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Expression of surfactant protein D in airways of asthmatics and interleukin-13 modulation of surfactant protein D in human models of airway epithelium.

Xu J, Singhera GK, Dorscheid DR - Respir. Res. (2015)

Bottom Line: Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC.Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.SP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Surfactant protein D (SP-D), a pattern recognition molecule, has been shown to play roles in host defense such as opsonisation, aggregation of pathogens, and modulation of the inflammatory response. In light of infection-induced exacerbations and damage to the airway epithelium from inflammation, these functions of SP-D make it relevant in the development and pathogenesis of asthma.

Methods: Expression of SP-D was examined in human airway sections and primary airway epithelial cells (AEC) grown in air-liquid interface (ALI) cultures and comparisons were made between those from asthmatic and non-asthmatic donors. ALI cultures of AEC from non-asthmatic donors were examined for SP-D, Mucin 5AC, and cytokeratin-5 expression at different stages of differentiation. Interleukin-13 (IL-13) treatment of airway epithelium and its effect on SP-D expression was studied using ALI and monolayer cultures of primary AEC from non-asthmatic and asthmatic donors.

Results: Airway epithelium of asthmatics, compared to that of non-asthmatics, expressed increased levels of SP-D as demonstrated in airway tissue sections (fraction of epithelium 0.66 ± 0.026 vs. 0.50 ± 0.043, p = 0.004) and ALI cultures (fraction of epithelium 0.50 ± 0.08 vs. 0.25 ± 0.07). SP-D expression decreased as ALI cultures differentiated from 7 days to 21 days (fraction of epithelium 0.62 ± 0.04 to 0.23 ± 0.03, p = 0.004). Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC. Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.

Conclusions: SP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics. This can have implications on the increased susceptibility to infections and altered inflammatory response in asthmatic patients. Future functional studies on the role of SP-D in asthma can provide better insight into defects in the structure and regulation of SP-D.

No MeSH data available.


Related in: MedlinePlus

SP-D expression in monolayer epithelial cultures treated by IL-13. SP-D mRNA was examined in non-asthmatic monolayer cultures treated with IL-13 (10 ng/mL) alone and in cultures pre-treated with either goat IgG isotype control (20 μg/mL), IL-13Rα1 neutralizing antibody (20 μg/mL), or IL-13Rα2 neutralizing antibody (2 μg/mL) one hour prior to IL-13 exposure. To obtain fold changes, ΔΔCT values were obtained by normalizing SP-D mRNA to HPRT mRNA and normalizing treatment groups to untreated control. A significant difference was observed between the IgG pre-treated group and IL-13Rα1 neutralizing antibody pre-treated group (n = 3, one-way ANOVA p = 0.01) [A]. SP-D protein levels in non-asthmatic monolayer cultures under the same treatment conditions were detected via Western blotting. A significant difference was found between the IL-13 treated group and IL-13Rα1 neutralizing antibody pre-treated group. Quantification was performed by densitometry and SP-D protein expression was normalized to untreated control (n = 6, one-way ANOVA p = 0.04) [B]. Phosphorylated-STAT6 (pSTAT6) expression in non-asthmatic monolayer cultures from the same experiment as panel [B] was examined via Western blotting. IL-13 treatment significantly increased the phosphorylation of STAT6 compared to control while pre-incubation with IL-13Rα1 neutralizing antibody significantly reduced the phosphorylation of STAT6 compared to IgG (n = 5, one-way ANOVA p = 0.005) [C]. pSTAT6 to STAT6 protein ratio in non-asthmatic ALI cultures treated with IL-13 (10 ng/mL) over the course of two weeks show significant increases relative to untreated control (n = 3, one-way ANOVA p = 0.001) [D]. IL-13 treatment resulted in a significant decrease in SP-D protein expression in non-asthmatic monolayer culture (n = 5, t-test p = 0.03) whereas no significant change was detected in asthmatic monolayer culture (n = 4) [E].
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Fig5: SP-D expression in monolayer epithelial cultures treated by IL-13. SP-D mRNA was examined in non-asthmatic monolayer cultures treated with IL-13 (10 ng/mL) alone and in cultures pre-treated with either goat IgG isotype control (20 μg/mL), IL-13Rα1 neutralizing antibody (20 μg/mL), or IL-13Rα2 neutralizing antibody (2 μg/mL) one hour prior to IL-13 exposure. To obtain fold changes, ΔΔCT values were obtained by normalizing SP-D mRNA to HPRT mRNA and normalizing treatment groups to untreated control. A significant difference was observed between the IgG pre-treated group and IL-13Rα1 neutralizing antibody pre-treated group (n = 3, one-way ANOVA p = 0.01) [A]. SP-D protein levels in non-asthmatic monolayer cultures under the same treatment conditions were detected via Western blotting. A significant difference was found between the IL-13 treated group and IL-13Rα1 neutralizing antibody pre-treated group. Quantification was performed by densitometry and SP-D protein expression was normalized to untreated control (n = 6, one-way ANOVA p = 0.04) [B]. Phosphorylated-STAT6 (pSTAT6) expression in non-asthmatic monolayer cultures from the same experiment as panel [B] was examined via Western blotting. IL-13 treatment significantly increased the phosphorylation of STAT6 compared to control while pre-incubation with IL-13Rα1 neutralizing antibody significantly reduced the phosphorylation of STAT6 compared to IgG (n = 5, one-way ANOVA p = 0.005) [C]. pSTAT6 to STAT6 protein ratio in non-asthmatic ALI cultures treated with IL-13 (10 ng/mL) over the course of two weeks show significant increases relative to untreated control (n = 3, one-way ANOVA p = 0.001) [D]. IL-13 treatment resulted in a significant decrease in SP-D protein expression in non-asthmatic monolayer culture (n = 5, t-test p = 0.03) whereas no significant change was detected in asthmatic monolayer culture (n = 4) [E].

