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Leishmania mexicana infection induces IgG to parasite surface glycoinositol phospholipids that can induce IL-10 in mice and humans.

Buxbaum LU - PLoS Negl Trop Dis (2013)

Bottom Line: These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control.Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG.Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

View Article: PubMed Central - PubMed

Affiliation: Philadelphia Research and Education Foundation, Philadelphia, Pennsylvania, United States of America. buxbaum17@hotmail.com

ABSTRACT
Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (10(7)-10(8) parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

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Phospholipase A2 abolishes IgG binding to the dominant GIPL.L. mexicana amastigote GIPLs (2×108 cell equivalents) were digested with bee venom phospholipase A2 (PLA2) and then partitioned into water-saturated n-butanol and water. The aqueous phase was purified by Sep-pak C18 column as described in the Materials and Methods. Samples were separated by TLC, and an immunoblot was performed as in Fig. 5. Lane 1, untreated L. mexicana amastigote GIPLs; 2, aqueous phase of mock digest; 3, n-butanol phase of mock digest; 4, aqueous phase of PLA2 digest; 5, n-butanol phase of PLA2 digest. The sample in lanes 4 and 5 was not digested to completion. Data represent 4 experiments with similar results.
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pntd-0002224-g007: Phospholipase A2 abolishes IgG binding to the dominant GIPL.L. mexicana amastigote GIPLs (2×108 cell equivalents) were digested with bee venom phospholipase A2 (PLA2) and then partitioned into water-saturated n-butanol and water. The aqueous phase was purified by Sep-pak C18 column as described in the Materials and Methods. Samples were separated by TLC, and an immunoblot was performed as in Fig. 5. Lane 1, untreated L. mexicana amastigote GIPLs; 2, aqueous phase of mock digest; 3, n-butanol phase of mock digest; 4, aqueous phase of PLA2 digest; 5, n-butanol phase of PLA2 digest. The sample in lanes 4 and 5 was not digested to completion. Data represent 4 experiments with similar results.

Mentions: To help determine if the lipid portion of the GIPL is required for antibody binding, we digested L. mexicana amastigote GIPLs with bee venom phospholipase A2 (PLA2), which specifically cleaves the sn-2 fatty acid from glycolipids. Binding of the immunodominant GIPL by antibodies was greatly diminished by PLA2 digestion (Fig. 7), although digestion was incomplete. As lyso-glycolipids (ones with only one fatty acid or fatty alkyl group and a free hydroxyl) are significantly less hydrophobic than ones with two lipid moieties, and depending on the hydrophobicity/chain length of the lipids, can be lost in the aqueous phase during butanol-water partitioning [31], we isolated the glycolipids from the aqueous phase using a Sep-pak C18 reverse phase syringe column [31]. Despite this maneuver, there was no antibody reactivity in the aqueous phase. This indicated either that the fatty acid itself is involved in binding, or at least that the conformation of the binding site on the glycolipid depends on the sn-2 fatty acid.


Leishmania mexicana infection induces IgG to parasite surface glycoinositol phospholipids that can induce IL-10 in mice and humans.

Buxbaum LU - PLoS Negl Trop Dis (2013)

Phospholipase A2 abolishes IgG binding to the dominant GIPL.L. mexicana amastigote GIPLs (2×108 cell equivalents) were digested with bee venom phospholipase A2 (PLA2) and then partitioned into water-saturated n-butanol and water. The aqueous phase was purified by Sep-pak C18 column as described in the Materials and Methods. Samples were separated by TLC, and an immunoblot was performed as in Fig. 5. Lane 1, untreated L. mexicana amastigote GIPLs; 2, aqueous phase of mock digest; 3, n-butanol phase of mock digest; 4, aqueous phase of PLA2 digest; 5, n-butanol phase of PLA2 digest. The sample in lanes 4 and 5 was not digested to completion. Data represent 4 experiments with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649955&req=5

pntd-0002224-g007: Phospholipase A2 abolishes IgG binding to the dominant GIPL.L. mexicana amastigote GIPLs (2×108 cell equivalents) were digested with bee venom phospholipase A2 (PLA2) and then partitioned into water-saturated n-butanol and water. The aqueous phase was purified by Sep-pak C18 column as described in the Materials and Methods. Samples were separated by TLC, and an immunoblot was performed as in Fig. 5. Lane 1, untreated L. mexicana amastigote GIPLs; 2, aqueous phase of mock digest; 3, n-butanol phase of mock digest; 4, aqueous phase of PLA2 digest; 5, n-butanol phase of PLA2 digest. The sample in lanes 4 and 5 was not digested to completion. Data represent 4 experiments with similar results.
Mentions: To help determine if the lipid portion of the GIPL is required for antibody binding, we digested L. mexicana amastigote GIPLs with bee venom phospholipase A2 (PLA2), which specifically cleaves the sn-2 fatty acid from glycolipids. Binding of the immunodominant GIPL by antibodies was greatly diminished by PLA2 digestion (Fig. 7), although digestion was incomplete. As lyso-glycolipids (ones with only one fatty acid or fatty alkyl group and a free hydroxyl) are significantly less hydrophobic than ones with two lipid moieties, and depending on the hydrophobicity/chain length of the lipids, can be lost in the aqueous phase during butanol-water partitioning [31], we isolated the glycolipids from the aqueous phase using a Sep-pak C18 reverse phase syringe column [31]. Despite this maneuver, there was no antibody reactivity in the aqueous phase. This indicated either that the fatty acid itself is involved in binding, or at least that the conformation of the binding site on the glycolipid depends on the sn-2 fatty acid.

Bottom Line: These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control.Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG.Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

View Article: PubMed Central - PubMed

Affiliation: Philadelphia Research and Education Foundation, Philadelphia, Pennsylvania, United States of America. buxbaum17@hotmail.com

ABSTRACT
Infection with the intracellular protozoan parasite Leishmania mexicana causes chronic disease in C57BL/6 mice, in which cutaneous lesions persist for many months with high parasite burdens (10(7)-10(8) parasites). This chronic disease process requires host IL-10 and FcγRIII. When Leishmania amastigotes are released from cells, surface-bound IgG can induce IL-10 and suppress IL-12 production from macrophages. These changes decrease IFN-γ from T cells and nitric oxide production in infected cells, which are both required for Leishmania control. However, antibodies targets and the kinetics of antibody production are unknown. Several groups have been unsuccessful in identifying amastigote surface proteins that bind IgG. We now show that glycoinositol phospholipids (GIPLs) of L. mexicana are recognized by mouse IgG1 by 6 weeks of infection, with a rapid increase between 12 and 16 weeks, consistent with the timing of chronic disease in C57BL/6 mice vs. healing in FcγRIII-deficient mice. A single prominent spot on TLC is recognized by IgG, and the glycolipid is a glycosyl phosphatidylinositol containing a branched mannose structure. We show that the lipid structure of the GIPL (the sn-2 fatty acid) is required for antibody recognition. This GIPL is abundant in L. mexicana amastigotes, rare in stationary-phase promastigotes, and absent in L. major, consistent with a role for antibodies to GIPLs in chronic disease. A mouse monoclonal anti-GIPL IgG recognizes GIPLs on the parasite surface, and induces IL-10 from macrophages. The current work also extends this mouse analysis to humans, finding that L. mexicana-infected humans with localized and diffuse cutaneous leishmaniasis have antibodies that recognize GIPLs, can bind to the surface of amastigotes, and can induce IL-10 from human monocytes. Further characterization of the target glycolipids will have important implications for drug and vaccine development and will elucidate the poorly understood role of glycolipids in the immunology of infections.

Show MeSH
Related in: MedlinePlus