Airway Surface Liquid Has Innate Antiviral Activity That Is Reduced in Cystic Fibrosis

Abstract

Although chronic bacterial infections and inflammation are associated with progressive lung disease in patients with cystic fibrosis (CF), much less is known regarding the contributions of respiratory viral infections to this process. Clinical studies suggest that antiviral host defenses may be compromised in individuals with CF, and CF airway epithelia exhibit impaired antiviral responses in vitro. Here, we used the CF pig model to test the hypothesis that the antiviral activity of respiratory secretions is reduced in CF. We developed an in vitro assay to measure the innate antiviral activity present in airway surface liquid (ASL) from CF and non-CF pigs. We found that tracheal and nasal ASL from newborn non-CF pigs exhibited dose-dependent inhibitory activity against several enveloped and encapsidated viruses, including Sendai virus, respiratory syncytial virus, influenza A, and adenovirus. Importantly, we found that the anti–Sendai virus activity of nasal ASL from newborn CF pigs was significantly diminished relative to non-CF littermate controls. This diminution of extracellular antiviral defenses appears to be driven, at least in part, by the differences in pH between CF and non-CF ASL. These data highlight the novel antiviral properties of native airway secretions and suggest the possibility that defects in extracellular antiviral defenses contribute to CF pathogenesis.

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians, with patients having a median life expectancy of 44 years (1). Although CF is a multiorgan disease, most morbidity is caused by chronic lung disease characterized by acute and chronic bacterial infections and neutrophil-dominated inflammation (2). CF is caused by mutations in the gene encoding CFTR (CF transmembrane conductance regulator) (3, 4), a nucleotide-phosphorylation–regulated anion channel that conducts Cl⁻, HCO₃⁻ (5), and SCN⁻ (6) across epithelia. Much is still unknown about the link between CFTR dysfunction and lung disease progression.

Although newborns with CF are commonly reported to have normal lungs, growing evidence indicates that airway host defenses are impaired as early as the first month after birth (7, 8). Studies using the CF pig model have provided insights into factors that initiate CF lung disease. Newborn CF pigs lack airway inflammation, but within hours of birth their airways exhibit impaired antibacterial defenses compared with non-CF littermates (9). Studies using several experimental approaches have shown that this defect in antibacterial defenses can be attributed to a reduced bacterial killing activity in CF airway secretions (10), suggesting that abnormalities in the extracellular compartment contribute to this defect. These findings suggest that defective innate immunity contributes to early lung disease onset and progression.

The contribution of respiratory viral infections to CF lung disease onset and progression is not well understood. Although viral infection rates are similar between infants and children with CF and their non-CF counterparts (11–13), the severity of disease from viral infections appears to be greater in children with CF. Compared with control subjects, children with CF exhibit a greater likelihood of lower-respiratory-tract involvement during viral infections (11, 13), higher rates of hospitalization for respiratory infections (11), and significant and persistent decreases in pulmonary function after viral infections (11, 14). In addition, epidemiological studies point to an important association between respiratory viral infections and bacterial colonization of the lungs in early childhood for children with CF (15–17). Increased viral loads have been observed in the lungs of respiratory syncytial virus (RSV)-infected children with CF relative to infected non-CF controls, despite a similar infection prevalence between the two groups (18), suggesting that host defense mechanisms for restricting viral replication may be less effective in CF lungs. This concept is further supported by in vitro studies of cultured CF and non-CF airway cells that demonstrated that several aspects of antiviral host defense, including IFN signaling and apoptotic responses to viral infection, are perturbed in CF airway epithelia (19–22).

