Effects of Highly Effective Modulator Therapy on the dynamics of the respiratory mucosal environment and inflammatory response in Cystic Fibrosis

Abstract

Background:

While the widespread initiation of elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic clinical improvements among persons with cystic fibrosis (pwCF), little is known about how ETI affects the respiratory mucosal inflammatory and physiochemical environment, or how these changes relate to lung function.

Methods:

We performed a prospective, longitudinal study of adults with CF and chronic rhinosinusitis (CF-CRS) followed at our CF Center (n=18). Endoscopic upper respiratory tract (paranasal sinus) aspirates from multiple visit dates, both pre and post ETI initiation, were collected and tested for cytokines, metals, pH, and lactate levels. Generalized estimating equations were used to identify relationships between ETI and upper respiratory tract (URT) biomarker levels, and between URT biomarkers and lung function or clinical sinus parameters.

Results:

ETI was associated with decreased upper respiratory mucosal cytokines B-cell activating factor (BAFF), IL-12p40, IL-32, IL-8, IL-22 and soluble tumor necrosis factor-1 (STNFR1), and an increase in a proliferation inducing ligand (APRIL) and IL-19. ETI was also associated with decreased URT levels of copper, manganese & zinc. In turn, lower URT levels of BAFF, IL-8, lactate, and potassium were each associated with ~1.5% to 4.3% improved FEV₁, while higher levels of IFNγ, iron, and selenium were associated with ~2% to 10% higher FEV₁.

Conclusions:

Our observations suggest a dampening of inflammatory signals and restriction in microbial nutrients in the upper respiratory tract with ETI. These findings improve our understanding of how ETI impacts the mucosal environment in the respiratory tract, and may give insight into the improved infectious and inflammatory status and the resulting clinical improvements seen in pwCF.

Introduction

Highly effective modulator therapy with the triple combination CFTR modulator Elexacaftor-Tezacaftor-Ivacaftor (ETI) was approved in 2019 for persons with CF (pwCF) who are ≥12 years of age with ≥1 copy of the F508del variant.(1) Since then, numerous studies have demonstrated significant clinical improvements shortly after initiating treatment with ETI, including enhanced lung function, reduced sweat chloride levels, decreased sputum production, improved infection burden and improved sinus disease.(2,3) While ETI is revolutionizing the care of pwCF, little is known about its effects on the respiratory mucosa and whether alterations in the host inflammatory response or physiochemical environment may underlie observed clinical improvements.

The host inflammatory response in pwCF is complex and multifactorial, characterized by increased levels of pro-inflammatory cytokines and impaired counter-regulatory defenses.(4) This dysregulated inflammatory response likely occurs early in pwCF, possibly even in utero.(5) Consequently, a detrimental cycle of chronic inflammation predisposing the airways to persistent infection and exacerbating inflammation can ensue.(4) Additionally, the physiochemical environment in CF is significantly altered. For instance, disruption in trace metal regulation may compromise the host’s ability to control microbial pathogenicity.(6) This concept, known as nutritional immunity, refers to the host’s ability to sequester bioavailable trace metals as a defense mechanism against invading microorganisms. In chronic lung disease, the regulation of trace metals becomes disrupted, altering nutrient availability for both commensal and invading microorganisms and potentially changing immune cell function.(6) Furthermore, lactate levels in the CF airways can be elevated, likely due to hypoxic conditions, bacterial infection and neutrophilic influx. This also promotes airway acidification, which impairs host defense mechanisms and increases airway bacterial burden.(7) How these inflammatory and physiochemical parameters are altered with ETI therapy is not well understood.

Further insight is needed into upper airway inflammation in pwCF, the majority of whom have chronic rhinosinusitis (CF-CRS).(8) The existing body of CF research has largely focused on systemic and lower airway inflammation despite significant morbidity related to sinus disease in CF.(9) However, the unified airway hypothesis, a conceptual framework which considers the entire respiratory tract from the nasal cavity to the distal lung as one functional unit, is becoming increasingly relevant in the CF literature.(10) For instance, the paranasal sinuses in CF have been identified as a reservoir for bacterial transmission to the lower airways,(11) and having a CF sinus exacerbation can increase the odds of a subsequent CF pulmonary exacerbation, further supporting the interconnectedness of these disease manifestations.(12)

The objectives of this study were therefore to investigate the longitudinal dynamics of the upper respiratory host inflammatory state in pwCF, assess the impact of ETI on the mucosal environment as a means of gaining insight into impacts on infection burden, and explore the relationships between upper respiratory mucosal biomarkers and lung function in CF.

