Increased MUC1 plus a larger quantity and complex size for MUC5AC in the peripheral airway lumen of long-term tobacco smokers

There is little information on mucins versus potential regulatory factors in the peripheral airway lumen of long-term smokers with (LTS+) and without (LTS − ) chronic obstructive pulmonary disease (COPD). We explored these matters in bronchoalveolar lavage (BAL) samples from two study materials, both including LTS+ and LTS − with a very similar historic exposure to tobacco smoke, and healthy non-smokers (HNSs; n = 4–20/group). Utilizing slot blot and immunodetection of processed (filtered and centrifuged), as well as unprocessed BAL samples from one of the materials, we compared the quantity and fraction of large complexes of mucins. All LTS displayed an enhanced (median) level of MUC5AC compared with HNS. LTS − displayed a higher level of large MUC5AC complexes than HNS while LTS+ displayed a similar trend. In all LTS, total MUC5AC correlated with blood leukocytes, BAL neutrophil elastase and net gelatinase activity. Large mucin complexes accounted for most MUC5B, without clear group differences. In all LTS, total MUC5B correlated with total MUC5AC and local bacteria. In the same groups, large MUC5B complexes correlated with serum cotinine. MUC1 was increased and correlated with BAL leukocytes in all LTS whereas MUC2 was very low and without clear group differences. Thus, the main part of MUC5AC and MUC5B is present as large complexes in the peripheral airway lumen and historic as well as current exposure to tobacco smoke emerge as potential regulatory factors, regardless of COPD per se . Bacteria, leukocytes and proteinases also constitute potential regulatory factors, of interest for future therapeutic strategies. determining the quality and/or quantity of mucins in the peripheral airway lumen of humans. In addition, the study forwards evidence that bacteria, leukocytes and proteinases may also constitute regulatory factors. Thus, these factors may represent targets for future therapy of pathological alterations in airway mucins, possibly regardless of COPD. The present study also indicates that a correct understanding of the pathological implications for mucins in long-term tobacco smokers requires the analysis of mucins in unprocessed BAL samples, allowing large gel forming mucins to be taken into account. We think that our novel findings motivate verification in larger study materials and a further elucidation of the underlying pathogenic mechanisms in translational models.


Introduction
Chronic obstructive pulmonary disease (COPD) is defined as a chronic airway disorder caused by inhalation of noxious particles or gases, including tobacco smoke; a disorder that is characterized by persistent respiratory symptoms and airflow limitation, as well as variable inflammatory patterns and tissue remodeling throughout the airways [1][2][3][4]. At the global level, COPD is predicted to become the third most common cause of disease-related mortality within a few years [1,5]. Still, COPD is largely a preventable disease, since long-term tobacco smoking remains the most common known cause [1].
There is a gradual decline in ventilatory capacity of the lungs in COPD, with more rapid loss of forced expiratory volume during 1 s (FEV 1 ) than vital capacity (VC), often associated with progressively impaired gas diffusion capacity (DLCO) over time [1,3]. The decline in FEV 1 correlates with the frequency of exacerbations, in particular among patients who also suffer from chronic productive cough caused by the comorbidity chronic bronchitis [1,3].
Among active smokers with COPD, smoking cessation remains the most effective treatment [1]. In addition, pharmacological treatments provide relief of symptoms and immunization against Influenza virus and Streptococcus pneumoniae reduces the risk for exacerbations [1]. However, there is an unmet medical need for more efficacious and tolerable therapy to improve the long-term prognosis of COPD. To develop such therapy, the understanding of pathogenic target mechanisms must be improved.
The increased accumulation of total mucus in the small airways correlates with enhanced mortality among human subjects in a statistically significant manner [6,7], suggesting that excess mucus per se is an important pathogenic factor. In population studies, it has been demonstrated that the decline in FEV 1 over time is enhanced in individuals with productive cough [8]. In addition, the risk of developing COPD is increased in smokers who early in life have increased airway phlegm and experience productive cough [9]. It is also known that the soluble mucins MUC5B and MUC5AC constitute the main components of mucus in humans under normal conditions [10]. Down to the tenth generation of bronchi, surface goblet cells account for most of the production of MUC5AC and submucosal glands account for most of the production of MUC5B, with some additional production in club cells [10,11]. However, from the tenth generation of bronchi and beyond where glands are lacking, surface goblet cells and club cells jointly account for the production of MUC5B [10,11] .
The membrane-bound mucins in the airways include MUC1 [12]; a mucin that may act as a 'decoy' when it is released, thereby limiting the exposure of epithelial cells to pathogens [12]. In experimental models in vivo, murine Muc1 may attenuate epithelial inflammation and injury in response to the opportunistic pathogen Pseudonomas aeruginosa [13], whereas murine Muc5b plays an important role in pulmonary host defense [14]. In patients with COPD, there is immunoreactivity for MUC5AC in lining epithelial cells and for MUC5B in submucosal gland cells [10]. However, it is still uncertain whether immunoreactivity for MUC5B is more prominent in the epithelium of patients with COPD than it is in clinically healthy smokers [10]. Moreover, there is evidence that the total area of MUC5AC immunoreactivity is increased in bronchial submucosal glands for patients with COPD, in comparison with smokers without COPD and healthy non-smokers (HNSs), respectively [15]. In terms of soluble mucins from the airway lumen, there is evidence for increased levels of MUC5AC and MUC5B in induced sputum from ever smokers with severe COPD, in comparison with non-smoking controls [16]. However, the underlying study did not specifically address historic (pack-years) or current (cotinine) exposure to tobacco smoke. In fact, this has been the case for several previous studies on mucins in the airways, including those claiming that the MUC5B/MUC5AC ratio is enhanced in sputum or bronchoalveolar lavage (BAL) samples from COPD patients, compared with smokers without COPD [10,17]. This means that it is difficult to judge the pathogenic significance of exposure to tobacco smoke per se in relation to qualitative and quantitative alterations in mucins from smokers with and without COPD. Moreover, relatively few studies have addressed extracellular mucins in BAL samples; samples that are likely to reflect the truly peripheral airway lumen.
It is evident that there is still an incomplete understanding of the quantitative and qualitative alterations among mucins in the peripheral airway lumen of smokers with and without COPD and even less known about potential regulatory factors, such as current and historic exposure to tobacco smoke or local and systemic signs of inflammation. Here, we addressed these matters using blood and BAL samples from current smokers with and without COPD, ascertaining that these particular study groups had a very similar historic exposure to tobacco smoke, and by comparing them with HNSs.

