smoke-induced effects, even though somewhat contradictory results have been published [19-21]. A small study on former smokers with COPD showed that the SPA content in PEx deceased with worsening lung-function [22]. SP-A levels in PEx have also been shown to be highly correlated with that of BAL fluid [23].

Albumin is also found at relatively high concentrations in small airways. A previous study showed that content of albumin in PEx was decreased in those subjects with asthma that also had small airway dysfunction [24]. Therefore, albumin may be of interest when studying the effect of tobacco smoke on small airway lining fluid.

Tobacco smoke is a mixture of thousands of toxic compounds, and the composition of small airway lining fluid is complex. There is likely to be an intricate interaction between respiratory irritants and small airway function. In addition, the influences of other factors on small airway lining fluid, such as age and lung function, increase the complexity. Based on this and on previous studies, we hypothesized that the effect of smoking varies across the distributions of surfactant lipids and proteins in small airway lining fluid and that the effects are associated with lung function.

The present study aimed to explore potential biomarkers for inflammation in small airway lining fluid; i.e. effects on the composition of lipids (DPPC and POPC) and proteins (SP-A and albumin) in PEx and on the PEx number concentration, in cigarette smokers. Furthermore, we wanted to examine if the novel PExA method could identify alterations, associated with tobacco smoking, before lung function is affected.

Materials and methods

Study population

102 subjects, aged 39–83 years, were recruited to participate in an extended study protocol in the follow-up study of the population-based INTERGENE-ADONIX cohort [25]. Recruited subjects had all given their written informed consent in the follow-up study to be contacted for participation in the extended study protocol. Eligible for inclusion were subjects whom had managed to perform spirometry and had reported their smoking status and smoking history in the follow-up study. Exclusion criteria were ongoing respiratory tract infection, myocardial infarction past month and/or pregnancy in last trimester. All current smokers were invited to participate, whereas former- and never-smokers were invited randomly. The subjects were classified as current-, former- and never smokers based on smoking history; current smokers reported to have smoked cigarettes on a regular daily basis for at least one year at the time of the clinical examination; former smokers had smoked on a regular basis, but had stopped smoking for one year or more before the clinical examination. Never smokers had never smoked on a regular basis.

The regional ethics board at Gothenburg University approved the study protocol (application number 626–13). Written informed consent was obtained from all participating subjects. The clinical characteristics of the study population, according to smoking status, are presented in Table 1.

Table 1

General and clinical characteristics of study population, subdivided according to smoking category.

	All	Never smokers	Former smokers	Current smokers
N	102	29	36	37
Sex females/males [n]	49/53	14/15	15/21	20/17
Age [years]	62.5 (51.8–69.0)	52.0 (47.0–66.5)	69.5 (61.3–72.0)	59.0 (53.0–66.5)
BMI [kg/m2]	25.3 (23.6–27.8)	25.3 (23.2–26.9)	26.7 (24.4–30.0)	24.7 (22.6–27.5)
Smoking history [pack years]	19.0 (0–33.0)	0	23.5 (15.3–35.3)	28.0 (18.0–40.0)
FENO50 [ppb]	18.0 (13.0–23.0)	19.0 (16.5–25.5)	19.0 (13.0–25.0)	16.0 (11.0–21.0)
CRP [mg/L]	1.3 (0.8–2.6)	1.0 (0.5–1.8)	1.3 (0.7–2.6)	1.7 (1.1–3.1)
N2-slope [%N2/L]	1.5 (1.1–3.5)	1.2 (0.8–1.5)	2.5 (1.4–4.4)	1.9 (1.2–3.5)
FEV1 [z-score]	-1.3 (-2.0- -0.4)	-0.8 (-1.7- -0.2)	-1.5 (-2.1- -0.8)	-1.2 (-2.1- -0.1)
FVC [z-score]	-0.5 (-1.4–0.1)	-0.1 (-1.3–0.3)	-1.2 (-1.5–0.0)	-0.6 (-1.4–0.3)
FEV1/FVC post BD [%]	0.73 (0.68–0.79)	0.78 (0.69–0.80)	0.73 (0.66–0.78)	0.73 (0.69–0.78)

Values are presented as median with inter quartile range, if nothing else stated. FENO50; Fraction of Exhaled Nitric Oxide at an expiratory flow of 50 mL s-1, CRP; C-Reactive Protein, N2-slope; slope III of single breath washout, FEV1; Forced Expiratory Volume in the 1st second, FVC; Forced Vital Capacity, FEV1; Forced Expiratory Volume in the 1st second, FVC; Forced Vital Capacity.

Study design

This cross-sectional study was conducted between June 2014 and September 2016 in Gothenburg, Sweden. The clinical examination comprised an exhaled air analysis, blood sample, lung function tests, and the completion of questionnaires. Current smokers were instructed to refrain from smoking 1 h prior to the clinical tests. All participants were instructed to withhold from taking long-acting β2-bronchodilators and inhaled gluco-corticoids for at least 24 h and short-acting β2-broncho-dilators for at least 12 h prior to the clinical tests. Included participants had no respiratory tract infections within three weeks prior to the clinical examination.

Measurements

FeNO

The Fraction of Exhaled Nitric Oxide (FENO) was measured at an expiratory flow of 50 mL/s with a chemiluminescence analyzer (NIOX VERO, Aerocrine AB, Stockholm, Sweden). Measurements were in accordance with American Thoracic Society (ATS) and European Respiratory Society (ERS) recommendations [26].

Spirometry

Spirometry was performed with a Spirare device (SPS3110 sensor and Spirare 3 software; Diagnostica AS, Oslo, Norway), before and after bronchodilation with 1.5 mcg terbutaline (Bricanyl; AstraZeneca; Sweden), in accordance with the ATS/ERS criteria [27]. The forced expired volume in the first second (FEV1) and the forced expiratory vital capacity (FVC) were measured, and the ratio FEV1/FVC was calculated. Predicted normal values were based on local reference values from Brisman et al [28, 29], and the results are expressed in terms of percent predicted (% pred), the z-score (z) or the Lower Limit of Normality (LLN).

Nitrogen single breath washout

Nitrogen single breath washout (N2SBW) was performed before bronchodilation, with an Exhalyzer D (Eco Medics AG). The target was three technically acceptable trials. Each trial included a full expiration to residual volume (RV), followed by a slow inspiration (maximum 500 mL/s) of 100% oxygen to TLC, and finally, a slow exhalation (maximum 500 mL/s) from TLC to RV. A linear regression of data between 25–75% of the exhaled volume was performed to obtain the alveolar nitrogen slope (N2-slope). Trials were acceptable when the coefficient of variation in the expiratory vital capacity was less than 10% between trials. The mean value of the accepted trials was used in the analyses.

