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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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IgG recognizes a single GIPL spot on TLC immunoblot.A. Purified GIPLs [2×108 cell equivalents (c.e.)] from L. mexicana amastigotes (lanes 1 and 4), L. mexicana stationary-phase promastigotes (lanes 2 and 5), and L. major stationary-phase promastigotes (lanes 3 and 6) were separated by TLC. Lanes 1–3 were stained by immunoblot with serum from L. mexicana-infected mice (from 29 wks). Lanes 4–6 were stained with orcinol to visualize GIPLs. O, origin; F, front; GIPL, the GIPL recognized by anti-serum. B. Immunoblot as in A: L. mexicana lesion amastigote GIPL (lane 1, 2×108 c.e.), L. mexicana axenic amastigote GIPL (lane 2, 108 c.e.). Data represent at least 12 experiments using 4 different sera, with similar results.
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pntd-0002224-g005: IgG recognizes a single GIPL spot on TLC immunoblot.A. Purified GIPLs [2×108 cell equivalents (c.e.)] from L. mexicana amastigotes (lanes 1 and 4), L. mexicana stationary-phase promastigotes (lanes 2 and 5), and L. major stationary-phase promastigotes (lanes 3 and 6) were separated by TLC. Lanes 1–3 were stained by immunoblot with serum from L. mexicana-infected mice (from 29 wks). Lanes 4–6 were stained with orcinol to visualize GIPLs. O, origin; F, front; GIPL, the GIPL recognized by anti-serum. B. Immunoblot as in A: L. mexicana lesion amastigote GIPL (lane 1, 2×108 c.e.), L. mexicana axenic amastigote GIPL (lane 2, 108 c.e.). Data represent at least 12 experiments using 4 different sera, with similar results.

Mentions: Next we separated the GIPLs by thin layer chromatography (TLC) and probed with anti-sera from L. mexicana-infected mice. We found that a single spot was recognized in L. mexicana axenic amastigote GIPL extract and to a much lesser extent in stationary-phase promastigotes (Fig. 5). L. mexicana-infected mouse serum did not recognize GIPLs from promastigotes of L. major (Fig. 5A), nor GIPLs from L. major amastigotes derived from IC-21 cells (data not shown). Uninfected mouse serum gave no binding to amastigote GIPLs in several similar experiments (data not shown). Staining with orcinol also demonstrated the strongly recognized GIPL molecule(s) in addition to other spots (Fig. 5A). These additional spots are also glycolipids, but they are not recognized by antibodies in the infected mouse sera. On immunoblot, GIPLs from lesion amastigotes had similar reactivity to GIPLs from axenic amastigotes (Fig. 5B).


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)

IgG recognizes a single GIPL spot on TLC immunoblot.A. Purified GIPLs [2×108 cell equivalents (c.e.)] from L. mexicana amastigotes (lanes 1 and 4), L. mexicana stationary-phase promastigotes (lanes 2 and 5), and L. major stationary-phase promastigotes (lanes 3 and 6) were separated by TLC. Lanes 1–3 were stained by immunoblot with serum from L. mexicana-infected mice (from 29 wks). Lanes 4–6 were stained with orcinol to visualize GIPLs. O, origin; F, front; GIPL, the GIPL recognized by anti-serum. B. Immunoblot as in A: L. mexicana lesion amastigote GIPL (lane 1, 2×108 c.e.), L. mexicana axenic amastigote GIPL (lane 2, 108 c.e.). Data represent at least 12 experiments using 4 different sera, with similar results.
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Related In: Results  -  Collection

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

pntd-0002224-g005: IgG recognizes a single GIPL spot on TLC immunoblot.A. Purified GIPLs [2×108 cell equivalents (c.e.)] from L. mexicana amastigotes (lanes 1 and 4), L. mexicana stationary-phase promastigotes (lanes 2 and 5), and L. major stationary-phase promastigotes (lanes 3 and 6) were separated by TLC. Lanes 1–3 were stained by immunoblot with serum from L. mexicana-infected mice (from 29 wks). Lanes 4–6 were stained with orcinol to visualize GIPLs. O, origin; F, front; GIPL, the GIPL recognized by anti-serum. B. Immunoblot as in A: L. mexicana lesion amastigote GIPL (lane 1, 2×108 c.e.), L. mexicana axenic amastigote GIPL (lane 2, 108 c.e.). Data represent at least 12 experiments using 4 different sera, with similar results.
Mentions: Next we separated the GIPLs by thin layer chromatography (TLC) and probed with anti-sera from L. mexicana-infected mice. We found that a single spot was recognized in L. mexicana axenic amastigote GIPL extract and to a much lesser extent in stationary-phase promastigotes (Fig. 5). L. mexicana-infected mouse serum did not recognize GIPLs from promastigotes of L. major (Fig. 5A), nor GIPLs from L. major amastigotes derived from IC-21 cells (data not shown). Uninfected mouse serum gave no binding to amastigote GIPLs in several similar experiments (data not shown). Staining with orcinol also demonstrated the strongly recognized GIPL molecule(s) in addition to other spots (Fig. 5A). These additional spots are also glycolipids, but they are not recognized by antibodies in the infected mouse sera. On immunoblot, GIPLs from lesion amastigotes had similar reactivity to GIPLs from axenic amastigotes (Fig. 5B).

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