Ethical review
The current study was based upon the analyses of samples from HNSs and long-term tobacco smokers (LTSs) with (LTS+) and without (LTS−) COPD, obtained from two study materials -from Stockholm (Material 1) and from Gothenburg (Material 2). The study populations were recruited and characterized after careful review and approval by the regional committee for ethical review in Stockholm (Material 1: Diary No. 2005/733-31/1-41) and Gothenburg (Material 2: Diary No. 968-11), respectively. After receiving oral and written information, all participating subjects gave oral and written informed consent, in accordance with the recommendations of the code of ethics of the World Medical Association (Declaration of Helsinki).

Material 2 HNS LTS− LTS+ (B)
Number  a negative history of atopy and, for Material 2 only, we also required a negative test for specific IgE against airborne allergens in blood. For all LTS, we required a tobacco load of at least 5 pack-years in Material 1 and at least 20 pack-years in Material 2. For all LTS, a history of chronic bronchitis was merely recorded. For HNSs and LTS−, the inclusion criterion was a post-bronchodilatory FEV 1 /FVC ratio of ≥70% and an FEV 1 of >80% of predicted whereas DLCO was merely recorded for all study groups. For LTS+, the inclusion criterion was a post-bronchodilatory FEV 1 /FVC ratio of <70% [1]. The X-ray had to be normal for HNS. For all LTS, it had to be without significant pathology beyond hyperinflation, direct signs of bronchitis and indirect signs of emphysema. To ascertain a high level of clinical safety during bronchoscopy, we required a post-bronchodilatory FEV 1 > 40% predicted. Clinical or laboratory signs of infection within 4 weeks prior to bronchoscopy resulted in a rescheduled investigation. The clinical characteristics for the included subjects in Material 1 and Material 2 is summarized separately (Supplementary Table S1A,B).

Bronchoscopy
For both Material 1 and Material 2, bronchoscopy was performed by an experienced pulmonologist during stable clinical conditions, in accordance with clinical routine of the interventional unit at respective university hospital (Supplement Material). Briefly, after premedication and local anesthesia, the bronchoscope was positioned and wedged in a subsegmental bronchus (middle lobe or lingula). The BAL was then performed by repeatedly instilling aliquots of sterile and body-tempered phosphate-buffered saline (PBS). The BAL samples were aspirated (yield presented in Table 1) in a sterile plastic bottle and kept on ice until transportation to the laboratory for processing (see below). During bronchoscopy, medication for bronchodilation and anesthesia was administered as needed.

