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Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

Wijayalath W, Majji S, Villasante EF, Brumeanu TD, Richie TL, Casares S - Malar. J. (2014)

Bottom Line: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries.Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria.Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: US Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA. sofia.a.casares.civ@mail.mil.

ABSTRACT

Background: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored.

Methods: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes.

Results: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge.

Conclusions: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

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DRAG mice develop human Kupffer cells and liver endothelial cells. A) Immunostaining of livers using anti-human CD68 (green fluorescence) and counterstained with DAPI (DNA, blue fluorescence). B) Frequency of human Kupffer cells in livers of DRAG mice as determined by FACS using anti-human CD45 and anti-human CD68 on cells gated on the FSC/SSC monocyte region. Middle and right panels show CD68 staining on human (gated CD45+) cells and mouse (gated CD45−) cells isolated from livers of HSC-infused DRAG mice. Data represent mean and interval frequencies from 4 mice analysed individually. Left panel shows binding of anti-human CD68 to monocytes from human peripheral blood. C) Immunostaining of livers using anti-human CD36 and counterstained with DAPI (DNA, blue fluorescence) showing presence of human endothelial cells in veins and sinusoids.
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Fig2: DRAG mice develop human Kupffer cells and liver endothelial cells. A) Immunostaining of livers using anti-human CD68 (green fluorescence) and counterstained with DAPI (DNA, blue fluorescence). B) Frequency of human Kupffer cells in livers of DRAG mice as determined by FACS using anti-human CD45 and anti-human CD68 on cells gated on the FSC/SSC monocyte region. Middle and right panels show CD68 staining on human (gated CD45+) cells and mouse (gated CD45−) cells isolated from livers of HSC-infused DRAG mice. Data represent mean and interval frequencies from 4 mice analysed individually. Left panel shows binding of anti-human CD68 to monocytes from human peripheral blood. C) Immunostaining of livers using anti-human CD36 and counterstained with DAPI (DNA, blue fluorescence) showing presence of human endothelial cells in veins and sinusoids.

Mentions: Prior to infection of hepatocytes, sporozoites must cross the sinusoidal cell layer and interact with endothelial cells and Kupffer cells [30, 31]. The presence of human Kupffer cells in livers of HSC-infused DRAG was revealed by immunohistochemistry using anti-human CD68 (Figure 2A). CD68 is a glycoprotein binding to low density lipoprotein (LDL) expressed on mature (phagocytic) human Kupffer cells [32]. To further estimate the frequency of human Kupffer cells, mononuclear cells isolated from livers that had been previously perfused to remove circulating blood cells, were analysed by FACS using anti-human CD45 (a marker expressed on human haematopoietic cells) [22] and anti-human CD68. As illustrated in Figure 2B (middle panel), the frequency of Kupffer cells (CD68+CD45+) averaged 11%. The specificity of the anti-human CD68 is shown by binding to monocytes from human peripheral blood (Figure 2B left panel) but not to mouse (CD45−) cells from livers of HSC-infused DRAG mice (right panel). Livers from HSC-infused DRAG mice also showed the presence of human endothelial cells lining the sinusoids, as indicated by histological examination using anti-human CD36 (Figure 2C). In aggregate, the results indicated that HSC-infused DRAG mice develop human hepatocytes, Kupffer cells, and liver endothelial cells.Figure 2


Humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice sustain the complex vertebrate life cycle of Plasmodium falciparum malaria.

Wijayalath W, Majji S, Villasante EF, Brumeanu TD, Richie TL, Casares S - Malar. J. (2014)

DRAG mice develop human Kupffer cells and liver endothelial cells. A) Immunostaining of livers using anti-human CD68 (green fluorescence) and counterstained with DAPI (DNA, blue fluorescence). B) Frequency of human Kupffer cells in livers of DRAG mice as determined by FACS using anti-human CD45 and anti-human CD68 on cells gated on the FSC/SSC monocyte region. Middle and right panels show CD68 staining on human (gated CD45+) cells and mouse (gated CD45−) cells isolated from livers of HSC-infused DRAG mice. Data represent mean and interval frequencies from 4 mice analysed individually. Left panel shows binding of anti-human CD68 to monocytes from human peripheral blood. C) Immunostaining of livers using anti-human CD36 and counterstained with DAPI (DNA, blue fluorescence) showing presence of human endothelial cells in veins and sinusoids.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197321&req=5