Exhaled particles

PEx collections were performed after bronchodilation, with the PExA instrument (PExA AB, Göteborg, Sweden), according to the method described in detail previously [3]. The PExA instrument does not collect all particle sizes found in the breath. In this study, particles in a size interval of 0.5–5.0 μm were collected and referred to as PEx. In brief, the PExA instrument included an optical particle counter (Grimm 1.108, Grimm Aerosol Technik GmbH, Ainring, Germany) and a collection plate covered with a thin membrane of hydrophilic polytetrafluorethylene (PTFE) (FHLC02500, Millipore, Billerica, MA, USA), in a modified multi-stage impactor (PM10 Impactor, Dekati Ltd., Tampere, Finland). With a diverter valve, the operator can divert the flow of the exhaled breath, either into the PExA instrument for sampling or back to ambient air, when sampling only selected exhalations. Subjects wore a nose clip and repeatedly performed a standardized breathing maneuver. The breathing maneuver started with an exhalation to residual volume (RV), a breath-hold for 5 s, a rapid inhalation to total lung capacity (TLC), and then, this was immediately followed by a deep exhalation at a spontaneous flow rate. PEx was sampled only during this final exhalation of the breathing maneuver. Between breathing maneuvers, subjects breathed tidally in filtered, particle-free air. The collection continued until 120 ng of PEx mass was obtained [30]. The PTFE membrane with sampled PEx was divided into two halves, which were transferred to separate, 2 mL polypropylene cryotubes (Sarsteds, Nümbrecht, Germany) and stored at -80°C, prior to chemical analysis. The PEx number concentrations are expressed as n *1000 (kn) per litre of exhaled breath, kn/L, and are referred to as number PEx.

Chemical analyses of exhaled particles

Chemical analyses of the proteins in PEx (SP-A and albumin) were performed with enzyme- linked immunosorbent assays (ELISAs), according to the protocol described by Kokelj and colleagues [31]. In brief, PEx were extracted from the PTFE membrane samples by adding extraction buffer. Levels of SP-A and albumin in extracted PEx samples were determined with a human SP-A ELISA kit (Lot nr; E-15-108, Product nr; RD191139200R, BioVendor, Brno, Czech Republic) and a human albumin ELISA kit (Lot nr; 19, Part number; E-80AL,Immunology Consultant Laboratory, Newberg, OR, USA), according to the manufacturer´s instructions, with minor modifications.

Chemical analyses of the phospholipids in PEx (DPPC and POPC) were performed with a triple quadrupole mass spectrometer (Sciex API3000, AB Sciex, Canada), equipped with an electrospray ion source operating in positive mode, as described in detail previously [7]. Briefly, internal standards were added to each sample before extraction. Extracted samples were introduced to the ion source with a flow gradient method, but without chromatography separation. DPPC and POPC were quantified based on a calibration curve with a linear regression model. In each run PTFE substrates spiked with 25 pico mol DPPC and POPC standards (an amount representative of the study samples) were analyzed to monitor method performance. Based on 22 measurements distributed throughout the study the average recovery was 81% and 104% for DPPC and POPC respectively. The reproducibility RSD% was 6.4 and 8.3 for DPPC and POPC respectively.

The concentrations of SP-A, albumin, DPPC, and POPC in PEx were calculated as the weight-percent of PEx (wt%), by dividing the mass of the analyte by the PEx mass. Number PEx and the concentrations of DPPC, POPC, SP-A, and albumin in PEx are all referred to as PEx variables.

Statistical methods

Statistical analyses were performed with IBM SPSS 26.0 software (SPSS, Chicago, IL). Number PEx were not normally distributed and therefore non-parametric tests were used to analyze both composition of PEx and number PEx. The Kruskal-Wallis test was used for testing differences between smoking categories. The Spearman rank correlation coefficient was employed to test linear relationships. Quantile regression was performed to assess associations between smoking status and different segments of the distribution of each PEx variable (i.e. number PEx and the composition of DPPC, POPC, SP-A, and albumin in PEx). Estimates of coefficient with confidence intervals in quantile regression indicate the change in the value at a modeled quantile of the dependent variable (e.g. number PEx) for each unit change in the independent variable (e.g. current smoker compared to never smoker). Age and sex were considered to have biological relevance with the outcomes and predictors, therefore; these factors were used as confounders in the quantile regression model. The effect of smoking status on the distribution of each PEx variable was considered significant when the confidence interval was separated from zero on the axis of the estimate. Each PEx variable was analyzed in independent models.

The association between smoking and the different PEx variables were also analysed in models stratified for lung function. Lung function was defined as normal when values of FEV1, FVC, and FEV1/FVC were ≥LLN. Abnormal lung function was defined as FEV1, FVC, and/or FEV1/FVC values

Results

In crude regression analysis, current smokers exhaled significantly higher concentration of number PEx and had higher concentration of DPPC and SP-A in PEx, compared to never smokers (Table 2). Current smokers also exhaled higher concentration of number PEx and had a higher concentration of POPC in PEx compared to former smokers. No significant differences in any of the PEx variables were found between never-smokers and former smokers.

Table 2

PEx-variables in study population, subdivided according to smoking category.

	Never smokers	Former smokers	Current smokers	p-value
number PEx [kn/L]	13.2 (7.8–20.5)	14.1 (10.7–20.8)	20.8 (12.4–35.7)a	0.011
DPPC [wt %]	10.3 (8.5–11.7)	10.6 (8.6–11.6)	11.3 (10.2–12.9)a	0.025
POPC [wt %]	2.9 (2.5–4.0)	3.1 (2.4–3.6)	3.7 (3.2–4.2)a,b	0.008
SP-A [wt %]	3.1 (2.4–3.5)	3.2 (2.4–3.9)	3.9 (2.6–4.4)a	0.037
Alb [wt %]	7.5 (5.4–8.8)	8.8 (6.4–10.3)	6.9 (5.4–10.0)	0.184

Data are presented as median with interquartile range (Q1-Q3). PEx: Particles in Exhaled Air; kn/L: thousand number exhaled PEx per liter exhaled air; DPPC: Dipalmiotoylphosphatidylcholine; wt%: weight percent of PEx; POPC: Palmitoyl-oleoylphosphatidylcholine; SP-A: Surfactant Protein A; Alb: Albumin. P-values based on Kruskal-Wallis test followed by Bonferronis multiple comparisons tests of significant difference (p<0.05)between

acurrent smokers and never smokers

bcurrent smokers and former smokers.

