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Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Beattie L, Peltan A, Maroof A, Kirby A, Brown N, Coles M, Smith DF, Kaye PM - PLoS Pathog. (2010)

Bottom Line: Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens.Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells.This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Centre for Immunology and Infection, Hull York Medical School, University of York, York, United Kingdom.

ABSTRACT
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

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Distribution of Leishmania amastigotes in the liver.C57BL/6 mice were infected with tdTom-L. donovani (A–F) or WT-L. donovani amastigotes (G–I) and 14 days later liver tissue was processed for confocal microscopy. A)–F) Intracellular amastigotes (red) are shown in combination with staining for F4/80 (white) and CD11c (green). A) Low magnification view to show diversity of the granulomatous response. B) High magnification image of single granuloma with macrophages containing numerous amastigotes. C) Infected F4/80+ cells with limited inflammatory cell recruitment. D) CD11c (green) and E) F4/80 (white) expression on amastigote (red) infected cells at the granuloma core. F) overlay of D) and E). G) CD11b+ cells (green) and H) F4/80+ cells (red) were predominantly localised to distinct sites within the granuloma and F4/80+ cells at the core did not express CD11b (I, overlay). DAPI was used as a nuclear counterstain (blue). Scale bars 50 µm for A) and 20 µm for B–F).
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ppat-1000805-g001: Distribution of Leishmania amastigotes in the liver.C57BL/6 mice were infected with tdTom-L. donovani (A–F) or WT-L. donovani amastigotes (G–I) and 14 days later liver tissue was processed for confocal microscopy. A)–F) Intracellular amastigotes (red) are shown in combination with staining for F4/80 (white) and CD11c (green). A) Low magnification view to show diversity of the granulomatous response. B) High magnification image of single granuloma with macrophages containing numerous amastigotes. C) Infected F4/80+ cells with limited inflammatory cell recruitment. D) CD11c (green) and E) F4/80 (white) expression on amastigote (red) infected cells at the granuloma core. F) overlay of D) and E). G) CD11b+ cells (green) and H) F4/80+ cells (red) were predominantly localised to distinct sites within the granuloma and F4/80+ cells at the core did not express CD11b (I, overlay). DAPI was used as a nuclear counterstain (blue). Scale bars 50 µm for A) and 20 µm for B–F).

Mentions: L. donovani amastigotes are usually identified in tissue based on their characteristic staining pattern after H&E staining of thin sections [14], with the sensitivity of detection, particularly for individual parasites being improved by immuno-histology using polyclonal or monoclonal antibodies [29]. To more readily observe parasites by fluorescent microscopy, we generated stable infective clones of L. donovani expressing tdTomato (tdTom; [30]), a fluorochrome amenable to both confocal and multi-photon imaging. We first infected mice with tdTom-L. donovani and examined their distribution in the liver at day 14 p.i. (Figure 1) in conjunction with staining for F4/80, a marker of mature KCs [31] and CD11c, a marker characteristically associated with DCs [32]. L. donovani amastigotes were readily apparent both at low magnification, where individual amastigotes within heavily-infected cells could not be resolved (Figure 1A), and at higher magnification, where individual parasites were easily distinguished (Figure 1B). Parasites were observed in two main anatomical locations: within granulomas, where they were predominantly associated with the core, and within the parenchyma, where by DAPI staining they appeared to be within isolated cells in areas largely devoid of local inflammatory reactions (Figure 1C). Almost invariably, amastigotes in either location were found within F4/80+ cells (Figure 1A–C). The close apposition and membrane interdigitation of F4/80+ cells made it difficult to score individual cells, so we did not attempt to calculate the percentage of F4/80+ cells that were infected within the core of the granuloma. Reminiscent of the pattern of staining with NLDC-145, a DEC 205-specific antibody [33], a diffuse but detectable level of CD11c expression was also observed on cells at the core of many, but not all, granulomas. These CD11c+ cells also expressed somewhat lower levels of F4/80, compared to the F4/80+ CD11c− cells that occupied the granuloma mantle (Figure 1D–F). Heterogeneity of expression of CD11c within granulomas did not correlate with the presence or absence of amastigotes. In contrast, CD11b+ cells were usually found in the granuloma mantle, with some clearly identifiable as neutrophils based on nuclear morphology. Importantly, the large amastigote-laden cells at the granuloma core that co-stained for F4/80 and CD11c were almost uniformly CD11b− (Figure 1G–I). These data, together with previously published studies [33] suggest that the majority of intra-granuloma amastigotes are found within F4/80+ cells, and some of these cells acquire markers in this local micro-environment that are often associated with DC.


Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Beattie L, Peltan A, Maroof A, Kirby A, Brown N, Coles M, Smith DF, Kaye PM - PLoS Pathog. (2010)

Distribution of Leishmania amastigotes in the liver.C57BL/6 mice were infected with tdTom-L. donovani (A–F) or WT-L. donovani amastigotes (G–I) and 14 days later liver tissue was processed for confocal microscopy. A)–F) Intracellular amastigotes (red) are shown in combination with staining for F4/80 (white) and CD11c (green). A) Low magnification view to show diversity of the granulomatous response. B) High magnification image of single granuloma with macrophages containing numerous amastigotes. C) Infected F4/80+ cells with limited inflammatory cell recruitment. D) CD11c (green) and E) F4/80 (white) expression on amastigote (red) infected cells at the granuloma core. F) overlay of D) and E). G) CD11b+ cells (green) and H) F4/80+ cells (red) were predominantly localised to distinct sites within the granuloma and F4/80+ cells at the core did not express CD11b (I, overlay). DAPI was used as a nuclear counterstain (blue). Scale bars 50 µm for A) and 20 µm for B–F).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837408&req=5

ppat-1000805-g001: Distribution of Leishmania amastigotes in the liver.C57BL/6 mice were infected with tdTom-L. donovani (A–F) or WT-L. donovani amastigotes (G–I) and 14 days later liver tissue was processed for confocal microscopy. A)–F) Intracellular amastigotes (red) are shown in combination with staining for F4/80 (white) and CD11c (green). A) Low magnification view to show diversity of the granulomatous response. B) High magnification image of single granuloma with macrophages containing numerous amastigotes. C) Infected F4/80+ cells with limited inflammatory cell recruitment. D) CD11c (green) and E) F4/80 (white) expression on amastigote (red) infected cells at the granuloma core. F) overlay of D) and E). G) CD11b+ cells (green) and H) F4/80+ cells (red) were predominantly localised to distinct sites within the granuloma and F4/80+ cells at the core did not express CD11b (I, overlay). DAPI was used as a nuclear counterstain (blue). Scale bars 50 µm for A) and 20 µm for B–F).
Mentions: L. donovani amastigotes are usually identified in tissue based on their characteristic staining pattern after H&E staining of thin sections [14], with the sensitivity of detection, particularly for individual parasites being improved by immuno-histology using polyclonal or monoclonal antibodies [29]. To more readily observe parasites by fluorescent microscopy, we generated stable infective clones of L. donovani expressing tdTomato (tdTom; [30]), a fluorochrome amenable to both confocal and multi-photon imaging. We first infected mice with tdTom-L. donovani and examined their distribution in the liver at day 14 p.i. (Figure 1) in conjunction with staining for F4/80, a marker of mature KCs [31] and CD11c, a marker characteristically associated with DCs [32]. L. donovani amastigotes were readily apparent both at low magnification, where individual amastigotes within heavily-infected cells could not be resolved (Figure 1A), and at higher magnification, where individual parasites were easily distinguished (Figure 1B). Parasites were observed in two main anatomical locations: within granulomas, where they were predominantly associated with the core, and within the parenchyma, where by DAPI staining they appeared to be within isolated cells in areas largely devoid of local inflammatory reactions (Figure 1C). Almost invariably, amastigotes in either location were found within F4/80+ cells (Figure 1A–C). The close apposition and membrane interdigitation of F4/80+ cells made it difficult to score individual cells, so we did not attempt to calculate the percentage of F4/80+ cells that were infected within the core of the granuloma. Reminiscent of the pattern of staining with NLDC-145, a DEC 205-specific antibody [33], a diffuse but detectable level of CD11c expression was also observed on cells at the core of many, but not all, granulomas. These CD11c+ cells also expressed somewhat lower levels of F4/80, compared to the F4/80+ CD11c− cells that occupied the granuloma mantle (Figure 1D–F). Heterogeneity of expression of CD11c within granulomas did not correlate with the presence or absence of amastigotes. In contrast, CD11b+ cells were usually found in the granuloma mantle, with some clearly identifiable as neutrophils based on nuclear morphology. Importantly, the large amastigote-laden cells at the granuloma core that co-stained for F4/80 and CD11c were almost uniformly CD11b− (Figure 1G–I). These data, together with previously published studies [33] suggest that the majority of intra-granuloma amastigotes are found within F4/80+ cells, and some of these cells acquire markers in this local micro-environment that are often associated with DC.

Bottom Line: Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens.Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells.This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Centre for Immunology and Infection, Hull York Medical School, University of York, York, United Kingdom.

ABSTRACT
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Show MeSH
Related in: MedlinePlus