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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

Lactoferrin co-migrates with SEA.(A) BALF-SEA was resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with anti-lactoferrin and anti-SEA antibodies as described in the Materials and Methods. Purified Lactoferrin and SEA (30 and 10 ng) were loaded in parallel to serve as positive controls. Specificity of the antibody was assessed using isotype control. The data are representative of 2 (lactoferrin) and 3 (SEA) independent experiments. (B) 16 h BALF-SEA was directly fractionated by cation exchange chromatography. Fractions were tested in a bioassay and immunoblotted for lactoferrin (inset). Chromatogram of protein absorption at 280nM (top panel) and bar graphs of IL-2 secretion (bottom panel) are shown. Representative of 1 out 3 experiments is shown. (C) BALF from naïve mice, and BALF from mice having received a time course of i.n. SEA and BSS were immunoblotted with anti-lactoferrin antibodies and 3 experiments are shown.
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pone.0141548.g005: Lactoferrin co-migrates with SEA.(A) BALF-SEA was resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with anti-lactoferrin and anti-SEA antibodies as described in the Materials and Methods. Purified Lactoferrin and SEA (30 and 10 ng) were loaded in parallel to serve as positive controls. Specificity of the antibody was assessed using isotype control. The data are representative of 2 (lactoferrin) and 3 (SEA) independent experiments. (B) 16 h BALF-SEA was directly fractionated by cation exchange chromatography. Fractions were tested in a bioassay and immunoblotted for lactoferrin (inset). Chromatogram of protein absorption at 280nM (top panel) and bar graphs of IL-2 secretion (bottom panel) are shown. Representative of 1 out 3 experiments is shown. (C) BALF from naïve mice, and BALF from mice having received a time course of i.n. SEA and BSS were immunoblotted with anti-lactoferrin antibodies and 3 experiments are shown.

Mentions: Lactoferrin was also detected in BALF-SEA which seemed to co-migrate with SEA since it was not detected by mass spectrometry in BALF-BSS (Table 1). It has been shown previously that lactoferrin can impede inflammation [24], attenuate SEB activity [25] and therefore we hypothesized it could also affect SEA activity. Immunoblotting showed that lactoferrin was present at high levels in BALF-SEA while SEA was undetectable (Fig 5a). To test if in BALF-SEA lactoferrin and SEA co-migrated, cation exchange chromatography was used and elution occurred between 1.03 M and 1.3 M NaCl (Fig 5b, top panel). Specifically, IL-2-inducing activity was found precisely where lactoferrin was detected (Fig 5b, bottom panel). Thus, lactoferrin may function as an innate factor. Lactoferrin was present in BALF-SEA and -BSS through a time course, and also detectable in BALF from naïve mice (Fig 5c). Thus, SEA fractionates at least with a portion or subspecies of lactoferrin. In sum, lactoferrin is in BALF-BSS and BALF-SEA before protein fractionation, but after fractionation a sub fraction of lactoferrin co-migrates with SEA from BALF-SEA but not in the equivalent fraction from BALF-BSS.


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)

Lactoferrin co-migrates with SEA.(A) BALF-SEA was resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with anti-lactoferrin and anti-SEA antibodies as described in the Materials and Methods. Purified Lactoferrin and SEA (30 and 10 ng) were loaded in parallel to serve as positive controls. Specificity of the antibody was assessed using isotype control. The data are representative of 2 (lactoferrin) and 3 (SEA) independent experiments. (B) 16 h BALF-SEA was directly fractionated by cation exchange chromatography. Fractions were tested in a bioassay and immunoblotted for lactoferrin (inset). Chromatogram of protein absorption at 280nM (top panel) and bar graphs of IL-2 secretion (bottom panel) are shown. Representative of 1 out 3 experiments is shown. (C) BALF from naïve mice, and BALF from mice having received a time course of i.n. SEA and BSS were immunoblotted with anti-lactoferrin antibodies and 3 experiments are shown.
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pone.0141548.g005: Lactoferrin co-migrates with SEA.(A) BALF-SEA was resolved by 4–15% gradient SDS-PAGE under reducing and denaturing conditions, transferred to nitrocellulose membrane and probed with anti-lactoferrin and anti-SEA antibodies as described in the Materials and Methods. Purified Lactoferrin and SEA (30 and 10 ng) were loaded in parallel to serve as positive controls. Specificity of the antibody was assessed using isotype control. The data are representative of 2 (lactoferrin) and 3 (SEA) independent experiments. (B) 16 h BALF-SEA was directly fractionated by cation exchange chromatography. Fractions were tested in a bioassay and immunoblotted for lactoferrin (inset). Chromatogram of protein absorption at 280nM (top panel) and bar graphs of IL-2 secretion (bottom panel) are shown. Representative of 1 out 3 experiments is shown. (C) BALF from naïve mice, and BALF from mice having received a time course of i.n. SEA and BSS were immunoblotted with anti-lactoferrin antibodies and 3 experiments are shown.
Mentions: Lactoferrin was also detected in BALF-SEA which seemed to co-migrate with SEA since it was not detected by mass spectrometry in BALF-BSS (Table 1). It has been shown previously that lactoferrin can impede inflammation [24], attenuate SEB activity [25] and therefore we hypothesized it could also affect SEA activity. Immunoblotting showed that lactoferrin was present at high levels in BALF-SEA while SEA was undetectable (Fig 5a). To test if in BALF-SEA lactoferrin and SEA co-migrated, cation exchange chromatography was used and elution occurred between 1.03 M and 1.3 M NaCl (Fig 5b, top panel). Specifically, IL-2-inducing activity was found precisely where lactoferrin was detected (Fig 5b, bottom panel). Thus, lactoferrin may function as an innate factor. Lactoferrin was present in BALF-SEA and -BSS through a time course, and also detectable in BALF from naïve mice (Fig 5c). Thus, SEA fractionates at least with a portion or subspecies of lactoferrin. In sum, lactoferrin is in BALF-BSS and BALF-SEA before protein fractionation, but after fractionation a sub fraction of lactoferrin co-migrates with SEA from BALF-SEA but not in the equivalent fraction from BALF-BSS.

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