Introduction: Cystic Fibrosis
Cystic fibrosis (CF) is a multi organ autosomal recessive genetic disorder that arises from mutations in the cystic fibrosis transmembrane regulator (CFTR) [42]. Though different areas of the body exhibit different symptoms due to this deficiency, most of the morbidity and mortality of CF is associated with respiratory complications and this is where my work will focus [43]. CFTR functions both in secretion of
chloride but also in the regulation of epithelial sodium channel (ENaC), which is responsible for sodium absorption [44]. Thus in CF there is a decrease in chloride secretion combined with an increase in sodium absorption ultimately resulting in a dehydrated mucus layer [45]. As discussed in the introduction, the mucus layer consists of a periciliary layer and a mobile mucus layer [2, 4]. The uppermost mobile mucus layer is the first layer to lose water as it has the lower osmotic pressure. As this layer becomes more dehydrated the gel forming mucins come into contact with membrane bound mucins present in the PCL and exhibit adhesion [46]. Secondly there is compression of the cilia within the PCL preventing regular movement of the overlying mucus layer by ciliary beating all of which lead to the development of a non mobile stagnant mucus layer or plaque [2]. Oxygen gradients and areas of hypoxia can develop within these mucus plaques creating a prime location for bacteria colonization, which will be discussed further below [47]. A natural response to this
bacterial infection is the host immune response, which includes heavy neutrophilic infiltration, dysregulated cytokine signaling, and also mucus hypersecretion, thus feeding into the ongoing cycle of dehydration, infection, inflammation, and mucus hypersecretion and eventually progressing to tissue destruction and fibrosis [1, 45, 48].
Numerous bacterial product and cytokine-mediated pathways leading to the upregulation of the gel forming mucins in cystic fibrosis have been described [49, 50]. In regards to MUC5AC, NF-κB has been implicated with several different upstream signaling partners including TNF-α, and MAP kinase (ERK), which can also signal through the cyclic AMP–responsive element binding protein (CREB) to increase MUC5AC transcription [51-54]. This pathway is responsive to neutrophil elastase, bacterial products, and inflammatory cytokines such as IL-1β. The epithelial growth factor receptor (EGFR) pathway has been shown to increase MUC5AC expression through downstream activation of the Sp1 and Fra-2 transcription factors and can be activated by NE and reactive oxygen species in addition to matrix metalloproteases all of which are present in the CF airways [55- 57]. MUC5B involves similar pathways as MUC5AC, though has been studied less extensively. MUC5B expression has been shown to be upregulated in response to inflammatory cytokines such as IL-6 and IL17 and signals through the MAPK pathway [58]. Nucleotide triphosphate signaling through P2Y2 receptor and ERK pathway has also been shown to increases transcription and secretion of MUC5B [59].
The development of mucostasis and bacterial infection presents in early childhood and persist into adulthood and until recently most of the focus was upon classical pathogenic strains such as Pseudomonas aeruginosa and Staphylococcus aureus [60, 61]. Application of 16S sequencing technologies to the lung microbiome has since revealed increased diversity including genera typically found in the oral cavity, which likely colonized the lung during infancy following aspiration, along with the pathogenic genera [62-64]. Also it has been shown that there is a progressive change in the CF microbiome that correlates with the age and disease severity [65, 66]. In early childhood (2 years) it was shown that mainly microbes belonging to the oral flora community colonized the lung and that several years later this shifted to a community dominated by classic CF pathogens which was accompanied by more severe inflammation and tissue damage [65].
There have been conflicting positions regarding the role and nature of mucins in the CF airways. Though numerous studies have shown at the transcript level that
MUC5B and MUC5AC are increased, initial reports suggested that these mucins were degraded by serine proteases within the airway and thus were not important in determining the mucus properties [67, 68]. This was refuted by Henderson et al which showed a several fold increase in total mucin in CF sputum using biophysical methods rather than antibody based [40]. The large size and significant glycosylation of mucins complicates their measurement and quantitation necessitating the need for less traditional techniques such as agarose gel electrophoresis western blot, multi-angle laser light scattering coupled with size exclusion chromatography to accurately measure concentration, and isopycnic density gradient ultra-
centrifugation to isolate and purify these macromolecules based on their buoyant density [69, 70]. At the other end of the spectrum, there are others that have reported that the mucins within the CF airways are cross-linked and this is responsible for the rheological changes of CF mucus [71]. Thus the question remains, in the highly proteolytic environment how is the macromolecular structure of the gel forming mucins altered and with what other proteins do they interact to create a pathologic CF mucus.
There are also many different hypotheses regarding the interaction and relationship between the microbiome and mucins. Some have suggested that the oral anaerobes are required to degrade mucins and provide metabolites for aerobes to use whereas others view mucin as a scaffold to separate different bacteria from each other and can alter their pathogenicity [72-74]. By combining comprehensive mucin characterization with microbiome data we hope to elucidate this relationship and also focus on specific bacterial genera and their effect on mucin.
