Surfactant Protein A Inhibits Human Rhinovirus C Binding and Infection of Airway Epithelial Cells from Pediatric Asthma

Abstract

Rhinovirus C (RV-C) infection can trigger asthma exacerbations in children and adults, and RV-C-induced wheezing illnesses in preschool children correlate with the development of childhood asthma. Surfactant protein A (SP-A) plays a critical role in regulating pulmonary innate immunity by binding to numerous respiratory pathogens. Mature SP-A consists of multiple isoforms that form the hetero-oligomers of SP-A1 and SP-A2, organized in 18-mers. In this report, we examined the efficacy of SP-A to antagonize RV-C infection using the wild-type (RV-C15) and reporter-expressing (RV-C15-GFP) viruses in differentiated nasal epithelial cells (NECs) from asthmatic and non-asthmatic children. We also determined the antiviral mechanism of action of SP-A on RV-C15 infection. The native SP-A was purified from alveolar proteinosis patients. The recombinant (r) SP-A1 and SP-A2 variants were expressed in FreeStyle™ 293-F cells. SP-A reduced the fluorescent focus-forming units (FFUs) after RV-C15-GFP infection of NECs by 99%. Both simultaneous and 4 h post-infection treatment with SP-A inhibited RV-C15 and RV-C15-GFP viral RNA load by 97%. In addition, the antiviral genes and chemokines (IFN-λ, IRF-7, MDA-5, and CXLC11) were not induced in the infected NECs due to the inhibition of RV-C propagation by SP-A. Furthermore, SP-A bound strongly to RV-C15 in a dose- and Ca²⁺-dependent manner, and this interaction inhibited RV-C15 binding to NECs. In contrast, rSP-A1 did not bind to solid-phase RV-C15, whereas the rSP-A2 variants, [A₉₁, K₂₂₃] and [P₉₁, Q₂₂₃], had strong binding affinities to RV-C15, similar to native SP-A. This study demonstrates that SP-A might have potential as an antiviral for RV infection and RV-induced asthma exacerbations.

Keywords: human rhinovirus C; innate immunity; pediatric asthma; pulmonary surfactant protein A.

Figure 1

Native SP-A reduces RV-C15-GFP infection in human NECs. Differentiated NECs were incubated with RV-C15-GFP for 4 h with or without SP-A at 50 µg/mL or 400 µg/mL followed by washing with DPBS and replacing medium with or without SP-A. At 24 h after viral challenge, the cultures were visualized by fluorescence microscopy ((a), upper row panel), or combined fluorescence and differential interference contrast (DIC) ((a), lower row panel). The scale bar indicates 50 μm. Quantitative data of fluorescent focus-forming units (FFUs; numbers/well) in cells from the non-asthmatic (n = 3) (b) and asthmatic subjects (n = 9) (c) are shown. Each experiment was performed in duplicate. Data are means ± SD from six fields taken in each well. RV-C15-GFP viral copy numbers were measured by RT-qPCR at 48 h p.i. using NECs from (d) non-asthmatic (n = 6), and (e) asthmatic (n = 10) subjects. Data are shown as means ± SD from two to three individual experiments per donor.

Figure 2

Native SP-A treatment at 4 h after viral challenge reduced viral copy numbers in a dose-dependent manner. Differentiated NECs were incubated with RV-C15 at 2 × 10⁴ viral copy numbers/well for 4 h, then the unbound virus was removed by washing with DPBS. Native SP-A treatment was started at 0 h, 4 h, or 24 h p.i. of viral challenge at indicated concentration (50, 100, 200, or 400 µg/mL). Viral RNA was quantitated at 4 h or 48 h p.i. by RT-qPCR. Data were means ± SD from 4 non-asthmatic donors. p-values were shown in comparison to RV-C15 48 h alone.

Figure 3

SP-A attenuates antiviral gene expression and chemokine secretion induced by RV-C15. Differentiated NECs were challenged with RV-C15-GFP as described in Figure 1. mRNA expression of MDA5, IRF7, IFNλ, and CXCL11 were quantified by RT-qPCR by using RNA of NECs from non-asthmatics (n = 6) (a) and asthmatics (n = 10) (b). Data are shown as means ± SD from duplicated well in each condition for each donor. Secretion of CXCL11 in apical media was quantified by ELISA in (c). The data are means ± SD using cells from 5 non-asthmatic and 6 asthmatic donors; experiments limited by cell availability. p-values were shown in comparison to the condition of RV-C15 48 h alone.

Figure 4

SP-A directly binds to RV-C15 and prevents its attachment to differentiated NECs in a dose-dependent manner. SP-A (0 to 10 µg/mL) was incubated for 1 h in RV-C15-precoated microtiter plate (a). The binding of SP-A and RV-C15 was quantified by anti-human SP-A HRP-conjugated antibody [27,30] in the presence of 5 mM EDTA () or 5 mM CaCl₂ (). The maximum binding (B_max) and dissociation constant (K_d) determined by nonlinear regression analysis are indicated. (b) RV-C15 attachment to NECs was examined in the presence of various concentrations of SP-A (0 to 400 µg/mL) using cells from 4 asthmatic participants. Bound RV-C15 was quantified by RT-qPCR and results are shown as a percentage of viral binding compared to control. The concentration of SP-A that could inhibit the binding by 50% (IC₅₀) is shown. The results represent means ± SD from 3 to 5 independent experiments.

Figure 5

Amino acids at positions 91 and 223 determine the binding affinity of SP-A2 to RV-C15. The schematic (a) illustrates variation at amino acid positions of SP-A1 and SP-A2. (b) The binding affinity of native SPA or recombinant SP-A variants at 10 µg/mL to the solid phase RV-C15 in the presence of 5 mM CaCl₂. The data are means ± SD from 3 independent experiments. p-values indicate samples that are significantly different from the native SP-A.

Figure 6

The SP-A inhibition of RV-C15-GFP infection corresponds to SP-A virus binding efficiency. (a) Differentiated NECs were infected for 48 h with RV-C15-GFP in the presence or absence of native SP-A, or recombinant SP-A1 [V₅₀, W₂₁₉] (SPA1VW) or SP-A2 [P₉₁, Q₂₂₃] (SPA2PQ) at 100 µg/mL. The results are shown in comparison to RV-C15-GFP control (expressed as 100%). (b) The cultures were infected as in panel A and treated with native SP-A, SP-A2 [A₉₁, Q₂₂₃], or SP-A2 [A₉₁, K₂₂₃] at 400 µg/mL. The data derived from cells obtained from 3 to 4 non-asthmatic participants. The data are means ± SD. p-values indicate samples that are significantly different from the RV-C15 GFP infection control.