Defensive responses to respiratory viruses are primarily mediated by the airway epithelium, a common site of virus replication and a first line of defense against inhaled microbes and other particulates. Serving as a vital barrier between the body and the external environment, the airway epithelium is home to cell-based mechanisms for recognizing and responding to viruses, including viral pattern recognition receptors and signaling pathways that initiate and amplify responses to viral pathogens. The airway epithelium is covered by an aqueous periciliary liquid bathing the cilia and mucus that traps microbes and allows for their removal from the airways, which is collectively termed airway surface liquid (ASL). Airway epithelia secrete a number of innate immune molecules into this ASL, including lysozyme, lactoferrin, defensins, cathelicidins, and many others (23). Many of these are multifunctional molecules that exhibit a diverse array of defensive functions, which can include antibacterial, antiviral, and antifungal activities; biofilm prevention; opsonization; pro-/antiinflammatory effects; inhibition of host and pathogen proteases; and chemokine activity, among other functions (reviewed in Reference 23). In addition, dendritic cells, neutrophils, and resident alveolar macrophages are present or may be recruited to the airways to participate in antiviral defenses and act as a bridge between the innate and adaptive immune responses to viruses.

Although several studies have described defects in the cellular responses of CF airway epithelia to viral pathogens, relatively little is known regarding the impact that genotype-dependent differences in the extracellular environment have on antiviral defenses. Using an assay developed to measure the native antiviral activity present in airway secretions, we found that ASL from humans and pigs exhibited innate antiviral properties against several relevant classes of respiratory viruses. We used this assay to test the hypothesis that extracellular antiviral host defenses are impaired in CF. We observed reduced antiviral activity in ASL from CF pigs relative to non-CF pigs, raising the possibility that defects in extracellular antiviral host defense may play a role in the establishment of CF lung disease.

This work was previously presented at the 2016 North American Cystic Fibrosis Conference (24). Portions of this article were previously published by the University of Iowa as part of A.R.B.’s Master of Science thesis (25).

Methods

Collection and Processing of ASL

Three-week-old pigs were anesthetized, and tracheal secretions were stimulated with methacholine as previously described (10). ASL was harvested by advancing a microsampling probe (model BC-401C; Olympus Optical) through a pediatric bronchoscope inserted into the trachea. ASL was then collected by centrifuging at 10,000 × g for 10 minutes, irradiated at 80 Gy, and stored at −80°C. Nasal ASL was collected from humans and newborn pigs by inserting a sterile polyester-tipped applicator (Puritan Medical Products) into each nostril for 5 minutes. Nasal ASL was collected by microcentrifugation, irradiated at 8 krad, and stored at −80°C until use. BAL was collected from newborn CF and non-CF pigs according to previously described protocols (9).

Viruses

Viral inactivation assays were performed using recombinant Sendai virus encoding an eGFP (enhanced GFP) reporter gene (SeV-eGFP) (26, 27) as well as a recombinant RSV encoding GFP (RSV-GFP) upstream of the NS1 open reading frame (28). Influenza A virus (A/Puerto Rico/8/1934) encoding the eGFP gene in the NS segment (IAV-eGFP) (29) was kindly provided by Kevin Legge (University of Iowa). Recombinant, replication-incompetent adenovirus encoding eGFP (Ad-eGFP) was prepared by the University of Iowa Viral Vector Core Facility.

Viral Inactivation Assay

To assess dose-dependent viral inactivation by airway secretions, increasing volumes of ASL were preincubated for 2 hours with 10⁶ fluorescence-forming units of SeV-eGFP, 4 × 10⁵ plaque-forming units of RSV-GFP, 2 × 10⁵ plaque-forming units of adenovirus (Ad-eGFP), or 10⁶ fluorescence-forming units of IAV-eGFP. Virus:ASL mixtures were then brought up to a volume of 250 μl in 100 mM HEPES buffer (pH 7.4) and applied at a multiplicity of infection of 5–10 to the appropriate cell lines (LLC-MK2 cells for SeV-eGFP, A549 cells for IAV-eGFP, and HEp-2 cells for RSV-GFP and Ad-eGFP). The cells were incubated with the virus:ASL mixtures for 1 hour at 37°C and then the mixtures were removed and replaced with cell culture medium. After overnight incubation at 37°C and 5% CO₂, the cells were harvested and GFP⁺ cells in each condition were quantified using a BD Accuri C6 flow cytometer (BD Biosciences). “Relative infectivity” represents the number of GFP⁺ cells that arose from infection with ASL-treated virus, expressed as a percentage of the number of GFP⁺ cells after infection with untreated virus (25).