In multiple regression analysis, adjusted for age and sex, the associations between smoking status and each PEx variable were estimated along the entire distribution of the different PEx variables (Fig 1). In current smokers, in comparison to never smokers, number PEx was increased, and the magnitude of the effect was amplified with increasing number PEx, as illustrated by the change in estimates between quantile 0.5 and 0.75 (Coef 11.1 kn/L, CI: 3.3–18.9 and Coef 19.3 kn/L, CI: 4.5–34.1, respectively) (Fig 1A). SP-A in PEx was also found to be higher in current smokers but only in the upper half of the quantiles, and the magnitude of the effect increased with increasing SP-A, as illustrated by the change in estimates between quantile 0.5 and 0.75 (Coef 0.9 wt%, CI: 0.1–1.6 and Coef 1.1 wt%, CI: 0.4–1.7, respectively) (Fig 1B). The estimates with confidence interval along the distribution of DPPC and POPC concentrations in PEx showed similar patterns for current and former smokers; however, only just about significantly increased in current smokers and only in restricted parts along the distributions. For instance, DPPC was significantly increased in quantile 0.25 (Coef 2.0 wt%, CI: 0.4–3.6), whereas POPC was significantly increased in quantile 0.5 (Coef 0.9 wt%, CI: 0.3–1.4), in current compared to never smokers (Fig 1D and 1E). No associations were found between albumin in PEx and current or former smokers, in comparison to never-smokers (Fig 1C). In parallel, no associations were found regarding former smokers and any of the PEx variables.

Fig 1

Levels of number PEx (kn/L), phospholipids and proteins in PEx (wt%) among current- and former smokers compared to never-smokers.

Plots illustrating age- and sex-adjusted quantile regression estimates of current- and former smokers to never-smokers (the red dotted line). (A) number PEx (kn/L), (B) SP-A (wt%), (C) Albumin (wt%), (D) DPPC and (E) POPC (wt%) were analyzed separately. Quantiles on x-axis refers to the distribution of the PEx-variable studied. Estimates from quantile regression denoted by black dotted line with blue confidence intervals.

Correlations between the different PEx variables and subject characteristics were evaluated in current smokers (Table 3). An increase in SP-A in current smokers was correlated with a decrease in the FEV1/FVC ratio. None of the PEx variables were correlated with either the N2-slope (%N2/L) or the smoking history (pack years).

Table 3

Correlation coefficients (spearman) between each PEx parameter and characteristics in current smokers (n = 37).

Variables	number PEx [kn/L]	DPPC (wt%)	POPC (wt%)	SP-A (wt%)	Alb (wt%)
Age [years]	-0,274	0,131	0,088	0,260	0,362*
smoking history [packyears]	-0,047	0,027	-0,058	0,145	0,062
FENO50 [ppb]	-0,336	0,045	0,047	0,077	-0,033
CRP [mg/L]	0,160	-0,357*	-0,321	0,054	-0,157
N2-slope [%N2/L]	-0,028	0,206	0,032	0,032	-0,091
FEV1 [z-score]	-0,034	-0,081	-0,077	-0,101	0,071
FVC [z-score]	-0,154	-0,140	-0,156	0,098	0,165
FEV1/FVC post BD	0,188	-0,020	0,119	-,411*	-0,282

Data expressed as spearman r value.

* p< 0.05.

Associations between PEx-variables and lung function

Among subjects with normal lung function (FEV1, FVC, and FEV1/FVC ≥LLN), no significant associations were found between number PEx and current smokers, in comparison to that of never-smokers, in multiple regression analysis adjusted for age and sex (Fig 2A, upper graph). However, when restricting the analysis to subjects with abnormal lung function (FEV1, FVC, and/or FEV1/FVC SP-A in these restricted analyses, showed similar patterns as in the entire study population, although only barely significantly increased in a small window of the higher quantiles of the SP-A distribution (Fig 2B). Current smokers with normal lung function had higher levels of DPPC than never-smokers along its entire distribution (Fig 2D, upper graph), and POPC levels, but with a large and not significant confidence interval in the upper quantiles (Fig 2E, upper graph). No significant associations were found regarding current smokers and either of the lipids, in the analysis restricted to subjects with abnormal lung function (Fig 2D and 2E, lower graphs). Albumin showed no effect of current smoking (Fig 2C).

Fig 2

PEx-variables among current- compared to never-smokers at normal and abnormal lung function.

Plots illustrating age- and sex-adjusted quantile regression estimates of current smokers to never smokers (the red dotted line), stratified on normal and abnormal lung function (n = 35 and n = 31, respectively). (A) number PEx (kn/L), (B) SP-A (wt%), (C) Albumin (wt%), (D) POPC (wt%) and (E) POPC (wt%) were analyzed separately, as were the sub-groups. Quantiles on x-axis refers to the distribution of the PEx-variable studied. Estimates from current smokers compared to never smokers presented at normal lung function (n = 19 and n = 16, respectively) and abnormal lung function (n = 18 and n = 13, respectively). Estimates from quantile regression denoted by black dotted line with blue confidence intervals.

Discussion

The effects of long-term smoking on the composition of the lining fluid of small airways, likewise the number of formed droplets of this fluid (i.e. PEx; particles in exhaled air), have been studied using the PExA method. Current smokers with normal lung function had increased concentrations of DPPC and POPC in PEx, in comparison to never smokers. Also, smokers tended to have higher levels of SP-A in PEx, irrespective of lung function. The number of PEx was markedly increased in current smokers compared to never smokers, and most apparent among subjects with abnormal lung function. The magnitude of these associations varied over the distribution of the different PEx variables.

We used quantile regression to study smoking and its association with the distribution of each PEx variable as the associations were suspected not to be equal along the different quantiles. It is well known that the effect of smoking differ between subjects, and in this cross-sectional setting, we assumed that the inflammatory responses and remodeling processes as a result of smoking, were unequal distributed among the included subjects. It seems likely that smoking may induce changes in the surfactant composition in small airways as a protective mechanism, but at certain stage this will not suffice in susceptible individuals, and pathological processes will take over. By this time the surfactant function will deteriorate, airways start to close, emphysema will develop and the lung function will start to decline. This was indicated by the present results, with a significant association between current smoking and higher DPPC in PEx among subjects with normal lung function as compared to no association between smoking and lipids in PEx in subjects with abnormal lung function (Fig 2D). Previous finding by Laerstad et al [22], that subjects with more severe COPD (GOLD II-IV) had lower number of exhaled particles, further support this.