Mentions: To study SP-D regulation in response to IL-13, monolayer cultures grown from non-asthmatic AEC were treated with IL-13 (10 ng/mL) for 24 hours. When normalized to untreated control, treatment with IL-13, either alone or after pre-incubation with IgG isotype control for 24 hours induced a reduction in SP-D mRNA (0.62 ± 0.05 and 0.60 ± 0.03 respectively, n = 3, Figure 5A). Cells pre-incubated with IL-13Rα1 neutralizing antibody prior to IL-13 treatment demonstrated a mitigation of the IL-13 effect on SP-D mRNA expression (0.99 ± 0.11). In contrast, cells pre-incubated with IL-13Rα2 neutralizing antibody demonstrated no significant difference in SP-D mRNA expression compared to cells pre-incubated with IgG isotype control (0.54 ± 0.07).Figure 5


Expression of surfactant protein D in airways of asthmatics and interleukin-13 modulation of surfactant protein D in human models of airway epithelium.

Xu J, Singhera GK, Dorscheid DR - Respir. Res. (2015)

SP-D expression in monolayer epithelial cultures treated by IL-13. SP-D mRNA was examined in non-asthmatic monolayer cultures treated with IL-13 (10 ng/mL) alone and in cultures pre-treated with either goat IgG isotype control (20 μg/mL), IL-13Rα1 neutralizing antibody (20 μg/mL), or IL-13Rα2 neutralizing antibody (2 μg/mL) one hour prior to IL-13 exposure. To obtain fold changes, ΔΔCT values were obtained by normalizing SP-D mRNA to HPRT mRNA and normalizing treatment groups to untreated control. A significant difference was observed between the IgG pre-treated group and IL-13Rα1 neutralizing antibody pre-treated group (n = 3, one-way ANOVA p = 0.01) [A]. SP-D protein levels in non-asthmatic monolayer cultures under the same treatment conditions were detected via Western blotting. A significant difference was found between the IL-13 treated group and IL-13Rα1 neutralizing antibody pre-treated group. Quantification was performed by densitometry and SP-D protein expression was normalized to untreated control (n = 6, one-way ANOVA p = 0.04) [B]. Phosphorylated-STAT6 (pSTAT6) expression in non-asthmatic monolayer cultures from the same experiment as panel [B] was examined via Western blotting. IL-13 treatment significantly increased the phosphorylation of STAT6 compared to control while pre-incubation with IL-13Rα1 neutralizing antibody significantly reduced the phosphorylation of STAT6 compared to IgG (n = 5, one-way ANOVA p = 0.005) [C]. pSTAT6 to STAT6 protein ratio in non-asthmatic ALI cultures treated with IL-13 (10 ng/mL) over the course of two weeks show significant increases relative to untreated control (n = 3, one-way ANOVA p = 0.001) [D]. IL-13 treatment resulted in a significant decrease in SP-D protein expression in non-asthmatic monolayer culture (n = 5, t-test p = 0.03) whereas no significant change was detected in asthmatic monolayer culture (n = 4) [E].
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Fig5: SP-D expression in monolayer epithelial cultures treated by IL-13. SP-D mRNA was examined in non-asthmatic monolayer cultures treated with IL-13 (10 ng/mL) alone and in cultures pre-treated with either goat IgG isotype control (20 μg/mL), IL-13Rα1 neutralizing antibody (20 μg/mL), or IL-13Rα2 neutralizing antibody (2 μg/mL) one hour prior to IL-13 exposure. To obtain fold changes, ΔΔCT values were obtained by normalizing SP-D mRNA to HPRT mRNA and normalizing treatment groups to untreated control. A significant difference was observed between the IgG pre-treated group and IL-13Rα1 neutralizing antibody pre-treated group (n = 3, one-way ANOVA p = 0.01) [A]. SP-D protein levels in non-asthmatic monolayer cultures under the same treatment conditions were detected via Western blotting. A significant difference was found between the IL-13 treated group and IL-13Rα1 neutralizing antibody pre-treated group. Quantification was performed by densitometry and SP-D protein expression was normalized to untreated control (n = 6, one-way ANOVA p = 0.04) [B]. Phosphorylated-STAT6 (pSTAT6) expression in non-asthmatic monolayer cultures from the same experiment as panel [B] was examined via Western blotting. IL-13 treatment significantly increased the phosphorylation of STAT6 compared to control while pre-incubation with IL-13Rα1 neutralizing antibody significantly reduced the phosphorylation of STAT6 compared to IgG (n = 5, one-way ANOVA p = 0.005) [C]. pSTAT6 to STAT6 protein ratio in non-asthmatic ALI cultures treated with IL-13 (10 ng/mL) over the course of two weeks show significant increases relative to untreated control (n = 3, one-way ANOVA p = 0.001) [D]. IL-13 treatment resulted in a significant decrease in SP-D protein expression in non-asthmatic monolayer culture (n = 5, t-test p = 0.03) whereas no significant change was detected in asthmatic monolayer culture (n = 4) [E].
Mentions: To study SP-D regulation in response to IL-13, monolayer cultures grown from non-asthmatic AEC were treated with IL-13 (10 ng/mL) for 24 hours. When normalized to untreated control, treatment with IL-13, either alone or after pre-incubation with IgG isotype control for 24 hours induced a reduction in SP-D mRNA (0.62 ± 0.05 and 0.60 ± 0.03 respectively, n = 3, Figure 5A). Cells pre-incubated with IL-13Rα1 neutralizing antibody prior to IL-13 treatment demonstrated a mitigation of the IL-13 effect on SP-D mRNA expression (0.99 ± 0.11). In contrast, cells pre-incubated with IL-13Rα2 neutralizing antibody demonstrated no significant difference in SP-D mRNA expression compared to cells pre-incubated with IgG isotype control (0.54 ± 0.07).Figure 5