Statistical Analysis

All data are presented as means ± SE about the mean. Statistical significance was determined using Student’s t tests or one-way ANOVA followed by Tukey’s post hoc tests, using Graphpad Prism (version 6.02).

Results

Airway Secretions Exhibit Antiviral Activity

Tracheal (10) and nasal airway (30, 31) secretions have previously been shown to have broad-spectrum antibacterial activity. However, the possible antiviral properties of airway secretions are less well established and, to our knowledge, have not been studied. To probe for such activity, we developed an assay to measure the innate antiviral activity of airway secretions, using nasal and tracheal ASL collected from 3-week-old methacholine-stimulated pigs as a model. Porcine nasal and tracheal ASL samples were incubated with SeV (a member of the Paramyxoviridae family of negative-sense, single-stranded, enveloped RNA viruses) for 2 hours at 37°C, 5% CO₂. For these studies, we used SeV-eGFP to assess viral infection. After the 2-hour incubation period, the virus:ASL mixtures were applied to LLC-MK2 cells, a rhesus monkey kidney epithelial cell line that is permissive for SeV. After ∼18 hours of incubation at 37°C, we quantified eGFP⁺ cells by flow cytometry. As shown in Figure 1A, preincubation with porcine nasal or tracheal ASL reduced infection by SeV-eGFP in a dose-dependent manner. In time-course studies with SeV-eGFP, we observed that the majority of antiviral activity occurred within the first 30 minutes of virus:ASL preincubation, with maximal viral inhibition consistently occurring after 2 hours of incubation (Figure E1 in the data supplement). For this reason, we selected a 2-hour incubation time for subsequent experiments.

Figure 1.

Porcine airway surface liquid (ASL) has innate antiviral activity against respiratory viruses. (A) Sendai virus–enhanced GFP (SeV-eGFP), (B) respiratory syncytial virus–GFP (RSV-GFP), (C) influenza A virus–eGFP (IAV-eGFP), or (D) adenovirus–eGFP (Ad-eGFP) was incubated with increasing amounts of porcine tracheal or nasal ASL for 2 hours at 37°C and 5% CO₂. Virus:ASL mixtures were then brought up to 250 μl volume in 100 mM HEPES buffer (pH 7.4) and used to infect the appropriate permissive cell lines. Viral infection was quantified 24 hours after infection via flow cytometry analysis of eGFP expression. Infectivity is expressed as the number of GFP⁺ cells observed in a given condition, as a percentage of the number of GFP⁺ cells seen for the untreated (virus alone) condition. Results are displayed as mean ± SE (n = 3–4 individual pig donors).

Using this experimental approach, we then asked whether ASL inhibited the infectivity of other respiratory viruses. We found that porcine nasal and tracheal ASL also had inhibitory activity against the related pneumovirus, RSV-GFP (Figure 1B). Similarly to the SeV-eGFP results, RSV-GFP infection was significantly reduced in the presence of nasal ASL. Tracheal ASL displayed a more modest effect against this virus, with an approximately 25% reduction in RSV-GFP infection levels. We also found that in addition to their effects against SeV and RSV, porcine nasal and tracheal ASL dose dependently reduced the infectivity of the orthomyxovirus, IAV-eGFP (Figure 1C), and serotype 5 Ad-eGFP (Figure 1D). Unlike SeV, RSV, and IAV, Ad is an encapsidated, nonenveloped DNA virus. These results suggest that ASL possesses inhibitory activity against a range of respiratory viral pathogens.

Of interest, we noted that similar antiviral activity could be detected in airway secretions from humans. We found that human nasal ASL reduced the infectivity of SeV-eGFP (Figure 2A), RSV-GFP (Figure 2B), IAV-eGFP (Figure 2C), and Ad-eGFP (Figure 2D) in a dose-dependent manner similar to that observed for porcine nasal and tracheal ASL, suggesting that this property is a conserved feature of ASL. For the remainder of our studies, we selected SeV as a model virus because of its close antigenic and genetic homology to human parainfluenza virus 1 (HPIV-1). HPIV-1, along with HPIV-3, causes one-third of the lower-respiratory-tract infections in children under the age of 5 in the United States (32). Of note, HPIVs are also implicated in pulmonary exacerbations in children with CF (7, 11, 33).