Growth of bacteria
For Material 2 only, a lower airway sample (Supplementary Material) was obtained from each subject and sent to the Department of Bacteriology at Sahlgrenska University Hospital, for a qualitative and quantitative analysis of the growth of aerobic bacteria. The principal bacteriological results are summarized separately ( Table 2).

Processing of BAL samples
The processing of BAL samples was performed according to clinical routine at the respective study site, as follows: after arrival to the laboratory, the entire BAL sample from Material 1 was filtered under sterile conditions and the yield was then centrifuged at low temperature to divert extracellular fluid (i.e., cell-free BAL fluid) from cells and debris. The cell pellet (i.e., BAL cells) was separated from the obtained supernatant and the latter was frozen. For Material 2 only, unprocessed BAL samples were also collected and, once retrieved, the cell pellet was resuspended in PBS and the cells were counted to determine total leukocyte concentrations. Smears for differential cell counts were prepared using cytocentrifugation and the obtained preparations were stained for cell differential counts. The principal findings in the BAL samples are presented separately (Table 1).

Processing of blood samples
For Material 2, venous blood samples were collected as plasma and as serum. Blood cell total (see Results below) and leukocyte differential (Supplementary Table S2) counts were determined utilizing an automated flow cytometer according to clinical routine. The obtained plasma and serum samples were frozen until further analysis.

Characterization and quantification of mucins
In brief, mucin analyses were performed as follows: a protease inhibitor cocktail was added to the BAL samples during thawing. The BAL samples were diluted in reduction buffer. To expose the MUC5B and MUC2 epitopes, the BAL samples were reduced and then alkylated for these specific analyses. Each sample was loaded on to a polyvinylidene difluoride membrane using a slot blot apparatus. In addition, serial dilutions of the mucin standards were also loaded.
Vacuum was applied to attach the mucins to the membrane and the membranes were then air-dried, pre-wetted in methanol, rinsed with water and incubated in PBS. The membranes to be analyzed for MUC1 were analyzed in duplicates, whereof one of the membranes was subject to periodate oxidation. The unspecific binding was attenuated by incubating with a blocking buffer. The membranes were then incubated with specific rabbit serum against MUC5B or MUC2 or with mouse monoclonal antibodies against MUC5AC or MUC1 in blocking buffer with gentle shaking. The membranes were then washed and were thereafter incubated with infrared-labeled secondary antibody diluted in blocking buffer. The membranes were washed again and the blots were subsequently visualized and quantified for determination of the type and level (i.e., concentration) of respective mucin.

Quantification of C-reactive protein
Protein concentrations of C-reactive protein (CRP) in serum were determined with a high-sensitivity commercial assay (Supplementary Material), utilizing the agglutination of CRP to latex particles. These particles were subsequently exposed to anti-CRP antibodies and then detected by a turbidimetric analysis instrument.

Quantification of cotinine
Protein concentrations of the nicotine metabolite cotinine were determined in serum samples utilizing a competitive immunochemical method with detection by a chemiluminescence instrument.

Quantification of neutrophil elastase
Levels (i.e., concentrations) of the neutrophil elastase-α-1 proteinase inhibitor complex in cell-free BAL fluid samples were determined utilizing ELISA.

Quantification of net gelatinase and serine proteinase activity
Fluorometric substrates were used to measure two types of net proteinase activity (Supplementary Material) in cell-free BAL fluid samples, where the increase in fluorescence is proportional to proteolytic activity.

Statistical analyses
Analytical statistics for key outcomes were addressed separately for Material 1 and Material 2 with a non-parametric approach, using GraphPad Prism 7.0 (GraphPad Software Inc. San Diego, U.S.A.) software package. Median (range) values are presented unless otherwise stated. Kruskal-Walllis one-way ANOVA on ranks with Dunn's multiple comparisons test was utilized for comparisons between groups. The Spearman's rank order correlation test was utilized to analyze associations between mucin levels and other parameters among LTS+ and LTS−. The HNS group was excluded from the analysis to avoid false positive correlations due to the mucin changes occurred with smoking per se. The level of statistical significance was set at P<0.05.