Bottom Line: Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC.Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.SP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Surfactant protein D (SP-D), a pattern recognition molecule, has been shown to play roles in host defense such as opsonisation, aggregation of pathogens, and modulation of the inflammatory response. In light of infection-induced exacerbations and damage to the airway epithelium from inflammation, these functions of SP-D make it relevant in the development and pathogenesis of asthma.

Methods: Expression of SP-D was examined in human airway sections and primary airway epithelial cells (AEC) grown in air-liquid interface (ALI) cultures and comparisons were made between those from asthmatic and non-asthmatic donors. ALI cultures of AEC from non-asthmatic donors were examined for SP-D, Mucin 5AC, and cytokeratin-5 expression at different stages of differentiation. Interleukin-13 (IL-13) treatment of airway epithelium and its effect on SP-D expression was studied using ALI and monolayer cultures of primary AEC from non-asthmatic and asthmatic donors.

Results: Airway epithelium of asthmatics, compared to that of non-asthmatics, expressed increased levels of SP-D as demonstrated in airway tissue sections (fraction of epithelium 0.66 ± 0.026 vs. 0.50 ± 0.043, p = 0.004) and ALI cultures (fraction of epithelium 0.50 ± 0.08 vs. 0.25 ± 0.07). SP-D expression decreased as ALI cultures differentiated from 7 days to 21 days (fraction of epithelium 0.62 ± 0.04 to 0.23 ± 0.03, p = 0.004). Treatment with IL-13 decreased SP-D expression in both ALI cultures (fraction of epithelium 0.21 ± 0.06 vs. 0.62 ± 0.04, p = 0.0005) and monolayer cultures (protein expression fold change 0.62 ± 0.05) of non-asthmatic AEC; however, IL-13 had no significant effect on SP-D expression in monolayer cultures of asthmatic AEC. Experiments with non-asthmatic monolayer cultures indicate IL-13 exert its effect on SP-D through the IL-13 receptor alpha1 and transcription factor STAT6.

Conclusions: SP-D is expressed differently in airways of asthmatics relative to that of non-asthmatics. This can have implications on the increased susceptibility to infections and altered inflammatory response in asthmatic patients. Future functional studies on the role of SP-D in asthma can provide better insight into defects in the structure and regulation of SP-D.

No MeSH data available.


Related in: MedlinePlus