Materials and Methods

Study Population:

We performed a prospective, longitudinal study of adults with CF-CRS followed in the CF Center at the University of Pittsburgh. The study population was recruited between November 2017 and June 2021 from a CF-focused otolaryngology clinic, following an IRB-approved protocol (CR19100149–006). After providing informed consent, 38 adults with symptomatic CF-CRS and prior functional endoscopic sinus surgery (FESS) were recruited to the original study. In addition to a diagnosis of CF, all participants met diagnostic criteria for chronic rhinosinusitis as per the Clinical Practice Guideline of the American Academy of Otolaryngology- Head and Neck Surgery. (13) Sinus aspirates were collected during quarterly clinic visits and unscheduled visits for acute sinus or pulmonary exacerbations. For the purposes of our study, we limited participants to those who had sinus aspirates collected prior to and post initiation of ETI (final n=18 participants).

Clinical Data:

Longitudinal clinical data with multiple assessment time points were collected at baseline and scheduled study visits, as well as unscheduled visits for exacerbations. Baseline demographics, including the presence of CF-related complications, were collected at enrollment. Additional clinical data including presence of sinus or pulmonary exacerbations, the use of modulator therapy, and patient reported quality of life measures (Sino-nasal Outcome Test i.e. SNOT-22 scores) were collected at initial and follow up visits. Additionally, endoscopic assessment of sinus disease via Modified Lund-Kennedy (MLK) scores was collected when available. Spirometry was obtained during clinic visits following American Thoracic Society (ATS) guidelines and percent-predicted values for spirometric indices were calculated using Morris-Polgar equations. Dates of ETI initiation were collected retrospectively through chart review. Clinical data was stored de-identified in a RedCAP database.

Sinus aspirate sampling:

Sinus aspirates were performed by endoscopically instilling 5mL of sterile saline into the right maxillary sinus and collecting samples endoscopically with a sterile trap. This method ensured no contamination of sinus aspirates with material in the nares. No additives were introduced. Samples were stored at −80°C within four hours of collection until time of analysis.

Biomarkers:

Cytokines:

Cytokine levels in 104 sinus wash samples (stored frozen at −80°C) were quantified using a Luminex MAGPIX system with a Bio-Plex Pro^™ Human Inflammation Panel 1, 37-Plex #171AL001M, following manufacturer’s guidelines.

Metallomics:

Metal levels in 99 sinus wash samples (stored frozen at −80°C) were quantified by Metabolon (Durham, North Carolina) using inductively coupled plasma-mass spectrometry (ICP-MS). The samples were diluted in deionized H₂O and introduced into the ICP-MS instrument via an Elemental Scientific (ESI) Prep-Fast autosampler including dilution with aqueous nitric acid. The Thermo Scientific™ iCAP^™ instrument was operated in positive ionization and used a kinetic energy discrimination (KED) cell to reduce polyatomic interferences. Quantitation was performed using a multi-point external calibration curve which preceded the samples into the instrument. This assay had not been previously conducted on sinus wash samples. Therefore, initial range-finder testing was completed using excess sinus wash samples from a previous study.

Lactate levels were measured on 104 sinus wash samples using Abcam colorimetric L-Lactate Assay Kit (ab65331), according to manufacturer’s guidelines. Once lactate levels were measured, remaining wash samples were allowed to reach room temperature and pH tested using a Mettler Toledo benchtop pH/ion meter (30019028).

Statistical Analysis:

All statistical analyses were completed using STATA XE, version 17 (StataCorp, College Station, TX). The raw data including the distribution of each biomarker was carefully assessed. Due to non-normal distribution, cytokine and lactate levels transformed to categorical variables (undetectable, below the median or above the median), for all statistical analyses. Metal concentrations were log transformed, which improved data distribution. Multivariable regression analyses using generalized estimating equations to explore relationships between ETI use (predictor variable) and biomarkers (dependent variable) adjusting for age, sex, pulmonary and sinus exacerbations, were completed. Subsequently, multivariable regression analyses using generalized estimating equations were completed to explore relationships between biomarker levels (predictor variable) and lung function (FEV1 as percent-of-predicted, dependent variable) or clinical upper respiratory parameters (SNOT-22, MLK scores, dependent variable), adjusting for age, sex, ETI use and presence of pulmonary exacerbation.