Clinical characteristics
In Material 1, the number (n) of included subjects ranged from 16 to 20 per study group and in Material 2, it ranged from 4 to 9 per study group (Supplementary Table S1A,B). Both genders were represented, the body mass index (BMI) was normal and similar among all study groups and the range of age was similar for all study groups. Importantly, the historic exposure to tobacco smoke (i.e., tobacco load) was very similar for all LTS (LTS− and LTS+) in both Material 1 and Material 2. For Material 2 only, we also monitored chronic bronchitis but only two subjects had chronic bronchitis. One such subject was included in the LTS− group and one in the LTS+ group. The results for cotinine and CRP in Material 2 are presented below. In terms of ventilatory capacity, the FEV 1 (% predicted) suggested mild to moderate chronic airflow obstruction in the LTS+ group of both study materials, with normal FEV 1 for the LTS− and HNS groups in these materials (Supplementary Table S1A,B). In terms of gas diffusion capacity, there was a modest reduction in DLCO (% predicted) for the LTS− group and a substantial reduction in the LTS+ group, with normal DLCO (% predicted) for the HNS group, in both study materials (Supplementary Table S1A,B).

Growth of bacteria
The lower airway samples harvested from Material 2 displayed growth of any bacteria in two subjects from the HNS group, in seven subjects from the LTS− group and in one subject from the LTS+ group (Table 2). Two subjects only displayed cultures positive for pathogenic species and these subjects belonged to the HNS and LTS− group, respectively.

Yield and viability for BAL samples
We obtained BAL samples from all included study subjects. The median volume yield was of a similar order of magnitude for the HNS, LTS− and LTS+ groups, respectively, in Material 1 (Table 1A) and Material 2 (Table 1B), with a gradual decline in BAL yield for these groups, as expected. There was a high cellular viability (>96%) among BAL cells in both study materials (Supplementary Material). In both study materials, the LTS groups tended to display a higher total leukocyte count in BAL samples than did the HNS group (Table 1A,B). However, this enhancement of leukocytes was not statistically significant for the LTS+ group in Material 2. Among the subpopulations of leukocytes in BAL samples, the percentage of neutrophils tended to be higher than that for eosinophils in both study materials, particularly so in the LTS+ groups. Moreover, in the BAL samples from Material 2, the percentage of neutrophils tended to be more increased in the LTS+ group compared with the HNS group in Material 2 whereas this increase was less evident in Material 1 (Table 1A,B). Finally, a similar pattern was observed for the percentage of eosinophils in BAL samples. However, in this case, it was for eosinophils only that the difference in percentage proved statistically significant.

Group-related differences for level and proportion of MUC5AC in large complexes
We quantified the level of MUC5AC in cell-free BAL fluid samples from both Material 1 and Material 2. The processed (filtered with a pore size of 70 μm) samples from Material 1 displayed a higher (P=0.046) level of MUC5AC in the pooled group of all LTS (254 [72-886] μg/ml; n=36) than in the HNS group (219 [72-305] μg/ml; n=20, Figure  1A). There was a trend in the same direction for MUC5AC in the LTS− group. Since a 2.5-fold increase in the level of MUC5AC in sputum has previously been observed in long-term smokers (LTSs) [18], we found the corresponding difference that we detected in BAL samples smaller than expected. We therefore hypothesized that a larger proportion of the secreted mucins forms large complexes in LTSs, with and without COPD, and therefore utilized the access to unprocessed material, that was available for the subjects in Material 2 only. In the unprocessed BAL samples from Material 2, the MUC5AC content was 8.5-fold higher in the pooled group of all LTS compared with the HNS group ( Figure 1B). There was a similar level for the LTS+ and LTS− groups. However, along the lines of the findings in Material 1, the level of MUC5AC in filtered BAL fluid from the pooled group of all LTS in Material 2 was only ∼35% higher than that in the HNS group ( Figure 1C). The proportions of mucins that were present as large complexes (i.e., those removed by processing including filtration) were indeed larger in the LTS− and LTS+ group, respectively, than in the HNS group from Material 2 ( Figure 1D).

Potential regulatory factors
Potential regulatory factors of mucins were analyzed in Material 2 only.

Cotinine
The level of cotinine in serum was markedly higher (P=0.00021; n=13) in the pooled group of all LTS (336  pg/ml) than in the HNS group (10 [10-10] pg/ml) (Figure 2A). This was also true for the LTS− group whereas the LTS+ group displayed a similar trend only.

Leukocytes
The leukocyte count in blood samples was higher in LTS+ than in HNS and tended to display an intermediate level in the LTS− group ( Figure 2B). The percentage of neutrophils tended to be higher in both LTS groups than in the HNS group. Moreover, this percentage of neutrophils tended to be higher than that for eosinophils in all study groups (Supplementary Table S2).