Methods Cell Culture
Primary human bronchial epithelial (HBE) cells passage 1 or 2 derived from non-cystic fibrosis and cystic fibrosis donors were grown at air liquid interface (ALI) and maintained according to previously established protocol [75]. Basal media was changed on alternating days and the apical surface washed with 37°C PBS 2-3 times each week. Challenges were performed on cultures once fully differentiated (21-28 days after confluence), which was verified by the presence of ciliation and mucus production. For the following experiments, treatments and controls were performed on cells derived from the same donor lung allowing for a paired statistical analysis.
CF cell culture models
To emulate the CF lung environment, two different reagents were used: culture filtrate from Pseudomonas aeruginosa (Ps.a.) or supernatant of
mucopurulent material (SMM), which were applied to the apical surface daily for 5 days. Ps.a. was prepared as previously described, 0.22μm filtered and diluted 1:5 in MEM [76]. SMM was isolated and prepared according to Ribiero et al and the resulting material pooled from 5 CF donors in order to generate sufficient volume for the challenges and reduce interpersonal variability [77]. For the large 30mm
collagen coated inserts, 100 μL of Ps.a. or SMM was added to the apical surface and for the smaller 12mm cultures, 40 μL was added. As controls, tripticase soy broth (TSB), the culture media used to grow the Pseudomonas cultures, or PBS
were used for the Ps.a. or SMM challenges, respectively. The apical surface was washed with warmed PBS prior to the start of and each day during the challenge. The large inserts were washed with 1mL of PBS and the small cultures with 350uL. These washings were stored at 4°C after addition of 8M GuHCl to achieve a final concentration of 4M in order to prevent further degradation. Basal media was collected at baseline, before start of treatment, and each day thereafter for cytokine analysis.
Mucin Isolation and Static Light Scattering
Isopycnic density gradient centrifugation was performed to isolate the gel forming mucins using 4mL of pooled apical secretions in 4M GuHCl at a starting density of 1.45g/ml CsCl in 4M GuHCl spun at 50,000 rpm in a fixed angle rotor (70.1 TI) for 60-70 hours at 14°C [78]. A slot blot with vacuum filtration was performed on the resulting twelve 750μL fractions, which were then probed with polyclonal MUC5B antibody and monoclonal MUC16 (CA125) antibody to identify the peak with the highest concentration of MUC5B and minimal contamination by membrane bound mucins. These fractions were pooled and subjected to CL2B size exclusion chromatography (2 x 5mL) coupled with matrix assisted laser light
scattering (Dawn Heleos II, Wyatt Technology) and refractometry (Optilab T-rEx, Wyatt Technology) (SEC-MALS/dRi) at a flow rate of 0.5mL/min to determine concentration and macromolecular properties’ of the gel forming mucins, as
Isolation and Analysis of Stored Gel Forming Mucins
After completion of the 5-day challenge, one large 30mm insert from
treatment and control for each donor was used for post nuclear supernatant isolation (PNS). After sequential gentle washings with prewarmed PBS minimizing
mechanical stimulation, freshly prepared homogenization buffer (20mM HEPES, 130mM glutamic acid, 0.1mM CaCl, 3mM EGTA, 10nM N-Ethylmaleimide, Turbo DNase reaction buffer/DNAse (according to manufacturer’s instructions, ambion), and cOmplete mini protease inhibitor tablets (according to manufacturer’s
instructions, Roche), pH 7.2) was added to the apical surface of the cell cultures. Cells were removed/scraped from the insert and homogenized with 50 strokes of the dounce homogenizer while on ice. An equal volume of 8M GuHCl was added to the resulting homogenate and then centrifuged at 200g for 10 minutes at 4°C to pellet any remaining cell debris. The supernatant was removed and subjected to isopycnic density gradient centrifugation at a starting density of 1.45g/ml CsCl in 4M GuHCl for 60-70 hours at 50,000 rpm with a fixed angle rotor (70.1 TI) [78]. Eighteen 500μL fractions were taken per gradient and analyzed for MUC5B and MUC16 reactively following slot blot with vacuum transfer. The MUC5B rich fractions were further analyzed by SEC-MALs/dRi as described above.