Results

Study Participant Characteristics:

The study included 53 visits from 18 participants (mean 2.9 visits per participant). Table 1 shows the clinical characteristics of our study cohort (n=18). The median age of the cohort was 33 years upon enrollment. Most participants were white (94%) and just under half were male (45%), with a mean BMI of 22.8 kg/m²; 61% of participants had CF-related diabetes (CFRD), 28% had a diagnosis of asthma or allergic rhinitis, and 28% had been on prior CFTR modulator therapy. The median baseline FEV1 was 60.5% predicted (range 25–96% predicted) and the median change in FEV1 post-ETI was +5% predicted (range −9% to +16% predicted) after an average of 10.8 months of ETI therapy (range 0.75 to 17.5 months).

Table 1 –

Clinical characteristics of the study cohort

	Study cohort (n=18)
Median age at enrollment, yrs (range)	33.0 (21.9–48.0)
Race, white (%)	17/18 (94.4)
Sex, male (%)	8/18 (44.5)
Mean BMI (SD)	22.8 (+/− 4.3)
dF508 homozygous (%)	12/18 (66.7)
CFRD (%)	11/18 (61.1)
Asthma (%)	5/18 (27.8)
Allergic rhinitis (%)	5/18 (27.8)
Prior CFTR modulator use (%)	5/18 (27.8)
Lung function:
Median baseline ppFEV1(range)	60.5 (25–96)
Delta ppFEV1, range (median)	−9–16 (5)

BMI: body mass index, kg/m². CFRD: cystic fibrosis related diabetes. CFTR: cystic fibrosis transmembrane conductance regulator. dF508: delta F508 homozygous. ppFEV1: forced expiratory volume in 1 second, percent predicted. deltaFEV1: change in ppFEV1 pre and post ETI. SD: standard deviation.

Alteration in biomarkers after initiation of ETI

To examine respiratory mucosal changes with ETI therapy, we examined sinus aspirates for mucosal cytokine, metal or metabolite changes associated with initiation of ETI treatment. Table 2 shows the biomarkers whose levels significantly changed with ETI in our multivariable models. Treatment with ETI was associated with increased levels of a proliferation induced ligand (APRIL, also known as tumor necrosis superfamily member 13 or TNFSF13) and interleukin (IL)-19 and decreased levels of B-cell activating factor (BAFF), IL-12p40, IL-22, IL-32, IL-8 and soluble tumor necrosis factor receptor 1 (sTNFR1). The use of ETI was also associated with decreased levels of three metals: Copper, Manganese and Zinc (all p<0.05). The results of the multivariable analyses for all cytokines, metals, lactate, and pH before vs after ETI initiation are shown in Supplemental Table 1.

Table 2 –

Effects of ETI on biomarker levels

Biomarker	Beta Coefficient	95% Confidence Interval	P-value
APRILa	0.63	(0.27, 0.99)	0.00*
BAFFa	−0.23	(−0.44, −0.01)	0.04
IL-12_p40a	−0.34	(−0.61, −0.06)	0.02
IL-19a	0.42	(0.06, 0.79)	0.02
IL-22a	−0.35	(−0.63, −0.06)	0.02
IL-32a	−0.42	(−0.72, −0.12)	0.01
IL-8a	−0.28	(−0.49, −0.06)	0.01
sTNFR1a	−0.35	(−0.57, −0.13)	0.00*
Lactatea	−0.29	(−0.59, 0.00)	0.05
Copperb	−3.27	(−5.11, −1.44)	0.00**
Manganeseb	−0.68	(−1.17, −0.18)	0.01
Zincb	−0.67	(−1.19, −0.16)	0.01

GEE model adjusted for age, sex, presence of pulmonary and sinus exacerbations.

^aCategorical cytokine levels (undetectable, < median or ≥ median), pg/ml.

^b(Log) metal levels, μg/L.

^*<0.005.