CRP
The level of CRP in serum tended to be higher in the LTS+ group than in the HNS group, with the LTS− group displaying an intermediate level ( Figure 2C).

Neutrophil elastase
The level of neutrophil elastase protein in cell-free BAL fluid samples did not markedly differ for either the LTS+ or the LTS− group, in comparison with the HNS group ( Figure 2D).

Net gelatinase activity
The level of net gelatinase activity in cell-free BAL fluid samples did not markedly differ for either the LTS+ or the LTS− group, compared with the HNS group ( Figure 2E).

Net serine proteinase activity
The level of net serine proteinase activity in cell-free BAL fluid samples did not markedly differ for either the LTS+ or the LTS− group, compared with the HNS group ( Figure 2F).

Associations between MUC5AC and potential regulatory factors
To identify likely causes of the increased level of MUC5AC among smokers, we performed statistical correlations between factors that may induce or be associated with the level of MUC5AC among LTSs with and without COPD in Material 2. We found that the leukocyte count in peripheral blood and the level of MUC5AC in BAL samples from the pooled group of all LTS displayed a positive correlation ( Figure 3A). In the same group, the level of the mucin secretagogue neutrophil elastase [19] and the level of MUC5AC in BAL samples displayed a positive correlation ( Figure 3B). A similar trend was present for the association between the net serine proteinase activity and the level of MUC5AC in BAL samples ( Figure 3C). Furthermore, the gelatinase metalloproteinase-9 has been experimentally implicated in the production of MUC5AC and accounts for net gelatinase activity in human airways [20]. In line with this, the level of net gelatinase activity and the level of MUC5AC displayed a positive correlation in BAL samples from all LTS ( Figure 3D). However, we detected no statistically significant correlations between these potentially regulatory factors and the proportion of MUC5AC present as large complexes in BAL samples from the pooled group of all LTS (data not shown).

Group-related differences for level and proportion of MUC5B in large complexes
We detected no statistically significant differences between the group of LTS+ and LTS− in terms of the total level of MUC5B in BAL samples from Material 2 ( Figure 4A). However, both LTS groups displayed a modest trend toward higher levels of MUC5B in comparison with HNS. Despite the lack of statistical significance, half of the subjects in the pooled group of all LTS displayed a 2-to 15-fold higher level of MUC5B in BAL samples than did the subject with the highest level of MUC5B in the HNS group, when comparing subjects without any detected colonization by pathogenic bacteria. Notably, the two subjects that had pathogenic colonization had very similar levels of MUC5B in BAL samples, although one belonged to the HNS group and one belonged to the LTS-group ( Figure 4A). Approximately 70% of all subjects in the study had 80% or more of their MUC5B present as large complexes in BAL samples ( Figure 4B). Therefore, we quantified MUC5B only in BAL samples from Material 2.

Associations between MUC5B and potential regulatory factors in all LTS
In Material 2, the level of MUC5B displayed a positive correlation with the level of MUC5AC in BAL samples from the pooled group of all LTS ( Figure 5A). In this material, the level of large complexes of these two mucins also correlated in a positive manner ( Figure 5B). It is known that MUC5B can be released from airway epithelial cells after stimulation by neutrophil elastase [21] and, along these lines, we detected a trend toward a positive correlation between the level of neutrophil elastase and the level of MUC5B in BAL samples from the pooled group of all LTS in Material 2 ( Figure  5C). Given that MUC5B is involved in antibacterial host defense in the airways and that bacterial products increase the mRNA for MUC5B [22], we also examined the association between MUC5B and the quantity of any bacteria in lower airway samples from the pooled group of all LTS in Material 2. Here, we detected a statistically significant positive correlation between the bacterial count (i.e., CFU) and the level of MUC5B ( Figure 5D). Moreover, we detected a correlation between the level of cotinine in serum and the proportion of MUC5B present as large complexes in BAL samples from the pooled group of all LTS from Material 2 ( Figure 5E).

Group-related levels and proportion of MUC2 in large complexes
Although the presence of significant amounts of MUC2 in the airways has been questioned [23], a recent study suggested the presence of large amounts of MUC2 in the airways of patients with COPD, including mainly former but also some current smokers [24]. Since MUC2 is a large oligomerizing mucin that is likely to be lost during sample filtration, we quantified MUC2 in Material 2 only, from which we harvested the unprocessed samples. Notably, the level of MUC2 in our BAL samples was very low and close to the detection limit of our assay, despite of the loading of a three-fold larger volume of BAL sample for its detection here, compared with the analyses of other mucins. Using this approach, the integrated density of MUC2 that we obtained ( Figure 6A) was nine-and seven-fold lower than that of MUC5AC and MUC5B, respectively. Moreover, the level of MUC2 in the collected BAL samples was close to the detection limit in the three study groups, making quantification uncertain in processed as well as unprocessed BAL samples. Therefore, we here present the data on MUC2 as integrated density only ( Figure 6A) and we did not detect any clear differences between the study groups for this mucin. Nor did we detect any corresponding difference in the proportion of large MUC2 complexes ( Figure 6B).