Whole Mount Immunohistochemistry (IHC)
After completion of challenge, the apical surface was washed gently and thoroughly with 37°C PBS prior to fixation with Carnoy’s Solution (60% Ethanol, 30% Chloroform, and 10% glacial Acetic Acid) applied to both basal and apical
with 0.2% Triton X in Tris Buffered Saline (TBS) for 30 minutes at room temperature and then the cultures were blocked overnight at 4°C with a solution of 1% BSA, 1% Fish Gelatin, 0.1% Triton X and 5% normal donkey serum in 1 X TBS. Primary antibodies against MUC5B (1:500, polyclonal), MUC5AC (4ug/mL, 45M1), and x- tubulin (3ug/mL) were prepared in blocking buffer and applied to apical and basal surfaces overnight at 4°C. After washing cultures with blocking buffer diluted 1:10, secondary antibodies diluted (1:1000) were added to both culture surfaces and incubated overnight at 4°C protected from light. The following day, the cultures were washed and counterstained with Hoechst, according to manufacturer’s instructions. The membrane was excised from the plastic insert and mounted on a slide with the apical surface facing upward. This was allowed to dry overnight before sealing and imaging.
Agarose Gel Electrophoresis
Daily apical secretions dialyzed into 6M urea both reduced (10mM DTT x 10 minutes at 95°C) and unreduced were loaded into a 0.7% (w/v) agarose gel using 6X bromophenol loading dye and subjected to electrophoresis until dye front was within ½ inch of lane end, using 1X Tris-acetate-EDTA (1xTAE) buffer with 1% SDS. To facilitate detection of the unreduced samples, the gel was incubated at room temp for 10 minutes in a solution of 10 mM DTT prior to transfer [40, 79]. This was followed by a 60-minute vacuum transfer (50mbar) onto a nitrocellulose membrane while submerged in 4× saline-sodium citrate (4xSSC) buffer. The membrane was blocked with 1% milk and probed with monoclonal and polyclonal antibodies against MUC5AC and MUC5B [40, 80-82]. Secondary IR-dye conjugated antibodies against
rabbit and mouse primary antibodies were applied and the resulting signal measured using Licor Odyssey Scanner and quantified via densitometric analysis using the software provided by manufacturer (Version 3.0.30). For each challenge and code, the intensity values were normalized to the highest value prior to statistical analysis to measure the change in mucin secretion and account for donor-to-donor variability.
Mass Spectrometry
Equal volumes (450μL) of daily apical secretion for treatment and control in 4M GuHCl were prepared for Liquid Chromatography Tandem Mass Spectrometry using a modified filter aided sample preparation (FASP) method [83]. Specifically, each sample solubilized in 4M GuHCL was reduced with DTT using a final
concentration of 20 mM for 1 hour and 65°C and then alkylated with 50 mM iodoacetamide for 1 hour at 25 °C protected from the light. The samples were centrifuged at 14,000g for 10 minutes and the 10kDA filter washed twice with 4M GuHCl and then an additional three times using 50mM ammonium hydrogen carbonate (NH4HCO3). The filter was placed in a new collection tube and 0.5 ug modified proteomic grade trypsin (Sigma) added and the samples were incubated in a humidified chamber for 18 hours at 37°C. The peptides were centrifuged and eluted from filter and then concentrated using vacuum centrifugation (Heto-vac). The peptides were then dissolved in 30 uL of 0.1% formic acid analyzed by liquid
chromatography-tandem mass spectrometry (data dependent analysis) using a Dionex ultimate 3000 RSLCnano system 6µl of samples were loaded in a trap column Acclaim PepMap 100, 100 µm x 2 cm, nanoViper C18 5 µm 100 Å, at 5 µL/min with aqueous solution containing 0.1 % (v/v) trifluoroacetic acid and 2 %
acetonitrile, while the column used for peptides separation is a Acclaim PepMap RSLC, 75 µm x 15 cm, nanoViper C18 2 µm 100 Å) coupled to a hybrid quadrupole orbitrap mass spectrometer with a Nano spray source (Q-Exactive, Thermo Fisher, Bremen, Germany).
Proteins were identified by searching against most current human database (Proteome Discoverer 1.4) and were quantified based on total precursor intensity using Scaffold, Version 4 (Proteome Software Inc). A paired students T-test was used to compare the treatment and controls from each donor.
For MUC5AC and MUC5B absolute quantification, an internal standard was prepared by spiking 3 heavy labeled internal peptide standards from each protein achieving a final concentration of 100 fmol /µl. All raw files obtained from tSIM-DIA analyses of sputum digest samples were processed by Skyline (version v1.4). For each peptide the ratio between the corresponding endogenous and internal standard peak areas of each precursor (MS) and top 3 most intensity product ions (MS/MS) was calculated. Ratios from the three peptides were averaged and MUC5B and MUC5AC concentrations were calculated with the following equation:
Protein concentration = [L/H × C × a/b * c/d]
Where L/H is the average area ratio between light and heavy peptides, C is the concentration of injected internal standard, a is the volume used to resuspend the peptides, b is the samples starting volume, c/d is the dilution factor for mixing sample and internal standard (10/8) [84].