Figure 2.

Human nasal ASL has innate antiviral activity against respiratory viruses. (A) SeV-eGFP, (B) RSV-GFP, (C) IAV-eGFP, or (D) Ad-eGFP was incubated with increasing amounts of human nasal ASL for 2 hours at 37°C and 5% CO₂. Virus:ASL mixtures were then brought up to 250 μl volume in 100 mM HEPES buffer (pH 7.4) and used to infect the appropriate permissive cell lines. Viral infection was quantified 24 hours after infection via flow cytometry analysis of eGFP expression. Infectivity is expressed as the number of GFP⁺ cells observed in a given condition, as a percentage of the number of GFP⁺ cells seen for the untreated (virus alone) condition. Results are displayed as mean ± SE (n = 3 adult donors).

To begin to characterize the antiviral activity we observed in porcine and human ASL, we tested whether the activity was heat labile. Porcine tracheal ASL was heated for 30 minutes at 56°C, 65°C, or 95°C, or left on ice for 30 minutes. Heat treatment at 56°C is sufficient to inactivate components of the complement system, and most proteins are denatured at 95°C; the 65°C treatment condition was chosen as an intermediate temperature. Heat-treated ASL samples were then incubated with SeV-eGFP for 2 hours before they were used to infect LLC-MK2 cells as described above. ASL incubated at 56°C exhibited a modest, nonsignificant reduction in anti-SeV activity compared with ASL incubated on ice (Figure 3A). Anti-SeV activity was significantly reduced when the ASL was heated to 65°C or 95°C, with the 95°C treatment causing the greatest reduction in ASL antiviral activity. The heat lability of ASL antiviral activity suggests that proteins are responsible for the activity. In addition, the partial reduction in ASL antiviral activity after incubation at 56°C and 65°C implies that multiple components contribute to the antiviral activity.

Figure 3.

The antiviral activity in porcine ASL is heat labile and does not represent transudated serum components. (A) Porcine tracheal ASL was heated at 56°C, 65°C, or 95°C, or incubated on ice, for 30 minutes. After they were cooled to room temperature, ASL samples (2.5 μl per condition) were incubated with 10⁶ fluorescence-forming units (ffu) of SeV-eGFP for 2 hours at 37°C, 5% CO₂. The virus:ASL mixtures were brought up to a volume of 250 μl in 100 mM HEPES buffer (pH 7.4) and used to infect LLC-MK2 cells. Viral infection was quantified 24 hours after infection via flow cytometry analysis of eGFP expression. Infectivity is expressed as the number of GFP⁺ cells observed in a given condition, as a percentage of the number of GFP⁺ cells seen for the untreated (virus alone) condition. Results are presented as mean ± SE (n = 3 replicate experiments). *P < 0.05 and ***P < 0.001 as determined by one-way ANOVA followed by Tukey’s multiple-comparisons test. Only statistically significant differences are noted. (B) Porcine serum was either heat treated at 56°C or left on ice for 30 minutes. The serum was then serially diluted and incubated with SeV-eGFP (10⁶ ffu) for 1 hour at 37°C, 5% CO₂. The samples were brought up to a volume of 250 μl in Opti-MEM medium and anti-SeV activity was assessed as described in A. Results are presented as mean ± SE (n = 3 replicate experiments).

Proteins from serum can reach the airways through transudation or, in the case of IgA, specific transcellular transport pathways. For this reason, many host defense proteins present in blood may be found in ASL or BAL fluid. We therefore considered that proteins such as complement and immunoglobulins might contribute to the antiviral activity of ASL. To test this hypothesis, we assessed antiviral activity in serial dilutions of serum from newborn pigs using the SeV-eGFP infectivity assay. For this study, the serum was either left untreated or heat treated for 30 minutes at 56°C to inactivate complement. Untreated serum had potent, dose-dependent antiviral activity against SeV-eGFP (Figure 3B). However, in contrast to tracheal ASL, the serum completely lost its anti-SeV activity after it was heated to 56°C. Together with the ASL heat-lability studies, these results suggest that the ASL antiviral components are not solely derived from serum (although we cannot exclude the possibility that serum-derived proteins make some contribution to the overall antiviral activity in ASL).