Consistent with these effects on the lipid homeostasis after smoking, previous experiments on smoke-exposed mice have shown that smoke disturbed the capacity of alveolar macrophages to take up oxidized lipids [15]. However, longitudinal studies are needed to elucidate these associations in humans, and the present findings only allows one to speculate about the effect of the irritants in tobacco smoke on the synthesis, secretion, and/or reuptake of surfactant lipids.

To date, knowledge is limited on changes in the small airways lining fluid in humans. In COPD, Agudelo and colleagues [17] recently showed a correlation between lung function and a reduction in surfactant lipids, based on the BAL fluids of former smokers. In present study, DPPC and POPC in PEx were found increased in current smokers throughout almost the entire distribution of these lipids, when restricting the analyses to subjects with normal lung function. Recent findings by Hussain-Alkhateeb et al [32], using quantile regression (50th percentile), also showed increased content of DPPC and POPC in PEx in current smokers (n = 17). Important to note, normal spirometry does not exclude subjects with affected lungs. A previous study on smokers with preserved spirometry (n = 4388) showed half of the smokers to have radiologic abnormalities, from which a large portion had evidence of emphysema or airway wall thickening [33]. This highlights the need for novel methods to easily detect changes in lung function.

The SP-A distribution shifted towards higher values in current smokers, compared to never smokers, and the association was stronger in the higher quantiles (Fig 1B). In a previous small exploratory study in former smokers with COPD, SP-A in PEx decreased significantly with worsening FEV1 [22]. In present study, SP-A correlated negatively to the FEV1/FVC ratio in current smokers (Table 3). In subjects with normal lung function, there was however no significant association between SP-A and smoking (Fig 2B, right hand graph), although the estimates were in the same direction as in subjects with abnormal lung function. SP-A seems thus to be associated with lung function, but also with on-going exposure.

SP-A is known to be important in enhancing the phagocytosis of inhaled toxins, and thus, it facilitates toxin clearance, for example, by alveolar macrophages. Accordingly, we speculated that high levels of SP-A in the surfactant of small airways lining fluid, here reflected as high content of SP-A in PEx, was a response to the inhalation of respiratory toxins from tobacco smoke. Thus, as high SP-A levels would speed up the removal of these toxins, it may be a favorable response.

The abundance of albumin in PEx was not affected by smoking status. One might assume it should increase with smoking, due to the increased leakage from the systemic circulation in smokers, but this did not seem to be the case. Our finding was supported by results from an earlier study by Schmekel et al [34].

The number PEx per liter of exhaled air was substantially increased in current smokers compared to never smokers (Fig 1A). This finding suggested that current smoking might increase the number of small airways that close and re-open. This hypothesis is consistent with previous findings, reporting increased closing volumes in smokers [35, 36]. The closing volume is the lung volume during an expiration, when a substantial number of airways close. It is highly likely that the larger the closing volume, the larger the number of airways that close and re-open. Thus, the high number PEx that we found in smokers might be explained by the extent of airway closure. The primary cause of increased closing volume in smokers has been attributed to smoking-induced loss of lung elastic recoil [37], which facilitates the closing of small airways.

Another possible mechanism underlying the increased number PEx in smokers might be the effects of smoking on the physical properties of surfactant per se. In computational studies on liquid film burst, an increased surface tension resulted in higher concentrations of droplets [38, 39]. Thus, if tobacco smoke increases the surface tension in surfactant, the number PEx might increase in smokers. Potentially, an increased number of exhaled particles might be due to both the increased number of airways that close and re-open and the altered surfactant homeostasis.

In analysis restricted to subjects with normal lung function, no effect of smoking on number PEx was shown. However, in the analysis on subjects with abnormal lung function, there was a strong association between current smokers and an increase in number PEx, indicating that small airways close and re-open easier in current smokers with abnormal lung function.

Intra-pulmonary ventilation inhomogeneity, assessed with the N2-slope (%N2/L), was not correlated with any PEx variables in current smokers (Table 3). In previous studies, the N2-slope were shown to correlate with pathological changes in the small airways in smokers [40, 41]. However, the N2-slope reflects structural changes that not necessarily overlap with inflammatory changes in small airways; potentially, this lack of overlap might explain the lack of correlation found in the present study.

Taken together, the increased number PEx and the altered levels of DPPC, POPC and SP-A in the PEx of current smokers may reflect a disturbed homeostasis of small airway lining fluid caused by smoking. Protein-lipid interactions are essential for maintaining the homeostasis of small airways lining fluid. For instance, SP-A promotes the re-uptake of inactivated surfactant lipids by alveolar type II cells [13]. The magnitudes of the effects of smoking varied along the distributions of each PEx variable, and we speculated that this variation might have reflected different stages of inflammatory response in the small airways lining fluid. These associations need however to be analyzed more in depth in a larger material with longitudinal design, before any stronger conclusions can be drawn.

Some strengths of the present study are worth pointing out. First, we measured PEx instead of BAL fluid. The PEx matrix consists of undiluted lining fluid from small airways that, due to the non-invasive methodology, provide a strong advantage over sampling and measuring potential biomarkers in BAL fluid, which contains diluted concentrations. Additionally, we expressed the concentrations of the different biomarkers in PEx in terms of the weight percent of the sample, thus, corrected for differences in the mass of the sample. Finally, to avoid systematic bias in present study, all samples were handled in the same way, but they were analyzed in a randomized order.

The present study also have limitations, the main being the small number of included subjects limiting the generalizability of the results. Especially the number of current smokers with abnormal lung function was low, which limited the analysis. The intra- and inter-individual variations in PEx-variables, collected with the standardized breathing maneuver, has not been addressed in current study. For practical reasons, it was not possible to perform repeated measures or measures at the same time-point during the day, which might have reduced the bias of diurnal variations in PEx variables if taken into account [31]. However, the recently published study by Hussain-Alkhateeb et al showed similar results as ours [32].

Conclusions

Tobacco smoking seems to influence on the composition of the lining fluid of small airways, and both the phospholipids DPPC and POPC as well as SP-A were increased in current smokers. The magnitude of these effects varied along the distribution of the PEx variables and seemed to be associated with lung function. The differences we observed between smokers and never smokers are likely to be associated with small airway inflammation, which is known to be induced by smoking, but further analyses are needed to explore these associations more in depth. As the PExA method is non-invasive and easy to apply, it may help us to identify novel biomarkers for disease at an early stage. At individual level, it is easy to neglect that smoking implies an increased risk for severe outcomes, but if there are signs of ongoing inflammation and an improved individual risk-assessment, it may help people to quit smoking. In a longer run, PExA might be a useful tool for the screening of large populations and may facilitate the identification of subjects at risk of developing severe diseases affecting small airways, such as COPD.