Proteomic Semi-tryptic peptide analysis
The label free proteomic spectrum files were uploaded and searched against the human database using Scaffold (Version 4) allowing for Semi-tryptic and fully tryptic cleavage sites. The unique MUC5B and MUC5AC Semi-tryptic peptides from the apical secretions following 120 hour challenge with Ps.a or TSB (control) were identified and aligned to the full mucin protein backbone to generate a percent coverage and localize the Semi-tryptic peptides to specific UniProt annotated
regions of MUC5B (Q9HC84) or MUC5AC (P98088). The frequency of each type of non-tryptic cleavage site was also calculated for MUC5AC and MUC5B for each sample.
Rate Zonal Centrifugation
Rate zonal centrifugation was performed using 200μL of apical secretions in 4M GuHCl layered on top of a 12 mL 6-8M GuHCl gradient spun at 40,000 rpm in swing out bucket rotor (SW40 Ti) for 2.5 hours at 14°C [85]. The 200μL of secretion was obtained through pooling 50μL per day of apical secretions challenge for each individual donor. The twelve 1 mL fractions were analyzed by slot blot with vacuum filtration and probed with antibodies against MUC5B. The MUC5B intensity was quantified using the Licor Odyssey scanner and software (version 3.0.30). The intensities of each individual gradient’s fractions were normalized to the highest value and plotted according to fraction number with 1 being the uppermost fraction and 12 the bottom.
Exosome Isolation
Exosomes were isolated from equal volumes of apical secretions by
differential centrifugation. Briefly the raw secretions were spun down at 3000g x 20 minutes at 4°C using a swing out bucket rotor (SW40 Ti) after which the supernatant was kept and centrifuged at 10,000 rpm x 110 minutes at 14°C. The supernatant was removed and centrifuged for a third time at 19,000 rpm x 1.5 hours at 14°C. After this step, the supernatant was discarded and the remaining pellet was washed with 10 mL of PBS prior to the final centrifugation at 25,000 rpm x 60 minutes at 14°C. The supernatant was removed and the remaining pellet was resuspended in 100mL of PBS. The freshly isolated exosomes were diluted 1:500 - 1:1000 in 0.22uM filtered PBS and analyzed by nanoparticle tracking analysis as described previously [33]. Based on the resulting particle concentration measurements,
volumes from each sample containing an equal number of particles were submitted for miRNA sequencing using the HTG EdgeSeq platform, the details for which have been described previously [39].
MUC5B and MUC5AC Standard In-Vitro Experimental Design
MUC5B and MUC5AC standards derived from healthy donor salivary secretions and apical washings from an A549 MUC5B knockdown cell line, a gift from Dr. Babu Subramani (UNC) respectively; that were subjected to two-
dimensional isopycnic centrifugation using a starting density of 1.35g/ml were
dialyzed into PBS. Ps.a. or TSB were diluted 1:5 in MEM and 0.22um filtered before adding to standards in a 1:4 ratio of Ps.a/TSB to standard. Standards were
120 minutes, and 24 hours after addition of the Ps. a. or TSB. For each of these timepoints, the first aliquot was added directly to premeasured urea to achieve a final concentration of 6M in preparation for agarose gel electrophoresis western blot using both monoclonal and polyclonal antibodies against the gel forming mucins. The second aliquot was added to an equal volume of 8M GuHCl in order to repurifiy the gel forming mucins by isopycnic density-gradient centrifugation under
dissociative conditions (4M GuHCl) using a starting density of 1.45 g/mL CsCl for further analysis by SEC-MALS.
Results:
CF Cell Culture Models: Immunohistochemistry and MUC5B and MUC5AC concentration quantitation
Cell cultures models of CF lung disease revealed significant hypersecretion of the gel forming mucins. MUC5AC and to a lesser extent MUC5B remained adherent to the apical surface after exposure to CF airway stimuli (Ps.a. and SMM) despite extensive washing as measured by whole mount immunohistochemistry (Figure 1.1A). Interestingly this increase in MUC5AC and MUC5B was not evident when the apical secretions of the Ps.a. treated cultures were analyzed by immunoblotting with antibodies against MUC5AC and MUC5B after agarose gel electrophoresis (Figure 1.1B and C, right panels) though MUC5AC and MUC5B did increase after the SMM challenge (Figure 1.1C, left panels).
In contrast to this, the proteomic analysis using absolute quantitative
methods (Figure1.1D) showed a significant increase in mean (±SE) MUC5B in both Ps.a. (7.481 ± 0.59 vs. 3.450 ± 0.92 pmol/mL) and SMM (32.70 ± 6.69 vs. 0.17 ± 0.03 pmol/mL) treated cultures as compared to their respective controls, TSB and PBS. This same trend was evident with label free LC-MS/MS analysis (Figure 1.1E)