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Bee venom processes human skin lipids for presentation by CD1a.

Bourgeois EA, Subramaniam S, Cheng TY, De Jong A, Layre E, Ly D, Salimi M, Legaspi A, Modlin RL, Salio M, Cerundolo V, Moody DB, Ogg G - J. Exp. Med. (2015)

Bottom Line: Patient studies showed that injected PLA2 generates lysophospholipids within human skin in vivo, and polyclonal T cell responses are dependent on CD1a protein and PLA2.These findings support a previously unknown skin immune response based on T cell recognition of CD1a proteins and lipid neoantigen generated in vivo by phospholipases.The findings have implications for skin barrier sensing by T cells and mechanisms underlying phospholipase-dependent inflammatory skin disease.

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Affiliation: Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, 02114.

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Wasp venom phospholipase is active in vivo in the skin. (A) Biotinylated CD1a proteins were coated on streptavidin plates. After washing, lipids were added at 10 µg/ml for 24–48 h. Lipids tested were as follows: phosphatidylcholine 18:1 (PC18:1), lysophosphatidylcholine 18:1 (LPC18:1), and oleic acid 18:1 (FA 18:1; left) or phosphatidic acid 16:0 (PA16:0), lysophosphatidic acid 18:1 (LPA18:1), and palmitic acid 16:0 (FA16:0; right). Phosphatidylcholine and lysophosphatidic acid were preincubated or not with PLA2 at 50 µg/ml for 1 h at 37°C. 24 h later, wells were washed and BC2 cells were added. Supernatants were collected after 24 h. IFN-γ in supernatants was measured by ELISA. Data are representative of three separate experiments. (B) Healthy donor epidermis was injected with 10 µg of wasp venom or saline. Skin blisters were raised and, 24 h later, the blister fluid was sampled. Lipids were extracted with chloroform, methanol, and water using the Bligh-Dyer method and analyzed on a quadruple time of flight mass spectrometer. Positive mode EIC-MS detected ions at m/z 758.5686, 786.6000, 810.5994, and 834.5993, corresponding to the homologous series of phosphatidylcholine C42H81NO8P+ (16:0/18:2), C44H85NO8P+ (18:0/18:2), C46H85NO8P+ (18:2/20:2), and C48H85NO8P+ (20:4/20:2). Positive mode EIC-MS detected ions at m/z 496.3374, 524.3683, and 544.3370, corresponding to the homologous series of lysophosphatidylcholine C24H51NO7P+ (16:0), C26H55NO7P+ (18:0), and C28H51NO7P+ (20:4). Data from one representative donor out of two are shown.
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fig5: Wasp venom phospholipase is active in vivo in the skin. (A) Biotinylated CD1a proteins were coated on streptavidin plates. After washing, lipids were added at 10 µg/ml for 24–48 h. Lipids tested were as follows: phosphatidylcholine 18:1 (PC18:1), lysophosphatidylcholine 18:1 (LPC18:1), and oleic acid 18:1 (FA 18:1; left) or phosphatidic acid 16:0 (PA16:0), lysophosphatidic acid 18:1 (LPA18:1), and palmitic acid 16:0 (FA16:0; right). Phosphatidylcholine and lysophosphatidic acid were preincubated or not with PLA2 at 50 µg/ml for 1 h at 37°C. 24 h later, wells were washed and BC2 cells were added. Supernatants were collected after 24 h. IFN-γ in supernatants was measured by ELISA. Data are representative of three separate experiments. (B) Healthy donor epidermis was injected with 10 µg of wasp venom or saline. Skin blisters were raised and, 24 h later, the blister fluid was sampled. Lipids were extracted with chloroform, methanol, and water using the Bligh-Dyer method and analyzed on a quadruple time of flight mass spectrometer. Positive mode EIC-MS detected ions at m/z 758.5686, 786.6000, 810.5994, and 834.5993, corresponding to the homologous series of phosphatidylcholine C42H81NO8P+ (16:0/18:2), C44H85NO8P+ (18:0/18:2), C46H85NO8P+ (18:2/20:2), and C48H85NO8P+ (20:4/20:2). Positive mode EIC-MS detected ions at m/z 496.3374, 524.3683, and 544.3370, corresponding to the homologous series of lysophosphatidylcholine C24H51NO7P+ (16:0), C26H55NO7P+ (18:0), and C28H51NO7P+ (20:4). Data from one representative donor out of two are shown.

Mentions: To directly test the hypothesis that bee venom PLA2 acts by cleaving intact cellular phospholipids to create neoantigens, we preincubated the enzyme with two synthetic substrates for bee venom PLA2, phosphatidylcholine comprised of singly unsaturated C18 fatty acyl chains (PC 18:1/18:1) and phosphatidic acid with C16 fatty acyl chain in sn-1 and unsaturated C18 fatty acyl chain in sn-2 (PA 16:0/18:1). We also tested for a T cell response to products of cleavage by PLA2 and control lipids (Fig. 5 A), including purified free fatty acids and lysophospholipids: lysophosphatidylcholine (LPC 18:1), oleic acid (FA 18:1), lysophosphatidic acid (LPA 18:1), and palmitic acid (FA 16:0). Higher production of IFN-γ was obtained in response to phospholipids when preincubated with PLA2. BC2 T cells responded to fatty acids to a greater extent than intact phospholipids or lysophospholipids. This result suggests that PLA2 activates CD1a-restricted T cells by cleaving nonantigenic phospholipids into lysophospholipids and antigenic fatty acid, in agreement with a recent study identifying free fatty acids as CD1-presented antigens (de Jong et al., 2014).


