SPLUNC1: a novel marker of cystic fibrosis exacerbations

Abstract

Background: Acute pulmonary exacerbations (AE) are episodes of clinical worsening in cystic fibrosis (CF), often precipitated by infection. Timely detection is critical to minimise morbidity and lung function declines associated with acute inflammation during AE. Based on our previous observations that airway protein short palate lung nasal epithelium clone 1 (SPLUNC1) is regulated by inflammatory signals, we investigated the use of SPLUNC1 fluctuations to diagnose and predict AE in CF.

Methods: We enrolled CF participants from two independent cohorts to measure AE markers of inflammation in sputum and recorded clinical outcomes for a 1-year follow-up period.

Results: SPLUNC1 levels were high in healthy controls (n=9, 10.7 μg·mL^-1), and significantly decreased in CF participants without AE (n=30, 5.7 μg·mL^-1; p=0.016). SPLUNC1 levels were 71.9% lower during AE (n=14, 1.6 μg·mL^-1; p=0.0034) regardless of age, sex, CF-causing mutation or microbiology findings. Cytokines interleukin-1β and tumour necrosis factor-α were also increased in AE, whereas lung function did not decrease consistently. Stable CF participants with lower SPLUNC1 levels were much more likely to have an AE at 60 days (hazard ratio (HR)±se 11.49±0.83; p=0.0033). Low-SPLUNC1 stable participants remained at higher AE risk even 1 year after sputum collection (HR±se 3.21±0.47; p=0.0125). SPLUNC1 was downregulated by inflammatory cytokines and proteases increased in sputum during AE.

Conclusion: In acute CF care, low SPLUNC1 levels could support a decision to increase airway clearance or to initiate pharmacological interventions. In asymptomatic, stable patients, low SPLUNC1 levels could inform changes in clinical management to improve long-term disease control and clinical outcomes in CF.

Figure 1.. SPLUNC1 is Decreased in the Sputum of Stable CF Patients.

A) SPLUNC1 levels (ELISA) in sputum samples from the Yale cohort of adult CF patients without respiratory symptoms (CF Stable) and healthy controls (HC). B) Total protein in sputum (BCA assay) from the same patients. C) Inflammatory cytokine levels (ELISA) in sputum from the same patients. Additional cytokines tested without significant difference: CXCL10, G-CSF, IFNλ, IL-6, IL-13, MCP1, MIP1α. CF Samples were obtained by voluntary expectoration during clinical assessment, HC samples obtained by sputum induction with nebulized normal saline solution. + = Mean; Bar inside box: Median; Whiskers: Minimum/Maximum. Mann-Whitney Test with Bonferroni correction; * = p<0.05; ** = p<0.01; **** = p<0.0001.

Figure 2.. SPLUNC1 is Decreased During Acute CF Exacerbations (AE).

Sputum SPLUNC1 and FEV₁ from two clinical cohorts including adult (Yale University, n=43) and mixed adult/pediatric (University of Minnesota, n=35) CF patients. Samples were obtained by voluntary expectoration during clinical assessment, A) SPLUNC1 quantified by ELISA, B) FEV₁ (Percent of Predicted, %) obtained by spirometry during clinical assessment; CF Stable: No symptoms of AE, no antibiotic treatment. AE: Acute CF exacerbation, symptoms of AE and ongoing antibiotic therapy; FEV₁: Forced Expiratory Volume in the first second; + = Mean; Bar inside box: Median; Whiskers: Minimum/Maximum. Mann-Whitney test; ** = p<0.005; *** = p<0.001; ns = not statistically significant.

Figure 3.. Individual-Specific SPLUNC1 and FEV₁ Decreases During AE.

A) Paired SPLUNC1 levels in sputum samples from the same individual with and without AE (ELISA); B) Paired FEV₁ measurements from the same individual with and without AE (Percent of Predicted, %) obtained by spirometry during clinical assessment; Samples from two clinical cohorts including adult (Yale University, n=8) and mixed adult/pediatric CF patients (University of Minnesota, n=11). Each vertical line and number represent a single patient that provided one Stable and one AE sample. CF Stable (Gray markers): No symptoms of AE, no antibiotic treatment. AE (Red markers): Acute CF Exacerbation, symptoms of AE and ongoing antibiotic therapy; When values were the same, these were represented by two overlapping diamonds along the patient’s line. Wilcoxon matched-pairs signed rank test; * = p<0.05, *** = p = 0.0001, ns=not statistically significant.

Figure 4.. SPLUNC1 Predicts AE-Free Time.

A) AE-Free time in Stable CF patients separated into SPLUNC1-High and SPLUNC1-Low groups over a 60-day follow-up period. SPLUNC1-high and –low groups were defined according to sputum concentration thresholds obtained from receiver-operator curves separating CF Stable and AE levels (Supplemental Figure 4). AE-Free time was defined as the number of days from sputum collection in Stable patients until the date of their next AE. B) AE-Free time in Stable CF patients separated into SPLUNC1-High and -Low groups over a 365-day follow up period. Cox Proportional Hazards model used to calculate AE-free intervals and adjust for age, sex, BMI, FEV₁, number of F508del mutations, presence of CF-related diabetes or pancreatic insufficiency, use of CFTR modulators, and microbiology for P. aeruginosa, A. xylosoxidans, H. parainfluenzae, Methicillin-sensitive S. aureus, and Methicillin-resistant S. aureus.

Figure 5.. Elastase Concentration and Activity are Increased in CF.

A) SPLUNC1 densitometry showing degradation by human neutrophil elastase (NE) relative to PBS control, at specified concentrations over 3 hours at 37°C. B) SPLUNC1 densitometry showing degradation by Elastase B (LasB) from P. aeruginosa relative to PBS control at specified concentrations over 8 hours at 37°C. C) NE densitometry in sputum from healthy controls (HC), Stable CF patients (CF Stable), and AE patients (AE) assessed by WB. D) Representative WB showing endogenous expression of SPLUNC1 (20–25 kD) and NE (25–30 kD) in HC and CF sputum samples from the same individuals. Membranes were probed for NE prior to stripping and re-probing for SPLUNC1. E) NE Activity in CF sputum: AMC formation from florigenic NE substrate MAA-3133 following 6 h incubation with HC, CF stable, and AE sputum at 37°C. For experiments in A and B: n = 4–5, 2 individual experiments; Mann-Whitney Test; + = Mean; Bar inside box: Median; Whiskers: Minimum/Maximum; * p < 0.05; ** = p < 0.01; *** = p < 0.005; ns: not statistically significant; HC: Healthy Control; ST: Stable CF; AE: CF exacerbation; m: marker; +ctl: recombinant protein positive control; OD: optic density.

Figure 6.. Cytokines IL-1β and TNF-α Increase During AE & Downregulate SPLUNC1 Expression.

A) Inflammatory cytokine levels in sputum from adult CF patients without respiratory symptoms (CF Stable) and with acute CF Exacerbation (AE). Additional cytokines tested without significant difference: CXCL10, G-CSF, IFNα2, IFNγ, IFNλ, IL-6, IL-8, IL-13, MCP1, MIP1α. Mann-Whitney Test with Bonferroni correction. B) Relative SPLUNC1 mRNA expression in mouse tracheal epithelial cells (Mouse epithelium) grown at air-liquid interface and in the NCI-H292 human airway epithelial cell line (Human epithelium) treated with recombinant TNFα and IL-1β (10 ng/mL) for 24 hours (2-way ANOVA); + = Mean; Bar inside box: Median; Whiskers: Minimum/Maximum; mRNA expression quantified by qPCR. Represents 2 experiments; * = p<0.05; ** = p<0.01.