Small Peptide Derivatives Within the Carbohydrate Recognition Domain of SP-A2 Modulate Asthma Outcomes in Mouse Models and Human Cells

Abstract

Surfactant Protein-A (SP-A) is an innate immune modulator that regulates a variety of pulmonary host defense functions. We have shown that SP-A is dysfunctional in asthma, which could be partly due to genetic heterogeneity. In mouse models and primary bronchial epithelial cells from asthmatic participants, we evaluated the functional significance of a particular single nucleotide polymorphism of SP-A2, which results in an amino acid substitution at position 223 from glutamine (Q) to lysine (K) within the carbohydrate recognition domain (CRD). We found that SP-A 223Q humanized mice had greater protection from inflammation and mucin production after IL-13 exposure as compared to SP-A-2 223K mice. Likewise, asthmatic participants with two copies the major 223Q allele demonstrated better lung function and asthma control as compared to asthmatic participants with two copies of the minor SP-A 223K allele. In primary bronchial epithelial cells from asthmatic participants, full-length recombinant SP-A 223Q was more effective at reducing IL-13-induced MUC5AC gene expression compared to SP-A 223K. Given this activity, we developed 10 and 20 amino acid peptides of SP-A2 spanning position 223Q. We show that the SP-A 223Q peptides reduce eosinophilic inflammation, mucin production and airways hyperresponsiveness in a house dust mite model of asthma, protect from lung function decline during an IL-13 challenge model in mice, and decrease IL-13-induced MUC5AC gene expression in primary airway epithelial cells from asthmatic participants. These results suggest that position 223 within the CRD of SP-A2 may modulate several outcomes relevant to asthma, and that short peptides of SP-A2 retain anti-inflammatory properties similar to that of the endogenous protein.

Keywords: SP-A; SP-A peptides; asthma; genetics; surfactant protein.

Figure 1

Genetic variation in SP-A2 determines extent of protection against IL-13 induced inflammation in humanized SP-A transgenic mice. BAL cells from (A) IL-13 challenged mice, which consisted of and (B) macrophages, (C) neutrophils and (D) eosinophils. (E) PAS scored lung histology from IL-13 challenged mice. N=12 WT; 15 SP-A^-/-; 8 SP-A223Q/Q; 12 SP-A223K/K per group from 3 separate experimental repeats. *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001 by One-way Anova with Dunnett’s test for multiple comparisons. (F) Representative PAS images of each genotype treated with IL-13. (G, H) Stat3 phosphorylation by Western blot and densitometry from representative lung samples; *p<0.05 by One-Way Anova with Tukey’s multiple comparisons.

Figure 2

Genetic variation in SP-A2 determines extent of protection against IL-13-induced inflammation from bronchial epithelial cells from asthmatic participants. (A) MUC5AC RNA expression from bronchial epithelial cells (n=3 normal, n=3 asthma) grown at ALI and treated with IL-13 for 5 days in the presence of absence of full-length recombinant SP-A2(223K) (20 μg/ml) or SP-A2(223Q) (20 μg/ml) that were added 30 min prior to challenge. After standardization to the housekeeper gene, data are displayed as fold relative to the non-IL-13 challenged control for each respective patient set, with the standard deviation shown. Average fold change and standard deviation are displayed. *p < 0.05. (B) Genetic variants of SP-A2 that differ only at position 223 (Q and K) were examined for relative binding to IL-13 relative to extracted human oligomeric control SP-A.

Figure 3

HDM-challenged SP-A deficient mice treated with SP-A peptides have reduced hallmarks of inflammation. (A) SP-A deficient mice challenged intra-nasally with HDM on days 0, 7, and 14. On day 15 mice were divided into groups and given either vehicle or SP-A peptides (10-mer or 20-mer) via oropharyngeal instillation at 25 μg/ml concentration (~1 mg/kg body weight). On day 19, mice were sacrificed and (B) eosinophils in the BAL and (C) mucin production in (D) lung histological sections were assessed. n = 10,10, **p < 0.01, ***p < 0.001 by One-way Anova for multiple comparisons.

Figure 4

HDM-challenged WT mice treated with SP-A peptides have reduced sensitivity to methacholine challenge. (A) WT male mice challenged intra-nasally with HDM on days 0, 7, and 14. On days 1, 8 and 15 mice were divided into groups and given either vehicle or SP-A peptides (10-mer, 25 μg/ml, ~1 mg/kg body weight) via oropharyngeal instillation. On day 19, pulmonary function tests during a methacholine challenge were performed while mice were under anesthesia. (B) Total airways resistance (Rrs), (C) Newtonian resistance (Rn), (D) total airways Elastance (Ers) and (E) tissue damping (G) were assessed by flexivent. Data graphed are mean +/- SEM. n = 12,12, *p < 0.05, **p < 0.01 by t-test at each indicated dose.

Figure 5

IL-13-challenged mice treated with SP-A 10-mer peptide have improved lung function. WT male mice were challenged with vehicle (saline) or IL-13 (3.9 μg) by oropharyngeal delivery for 3 consecutive days. Two hours after each IL-13 challenge, mice received either vehicle (saline) or SP-A 10-mer peptide (25 μg/ml; ~1mg/kg body weight) via oropharyngeal delivery. Pulmonary function tests were conducted on day 4 on a flexiVent machine (SCIREQ) with the negative pressure-driven forced expiration (NPFE) extension. IL-13 challenge resulted in significantly increased Newtonian resistance (Rn) and decreased forced expiratory volumes (FEV) at 0.05 seconds. Treatment with SP-A 10-mer peptide protected against IL-13 induced increase in Rn and decreases in FEV. Average mean +/- SEM is graphed, n= as shown from 2 independent experiments. *p < 0.05, ***p < 0.001 by ANOVA for multiple comparisons.

Figure 6

Primary human lung epithelial cells treated with SP-A peptides have reduced IL-13 induced MUC5AC gene expression. Primary human bronchial epithelial cells derived from normal and asthmatic participants were incubated for 30 minutes with either 20-mer or 10-mer SP-A peptide (20 μg/ml) before stimulation with IL-13 (10 ng/ml) for 5 days. When taken together (asthma and normal cells), MUC5AC gene expression was significantly reduced in the SP-A 20-mer treatment group compared with IL-13 alone (p=0.004).