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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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Patients with L. mexicana infection have anti-GIPL antibodies.A. Serum from Mexican patients infected with L. mexicana with localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), or Mexican controls without disease (nl) were analyzed by GIPL ELISA, and mean OD values for groups of 4 individuals are shown with SEM. *, P<0.01 for all pair-wise comparisons. B. Sera from four DCL patients (colored lines) and a Mexican normal control (black line) were bound to L. mexicana axenic amastigotes, with IgG detected using PE-anti-human IgG and flow cytometry. C. Same as B, but showing four LCL sera. D. Same as B, but showing one Philadelphia control serum (black line) and four Mexican endemic control sera (colored lines). E. A comparison of geometric mean fluorescence intensity (GMFI) of DCL, LCL, and control (nl) sera (four per group) labeled with PE anti-human IgG (data from B, C, and D analyses) is shown. Sera are designated A–D for each group of subjects. Data represent 2 experiments using the same sera with similar results.
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pntd-0002224-g009: Patients with L. mexicana infection have anti-GIPL antibodies.A. Serum from Mexican patients infected with L. mexicana with localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), or Mexican controls without disease (nl) were analyzed by GIPL ELISA, and mean OD values for groups of 4 individuals are shown with SEM. *, P<0.01 for all pair-wise comparisons. B. Sera from four DCL patients (colored lines) and a Mexican normal control (black line) were bound to L. mexicana axenic amastigotes, with IgG detected using PE-anti-human IgG and flow cytometry. C. Same as B, but showing four LCL sera. D. Same as B, but showing one Philadelphia control serum (black line) and four Mexican endemic control sera (colored lines). E. A comparison of geometric mean fluorescence intensity (GMFI) of DCL, LCL, and control (nl) sera (four per group) labeled with PE anti-human IgG (data from B, C, and D analyses) is shown. Sera are designated A–D for each group of subjects. Data represent 2 experiments using the same sera with similar results.

Mentions: Human infection with L. mexicana can cause localized cutaneous leishmaniasis (LCL), a disseminated skin disease called diffuse cutaneous leishmaniasis (DCL), and very infrequently visceral disease or mucocutaneous disease. Although high antibody titers are seen in visceral leishmaniasis, antibody titers in cutaneous leishmaniasis are not generally sensitive, with visualization of parasites from skin scrapings or biopsies being the main diagnostic tool [34]. In order to determine if human infection leads to anti-GIPL antibodies, we obtained human sera from Mexican patients with L. mexicana infection and LCL or DCL. We analyzed these sera using the GIPL ELISA and found that patients with LCL, and to a greater extent those with DCL, have antibodies to GIPL molecules (Fig. 9A). Background values from uninfected sera may represent low levels of natural IgG antibodies or non-specific binding. We also bound the sera from DCL and LCL patients to L. mexicana amastigotes and found that IgG was detectible on the parasite surface (Fig. 9B and C), with all uninfected controls (both endemic controls and one random serum sample from Pennsylvania) having lower mean fluorescence (Fig. 9D). Geometric mean fluorescence was significantly greater in DCL and LCL patients than in controls, but these groups were not significantly different from each other (Fig. 9E). Thus L. mexicana-infected people have antibodies that bind the parasite surface and that bind GIPLs in an ELISA format. Similar results were obtained from true L. mexicana amastigotes derived from the IC21 macrophage cell line as with axenic amastigote-like forms (data not shown).


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)

Patients with L. mexicana infection have anti-GIPL antibodies.A. Serum from Mexican patients infected with L. mexicana with localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), or Mexican controls without disease (nl) were analyzed by GIPL ELISA, and mean OD values for groups of 4 individuals are shown with SEM. *, P<0.01 for all pair-wise comparisons. B. Sera from four DCL patients (colored lines) and a Mexican normal control (black line) were bound to L. mexicana axenic amastigotes, with IgG detected using PE-anti-human IgG and flow cytometry. C. Same as B, but showing four LCL sera. D. Same as B, but showing one Philadelphia control serum (black line) and four Mexican endemic control sera (colored lines). E. A comparison of geometric mean fluorescence intensity (GMFI) of DCL, LCL, and control (nl) sera (four per group) labeled with PE anti-human IgG (data from B, C, and D analyses) is shown. Sera are designated A–D for each group of subjects. Data represent 2 experiments using the same sera with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649955&req=5

pntd-0002224-g009: Patients with L. mexicana infection have anti-GIPL antibodies.A. Serum from Mexican patients infected with L. mexicana with localized cutaneous leishmaniasis (LCL), diffuse cutaneous leishmaniasis (DCL), or Mexican controls without disease (nl) were analyzed by GIPL ELISA, and mean OD values for groups of 4 individuals are shown with SEM. *, P<0.01 for all pair-wise comparisons. B. Sera from four DCL patients (colored lines) and a Mexican normal control (black line) were bound to L. mexicana axenic amastigotes, with IgG detected using PE-anti-human IgG and flow cytometry. C. Same as B, but showing four LCL sera. D. Same as B, but showing one Philadelphia control serum (black line) and four Mexican endemic control sera (colored lines). E. A comparison of geometric mean fluorescence intensity (GMFI) of DCL, LCL, and control (nl) sera (four per group) labeled with PE anti-human IgG (data from B, C, and D analyses) is shown. Sera are designated A–D for each group of subjects. Data represent 2 experiments using the same sera with similar results.
Mentions: Human infection with L. mexicana can cause localized cutaneous leishmaniasis (LCL), a disseminated skin disease called diffuse cutaneous leishmaniasis (DCL), and very infrequently visceral disease or mucocutaneous disease. Although high antibody titers are seen in visceral leishmaniasis, antibody titers in cutaneous leishmaniasis are not generally sensitive, with visualization of parasites from skin scrapings or biopsies being the main diagnostic tool [34]. In order to determine if human infection leads to anti-GIPL antibodies, we obtained human sera from Mexican patients with L. mexicana infection and LCL or DCL. We analyzed these sera using the GIPL ELISA and found that patients with LCL, and to a greater extent those with DCL, have antibodies to GIPL molecules (Fig. 9A). Background values from uninfected sera may represent low levels of natural IgG antibodies or non-specific binding. We also bound the sera from DCL and LCL patients to L. mexicana amastigotes and found that IgG was detectible on the parasite surface (Fig. 9B and C), with all uninfected controls (both endemic controls and one random serum sample from Pennsylvania) having lower mean fluorescence (Fig. 9D). Geometric mean fluorescence was significantly greater in DCL and LCL patients than in controls, but these groups were not significantly different from each other (Fig. 9E). Thus L. mexicana-infected people have antibodies that bind the parasite surface and that bind GIPLs in an ELISA format. Similar results were obtained from true L. mexicana amastigotes derived from the IC21 macrophage cell line as with axenic amastigote-like forms (data not shown).

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