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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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Related in: MedlinePlus

Recruitment of local Kupffer Cells results in a redistribution of nanobeads.Distribution of nanobeads (red) in naïve liver following pre-injection showing A) F4/80+ Kupffer cells (green), B) Desmin+ stellate cells (white) and C) CD11b+ monocytes (green). Distribution of nanocrystals (red), in liver of mice pre-injected with NBs and then infected with L. donovani, in D) F4/80+ Kupffer cells (green), E) Desmin+ stellate cells (white) and F) CD11b+ monocytes (green). DAPI was used as a nuclear counterstain (blue). Scale bars for A–F 20 µm.
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ppat-1000805-g003: Recruitment of local Kupffer Cells results in a redistribution of nanobeads.Distribution of nanobeads (red) in naïve liver following pre-injection showing A) F4/80+ Kupffer cells (green), B) Desmin+ stellate cells (white) and C) CD11b+ monocytes (green). Distribution of nanocrystals (red), in liver of mice pre-injected with NBs and then infected with L. donovani, in D) F4/80+ Kupffer cells (green), E) Desmin+ stellate cells (white) and F) CD11b+ monocytes (green). DAPI was used as a nuclear counterstain (blue). Scale bars for A–F 20 µm.

Mentions: To determine if the aggregation of KCs in granulomas was due to a re-distribution of liver-resident KCs, or whether this reflected the recruitment/differentiation of blood or BM-derived precursors after infection had been established, we used fluorescent nanobeads (NBs) to label KCs (and other potential liver-resident phagocytic cells) prior to infection. Such cells could then be subsequently discriminated from inflammatory phagocytes recruited after infection (Figure 3A–F). We first analysed the distribution of these NBs after intravenous injection into naïve mice. As shown in Figure 3A, NBs were readily ingested by liver-resident F4/80+ KC in uninfected mice, providing a readily detectable measure of their phagocytic activity. Most KCs were phagocytic (∼74%, n = 42), with a variable phagocytic load of NBs. Within individual KCs, multiple ‘patches’ of NB labeling could often be observed, presumably reflecting uptake of NBs into discrete phagosomes. These patches also varied in size, a result that might reflect either aggregation of NBs during injection and/or coalescence of multiple phagosomes each containing small numbers of NBs. NBs were also phagocytosed by desmin+ hepatic stellate cells in naïve mice (∼66% of desmin+ cells contained NBs, n = 90), but large aggregates were rarely observed in these cells (Figure 3B). CD11b+ cells are rare in the resting liver as determined by immuno-histochemistry [35], and when observed, these cells did not contain NBs (Figure 3C). We then injected mice with NBs and 4–12 h later, infected them with L. donovani. The distribution of NB+ cells was then observed at both day 14 p.i. (Figure 3D–F) and at d28 p.i. (data not shown), with similar results being obtained at each time point. NBs were readily observed in L. donovani- infected mice, confirming their value as a long-term cell tracer. NBs were highly concentrated in granulomas, largely at the core, and almost exclusively within F4/80+ KCs (Figure 3D). In contrast, although occasionally present within granulomas, hepatic stellate cells were normally excluded from the core of the granuloma and usually did not contain readily distinguishable NBs (Figure 3E). Strikingly, NBs were also not observed in CD11b+ cells (presumptive monocytes, DC and neutrophils) either at the core of the granuloma or when more peripherally dispersed at the granuloma mantle (Figure 3F). To confirm that the distribution of NBs in granulomas was not the result of rapidly recruited inflammatory cells, NBs were injected and the mice infected with L. donovani 12 hours later as described above. No significant infiltration of inflammatory cells was observed 6 hours after infection, with the proportions of CD11b−, CD11bint and CD11bhi cells being similar between mice that received NBs only or mice that received NBs and L. donovani, whether measured in terms of either the frequency or absolute number of cells (Figure S2). These data suggest that NB distribution after infection reflects KC redistribution and is not influenced by rapidly recruited inflammatory cells. Collectively, these data therefore strongly support the contention that the core of the granuloma is derived almost exclusively from resident KCs recruited from the sinusoids early during the inflammatory process.