Data set.

(PDF)

Click here for additional data file.

12 Apr 2021

PONE-D-21-07178

Current smoking alter phospholipid- and surfactant protein A levels in small airway lining fluid: an explorative study on exhaled breath

PLOS ONE

Dear Dr. Viklund,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers raised some signifcant concenrs which are mainly attibuted to methodological issues as well as to the way that results were interpreted. Please also revse the statistica analysis.

Please submit your revised manuscript by May 27 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Stelios Loukides

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as: a) the recruitment date range (month and year), b) a description of any inclusion/exclusion criteria that were applied to participant recruitment, d) a description of how participants were recruited, and e) descriptions of where participants were recruited and where the research took place.

3. Please provide a sample size and power calculation in the Methods, or discuss the reasons for not performing one before study initiation.

4. Please provide the product number and any lot numbers of the ELISA kits purchased for your study.

5. Please note that PLOS does not permit references to 'data not shown.' Authors should provide the relevant data within the manuscript, the Supporting Information files, or in a public repository. If the data are not a core part of the research study being presented, we ask that authors remove any references to these data.

Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data.

6. During your revisions, please confirm whether the wording in the title is correct and update it in the manuscript file and online submission information if needed. Specifically, please consider whether it should read "Current smoking alters..." rather than "alter".

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Regarding the submitted manuscript Current smoking alter phospholipid- and surfactant protein A levels in small airway lining fluid: an explorative study on exhaled breath-

The authors aimed to explore the effect of smoking on the composition and number of exhaled particles in a smoker-enriched study population.

The study consists of 102 subjects (29 never smokers, 36 former smokers and 37 current smokers) aged 39 to 83 years (median 63). Samples were optioned at one time point for all included patients.

The contents of surfactant protein A and albumin in exhaled particles was quantified with immunoassays and the contents of the phospholipids dipalmitoyl- and palmitoyl-oleoyl- phosphatidylcholine with mass spectrometry.

I have some major concern regarding the current manuscript:

1. Please explain the rational for analyzing number of exhaled particles expressed av particles per liter. It seems possible that even small variations in the standard breathing maneuver could result in significant changes.

2. The amount of analyzed particles are in exceptionally low range – nanogram. The membrane collecting the particles were divided in 2 by hand after collection. One of the samples were used for SpA/Albumin and one sample was used for MS. Seems highly likely that personal differences and imprecise cutting of the membrane could potentially be a highly likely source of source of error. Was the amount off particles confirmed on the membrane after dividing by hand? Dividing 120ng into exactly 60 ng in each seems highly unlikely.

3. The results are somehow contradictory. In the introduction the authors discuss previous findings. COPD patients has been found to have less surfactant, DPPC, and POPC. However, it seems like the results form the current study implicate the opposite.

4. Age is well known to affect the lung. In the current study the age ranges from 38 - 83. The authors state that has taken the age into consideration in the statistical regression model. Please explain how the age was considered using their pexa method. Known age differences using the pexa method for sampling etc.

5. The pack years of the patients are presented in the supplement and taken into consideration by the authors in their analyses. Patient number 50, 53 and 65 have a reported pack year of 999 years. Seems unlikely and probably an error. However, the analyses seem to be based on those numbers why one should consider a recalculation of the study.

6. The study is based on a rather small cohort, which could be totally fine. However, the group seems to be highly heterogenous. For example, pack year range between the groups: 3-71 pack years in the former smoking group and 1-55 pack years in the current smokers group. Please explain the selection of groups and how these differences might have inflicted the results.

7. Please add information regarding the former smoker group - time from last smoke until the sampling. Could the results also alter if the patient smoked directly before? Do the authors have information about time from last smoke until sampling in the smokers group? This should be added and taken into consideration.

8. Why was the biomarkers SpA, albumin, DPPC, and POPC selected? Looking at the authors publication list and history it seems that the authors have invented the pexa method some years ago and these are the only biomarkers that have ever been able to be analyzed using this method.

9. A main issue regarding the current study is that the biomarkers SpA, albumin, DPPC, and POPC measured in the study is not validated with for example another method. This should be added.

Reviewer #2: Peer review of manuscript ID: PONE-D-21-07178

Current smoking alter phospholipid- and surfactant protein A levels in small airway lining fluid: an explorative study on exhaled breath

COMMENTS TO THE AUTHORS

First of all I’d like to compliment the authors with the well executed study and the excellent way they reported their results. The field of exhaled breath research holds great promise for the future of (clinical) medicine. And as the results of this study have shown, also for basic understanding of human physiology. My compliments and thank you for inviting me to have a say about it.

The manuscript covers all essential criteria: original research is presented; collection of data, statistics and analyses are performed well and described in sufficient detail; conclusions are supported by the data; and the data set has been made available.

Nevertheless I do have a very small amount of issues which – when addressed – may improve the quality of this manuscript to some extent:

1. Subjects were classified as ‘current’, ‘former’ and ‘never’ smokers. Current smokers are defined as ‘having smoked cigarettes on a regular daily basis for at least a year’. I’m wondering: do you have any data on how many cigarettes a day they actually smoked, since 20 cigarettes a day versus 2 cigarettes a day may make a lot of difference within this group? How may this binary approach of current smoking (smoking “yes” or “no”) influence your results? If no data is available, this could represent a limitation of your study and should thus be mentioned.

2. Line 91 states “The present study aimed to explore the long-term effects of tobacco smoking on [….]”. What do you mean by long-term? You did not specify how long patients in the ‘former smoker’ group had quit smoking, again you chose a binary approach: former smoking yes or no, instead of making sub-divisions within the group. Because of that, I think you cannot say anything about long-term effects. Maybe just leave out the word ‘long-term’ in this sentence, or otherwise try to specify.

3. You state that your findings might lead to a useful tool for the identification of patients at risk of developing diseases affecting small airways such as COPD. As a clinician I am very interested in the clinical application and value of a new biomarker and/or test. Therefore I’d like to invite you to take it one step further and elaborate on how this breath test would benefit future early COPD patients..? Could be in one or two sentences. I think you can really point out to your readers why exhaled breath analysis can make a great contribution to future clinical practice.

4. Line 139, last word: ‘was’ should be ‘were’.

5. Line 357, sixth word: ‘needs’ should be ‘need’.

Again, thank you for your excellent work and please continue your research on breath analysis.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Dr PMP van Oort, MD PhD

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

27 May 2021

EDITOR comments and our replies:

Comment 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf

and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Reply: We have tried to follow your style requirements of the main body of the manuscript, likewise file naming have been updated according to PLOS ONE´s style requirements.