^**<0.001

Biomarkers associated with changes in lung function

We next examined the interaction of upper respiratory biomarkers with changes in pulmonary function. Biomarkers which were significantly associated with FEV1 (p <0.05) are shown in Table 3. Among the cytokine biomarkers, BAFF and IL-8 were inversely associated with FEV1: higher levels of BAFF were associated with a 3.2% decline in FEV1 after ETI initiation, while individuals with detectable IL-8 levels had 4.3% lesser improvement in FEV1 than those with undetectable levels. In contrast, detectable levels of IFNγ were associated with a 10.5% increase in FEV1, as compared to those with undetectable levels. Sinus lactate was also inversely associated with FEV1, with higher levels of sinus lactate associated with 3.4% decline in FEV1 after ETI initiation.

Table 3 –

Biomarkers associated with FEV1

Biomarker	Beta Coefficient	95% Confidence Interval	P-value
BAFFa	−3.20	(−6.29, −0.10)	0.04
IFNγa	10.47	(1.69, 19.26)	0.02
IL-8a	−4.30	(−7.51, −1.08)	0.01
Lactatea	−3.38	(−5.51, −1.24)	0.00*
Ironb	2.01	(0.53, 3.48)	0.01
Seleniumb	1.84	(0.28, 3.40)	0.02
Potassiumb	−1.52	(−2.60, −0.44)	0.01

GEE model adjusted for age, sex and the presence of pulmonary exacerbation

^aCategorical cytokine/lactate levels (undetectable, < median or ≥ median), pg/ml

^b(Log) metal levels, μg/L

^*<0.005

Among the metal biomarkers, iron and selenium were positively associated with FEV1: each 1.0-log-increase in iron with ETI therapy was associated with a 2% improvement in FEV1, while each log-increase in selenium with ETI therapy was associated with a 1.8% improvement in FEV1. On the other hand, sinus potassium was inversely associated with FEV1: each 1.0-log increase in potassium on ETI was associated with a 1.5% decline in FEV1. Results of all other variables tested are listed in Supplemental Table 2.

Biomarkers associated with upper respiratory clinical outcomes

Amongst our study population, the use of ETI was associated with reduced (improved) SNOT22 scores (coefficient −11.0, p 0.012, C.I. −19.6 to −2.4) and MLK scores (coefficient −2.8, p 0.006, C.I −4.8 to −0.79). We were additionally interested exploring the interaction between upper respiratory biomarkers and changes in SNOT-22 and MLK scores. Among the cytokine biomarkers examined, we found that higher levels of osteopontin were associated with 8.9% decline in SNOT-22 scores after ETI initiation (p 0.013, C.I −15.8 to −1.8), when correcting for age, sex and presence of sinus exacerbation. Additionally, detectable levels of IFNγ were associated with 5% decline in MLK scores (p 0.049, C.I. −9.9, −0.02), and detectable levels of IL-34 were associated with a 2.9% decline in MLK score after initiation of ETI, when correcting for age, sex and presence of sinus exacerbation. No significant associations were found between metal biomarkers, lactate and SNOT-22 or MLK scores. Results of all variables tested are included in Supplemental Tables 3 and 4.

Discussion

There is robust evidence that ETI leads to profound improvements in pulmonary, upper respiratory and nutritional outcomes for pwCF(14). Despite these dramatic clinical improvements, little is known about the effects of ETI on the respiratory mucosa and whether alterations in the host inflammatory response or physiochemical environment may underlie observed clinical improvements. Therefore, the results of this study, which aimed to examine these mucosal changes using the upper respiratory tract as a survey site, begin to lay the groundwork for elucidating the underlying mechanisms behind these noted clinical improvements.