Bee venom processes human skin lipids for presentation by CD1a.

Bourgeois EA, Subramaniam S, Cheng TY, De Jong A, Layre E, Ly D, Salimi M, Legaspi A, Modlin RL, Salio M, Cerundolo V, Moody DB, Ogg G - J. Exp. Med. (2015)

Wasp venom phospholipase is active in vivo in the skin. (A) Biotinylated CD1a proteins were coated on streptavidin plates. After washing, lipids were added at 10 µg/ml for 24–48 h. Lipids tested were as follows: phosphatidylcholine 18:1 (PC18:1), lysophosphatidylcholine 18:1 (LPC18:1), and oleic acid 18:1 (FA 18:1; left) or phosphatidic acid 16:0 (PA16:0), lysophosphatidic acid 18:1 (LPA18:1), and palmitic acid 16:0 (FA16:0; right). Phosphatidylcholine and lysophosphatidic acid were preincubated or not with PLA2 at 50 µg/ml for 1 h at 37°C. 24 h later, wells were washed and BC2 cells were added. Supernatants were collected after 24 h. IFN-γ in supernatants was measured by ELISA. Data are representative of three separate experiments. (B) Healthy donor epidermis was injected with 10 µg of wasp venom or saline. Skin blisters were raised and, 24 h later, the blister fluid was sampled. Lipids were extracted with chloroform, methanol, and water using the Bligh-Dyer method and analyzed on a quadruple time of flight mass spectrometer. Positive mode EIC-MS detected ions at m/z 758.5686, 786.6000, 810.5994, and 834.5993, corresponding to the homologous series of phosphatidylcholine C42H81NO8P+ (16:0/18:2), C44H85NO8P+ (18:0/18:2), C46H85NO8P+ (18:2/20:2), and C48H85NO8P+ (20:4/20:2). Positive mode EIC-MS detected ions at m/z 496.3374, 524.3683, and 544.3370, corresponding to the homologous series of lysophosphatidylcholine C24H51NO7P+ (16:0), C26H55NO7P+ (18:0), and C28H51NO7P+ (20:4). Data from one representative donor out of two are shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Wasp venom phospholipase is active in vivo in the skin. (A) Biotinylated CD1a proteins were coated on streptavidin plates. After washing, lipids were added at 10 µg/ml for 24–48 h. Lipids tested were as follows: phosphatidylcholine 18:1 (PC18:1), lysophosphatidylcholine 18:1 (LPC18:1), and oleic acid 18:1 (FA 18:1; left) or phosphatidic acid 16:0 (PA16:0), lysophosphatidic acid 18:1 (LPA18:1), and palmitic acid 16:0 (FA16:0; right). Phosphatidylcholine and lysophosphatidic acid were preincubated or not with PLA2 at 50 µg/ml for 1 h at 37°C. 24 h later, wells were washed and BC2 cells were added. Supernatants were collected after 24 h. IFN-γ in supernatants was measured by ELISA. Data are representative of three separate experiments. (B) Healthy donor epidermis was injected with 10 µg of wasp venom or saline. Skin blisters were raised and, 24 h later, the blister fluid was sampled. Lipids were extracted with chloroform, methanol, and water using the Bligh-Dyer method and analyzed on a quadruple time of flight mass spectrometer. Positive mode EIC-MS detected ions at m/z 758.5686, 786.6000, 810.5994, and 834.5993, corresponding to the homologous series of phosphatidylcholine C42H81NO8P+ (16:0/18:2), C44H85NO8P+ (18:0/18:2), C46H85NO8P+ (18:2/20:2), and C48H85NO8P+ (20:4/20:2). Positive mode EIC-MS detected ions at m/z 496.3374, 524.3683, and 544.3370, corresponding to the homologous series of lysophosphatidylcholine C24H51NO7P+ (16:0), C26H55NO7P+ (18:0), and C28H51NO7P+ (20:4). Data from one representative donor out of two are shown.
Mentions: To directly test the hypothesis that bee venom PLA2 acts by cleaving intact cellular phospholipids to create neoantigens, we preincubated the enzyme with two synthetic substrates for bee venom PLA2, phosphatidylcholine comprised of singly unsaturated C18 fatty acyl chains (PC 18:1/18:1) and phosphatidic acid with C16 fatty acyl chain in sn-1 and unsaturated C18 fatty acyl chain in sn-2 (PA 16:0/18:1). We also tested for a T cell response to products of cleavage by PLA2 and control lipids (Fig. 5 A), including purified free fatty acids and lysophospholipids: lysophosphatidylcholine (LPC 18:1), oleic acid (FA 18:1), lysophosphatidic acid (LPA 18:1), and palmitic acid (FA 16:0). Higher production of IFN-γ was obtained in response to phospholipids when preincubated with PLA2. BC2 T cells responded to fatty acids to a greater extent than intact phospholipids or lysophospholipids. This result suggests that PLA2 activates CD1a-restricted T cells by cleaving nonantigenic phospholipids into lysophospholipids and antigenic fatty acid, in agreement with a recent study identifying free fatty acids as CD1-presented antigens (de Jong et al., 2014).

Bottom Line: Patient studies showed that injected PLA2 generates lysophospholipids within human skin in vivo, and polyclonal T cell responses are dependent on CD1a protein and PLA2.These findings support a previously unknown skin immune response based on T cell recognition of CD1a proteins and lipid neoantigen generated in vivo by phospholipases.The findings have implications for skin barrier sensing by T cells and mechanisms underlying phospholipase-dependent inflammatory skin disease.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, 02114.

Show MeSH
Related in: MedlinePlus