Comment 2. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as: a) the recruitment date range (month and year), b) a description of any inclusion/exclusion criteria that were applied to participant recruitment, d) a description of how participants were recruited, and e) descriptions of where participants were recruited and where the research took place.

Reply: Thank you! We have modified the information about the participant recruitment method and the demographic details of the study participants as suggested under the method section.

Comment 3. Please provide a sample size and power calculation in the Methods, or discuss the reasons for not performing one before study initiation.

Reply: In view of this novel explorative study, where no a priori information on the variation of most PEx-variables and the expected difference between smoking groups were available, a sample size estimation was not possible to perform. Thus, the sample size was based on general idea of what sufficient analytical sample is needed from previous pilots and studies conducted within the team in this research context.

Comment 4. Please provide the product number and any lot numbers of the ELISA kits purchased for your study.

Reply: The product number and lot numbers of the ELISA kits used have been updated in the method section in the manuscript.

Comment 5. Please note that PLOS does not permit references to 'data not shown.' Authors should provide the relevant data within the manuscript, the Supporting Information files, or in a public repository. If the data are not a core part of the research study being presented, we ask that authors remove any references to these data. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data.

Reply: Data previously referred to as “data not shown” has now been included in the already existing figures.

Comment 6. During your revisions, please confirm whether the wording in the title is correct and update it in the manuscript file and online submission information if needed. Specifically, please consider whether it should read "Current smoking alters..." rather than "alter".

Reply: The title has been updated and now reads “Current smoking alters phospholipid- and surfactant protein A levels in small airway lining fluid: an explorative study on exhaled breath”.

REVIEWER #1 comments and our replies:

Comment 1: Regarding the submitted manuscript Current smoking alter phospholipid- and surfactant protein A levels in small airway lining fluid: an explorative study on exhaled breath-

The authors aimed to explore the effect of smoking on the composition and number of exhaled particles in a smoker-enriched study population.

The study consists of 102 subjects (29 never smokers, 36 former smokers and 37 current smokers) aged 39 to 83 years (median 63). Samples were optioned at one time point for all included patients.

The contents of surfactant protein A and albumin in exhaled particles was quantified with immunoassays and the contents of the phospholipids dipalmitoyl- and palmitoyl-oleoyl- phosphatidylcholine with mass spectrometry.

I have some major concern regarding the current manuscript

Reply: We sincerely thank you for reading and reviewing our manuscript. We have tried to address your major concerns, reflected by your comments below, and hope that this have clarified and improved the manuscript.

Comment 2: Please explain the rational for analyzing number of exhaled particles expressed as particles per liter. It seems possible that even small variations in the standard breathing maneuver could result in significant changes.

Reply: Thank you for this relevant reflection regarding how to address number of exhaled particles (PEx), and likewise the repeatability and reproducibility of using the presented standard breathing maneuver. How to best address number PEx to make it comparable between subjects is still not fully clear. By dividing number PEx with total volume of exhaled air, we try to normalize PEx based on different lung sizes. PEx/exhalation is an alternative way of expressing number PEx, reducing the variation between gender. In present study, we chose to use number PEx/L and adjusted for age and gender in the regression analysis.

Kokelj and colleagues recently presented data on variability in number of particles, expressed as both per exhalation and per Liter, in healthy subjects [1]. The intra- and inter-individual variation in both these PEx-variables, collected with the standardized breathing maneuver, is only partly explained by age, gender and lung size [2]. We try, within reasonable efforts, to minimize the impact of factors in the breathing maneuver that we think can influence the composition of exhaled particles. For instance, inhalation- and exhalation flow, likewise the breath-hold at residual volume, are therefore carefully monitored by the instructor.

We have added to the discussion-section, the following limitations (page 18, line 376-380); “The intra- and inter-individual variation in PEx-variables, collected with the standardized breathing maneuver has not been addressed in current study. For practical reasons, it was not possible to perform repeated measures or measures at the same time-point during the day, which might have reduced the bias of diurnal variations in PEx-variables if taken into account [1]. However, the recently published study by Hussain-Alkhateeb et al showed similar results as ours [3].”

Comment 3: The amount of analyzed particles are in exceptionally low range – nanogram. The membrane collecting the particles were divided in 2 by hand after collection. One of the samples were used for SpA/Albumin and one sample was used for MS. Seems highly likely that personal differences and imprecise cutting of the membrane could potentially be a highly likely source of source of error. Was the amount off particles confirmed on the membrane after dividing by hand? Dividing 120ng into exactly 60 ng in each seems highly unlikely.

Reply: The impactor is designed with 10 nozzles through which particles of a certain size passes and impacts on the membrane as 10 spots, see fig 1. These spots are separated with a space in between them, with 5 placed to the left and 5 to the right, making it easy to separate the two halves without being in contact with any of the spots. The nozzles of the impactor was designed with the intention of having the possibility to split the sample in half without touching the sample. The splitting of the sample in two equal parts allow the use of different extraction protocols required for lipid-analysis (organic) and protein-analysis (aqueous). We have extensive data of how the particles deposit on the collection surface based on for example TOF-SIMS analysis (a method where lipids are analyzed directly on the collection substrate without extraction). On the mirror like finish of silicon wafers, it is quite apparent visually (fig.1). Furthermore, during method development for analytical assays, identical samples are needed for evaluating different protocols of extraction and reproducibility, it is imperative to distinguish sampling errors to analytical errors to identify the weakest link. This testing have confirmed that a sample on PTFE substrate split in half will have very similar analyte amounts. This was evaluated for three consecutive collections where substrate was split in half, RSD% for split samples were only 5-10% whereas RSD% for the three samples in total were 10-15%, based on lipids PC16:0/16:0 and PC16:0/18:1 analyzed with LC-MSMS.

Figure 1. A 25 mm Silcon wafer with sample spots

Comment 4: The results are somehow contradictory. In the introduction the authors discuss previous findings. COPD patients has been found to have less surfactant, DPPC, and POPC. However, it seems like the results form the current study implicate the opposite.