Group-related differences for level and proportion of MUC1 in large complexes
In Material 2, the level of MUC1 in BAL samples from the LTS-group was 20% higher than that of HNS ( Figure 7A). However, MUC1 is a relatively small mucin and does not form the same type of large oligomeric structures as the other gel forming mucins analyzed here [12]. To verify this, we compared the levels of MUC1 in unprocessed ( Figure  7A) and processed ( Figure 7B) BAL samples from Material 2, and indeed, less than 3% of MUC1 in the HNS group, and less than 8% in the LTS+ and LTS− groups, was lost due to sample processing ( Figure 7C). Since a relatively low proportion of MUC1 was lost in the mesh spacer and centrifugation steps here, we analyzed MUC1 in Material 1 as well. We found an increased level of MUC1 being present in the BAL samples from all LTS in this larger material as well ( Figure 7D).
It seems likely that the small loss of MUC1 due to sample processing was due to a portion of MUC1 interacting with the other and larger mucin complexes, as in Material 2, the quantity of MUC1 in large complexes tended to correlate with the quantity of MUC5AC large complexes ( Figure 8A) in a positive manner and clearly correlated in this manner with MUC5B in large complexes ( Figure 8B). The level of MUC1 did not correlate (r = −0.13; P=0.60; n=18) with the level of net gelatinase activity for all study subjects in Material 2, in spite of previous studies indicating that certain gelatinases, such as MMP-14, are involved in the shedding of the MUC1 in response to bacteria [12]. However, the level of MUC1 did correlate in a positive manner with the total leukocyte count per subject in BAL samples from the pooled group of all LTS from Material 2 ( Figure 8C). Together, this suggests that some of the MUC1 detected may originate from leukocytes, especially in the case of unprocessed BAL samples.

Association between MUC1 and DLCO
We detected a moderate and negative correlation between the level of MUC1 and DLCO among all study subjects in Material 1 only (Supplementary Figure S1). We were unable to detect such a correlation in the substantially smaller dataset from Material 2 (data not shown).