Fig2: DRAG mice develop human Kupffer cells and liver endothelial cells. A) Immunostaining of livers using anti-human CD68 (green fluorescence) and counterstained with DAPI (DNA, blue fluorescence). B) Frequency of human Kupffer cells in livers of DRAG mice as determined by FACS using anti-human CD45 and anti-human CD68 on cells gated on the FSC/SSC monocyte region. Middle and right panels show CD68 staining on human (gated CD45+) cells and mouse (gated CD45−) cells isolated from livers of HSC-infused DRAG mice. Data represent mean and interval frequencies from 4 mice analysed individually. Left panel shows binding of anti-human CD68 to monocytes from human peripheral blood. C) Immunostaining of livers using anti-human CD36 and counterstained with DAPI (DNA, blue fluorescence) showing presence of human endothelial cells in veins and sinusoids.
Mentions: Prior to infection of hepatocytes, sporozoites must cross the sinusoidal cell layer and interact with endothelial cells and Kupffer cells [30, 31]. The presence of human Kupffer cells in livers of HSC-infused DRAG was revealed by immunohistochemistry using anti-human CD68 (Figure 2A). CD68 is a glycoprotein binding to low density lipoprotein (LDL) expressed on mature (phagocytic) human Kupffer cells [32]. To further estimate the frequency of human Kupffer cells, mononuclear cells isolated from livers that had been previously perfused to remove circulating blood cells, were analysed by FACS using anti-human CD45 (a marker expressed on human haematopoietic cells) [22] and anti-human CD68. As illustrated in Figure 2B (middle panel), the frequency of Kupffer cells (CD68+CD45+) averaged 11%. The specificity of the anti-human CD68 is shown by binding to monocytes from human peripheral blood (Figure 2B left panel) but not to mouse (CD45−) cells from livers of HSC-infused DRAG mice (right panel). Livers from HSC-infused DRAG mice also showed the presence of human endothelial cells lining the sinusoids, as indicated by histological examination using anti-human CD36 (Figure 2C). In aggregate, the results indicated that HSC-infused DRAG mice develop human hepatocytes, Kupffer cells, and liver endothelial cells.Figure 2

Bottom Line: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries.Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria.Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: US Military Malaria Vaccine Program, Naval Medical Research Center/Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA. sofia.a.casares.civ@mail.mil.

ABSTRACT

Background: Malaria is a deadly infectious disease affecting millions of people in tropical and sub-tropical countries. Among the five species of Plasmodium parasites that infect humans, Plasmodium falciparum accounts for the highest morbidity and mortality associated with malaria. Since humans are the only natural hosts for P. falciparum, the lack of convenient animal models has hindered the understanding of disease pathogenesis and prompted the need of testing anti-malarial drugs and vaccines directly in human trials. Humanized mice hosting human cells represent new pre-clinical models for infectious diseases that affect only humans. In this study, the ability of human-immune-system humanized HLA-DR4.RagKO.IL2RγcKO.NOD (DRAG) mice to sustain infection with P. falciparum was explored.

Methods: Four week-old DRAG mice were infused with HLA-matched human haematopoietic stem cells (HSC) and examined for reconstitution of human liver cells and erythrocytes. Upon challenge with infectious P. falciparum sporozoites (NF54 strain) humanized DRAG mice were examined for liver stage infection, blood stage infection, and transmission to Anopheles stephensi mosquitoes.

Results: Humanized DRAG mice reconstituted human hepatocytes, Kupffer cells, liver endothelial cells, and erythrocytes. Upon intravenous challenge with P. falciparum sporozoites, DRAG mice sustained liver to blood stage infection (average 3-5 parasites/microlitre blood) and allowed transmission to An. stephensi mosquitoes. Infected DRAG mice elicited antibody and cellular responses to the blood stage parasites and self-cured the infection by day 45 post-challenge.

Conclusions: DRAG mice represent the first human-immune-system humanized mouse model that sustains the complex vertebrate life cycle of P. falciparum without the need of exogenous injection of human hepatocytes/erythrocytes or P. falciparum parasite adaptation. The ability of DRAG mice to elicit specific human immune responses to P. falciparum parasites may help deciphering immune correlates of protection and to identify protective malaria antigens.

Show MeSH
Related in: MedlinePlus