Reply: Thank you for pointing this out, the comparison between current and previous studies seem indeed somewhat contradictory. To make this comparison less inconsistent, we have tried to clarify our hypothesis in the discussion section, as follows (page 14, line 295-303); “It seems likely that smoking may induce changes in the surfactant composition in small airways as a protective mechanism, but at certain stage this will not suffice in susceptible individuals, and pathological processes will take over. By this time the surfactant function will deteriorate, airways start to close, emphysema will develop and the lung function will start to decline. This was indicated by the present results, with a significant association between current smoking and higher DPPC in PEx among subjects with normal lung function as compared to no association between smoking and lipids in PEx in subjects with abnormal lung function (Fig 2D). Previous finding by Laerstad et [4], that subjects with more severe COPD (Gold II-IV) had lower number of exhaled particles, further support this.

Comment 5: Age is well known to affect the lung. In the current study the age ranges from 38 - 83. The authors state that has taken the age into consideration in the statistical regression model. Please explain how the age was considered using their pexa method. Known age differences using the pexa method for sampling etc.

Reply: Age was included as an adjustment factor in the quantile regression, which showed significant contribution to the model. Previous studies have shown age to be a significant predictor of certain PEx-variables [2, 3]. In current study, the number of subjects were too small to draw any further conclusions on how age, in relation to smoking, affects the PEx-variables, wherefore we only chose to adjust for age to minimize the associated heterogeneity in the smoking groups.

Comment 6: The pack years of the patients are presented in the supplement and taken into consideration by the authors in their analyses. Patient number 50, 53 and 65 have a reported pack year of 999 years. Seems unlikely and probably an error. However, the analyses seem to be based on those numbers why one should consider a recalculation of the study.

Reply: 999 indicated missing values and have been treated as such in the analysis.

Comment 7: The study is based on a rather small cohort, which could be totally fine. However, the group seems to be highly heterogenous. For example, pack year range between the groups: 3-71 pack years in the former smoking group and 1-55 pack years in the current smokers group. Please explain the selection of groups and how these differences might have inflicted the results.

Reply: Thank you for this comment. The recruiting procedure have been clarified and now reads (page 5, line 100-106); 102 subjects, aged 39-83 years, were recruited to participate in an extended study protocol in the follow-up study of the population-based INTERGENE-ADONIX cohort [5]. Recruited subjects had all given their written informed consent in the follow-up study to be contacted for participation in the extended study protocol. Eligible for inclusion were subjects whom had managed to perform spirometry and had reported their smoking status and smoking history in the follow-up study. Exclusion criteria were ongoing respiratory tract infection, myocardial infarction past month, and/or pregnancy in last trimester. All current smokers were invited to participate, whereas former- and never-smokers were invited randomly.

The selection of groups was mainly done with the intention to see whether an ongoing tobacco exposure may be associated with altered PEx-variables concentrations, in comparison to that of never-smokers. Furthermore, we aimed to explore whether this potential alteration would remain after smoking cessation. It would of course be of interest to further explore how the amount smoked affects the PEx-variables, but for doing that we believe we need to have a larger study population. Anyhow, with age-adjustment in current study, we have minimized the associated heterogeneity in the groups. It is quite likely that higher pack-year range in former smoker is due to that older group present in that group.

Comment 8: Please add information regarding the former smoker group - time from last smoke until the sampling. Could the results also alter if the patient smoked directly before? Do the authors have information about time from last smoke until sampling in the smokers group? This should be added and taken into consideration.

Reply: Thank you for this interesting reflection regarding the acute and time-course of effect of smoking on PEx-variables. Of course this would be of interest to study further, but that would demand another study-setup which hopefully could be done in a near future. In current study, smokers were “restricted to withdraw from smoking 1 hour prior to the test.” Unfortunately, no more information about last smoke was collected. The definition of former smokers in current study was that they “had smoked on a regular basis, but had stopped smoking for one year or more before the clinical examination.” Due to the fairly low number of former smokers, likewise the wide range of pack-years among smokers (as discussed in reply to comment 7), time since smoking cessation will not be addressed in current study but instead more in depth in an upcoming study with a larger number of former smokers.

Comment 9: Why was the biomarkers SpA, albumin, DPPC, and POPC selected? Looking at the authors publication list and history it seems that the authors have invented the pexa method some years ago and these are the only biomarkers that have ever been able to be analyzed using this method.

Reply: Findings from earlier study by Laerstad et al [4] indicated that SP-A and albumin in PEx potentially are interesting biomarkers for smoke pathology but further studies including more subjects were asked for, and also comparison of PEx-variables in smokers without COPD. Methods for analyzing surfactant protein A, albumin, DPPC and POPC have successfully been developed and shown high reproducibility at the present small lab. Concerning lipids in the surfactant, there are animal studies (mice) indicating a rather large effect on lipid composition after exposure to cigarette smoke [6]. We therefore chose these biomarkers, and the number of PEx, to further explore their potential as biomarkers for smoke pathology, with special focus on current smokers without COPD.

Comment 10: A main issue regarding the current study is that the biomarkers SpA, albumin, DPPC, and POPC measured in the study is not validated with for example another method. This should be added.

Reply: Thank you for this comment. The PExA method have previously been compared with broncho-alveolar lavage (BAL) fluid and bronchial wash (BW) in a study of Behndig and colleagues who compared SP-A and albumin in PEx to BAL fluid and to BW [7]. The results showed PEx-content to be similar to BAL but not to BW. We have added this reference to the introduction section (page 4, line 81-82); SP-A levels in PEx have also been shown to be highly correlated with that of BAL fluid [7].

In an early study, SP-A in PExA were compared to that in Exhaled breath condensate (EBC) and in serum [8]. We are currently working on a manuscript comparing lipids in BAL and PExA, taken from the same subject, that will be submitted within short, showing in general extremely good correlation for PC class of lipids. Pearson correlation of log transformed data (for normal distribution of model residuals) ranged between 0.68-0.9, and was recently presented at American Respiratory Society (ATS) [9]. Nevertheless, the PEx-matrix is neither collected in the same way, nor at the same site as for instance BAL or blood, wherefore one would not suspect these different matrixes to reflect the same thing equally.

REVIEWER #2 comments and our replies:

Comment 1: First of all I’d like to compliment the authors with the well executed study and the excellent way they reported their results. The field of exhaled breath research holds great promise for the future of (clinical) medicine. And as the results of this study have shown, also for basic understanding of human physiology. My compliments and thank you for inviting me to have a say about it. The manuscript covers all essential criteria: original research is presented; collection of data, statistics and analyses are performed well and described in sufficient detail; conclusions are supported by the data; and the data set has been made available. Nevertheless I do have a very small amount of issues which – when addressed – may improve the quality of this manuscript to some extent.

Reply: Thank you so much for this supportive comments and shared enthusiasm about the field of exhaled breath research. We have tried to answer the highly relevant comments regarding some issues that should be addressed and we hope that this has improved the quality of the manuscript.