Discussion
The results from several previous studies on mammalian airways are suggestive of a pathological up-regulation of MUC5AC in a variety of airway disorders, including COPD, but the role of exposure to tobacco smoke per se has not been thoroughly addressed in these studies [17,[25][26][27]. Given this, we think that the most important aspect of the current study is the novel evidence that historic and current exposure to tobacco smoke constitute two potentially important regulatory factors, explaining both qualitative and/or quantitative alterations of mucins in the peripheral airway lumen of humans. As judged from the current data, the exposure to tobacco smoke emerges as more important than COPD per se and we think that this principal finding was facilitated by three key aspects of our study design. First, we investigated LTS+ and LTS− with a very similar tobacco load and HNS without any substantial tobacco load. Second, we compared mucins in unprocessed with those in processed BAL samples. Third, we explored BAL samples originating from the peripheral airways and not induced sputum sample originating from the more proximal airways. Importantly, this allowed us to characterize alterations of mucins among smokers in the most critical compartment in terms of typical pathology.
Despite limitations in statistical power due to a modest sample size for our materials, we detected quantitative differences in terms of enhanced levels in all LTS for MUC5AC and MUC1 in comparison with HNS. In contrast, we failed to do this for MUC5B and MUC2. Moreover, we detected an enhanced proportion of large MUC5AC complexes among all LTS. When we addressed associations with potential regulatory factors, we detected positive correlations between blood leukocytes and MUC5AC as well as between BAL leukocytes and MUC1 in BAL samples from all LTS. This is compatible with a mechanistic link to systemic inflammation as well as with a causative relationship, since neutrophils can produce neutrophil elastase and other proteinases that may act as secretagogues [19][20][21]. Along these lines, we detected positive correlations between two different types of net proteinase activity and MUC5AC in BAL samples from all LTS. Taken together, these observations are in line with the previously suggested association of MUC5AC with acute lung injury, neutrophil trafficking and inflammation [28].
From previous studies, it is known that the mucins MUC5AC and MUC5B constitute the major secretory mucins in the airways. It is also known that both these mucins form large oligomeric complexes and although the complexes are normally large, they can become even larger in chronic inflammatory airway disease, such as asthma [29]. We therefore quantified the mucins in processed (filtration and centrifugation) and unprocessed (no filtration and no centrifugation) BAL samples for each of the three study groups in the one material where this was possible (Material 2). From a methodological point-of-view, our results clearly suggest that the combination of centrifugation and filtration with a large mesh does affect the content of mucins in a detrimental manner. To us, the most obvious explanation for this is that the large mucin complexes are blocked by the filter membrane and/or lost due to centrifugation; a mild centrifugation step that was originally designed to precipitate large objects such as cells. Notably, in some samples, the processing of BAL samples removed as much as 95% of the mucins, by average 77% of MUC5AC, 47% of MUC5B and 5 % of MUC1. These observations highlight the importance of awareness of the impact of this type of processing of BAL samples in future studies of the airways. Given the outcome of our comparison between processed and unprocessed samples, we suspect that the 'methodological confounders' filtering and centrifugation may account for the discrepancy between some of our current findings and previous findings [18]. Furthermore, our methodological approach allowed the identification of the proportion of mucins present in large complexes, a factor of potential mechanistic importance for the clearance of mucus. In addition, the qualitative similarities in group-related differences for the level of MUC5AC in Material 1 and Material 2 do support that the results obtained in Material 2 are representative, despite of the limited sample size of this material.
Interestingly, the level of MUC5B tended to follow a similar trend to that of MUC5AC, albeit the group-related differences were not statistically significant in our study, presumably due to limitations in statistical power. In a previous publication, it was reported that the total amount of mucin and goblet cells of the bronchiolar epithelium in peripheral airways were similar in HNSs, smokers and patients with COPD [15]. However, the investigators concluded that the presence of intraluminal mucin was more frequent among the patients with COPD and that the predominant intraluminal mucin was MUC5B, which was also more frequent among the patients with COPD. Given that there was more historic smoking (i.e., higher tobacco load) in the group of patients with COPD than in the group of smokers (without COPD), it seems feasible that tobacco load per se was a confounder that contributed to the results in the referred study. Moreover, tethering of MUC5AC to epithelial mucous cells can lead to the accumulation of luminal mucins in the airways of patients with the chronic inflammatory airway disorder asthma and this impairs mucociliary clearance [30]. Collectively, these results suggest that it may not solely be the quantity of mucin produced but also the quality and clearance of mucus from the lumen that constitute keys to an improved understanding pathology in chronic inflammatory disorders of the lungs.
When addressing associations with potential regulatory factors in our current study, we detected a positive correlation between the level of MUC5B large complexes in BAL samples and the level of serum cotinine in samples from all LTS. Given that cotinine is a relatively short-lived metabolite of nicotine, this forward current smoking as a potential regulatory factor; in line with clinical data suggesting that current smoking drives chronic bronchitis [31]. Given our observation, it is interesting to note that nicotine bears the mechanistic potential to hinder post-exocytotic swelling and hydration, leading to higher viscosity and enhanced self-aggregation of the mucin polymer [32].