ETI decreases upper respiratory tract inflammation in CF

Our study showed that ETI is associated with a reduction in upper respiratory tract mucosal levels of multiple pro-inflammatory cytokines: BAFF, IL-12p40, IL-32, IL-8 and sTNFR1. IL-8 and IL-6 are two of the major components of the pro-inflammatory response in CF.(6) Previous research has shown that the detrimental effects of IL-6 are driven by the metalloprotease ADAM-17, and levels of sTNFR1 have served as a bioassay of ADAM-17 activity and IL-6-mediated harm.(15–16) Therefore, the reductions of IL-8 and sTNFR1 seen in our study likely reflect important reductions of inflammation in the CF URT with ETI. These findings are consistent with one recent study by Casey et al, which showed that ETI was associated with reduction in various systemic and airway cytokines, including sputum IL8 and serum levels of IL-6 and sTNFR1.(15) While IL-12p40 and IL-32 have not been extensively described in the CF literature, their effects are largely proinflammatory and have been associated with pulmonary fibrosis.(17,18) More exploration is required to determine their effects in CF; however, it remains possible that declines in these cytokines with ETI therapy also contribute to improvements in respiratory mucosal inflammation. Interestingly, our study demonstrated a significant decrease in BAFF in response to ETI. BAFF levels have been shown to be elevated in bronchoalveolar lavage fluid of children with CF.(19) Additionally, prior in vitro studies have demonstrated that BAFF promotes B cell survival in ectopic lymphoid tissue, thereby inhibiting nasal polyp apoptosis.(20) It is therefore possible that an ETI-mediated reduction in BAFF allows for improvements in nasal polyposis, a significant burden in CF sinus disease. We hypothesize that the reductions in proinflammatory biomarkers shown here play a mechanistic role in the post-ETI clinical improvements seen in prior studies.

Our study also showed that ETI was associated with an increase in APRIL, a member of the TNF superfamily implicated in tumor growth and autoimmune disease.(21) APRIL has not been previously reported in the CF literature and its effects on the sinomucosal environment remain unknown. ETI was also associated with an increase in IL-19 and a decrease in IL-22, both members of the IL-10 superfamily. While previously thought to have a largely anti-inflammatory role, increasing evidence has implicated IL-19 in the TH2 response pathway, and systemic levels of IL-19 have found to be increased in asthma, suggesting a TH2-mediated pro-inflammatory role.(22) Conversely, one recent study from our lab showed that increased IL-19 in the CF sinuses was associated with a decrease in the relative abundance of Haemophilus spp., suggesting a possible anti-inflammatory role.(23) Similarly, multiple studies have shown both pro and anti-inflammatory effects of IL-22 in the airways. (24, 25) While an anti-inflammatory effect has been favored in CF, a reduction in IL-22 may represent an upstream reduction in TH17-mediated inflammation, which has been associated with negative pulmonary outcomes in CF.(26) Additionally, it is possible that the post-ETI decrease in IL-22 shown in this study, may be secondary to overall reduction in sinus exacerbations and reduced need for counter-regulatory host defenses.

Our study also showed that ETI was associated with a statistically significant decrease in sinus lactate, which was used as a marker of hypoxia, consistent with previous work by Ester et al.(27) While there is minimal existing literature on sinus lactate levels, one study, which explored energy metabolism in chronic maxillary sinusitis, showed that individuals with chronic sinusitis had higher levels of sinomucosal lactate than those without chronic sinus disease, possibly a result of increased glycolysis or impaired mucosal diffusion.(28) We hypothesize that the reduction of lactate seen with ETI may be due to improved oxygenation and reduced epithelial damage secondary to chronic inflammation.

In sum, the inflammatory environment in CF is determined by an intricate balance of both pro and anti-inflammatory mediators, with dysregulation of the host inflammatory response related to underlying CFTR dysfunction and chronic infection(6). While the interplay between these cytokines in the airways is complex and often context dependent, our results highlight a role for ETI in improving this dysregulated inflammatory response and reducing upper respiratory inflammation in CF, consistent with clinically observed improvements in CF-CRS. These clinical improvements were also reflected in our study by the association between ETI use and improved patient reported symptoms via SNOT-22 scores and endoscopic parameters of sinus disease (MLK scores).

ETI use is associated with changes in respiratory mucosal metal levels

In our study, ETI use was associated with reductions in mucosal levels of zinc, manganese and copper. While previous research has shown an overall systemic zinc deficiency in pwCF, other studies have shown increased levels of zinc in the sputum of pwCF.(8) Furthermore, sputum zinc concentration has been positively correlated with IL-8 concentration in prior work.(29) While levels of metals in the upper airways of pwCF have yet to be explored, we hypothesize that post-ETI reduction in sinus zinc concentration leads to overall reduction in sinus inflammation, improving CF-CRS outcomes. However, there is some conflicting data as other studies have shown that Pseudomonas aeruginosa in the CF airways have transcriptomic profiles suggesting a likely zinc-starved state.(30) Furthermore, another study showed that zinc and manganese depleted portions of the P. aeruginosa biofilm repressed the production of anti-staphylococcal molecules. In said study, the presence of calprotectin, which chelates both zinc and manganese, promoted co-colonization of the murine lung, and additionally P. aeruginosa and S. aureus were found to co-exist within a calprotectin-enriched airspace of a human CF lung explant.(31) More research is needed to determine if the reductions in zinc & manganese associated with ETI alter microbial interactions and/or persistence of P. aeruginosa in the CF airways. Finally, although some studies have suggested reduced activity of copper enzymes in blood neutrophils, erythrocytes and monocytes in pwCF, indicating systemic deficiency, other data has indicated elevated sputum copper levels in pwCF.(8) The cause of altered metal homeostasis in CF is not fully understood, and this is the first study to report changes in respiratory mucosal metal levels associated with ETI use. As nutritional immunity becomes increasingly recognized as an important driver of the host inflammatory response and the airway microbiome in respiratory disease, including CF, further exploration of these relationships is warranted.