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)

Recruitment of local Kupffer Cells results in a redistribution of nanobeads.Distribution of nanobeads (red) in naïve liver following pre-injection showing A) F4/80+ Kupffer cells (green), B) Desmin+ stellate cells (white) and C) CD11b+ monocytes (green). Distribution of nanocrystals (red), in liver of mice pre-injected with NBs and then infected with L. donovani, in D) F4/80+ Kupffer cells (green), E) Desmin+ stellate cells (white) and F) CD11b+ monocytes (green). DAPI was used as a nuclear counterstain (blue). Scale bars for A–F 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837408&req=5

ppat-1000805-g003: Recruitment of local Kupffer Cells results in a redistribution of nanobeads.Distribution of nanobeads (red) in naïve liver following pre-injection showing A) F4/80+ Kupffer cells (green), B) Desmin+ stellate cells (white) and C) CD11b+ monocytes (green). Distribution of nanocrystals (red), in liver of mice pre-injected with NBs and then infected with L. donovani, in D) F4/80+ Kupffer cells (green), E) Desmin+ stellate cells (white) and F) CD11b+ monocytes (green). DAPI was used as a nuclear counterstain (blue). Scale bars for A–F 20 µm.
Mentions: To determine if the aggregation of KCs in granulomas was due to a re-distribution of liver-resident KCs, or whether this reflected the recruitment/differentiation of blood or BM-derived precursors after infection had been established, we used fluorescent nanobeads (NBs) to label KCs (and other potential liver-resident phagocytic cells) prior to infection. Such cells could then be subsequently discriminated from inflammatory phagocytes recruited after infection (Figure 3A–F). We first analysed the distribution of these NBs after intravenous injection into naïve mice. As shown in Figure 3A, NBs were readily ingested by liver-resident F4/80+ KC in uninfected mice, providing a readily detectable measure of their phagocytic activity. Most KCs were phagocytic (∼74%, n = 42), with a variable phagocytic load of NBs. Within individual KCs, multiple ‘patches’ of NB labeling could often be observed, presumably reflecting uptake of NBs into discrete phagosomes. These patches also varied in size, a result that might reflect either aggregation of NBs during injection and/or coalescence of multiple phagosomes each containing small numbers of NBs. NBs were also phagocytosed by desmin+ hepatic stellate cells in naïve mice (∼66% of desmin+ cells contained NBs, n = 90), but large aggregates were rarely observed in these cells (Figure 3B). CD11b+ cells are rare in the resting liver as determined by immuno-histochemistry [35], and when observed, these cells did not contain NBs (Figure 3C). We then injected mice with NBs and 4–12 h later, infected them with L. donovani. The distribution of NB+ cells was then observed at both day 14 p.i. (Figure 3D–F) and at d28 p.i. (data not shown), with similar results being obtained at each time point. NBs were readily observed in L. donovani- infected mice, confirming their value as a long-term cell tracer. NBs were highly concentrated in granulomas, largely at the core, and almost exclusively within F4/80+ KCs (Figure 3D). In contrast, although occasionally present within granulomas, hepatic stellate cells were normally excluded from the core of the granuloma and usually did not contain readily distinguishable NBs (Figure 3E). Strikingly, NBs were also not observed in CD11b+ cells (presumptive monocytes, DC and neutrophils) either at the core of the granuloma or when more peripherally dispersed at the granuloma mantle (Figure 3F). To confirm that the distribution of NBs in granulomas was not the result of rapidly recruited inflammatory cells, NBs were injected and the mice infected with L. donovani 12 hours later as described above. No significant infiltration of inflammatory cells was observed 6 hours after infection, with the proportions of CD11b−, CD11bint and CD11bhi cells being similar between mice that received NBs only or mice that received NBs and L. donovani, whether measured in terms of either the frequency or absolute number of cells (Figure S2). These data suggest that NB distribution after infection reflects KC redistribution and is not influenced by rapidly recruited inflammatory cells. Collectively, these data therefore strongly support the contention that the core of the granuloma is derived almost exclusively from resident KCs recruited from the sinusoids early during the inflammatory process.

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