Currently, a common perception is that the main role of both MUC5AC and MUC5B is to facilitate mucociliary clearance and that MUC5B is required to maintain immune homeostasis and antibacterial host defense [33]. For example, the trachea is cleaned by MUC5B mucin bundles coated with MUC5AC and mice lacking Muc5b have inflammatory infiltrates and bacteria, such as Stapylococcus aureus in the lower airways [14,34]. The results from our current study support the role of antimicrobial host defense given the positive correlation between the level of MUC5B and the quantity of any bacteria present in lower airway samples from the pooled group of all LTS displaying growth of bacteria. This is in line with previous observations, showing that microbial products and inflammatory cytokines can cause a massive discharge of mucins [35]. In addition, published datasets indicate that microbes affect goblet cell dynamics and the mucus layer directly via local release of bioactive factors, in addition to an indirect effect via the activation of leukocytes [19,35,36]. In this context, it is interesting that a previous publication on BAL fluid from COPD patients, with and without colonization by bacterial pathogens, indicated no difference in the levels of MUC5AC and MUC5B [24]. However, in that study, current and former smokers were mixed, so current smoking was not specifically addressed, and there was no matching for anti-inflammatory treatment with steroids either. Moreover, similar reservations can be forwarded against the claims that there is a high level of MUC2 in the sputum and BAL samples of COPD patients, with a lower amount of MUC2 in the colonized group. In addition, the referred results regarding MUC2 should be considered with caution due to concerns with potential cross-reactivity of antibodies in the quantification assay with other mucins [23]. In fact, most recent studies suggest that there is very little or no MUC2 in human airways [10,26]. The latter is in line with our current finding of a very low level of MUC2 in the BAL samples, with no clear difference between study groups or in relation to the presence of local bacteria.
It is widely accepted that cell-surface mucins play an important role in mucosal host defense since they constitute both a barrier and a messenger. Notably, these intriguing filamentous molecules extend further than most other cell surface structures [35]. Moreover, several membrane-bound mucins are produced in mammalian airways, including MUC1, a mucin that has been shown to act as a releasable decoy that limits the pathogen's access to epithelial cells in the stomach, as indicated in human cells in vitro and knockout mice in vivo [12]. In addition, it has been reported that there is decreased mRNA for MUC1 in lung tissue, bronchial epithelial cells and neutrophil samples from smokers and clinically stable patients with COPD compared with HNSs [37]. In contrast, in other studies, enhanced MUC1 levels have been detected in airway epithelial cells and sputum samples from patients with clinically stable COPD compared with the corresponding samples from HNSs [38]. This has also been the case for sputum samples from patients with exacerbations of COPD [39]. These differences may be explained by the high degree of post-translational regulation of mucins since they mainly comprise carbohydrate, and as a consequence, the levels of mucin mRNA levels do not reflect mucin glycoprotein levels [36,40]. In our current study, we detected an increased level of MUC1 in the LTS-group and a trend toward a similar increase in the LTS+ group, compared with the HNS group, in the samples from Material 2. Of note here, compared with MUC5AC and MUC5B, MUC1 is a smaller, non-oligomerizing mucin [12,35]. Given this, the quantification of MUC1 is much less likely to be affected by filtering of BAL samples, which presumably made it possible to detect increased MUC1 levels in both LTS groups in the filtered samples from Material 1.
Several studies provide evidence of the anti-inflammatory role of MUC1 in the airways [41,42]. Thus, murine Muc1 can attenuate epithelial inflammation and injury in response to Pseudomonas aeruginosa [13]. Moreover, there is both clinical and experimental evidence that the level of MUC1 correlates with the severity of airway inflammation [39] as well as with the levels of TNF-α [43] and neutrophil elastase [44]. In our study, the level of MUC1 correlated in a positive manner with total leukocyte count per subject in BAL samples. This is a finding compatible with luminal MUC1 originating from local leukocytes responding to inflammatory stimuli or inflammatory mediators from leukocytes increasing the epithelial shedding of MUC1 [12].
Given that we performed our current pilot study on two clinical materials of modest sample size, it could be argued that variations in these limited materials due to mere chance would prevent us from making compatible findings. However, even if there was some variation in the quantitative outcomes of Material 1 and Material 2, such as the BAL leukocyte counts, these outcomes displayed quite similar patterns for group-related differences from a qualitative point-of-view, including the trends toward group-related differences in BAL neutrophil and eosinophil counts. Clearly, these variations were within what could be expected given the previous literature [45][46][47].
In conclusion, the present study forwards evidence that both historic and current exposure to tobacco smoke constitute regulatory factors determining the quality and/or quantity of mucins in the peripheral airway lumen of humans. In addition, the study forwards evidence that bacteria, leukocytes and proteinases may also constitute regulatory factors. Thus, these factors may represent targets for future therapy of pathological alterations in airway mucins, possibly regardless of COPD. The present study also indicates that a correct understanding of the pathological implications for mucins in long-term tobacco smokers requires the analysis of mucins in unprocessed BAL samples, allowing large gel forming mucins to be taken into account. We think that our novel findings motivate verification in larger study materials and a further elucidation of the underlying pathogenic mechanisms in translational models.

Clinical perspectives
• Mucins have been attributed a pathogenic role in COPD but there is an incomplete understanding of how mucins relate to potential regulatory factors in current tobacco smokers with and without COPD but yet a similar exposure history.
• The current results show that not only the quantity of specific mucins but also the proportion of large mucin complexes in the peripheral airways relate to tobacco smoking per se and is suggestive that local bacteria and proteinases, as well as systemic leukocytes, exert an impact too. Moreover, critical information on these mucins can be obtained by comparing processed and unprocessed samples.
• The most important clinical implication is that smoking per se, rather than COPD alone, accounts for important alterations in mucins located in the peripheral airway lumen.