Upper respiratory mucosal biomarkers predict lung function in CF

We have thus far shown that ETI is associated with alterations in upper respiratory biomarkers. We were further interested in exploring whether these biomarkers were also associated with lung function, specifically the forced expiratory volume in 1 second (FEV1), which is the primary spirometric index used to evaluate CF lung disease progression.

Our results showed that of the respiratory biomarkers altered by ETI use, two pro-inflammatory cytokines: IL-8 and BAFF, were able to significantly predict lung function in pwCF. Sinus levels of IL-8, a major component of the pro-inflammatory response in CF, were negatively correlated with FEV1. Given that our study demonstrated reduced IL-8 levels in response to ETI, it is possible that ETI improves both sinus and pulmonary outcomes through this pathway.

We also demonstrated a significant reduction in sinus BAFF with ETI, and subsequently demonstrated a negative correlation between sinus BAFF and lung function. While sputum BAFF has been negatively correlated with lung function in asthma (32), the association between upper airway BAFF levels and lung function has not been explored. Lactate levels were negatively correlated with FEV1 in our study. Sinus lactate levels have not been previously correlated with lung function in CF. However, sputum lactate concentrations have been shown to be elevated in pwCF, likely related to underlying chronic inflammation, neutrophilic degradation and tissue hypoxia and further increased during pulmonary exacerbations.(33) In our study, ETI was associated with a near significant reduction in sinus lactate levels (p=0.05, supplemental Table 1), and we suspect that a reduction in chronic inflammation and neutrophilic degradation in the setting of ETI use may also contribute to improvements in lung function. In light of the unified airway hypothesis, these findings altogether provide further evidence that upper airways disease may influence lower airways outcomes. Whether this is related to generalized airway inflammation affecting the respiratory tract in unison, or initial colonization of the sinuses with CF pathogens with subsequent lower airways dissemination, or both, is yet to be determined.

Additional biomarkers which predicted lung function included potassium, which was associated with a decrease in FEV1, and IFNγ, iron and selenium which were associated with an increase in FEV1. Recent data has shown that increased potassium flux promotes P. aeruginosa biofilm formation on airway epithelial cells.(34) This may be a mechanism by which upper respiratory mucosal potassium is associated with reduced FEV1 in pwCF, as P. aeruginosa persistence is known to be a significant contributor to lung function decline. Multiple studies have reported persistence of P. aeruginosa and other pathogens in the airways following initiation of ETI,(35) despite overall reductions in bacterial burden, exacerbations and improvement in lung function. Furthermore, serum IFNγ levels in pwCF have been positively correlated with FEV1 in prior research, despite its pro-inflammatory effects.(36) Therefore, it is possible that the mucosal biomarker changes observed in the URT are signatures of improvements in microbial control by the respiratory mucosa with ETI treatment. It will be important to follow these biomarkers and respiratory microbial changes over time with ETI to ascertain if these improvements persist.

In summary, we have shown that ETI leads to alterations in the complex balance of pro and anti-inflammatory cytokines and metals, in the CF upper airways. Our data suggest that these alterations contribute to the clinical relationships observed between upper & lower airways disease in CF. The major limitations of this study include our sample size (n=18), which was significantly limited by the COVID-19 pandemic and subsequent reduction in follow up visits. The study may therefore be underpowered to identify all significant associations between ETI use, sinus biomarkers and lung function. Our results suggest the opportunity for a future, larger multicenter study for further exploration of these relationships. Additionally, future investigation of CF airway microbiome changes in response to these host parameters will be of great clinical importance.