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Exogenous pathogen and plant 15-lipoxygenase initiate endogenous lipoxin A4 biosynthesis.

Bannenberg GL, Aliberti J, Hong S, Sher A, Serhan C - J. Exp. Med. (2004)

Bottom Line: Hence, we incubated STAg itself with arachidonic acid and found using LC-UV-MS-MS-based lipidomics that STAg produced both 15-HETE and 5,15-diHETE, indicating that T. gondii carries 15-lipoxygenase activity.Local administration of a plant (soybean) lipoxygenase itself reduced neutrophilic infiltration in murine peritonitis, demonstrating that 15-lipoxygenase possesses antiinflammatory properties.Together, these results indicate that 15-lipoxygenase expressed by a pathogen as well as exogenously administered 15-lipoxygenase can interact with host biosynthetic circuits for endogenous "stop signals" that divert the host immune response and limit acute inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Lipoxin A4 (LXA4) is a potent endogenous lipoxygenase-derived eicosanoid with antiinflammatory and proresolving properties. Supraphysiological levels of LXA4 are generated during infection by Toxoplasma gondii, which in turn reduces interleukin (IL) 12 production by dendritic cells, thus dampening Th1-type cell-mediated immune responses and host immunopathology. In the present work, we sought evidence for the structural basis of T. gondii's ability to activate LXA4 biosynthesis. Proteomic analysis of T. gondii extract (soluble tachyzoite antigen [STAg]), which preserves the immunosuppressive and antiinflammatory activity of the parasite, yielded several peptide matches to known plant lipoxygenases. Hence, we incubated STAg itself with arachidonic acid and found using LC-UV-MS-MS-based lipidomics that STAg produced both 15-HETE and 5,15-diHETE, indicating that T. gondii carries 15-lipoxygenase activity. In addition, T. gondii tachyzoites (the rapidly multiplying and invasive stage of the parasite) generated LXA4 when provided with arachidonic acid. Local administration of a plant (soybean) lipoxygenase itself reduced neutrophilic infiltration in murine peritonitis, demonstrating that 15-lipoxygenase possesses antiinflammatory properties. Administration of plant 15-lipoxygenase generated endogenous LXA4 and mimicked the suppression of IL-12 production by splenic dendritic cells observed after T. gondii infection or STAg administration. Together, these results indicate that 15-lipoxygenase expressed by a pathogen as well as exogenously administered 15-lipoxygenase can interact with host biosynthetic circuits for endogenous "stop signals" that divert the host immune response and limit acute inflammation.

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Lipidomic analysis. Profile of lipoxygenase products formed by STAg extracts. (A) Selected ion chromatogram for 15-HETE, 12-HETE, 5-HETE, and 5,15-diHETE formed by 15 μg STAg incubated with 30 μg arachidonic acid in 0.5 ml DPBS, pH 7.4, for 30 min (see Materials and Methods). Tandem mass spectra for the lipoxygenase products 15-HETE (B) and 5,15-diHETE (C). Results are representative of three independent incubations.
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fig2: Lipidomic analysis. Profile of lipoxygenase products formed by STAg extracts. (A) Selected ion chromatogram for 15-HETE, 12-HETE, 5-HETE, and 5,15-diHETE formed by 15 μg STAg incubated with 30 μg arachidonic acid in 0.5 ml DPBS, pH 7.4, for 30 min (see Materials and Methods). Tandem mass spectra for the lipoxygenase products 15-HETE (B) and 5,15-diHETE (C). Results are representative of three independent incubations.

Mentions: To assess lipoxygenase activity in STAg, 15 μg STAg total protein extract was added to 0.5 ml DPBS containing 0.88 mM CaCl2 and 0.49 mM MgCl2, pH 7.4, with or without 30 μg arachidonic acid (Cayman Chemical). After incubating for 30 min at room temperature, two volumes of ice-cold methanol were added, and samples were stored at −80°C. The samples were extracted using solid phase extraction (reversed phase Extract Clean/RC; Alltech), as described previously (10, 13). In brief, the eicosanoids generated by STAg were identified using liquid chromatography–photodiode array detector–tandem mass spectrometry (ThermoFinnigan). Reverse-phase LC was conducted on a Discovery C18 (100 × 2 mm, particle size 5 μm; Supelco) column at a flow rate of 0.2 ml/min. The mobile phase consisted of methanol/water/acetic acid (65:35:0.01 vol/vol/vol) with an isocratic elution for 8 min, followed by a linear gradient to 100% methanol from 8 to 30 min, and isocratic elution at 100% methanol from 30 to 38 min. Tandem mass spectra were recorded with an electrospray ionization mass spectrometer (LCQ; ThermoFinnigan). The characteristic parent ions for the arachidonic acid–derived lipoxygenase products monoHETE (m/z = 319) and diHETEs (m/z = 335) were monitored, and tandem mass spectra of these ions were recorded. Ions of diagnostic value or signature ions for 5-HETE, 12-HETE, 15-HETE, and 5,15-diHETE were as follows: 5-HETE, m/z = 319 [M-H], 301, 275, 257, 203, and 119; 12-HETE, m/z = 319 [M-H], 301, 275, 257, 207, 179, and 163; 15-HETE, m/z = 319 [M-H], 301, 275, 257, 219, and 204; and 5,15-diHETE, m/z = 335, 317, 299, 291, 273, 253, 235, and 217 (Fig. 2; compare with reference 10).