ASL from CF Pigs Has Reduced Antiviral Activity

Earlier studies indicated that CF pigs have impaired antimicrobial defenses at birth, and that a relatively acidic ASL pH leads to reduced antibacterial properties of their airway secretions (10). We hypothesized that CF pig ASL would similarly have reduced antiviral activity compared with non-CF ASL. To test this, we collected nasal ASL from newborn CFTR^−/− pigs (CF pigs) and non-CF littermates (CFTR^+/+ and CFTR^+/−). As shown in Figure 4, CF pig nasal ASL exhibited significantly reduced antiviral activity against SeV-eGFP relative to that of non-CF pig nasal ASL. This reduced antiviral activity was seen consistently throughout a range of tested volumes of ASL (Figure 4). We confirmed that nasal ASL from newborn CF pigs had a reduced pH compared with that from their non-CF littermates (Figure 5A), in agreement with previous reports regarding newborn CF pig tracheal ASL (10), newborn human nasal fluid (34), airway breath condensate (35), human primary airway epithelial cells (36), and human submucosal gland secretions (37).

Figure 4.

The innate antiviral activity of ASL from newborn cystic fibrosis (CF) pigs is reduced compared with non-CF pig secretions. SeV-eGFP was incubated with increasing amounts of nasal ASL from newborn CF and non-CF pigs for 2 hours at 37°C, 5% CO₂. The virus:ASL mixtures were then brought up to a volume of 250 μl in 100 mM HEPES buffer (pH 7.4) and used to infect LLC-MK2 cells. Viral infection was quantified 24 hours after infection via flow cytometry analysis of eGFP expression. Infectivity is expressed as the number of GFP⁺ cells observed in a given condition, as a percentage of the number of GFP⁺ cells seen for the untreated (virus alone) condition. Results are presented as mean ± SE. *P < 0.05 for area under the curve as determined by Student’s t test (n = 6 non-CF, 8 CF).

Figure 5.

pH dependence of antiviral activity in CF and non-CF nasal ASL. (A) The pH of newborn CF and non-CF pig nasal ASL was measured ex vivo with a Restech Dx-pH probe immediately before antiviral activity measurements were obtained (n = 6 non-CF, 8 CF). Results are presented as mean ± SE. *P < 0.05 as determined by Student’s t test. (B) The pH of newborn CF and non-CF nasal ASL was adjusted by mixing 1 μl of each sample with 25 μl 100 mM HEPES buffer at pH 6.8, 7.4, or 8.0. A viral inactivation assay was then performed as described in Methods (n = 16 non-CF, 13 CF). Results are presented as mean ± SE.

To test the hypothesis that the reduced ASL pH in CF impairs antiviral activity, we predicted that increasing the pH of CF nasal ASL would shift the antiviral activity toward non-CF levels. We tested this by measuring the antiviral activity of CF and non-CF nasal ASL at three defined pHs (pH 6.8, 7.4, and 8.0). In contrast to the experiments with unmodified ASL shown in Figures 1–4, here the pH was clamped in each condition using buffer. As shown in Figure 5B, although the CF ASL continued to display a slight (nonsignificant) trend toward reduced antiviral activity relative to the non-CF ASL at each pH, the genotype-dependent differences in ASL antiviral activity largely disappeared when the pH was clamped in this way.

We also addressed this hypothesis by asking whether altered pH might impact the antiviral functions of individual antimicrobial proteins and antimicrobial peptides (AMPs) with known antiviral activity in the airways. (Of note, the overall protein abundance did not differ significantly between CF and non-CF nasal ASL; Figure E2). The human and porcine cathelicidins LL-37 and protegrin-1, human β-defensin 3 (HBD-3), and lysozyme all displayed antiviral activity against SeV-eGFP in our infectivity assay (Figure 6). All of these peptides, except for lysozyme, had activity that was increased at pH 6.8, although statistically significant differences between the different pH conditions were only observed for HBD-3. HBD-2 and lactoferrin did not exhibit significant antiviral activity against SeV-eGFP in this assay.

