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Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

Ménoret A, Svedova J, Behl B, Vella AT - PLoS ONE (2015)

Bottom Line: Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis.Higher doses of lactoferrin were required to inhibit effector compared to resting T cells.These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology MC3710. University of Connecticut Health, 263 Farmington Avenue, Farmington, CT 06032, United States of America.

ABSTRACT
Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

No MeSH data available.


Related in: MedlinePlus

The inflammatory activity induced by BALF-SEA is T cell dependent.BALFs obtained from mice 16 h after i.n. SEA and BSS were tested in a bioassay using C57BL/6 splenocytes (top panel) and TCR β/γ-/- splenocytes (bottom panel) and supernatants were assayed for IFN-γ (A) and IL-2 (B). Bar graphs show cytokines secretion measured in triplicate with n = 3. The data are representative of 3 independent experiments. Statistical significance between BALF-BSS and BALF-SEA was evaluated by two-tailed Student’s t tests. The error bars indicate the standard error of the mean between triplicates.
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pone.0141548.g002: The inflammatory activity induced by BALF-SEA is T cell dependent.BALFs obtained from mice 16 h after i.n. SEA and BSS were tested in a bioassay using C57BL/6 splenocytes (top panel) and TCR β/γ-/- splenocytes (bottom panel) and supernatants were assayed for IFN-γ (A) and IL-2 (B). Bar graphs show cytokines secretion measured in triplicate with n = 3. The data are representative of 3 independent experiments. Statistical significance between BALF-BSS and BALF-SEA was evaluated by two-tailed Student’s t tests. The error bars indicate the standard error of the mean between triplicates.

Mentions: BAL was collected by lavage of mouse lung with sterile PBS. BAL was centrifuged at 1,000 rpm at 4°C to separate cells from BALF and 0.2 micron filtered as described earlier [18]. The immunostimulatory activity contained in the BALF was measured using a bioassay adapted from a technique used to measure IL-18 bioactivity [19]. Briefly, RBC depleted mouse splenocytes were incubated overnight with IL-12 (2 ng/ml) in Figs 1 and 2 only, washed and incubated in tissue culture medium without IL-12. After 5 h, splenocytes were seeded in 96-well flat-bottom plates (2 x 106 per well in triplicate) and stimulated with 20 μlBALF from SEA or vehicle (alone) exposed mice for 16–18 h. Supernatants were measured for IL-2 and IFN-γ levels by ELISA.


Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

Ménoret A, Svedova J, Behl B, Vella AT - PLoS ONE (2015)

The inflammatory activity induced by BALF-SEA is T cell dependent.BALFs obtained from mice 16 h after i.n. SEA and BSS were tested in a bioassay using C57BL/6 splenocytes (top panel) and TCR β/γ-/- splenocytes (bottom panel) and supernatants were assayed for IFN-γ (A) and IL-2 (B). Bar graphs show cytokines secretion measured in triplicate with n = 3. The data are representative of 3 independent experiments. Statistical significance between BALF-BSS and BALF-SEA was evaluated by two-tailed Student’s t tests. The error bars indicate the standard error of the mean between triplicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4625020&req=5

pone.0141548.g002: The inflammatory activity induced by BALF-SEA is T cell dependent.BALFs obtained from mice 16 h after i.n. SEA and BSS were tested in a bioassay using C57BL/6 splenocytes (top panel) and TCR β/γ-/- splenocytes (bottom panel) and supernatants were assayed for IFN-γ (A) and IL-2 (B). Bar graphs show cytokines secretion measured in triplicate with n = 3. The data are representative of 3 independent experiments. Statistical significance between BALF-BSS and BALF-SEA was evaluated by two-tailed Student’s t tests. The error bars indicate the standard error of the mean between triplicates.
Mentions: BAL was collected by lavage of mouse lung with sterile PBS. BAL was centrifuged at 1,000 rpm at 4°C to separate cells from BALF and 0.2 micron filtered as described earlier [18]. The immunostimulatory activity contained in the BALF was measured using a bioassay adapted from a technique used to measure IL-18 bioactivity [19]. Briefly, RBC depleted mouse splenocytes were incubated overnight with IL-12 (2 ng/ml) in Figs 1 and 2 only, washed and incubated in tissue culture medium without IL-12. After 5 h, splenocytes were seeded in 96-well flat-bottom plates (2 x 106 per well in triplicate) and stimulated with 20 μlBALF from SEA or vehicle (alone) exposed mice for 16–18 h. Supernatants were measured for IL-2 and IFN-γ levels by ELISA.

Bottom Line: Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis.Higher doses of lactoferrin were required to inhibit effector compared to resting T cells.These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology MC3710. University of Connecticut Health, 263 Farmington Avenue, Farmington, CT 06032, United States of America.

ABSTRACT
Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

No MeSH data available.


Related in: MedlinePlus