Surfactant Protein-A Protects against IL-13-Induced Inflammation in Asthma

Abstract

The lung surfactant proteins are recognized as critical not only for their role in lowering lung surface tension but also in innate host defense. Reports have shown that some asthmatic patients have decreased levels of one member of this protein family in particular, surfactant protein-A (SP-A). Our studies set out to determine the contribution of SP-A to the response of a key effector cytokine in asthma, IL-13. Our studies employ both animal models sufficient and deficient in SP-A challenged with IL-13 and primary epithelial cells from participants with asthma that are exogenously treated with SP-A in the context of IL-13 challenge. The inflammatory response and mucin production were assessed in both model systems. As compared with WT mice, we show that the activity of IL-13 is dramatically augmented in SP-A^-/- mice, which have significantly increased neutrophil and eosinophil recruitment, mucin production and asthma-associated cytokines in the bronchoalveolar lavage fluid. In parallel, we show asthma-associated factors are attenuated in human cells from asthma subjects when exogenous SP-A is added during IL-13 challenge. Although many of these phenotypes have previously been associated with STAT6 signaling, SP-A inhibited IL-13-induced STAT3 phosphorylation in mice and in human epithelial cells while having little effect on STAT6 phosphorylation. In addition, when either STAT3 or IL-6 were inhibited in mice, the phenotypes observed in SP-A^-/- mice were significantly attenuated. These studies suggest a novel mechanism for SP-A in asthma as a modulator of IL-13-induced inflammation via mediating downstream IL-6/STAT3 signaling.

Figure 1,. SP-A deficient mice are more susceptible to IL-13 challenge than WT mice.

A) WT and SP-A mice were challenged with IL-13 for 3 consecutive days via oropharyngeal instillation. B) Total cells present in the BAL were examined on day 4 in saline challenged versus IL-13 challenged mice. Differential cell populations were determined by cell staining for macrophages, neutrophils and eosinophils in BAL of C) saline challenged or D) IL-13 challenged mice. E) Muc5AC gene expression was quantified by RT-PCR of lung tissue and represented as fold of the saline treated control. F) PAS scores from IL-13 challenged mice were quantified by lung histological staining. G) KC, H) Eotaxin and I) IL-6 in BALF were quantified by ELISA. J) Representative micrographs of PAS stained lungs in WT and SP-A^−/− mice after IL-13 challenge. B-I data were obtained from n=8,8 saline treated and n=20,20 IL-13 treated mice across three separate experiments, *p<0.05, **p<0.01, ***p<0.001.

Figure 2,. Inhibition of Stat3 signaling attenuates IL-13 driven inflammation in SP-A^−/− mice.

A) WT, SP-A^−/− mice were exposed to IL-13 for 3 days and Stat3 and Stat6 phosphorylation was assessed from whole lung lystates by Western blot. n=8,8 lysates per group; representative bands shown from two independent blots. No Stat3 or Stat6 phosphorylation was detected in saline control treated mice. B-I) WT (black bars) or SP-A^−/− (white bars) mice received vehicle or Stat3 inhibitor (5 mg/kg body weight) via ip injection 2 hrs prior to a one time airway challenge with IL-13. Twenty-four hours later, B) total cells in BAL were counted and C) mucin production was assessed from PAS stained histological sections. By differential staining, BAL cells consisted of D) macrophages (Macs), E) neutrophils (PMNs), and F) eosinophils (Eos). G) KC, H) Eotaxin and I) IL-6 were determined in BAL of SP-A^−/− mice treated with and without Stat3 inhibitor prior to IL-13 by ELISA. n=8-12/per group, *p<0.05, **p<0.01, ***p<0.001.

Figure 3,. Inhibition of IL-6 signaling attenuates IL-13 driven inflammation in SP-A^−/− mice.

A-F) WT (black bars) and SP-A^−/− (white bars) mice were injected with either an IL-6 inhibitor (or vehicle control) 2 hrs prior to a one time airway challenge with IL-13. Twenty-four hours post challenge A) total cells in the BAL which consisted of B) macrophages (Macs), C) neutrophils (PMNs), D) eosinophils (Eos) were examined. E) Muc5AC gene expression was examined by RT-PCR in lung tissue. F) KC, and G) Eotaxin levels were examined by ELISA. n=9,9/per group, *p<0.05, **p<0.01, ***p<0.001.

Figure 4,. SP-A attenuates IL-13 induced inflammation in cultured bronchial epithelial cells from asthmatic participants.

BECs from asthmatic and normal participants obtained by bronchoscopy were cultured at ALI for 2 weeks after which they were stimulated with IL-13 in the presence or absence of oligomeric full-length SP-A isolated from APP patients (20 μg/ml), which was added 30 min prior to each IL-13 challenge. A) IL-8 in AS was determined by ELISA 48 hrs after IL-13 challenge (10 ng/ml) from cells of asthmatic and normal participants. B) IL-8 in AS from IL-13 stimulated asthma cells, in the presence or absence of SP-A. IL-8 data are displayed as fold relative to the non-IL-13 challenged control for each respective patient set, with the standard error of the mean. C) Muc5AC gene expression was determined by RT-PCR relative to GAPDH after chronic IL-13 challenge for 5 days (10 ng/ml) from BEC of asthmatic and normal participants. D) Muc5AC gene expression from IL-13 challenged BEC from asthmatic participants in the presence or absence of SP-A. Experiments were performed in triplicate for each set of patient cells based upon availability of adequate cell number for experiments. Each dot represents the average value for one patient, *p<0.05,**p<0.01.

Figure 5,. SP-A inhibits IL-13-induced STAT3 phosphorylation in asthma.

Human bronchial epithelial cells (BECs) obtained from asthmatic and normal participants were grown at an ALI for 2 weeks prior to IL-13 challenge (10 ng/ml) in the presence or absence of control oligomeric SP-A (20 μg/ml). A) Total cell lysates were analyzed for STAT3 or STAT6 phosphorylation determined by Western blot and compared to total STAT3 or STAT6 30 min after IL-13 challenge. Western blots are representative of n=3 asthma and 3 normal participants on two independent blots. B) Muc5AC gene expression was determined after stimulation with IL-13 using a 5 day protocol (10 ng/ml at 48 hours x 2, with trypsinization at 5 days) and C) IL-8 secretion were examined 48 hours from BEC that were challenged with IL-13 in the presence or absence of either SP-A or a STAT3 inhibitor added 30 minutes prior to IL-13.

Figure 6,. Increased IL-6 production from IL-13 challenged MTECs.

Mouse tracheal epithelial cells were harvested from WT and SP-A^−/− mice and grown at an ALI for 2 weeks prior to IL-13 challenge (10 ng/ml, basolaterally). Apical and basolateral supernatants were harvested for analysis after 48 hrs of challenge. IL-6 levels were determined by ELISA. *p<0.05, n=3 for each condition.

Figure 7,. SP-A binds to IL-6 in a dose-dependent manner.

A 96-well plate was coated with IL-6 and increasing concentrations of SP-A protein were added. An HRP-conjugated anti-SP-A antibody and TMB substrate were used to detect SP-A binding at an absorbance of 490 nM.