Figure 6.

Antiviral activity of individual host defense molecules at varying pHs. SeV-eGFP was incubated with increasing amounts of (A) human LL-37, (B) porcine protegrin-1, (C) human β-defensin 3 (HBD-3), (D) human lysozyme, (E) HBD-2, or (F) human lactoferrin for 2 hours at 37°C and 5% CO_2. Host defense proteins were suspended in 100 mM HEPES buffers at pH 6.8, 7.4, or 8.0. After the 2-hour incubation, SeV-eGFP infectivity was determined as described in the Methods. Results are presented as mean ± SE (n = 3 replicate experiments). **P < 0.01 and ***P < 0.001 for area under the curve as determined by one-way ANOVA followed by Tukey’s multiple-comparisons test.

Discussion

Here, we report that ASL from the airways of pigs and humans exhibits antiviral properties against a range of clinically relevant respiratory viruses, including RSV, influenza, and adenovirus. Although it has long been recognized that airway epithelia mediate defensive responses to viral pathogens through cell-based signaling pathways, such as IFN production and inflammasome activation (38), there is less appreciation of the role that secreted proteins and peptides in the extracellular environment play in antiviral defenses. Our findings in pigs and humans suggest that, similar to what is known regarding antibacterial defenses, secretion of a suite of antiviral effector molecules into the ASL is likely a conserved mechanism that forms an “antiviral shield” to protect the airways from inhaled viruses.

Based on our heat-lability studies, we propose that the antiviral activity of porcine ASL is largely protein mediated and represents the combined activities of multiple secreted factors. Antiviral properties have been described for a number of individual proteins and peptides found in human ASL, including LL-37 (39), HBD-2 (40), HBD-3 (41), lactoferrin (42), lysozyme (43), SLPI (secretory leukocyte protease inhibitor) (44), and the collectins SP-A (surfactant protein A) (45) and SP-D (46). Several mechanisms are responsible for the antiviral actions of these proteins and peptides, including direct binding to and aggregation of viruses through interactions with lipid bilayers and/or viral glycoproteins, and in some cases disruption of viral membranes (39, 40). In contrast, lactoferrin inhibits PIV2 infection by binding to cellular proteins rather than through direct interaction with the virus (42). Additionally, airway mucins are believed to contribute to antiviral defenses by interacting with viruses in ways that impede their movement and/or prevent their uptake (47–49). Proteomic analysis indicates that many of the antimicrobial factors found in human airways can also be detected in porcine ASL, such as lactoferrin, lysozyme, SP-A, SP-D, SLPI, and numerous mucins, including MUC1, -2, -4, -5B, -5AC, -13, -16, and -19 (50). Although LL-37 is unique to humans, porcine ASL contains a related molecule, the cathelicidin protegrin-1 (50), which displays antiviral activity against several different viruses (51, 52). We expect that these proteins, and likely others, contribute to the broad-spectrum antiviral activity of porcine ASL.

Our studies demonstrate that this innate antiviral property of ASL is diminished in CF. This is consistent with a growing body of literature supporting the idea that antiviral defenses are impaired in CF airways. Compared with non-CF cells, CF airway epithelia have been shown to support increased replication of PIV3 (22), influenza (21), and rhinovirus (19, 20), and viral loads were found to be increased in the lungs of CF mice after experimental infection with RSV (53). To date, studies have focused on dysregulated IFN signaling, possibly as a consequence of chronic inflammation in the airway tissues from which the cells were derived, as a central mechanism for this defect. In support of this, Xu and colleagues reported delayed production of IFN-β in CF cells upon infection with influenza virus, along with altered expression of a number of IFN-inducible and antiviral response genes (21). Another report described blunted expression of the intracellular molecules 2′,5′-oligoadenylate synthetase 1 and nitric oxide synthase 2 in CF airway epithelia, both at baseline and after infection with PIV3 (22).

