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.

Keywords: airway secretions; airway surface liquid; antiviral proteins; cystic fibrosis; host defense.

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).

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).

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).

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.

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.