Comment 2: Subjects were classified as ‘current’, ‘former’ and ‘never’ smokers. Current smokers are defined as ‘having smoked cigarettes on a regular daily basis for at least a year’. I’m wondering: do you have any data on how many cigarettes a day they actually smoked, since 20 cigarettes a day versus 2 cigarettes a day may make a lot of difference within this group? How may this binary approach of current smoking (smoking “yes” or “no”) influence your results? If no data is available, this could represent a limitation of your study and should thus be mentioned.

Reply: Thank you for this comment on the complexity of how to target the exposure cigarette smoke in this context. The intensity in smoking can of course be analyzed in different ways, as for example in pack years or in cigarettes per day or in years smoked. In current study, the subjects reported the year when starting to smoke and the number of cigarettes smoked per day during the different years when smoking, and from that, we have calculated pack-years.

In this exploratory set-up, with a limited number of subjects with history of smoking, we mainly used pack years as a variable to describe the smoking load. Neverteless, in a sensitivity study, we tested both pack years and smoking status as variables in the regression analysis and found that being a current smoker affected the PEx-variables stronger than an increase in pack years, even if the latter also was significant, which is why we used smoking status in the presented analysis. In an upcoming study with more subjects, we will address both smoking status and the smoking intensity likewise the duration of smoking more thoroughly.

Comment 3: Line 91 states “The present study aimed to explore the long-term effects of tobacco smoking on [….]”. What do you mean by long-term? You did not specify how long patients in the ‘former smoker’ group had quit smoking, again you chose a binary approach: former smoking yes or no, instead of making sub-divisions within the group. Because of that, I think you cannot say anything about long-term effects. Maybe just leave out the word ‘long-term’ in this sentence, or otherwise try to specify.

Reply: We have, as suggested, leaved out the word “long-term” and rephrased the sentence which now reads (page 5, line 93-95); “The present study aimed to explore potential biomarkers for inflammation in small airway lining fluid; i.e. lipids (DPPC and POPC) and proteins (SP-A and albumin) in PEx and the PEx number concentration, in cigarette smokers.”

Comment 4: You state that your findings might lead to a useful tool for the identification of patients at risk of developing diseases affecting small airways such as COPD. As a clinician I am very interested in the clinical application and value of a new biomarker and/or test. Therefore I’d like to invite you to take it one step further and elaborate on how this breath test would benefit future early COPD patients..? Could be in one or two sentences. I think you can really point out to your readers why exhaled breath analysis can make a great contribution to future clinical practice.

Reply: Thank you for showing your enthusiasm in the potential of exhaled breath analysis, and for giving us the push forward to take the chance to further elaborate on the potential usefulness of the PExA method and its potential in the field of early COPD.

In the discussion section, we already wrote (page 18, line 390-392); “In a longer run, it might be a useful tool for the screening of large populations and may facilitate the identification of subjects at risk of developing severe diseases affecting small airways, such as COPD.”

We have now also added the following sentence to the discussion section (page 18, line 388-390); “At individual level, it is easy to neglect that smoking implies an increased risk for severe outcomes, but if there are signs of ongoing inflammation and an improved individual risk-assessment, it may help people to quit smoking.”

Comment 5: Line 139, last word: ‘was’ should be ‘were’.

Reply: Thank you for pointing this out. This is corrected.

Comment 6: Line 357, sixth word: ‘needs’ should be ‘need’.

Reply: Thank you for pointing this out. This is corrected.

Comment 7: Again, thank you for your excellent work and please continue your research on breath analysis.

Reply: We are so grateful for this positive feedback! We will of course continue with our research on breath analysis, as there are so many interesting and possible studies yet to be done.

REFERENCES

1. Kokelj S, Kim JL, Andersson M, Runstrom Eden G, Bake B, Olin AC. Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin. PLoS One. 2020;15(1):e0227980.

2. Bake B, Ljungström E, Claesson A, Carlsen HK, Holm M, Olin AC. Exhaled Particles After a Standardized Breathing Maneuver. J Aerosol Med Pulm Drug Deliv. 2017;30(4):267-73.

3. Hussain-Alkhateeb L, Bake B, Holm M, Emilsson Ö, Mirgorodskaya E, Olin AC. Novel non-invasive particles in exhaled air method to explore the lining fluid of small airways-a European population-based cohort study. BMJ open respiratory research. 2021;8(1).

4. Larstad M, Almstrand AC, Larsson P, Bake B, Larsson S, Ljungstrom E, et al. Surfactant Protein A in Exhaled Endogenous Particles Is Decreased in Chronic Obstructive Pulmonary Disease (COPD) Patients: A Pilot Study. PLoS One. 2015;10(12):e0144463.

5. Mehlig K, Berg C, Bjorck L, Nyberg F, Olin AC, Rosengren A, et al. Cohort Profile: The INTERGENE Study. International journal of epidemiology. 2017;46(6):1742-3h.

6. Morissette MC, Shen P, Thayaparan D, Stampfli MR. Disruption of pulmonary lipid homeostasis drives cigarette smoke-induced lung inflammation in mice. Eur Respir J. 2015;46(5):1451-60.

7. Behndig AF, Mirgorodskaya E, Blomberg A, Olin AC. Surfactant Protein A in particles in exhaled air (PExA), bronchial lavage and bronchial wash - a methodological comparison. Respir Res. 2019;20(1):214.

8. Larsson P, Mirgorodskaya E, Samuelsson L, Bake B, Almstrand AC, Bredberg A, et al. Surfactant protein A and albumin in particles in exhaled air. Respir Med. 2012;106(2):197-204.

9. Larsson P, Biller H, Koster G, Postle A, Olin A-CC, Hohlfeld JM. Exhaled Breath Particles as a Novel Tool to Study Lung Lipid Composition. C31 COPD BASIC MECHANISMS. p. A4742-A.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

14 Jun 2021

Current smoking alters phospholipid- and surfactant protein A levels in small airway lining fluid: An explorative study on exhaled breath

PONE-D-21-07178R1

Dear Dr. Viklund,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stelios Loukides

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have adressed all my questions and given answers in a point by point letter. The senior authors seem to be the owner of the PExA company, and some of the co authors seems to be stake holders in the same company, therefore it should be clear for the readers that authors do have a conflict of interest.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

17 Jun 2021

PONE-D-21-07178R1

Current smoking alters phospholipid- and surfactant protein A levels in small airway lining fluid:  An explorative study on exhaled breath

Dear Dr. Viklund:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stelios Loukides

Academic Editor

PLOS ONE