Our results suggest that in addition to this defect in the cellular IFN response, compromised antiviral activity in the extracellular compartment is also a likely contributor to impaired antiviral defenses in CF. Of importance, we performed our experiments using ASL from newborn CF pigs. CF pig airways lack inflammation at birth (9) and would have had minimal exposure to infectious or other immunostimulatory agents at the time of sample collection. Therefore, the observation of decreased antiviral activity in newborn airway secretions argues against the idea that this phenotype is a secondary consequence of inflammation, and instead suggests that diminished antiviral capacity is an inherent property of CF ASL.

The finding that CF pig ASL is intrinsically less antiviral than non-CF ASL echoes the observation that bacterial killing is impaired in the ASL of CF pigs (10). In the case of this bacterial killing defect, antibacterial activity can be restored by adjusting the pH of CF ASL to non-CF levels (10), indicating that loss of antibacterial activity in these secretions is primarily due to altered function of secreted antimicrobial proteins and AMPs in the lower pH of CF ASL. In the current study, the finding that CF and non-CF nasal ASL exhibited similar antiviral activity when tested at equivalent pHs suggests that the disease-related reduction in pH likely contributes to the reduced antiviral activity of CF ASL.

However, our experiments also suggest a somewhat more complicated picture with respect to the effect of pH on the antiviral activity of native secretions. In the airways, ASL pH is regulated by secretion of HCO₃⁻ and H⁺ (54). Contrary to our hypothesis, reducing the pH of non-CF nasal ASL to a lower, more “CF-like” level had only a very modest effect on overall antiviral activity. Furthermore, the effect of pH on the activities of individual antimicrobial molecules was variable: some molecules (LL-37, protegrin-1, and HBD-3) exhibited increased antiviral activity at lower pH, whereas others (lysozyme) were unaffected by pH changes. We speculate that this lack of consistent pH dependence reflects the fact that the secreted antimicrobial factors in ASL inactivate bacteria and viruses by distinct mechanisms.

We note that although adjusting the pH in the SeV infectivity assay significantly diminished the magnitude of the CF/non-CF difference in antiviral activity, CF ASL still showed a slight impairment (albeit not statistically significant) in activity with respect to non-CF ASL at all pHs tested (Figure 5B). This observation suggests that pH differences may provide only a partial explanation for the defect in CF secretions. It is possible that differences in the composition of CF and non-CF ASL may additionally influence antiviral activity. For example, although the overall protein abundance between CF and non-CF nasal ASL was not significantly different (Figure E2), it is possible that CF ASL lacks a specific factor (or several factors) that accounts for some proportion of the antiviral activity in airway secretions. In an earlier study, no significant differences were noted in the abundance of selected AMPs (lysozyme, lactoferrin, PLUNC [palate, lung, and nasal epithelial clone], and SP-A) in tracheal ASL from newborn CF and non-CF pigs (10). However, it is currently unknown whether levels of other antimicrobial agents may be altered in CF secretions. Additionally, it has been reported that the protease/antiprotease balance is altered in CF ASL, which could potentially impact innate immunity by causing aberrant cleavage and inactivation of secreted antiviral factors (55–57). Further studies will be needed to elucidate whether these additional mechanisms contribute to the reduced antiviral activity in CF ASL.

In conclusion, we found that human and porcine ASL is innately antiviral, with broad-spectrum activity against representative enveloped and encapsidated respiratory viruses. Furthermore, the potency of this antiviral activity was reduced in ASL from CF pigs relative to non-CF controls. These findings implicate extracellular antiviral mechanisms in defending the airways from inhaled viral pathogens, and also suggest that altered extracellular defenses may provide an advantage to invading viruses in CF airways. The activities of secreted antiviral molecules in the extracellular milieu are part of a larger, multilayered antiviral defense system, and further studies are needed to determine the overall impact of this defect in CF. It is possible that this reduction in the antiviral properties of CF ASL leads to a weakening of the antiviral host defense in CF airways, which is then further impaired by chronic inflammation associated with the progression of CF lung disease. This reveals a previously underappreciated aspect of airway innate immunity and provides insight into how defects in this system may contribute to dysfunctional host defenses in CF.