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Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs.

Ledford JG, Mukherjee S, Kislan MM, Nugent JL, Hollingsworth JW, Wright JR - PLoS ONE (2012)

Bottom Line: Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products.In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity.These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America. j.ledford@cellbio.duke.edu

ABSTRACT
Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A(-/-) allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/-) mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.

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Eosinophil-mediated Mp killing is attenuated by exogenous SP-A.A) Purified eosinophils were added to Mp (10∶1) for 1 hr and aliquots were diluted on PPLO agar plates for CFU counts. B) Mp and C) Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the killing assay. D–F) SP-D (0.5–5 µg/ml) or SP-A (5–10 µg/ml) was pre-incubated with eosinophils prior to the addition of Mp MOI 10∶1. EPO was measured in the supernatant after 1 hr of stimulation. G) Mp or Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the EPO stimulation assay *p<.05, **p<.01, n = 3 experiments.
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pone-0032436-g005: Eosinophil-mediated Mp killing is attenuated by exogenous SP-A.A) Purified eosinophils were added to Mp (10∶1) for 1 hr and aliquots were diluted on PPLO agar plates for CFU counts. B) Mp and C) Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the killing assay. D–F) SP-D (0.5–5 µg/ml) or SP-A (5–10 µg/ml) was pre-incubated with eosinophils prior to the addition of Mp MOI 10∶1. EPO was measured in the supernatant after 1 hr of stimulation. G) Mp or Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the EPO stimulation assay *p<.05, **p<.01, n = 3 experiments.

Mentions: In order to determine if eosinophils kill Mp, eosinophils were purified from the blood of IL-5 transgenic mice and co-incubated with Mp for 1 hour. In the first 15 minutes, ∼75% of the Mp was killed by eosinophil-mediated mechanisms, and by 60 minutes less than 10% of Mp remained viable (fig. 5A). Additionally, pre-incubation of eosinophils with a physiological concentration of SP-A prior to Mp addition, interfered with their killing mechanisms as compared to control eosinophils incubated with control buffer (fig. 5B). These experiments support the in vivo data where we observed less Mp burden when more eosinophils are present (as in the SP-A−/− mice). Since SP-A is known to bind Mp, we did the reverse experiment in which SP-A was pre-incubated with Mp prior to addition to eosinophils in culture. SP-A bound to Mp also decreased the ability of eosinophils to kill Mp optimally when compared to the Mp that was pre-incubated with the buffer control (fig. 5C). Since both sets of experiments, eosinophils pre-incubated with SP-A and Mp pre-incubated with SP-A, were centrifuged after the pre-incubation to remove any unbound SP-A, our findings suggest the binding of SP-A to either eosinophils or Mp is sufficient to interfere with eosinophil-mediated Mp killing. Pre-incubation with SP-D did not affect the ability of eosinophils to kill Mp (fig. 5D).


Surfactant protein-A suppresses eosinophil-mediated killing of Mycoplasma pneumoniae in allergic lungs.

Ledford JG, Mukherjee S, Kislan MM, Nugent JL, Hollingsworth JW, Wright JR - PLoS ONE (2012)

Eosinophil-mediated Mp killing is attenuated by exogenous SP-A.A) Purified eosinophils were added to Mp (10∶1) for 1 hr and aliquots were diluted on PPLO agar plates for CFU counts. B) Mp and C) Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the killing assay. D–F) SP-D (0.5–5 µg/ml) or SP-A (5–10 µg/ml) was pre-incubated with eosinophils prior to the addition of Mp MOI 10∶1. EPO was measured in the supernatant after 1 hr of stimulation. G) Mp or Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the EPO stimulation assay *p<.05, **p<.01, n = 3 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285686&req=5