Exogenous pathogen and plant 15-lipoxygenase initiate endogenous lipoxin A4 biosynthesis.

Bannenberg GL, Aliberti J, Hong S, Sher A, Serhan C - J. Exp. Med. (2004)

Lipidomic analysis. Profile of lipoxygenase products formed by STAg extracts. (A) Selected ion chromatogram for 15-HETE, 12-HETE, 5-HETE, and 5,15-diHETE formed by 15 μg STAg incubated with 30 μg arachidonic acid in 0.5 ml DPBS, pH 7.4, for 30 min (see Materials and Methods). Tandem mass spectra for the lipoxygenase products 15-HETE (B) and 5,15-diHETE (C). Results are representative of three independent incubations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211821&req=5

fig2: Lipidomic analysis. Profile of lipoxygenase products formed by STAg extracts. (A) Selected ion chromatogram for 15-HETE, 12-HETE, 5-HETE, and 5,15-diHETE formed by 15 μg STAg incubated with 30 μg arachidonic acid in 0.5 ml DPBS, pH 7.4, for 30 min (see Materials and Methods). Tandem mass spectra for the lipoxygenase products 15-HETE (B) and 5,15-diHETE (C). Results are representative of three independent incubations.
Mentions: To assess lipoxygenase activity in STAg, 15 μg STAg total protein extract was added to 0.5 ml DPBS containing 0.88 mM CaCl2 and 0.49 mM MgCl2, pH 7.4, with or without 30 μg arachidonic acid (Cayman Chemical). After incubating for 30 min at room temperature, two volumes of ice-cold methanol were added, and samples were stored at −80°C. The samples were extracted using solid phase extraction (reversed phase Extract Clean/RC; Alltech), as described previously (10, 13). In brief, the eicosanoids generated by STAg were identified using liquid chromatography–photodiode array detector–tandem mass spectrometry (ThermoFinnigan). Reverse-phase LC was conducted on a Discovery C18 (100 × 2 mm, particle size 5 μm; Supelco) column at a flow rate of 0.2 ml/min. The mobile phase consisted of methanol/water/acetic acid (65:35:0.01 vol/vol/vol) with an isocratic elution for 8 min, followed by a linear gradient to 100% methanol from 8 to 30 min, and isocratic elution at 100% methanol from 30 to 38 min. Tandem mass spectra were recorded with an electrospray ionization mass spectrometer (LCQ; ThermoFinnigan). The characteristic parent ions for the arachidonic acid–derived lipoxygenase products monoHETE (m/z = 319) and diHETEs (m/z = 335) were monitored, and tandem mass spectra of these ions were recorded. Ions of diagnostic value or signature ions for 5-HETE, 12-HETE, 15-HETE, and 5,15-diHETE were as follows: 5-HETE, m/z = 319 [M-H], 301, 275, 257, 203, and 119; 12-HETE, m/z = 319 [M-H], 301, 275, 257, 207, 179, and 163; 15-HETE, m/z = 319 [M-H], 301, 275, 257, 219, and 204; and 5,15-diHETE, m/z = 335, 317, 299, 291, 273, 253, 235, and 217 (Fig. 2; compare with reference 10).

Bottom Line: Hence, we incubated STAg itself with arachidonic acid and found using LC-UV-MS-MS-based lipidomics that STAg produced both 15-HETE and 5,15-diHETE, indicating that T. gondii carries 15-lipoxygenase activity.Local administration of a plant (soybean) lipoxygenase itself reduced neutrophilic infiltration in murine peritonitis, demonstrating that 15-lipoxygenase possesses antiinflammatory properties.Together, these results indicate that 15-lipoxygenase expressed by a pathogen as well as exogenously administered 15-lipoxygenase can interact with host biosynthetic circuits for endogenous "stop signals" that divert the host immune response and limit acute inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Lipoxin A4 (LXA4) is a potent endogenous lipoxygenase-derived eicosanoid with antiinflammatory and proresolving properties. Supraphysiological levels of LXA4 are generated during infection by Toxoplasma gondii, which in turn reduces interleukin (IL) 12 production by dendritic cells, thus dampening Th1-type cell-mediated immune responses and host immunopathology. In the present work, we sought evidence for the structural basis of T. gondii's ability to activate LXA4 biosynthesis. Proteomic analysis of T. gondii extract (soluble tachyzoite antigen [STAg]), which preserves the immunosuppressive and antiinflammatory activity of the parasite, yielded several peptide matches to known plant lipoxygenases. Hence, we incubated STAg itself with arachidonic acid and found using LC-UV-MS-MS-based lipidomics that STAg produced both 15-HETE and 5,15-diHETE, indicating that T. gondii carries 15-lipoxygenase activity. In addition, T. gondii tachyzoites (the rapidly multiplying and invasive stage of the parasite) generated LXA4 when provided with arachidonic acid. Local administration of a plant (soybean) lipoxygenase itself reduced neutrophilic infiltration in murine peritonitis, demonstrating that 15-lipoxygenase possesses antiinflammatory properties. Administration of plant 15-lipoxygenase generated endogenous LXA4 and mimicked the suppression of IL-12 production by splenic dendritic cells observed after T. gondii infection or STAg administration. Together, these results indicate that 15-lipoxygenase expressed by a pathogen as well as exogenously administered 15-lipoxygenase can interact with host biosynthetic circuits for endogenous "stop signals" that divert the host immune response and limit acute inflammation.

Show MeSH
Related in: MedlinePlus