pone-0032436-g005: Eosinophil-mediated Mp killing is attenuated by exogenous SP-A.A) Purified eosinophils were added to Mp (10∶1) for 1 hr and aliquots were diluted on PPLO agar plates for CFU counts. B) Mp and C) Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the killing assay. D–F) SP-D (0.5–5 µg/ml) or SP-A (5–10 µg/ml) was pre-incubated with eosinophils prior to the addition of Mp MOI 10∶1. EPO was measured in the supernatant after 1 hr of stimulation. G) Mp or Eosinophils were pre-incubated with SP-A (10 µg/ml) for 30 minutes to allow for binding and centrifuged prior to their addition to the EPO stimulation assay *p<.05, **p<.01, n = 3 experiments.
Mentions: In order to determine if eosinophils kill Mp, eosinophils were purified from the blood of IL-5 transgenic mice and co-incubated with Mp for 1 hour. In the first 15 minutes, ∼75% of the Mp was killed by eosinophil-mediated mechanisms, and by 60 minutes less than 10% of Mp remained viable (fig. 5A). Additionally, pre-incubation of eosinophils with a physiological concentration of SP-A prior to Mp addition, interfered with their killing mechanisms as compared to control eosinophils incubated with control buffer (fig. 5B). These experiments support the in vivo data where we observed less Mp burden when more eosinophils are present (as in the SP-A−/− mice). Since SP-A is known to bind Mp, we did the reverse experiment in which SP-A was pre-incubated with Mp prior to addition to eosinophils in culture. SP-A bound to Mp also decreased the ability of eosinophils to kill Mp optimally when compared to the Mp that was pre-incubated with the buffer control (fig. 5C). Since both sets of experiments, eosinophils pre-incubated with SP-A and Mp pre-incubated with SP-A, were centrifuged after the pre-incubation to remove any unbound SP-A, our findings suggest the binding of SP-A to either eosinophils or Mp is sufficient to interfere with eosinophil-mediated Mp killing. Pre-incubation with SP-D did not affect the ability of eosinophils to kill Mp (fig. 5D).

Bottom Line: Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products.In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity.These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, United States of America. j.ledford@cellbio.duke.edu

ABSTRACT
Surfactant protein-A (SP-A) has well-established functions in reducing bacterial and viral infections but its role in chronic lung diseases such as asthma is unclear. Mycoplasma pneumoniae (Mp) frequently colonizes the airways of chronic asthmatics and is thought to contribute to exacerbations of asthma. Our lab has previously reported that during Mp infection of non-allergic airways, SP-A aides in maintaining airway homeostasis by inhibiting an overzealous TNF-alpha mediated response and, in allergic mice, SP-A regulates eosinophilic infiltration and inflammation of the airway. In the current study, we used an in vivo model with wild type (WT) and SP-A(-/-) allergic mice challenged with the model antigen ovalbumin (Ova) that were concurrently infected with Mp (Ova+Mp) to test the hypothesis that SP-A ameliorates Mp-induced stimulation of eosinophils. Thus, SP-A could protect allergic airways from injury due to release of eosinophil inflammatory products. SP-A deficient mice exhibit significant increases in inflammatory cells, mucus production and lung damage during concurrent allergic airway disease and infection (Ova+Mp) as compared to the WT mice of the same treatment group. In contrast, SP-A deficient mice have significantly decreased Mp burden compared to WT mice. The eosinophil specific factor, eosinophil peroxidase (EPO), which has been implicated in pathogen killing and also in epithelial dysfunction due to oxidative damage of resident lung proteins, is enhanced in samples from allergic/infected SP-A(-/-) mice as compared to WT mice. In vitro experiments using purified eosinophils and human SP-A suggest that SP-A limits the release of EPO from Mp-stimulated eosinophils thereby reducing their killing capacity. These findings are the first to demonstrate that although SP-A interferes with eosinophil-mediated biologic clearance of Mp by mediating the interaction of Mp with eosinophils, SP-A simultaneously benefits the airway by limiting inflammation and damage.

Show MeSH
Related in: MedlinePlus