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Specific depletion of Ly6C(hi) inflammatory monocytes prevents immunopathology in experimental cerebral malaria.

Schumak B, Klocke K, Kuepper JM, Biswas A, Djie-Maletz A, Limmer A, van Rooijen N, Mack M, Hoerauf A, Dunay IR - PLoS ONE (2015)

Bottom Line: Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage.Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development.Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany.

ABSTRACT
Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6C(hi) inflammatory monocytes (by anti-CCR2), Ly6G+ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C(hi) inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ+CD8+ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C(hi) inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C+ monocytes in ECM inflammatory processes.

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Histological analysis of brains of PbTg infected mice upon early depletion.C57BL/6 mice were infected i.v. with 5*104 PbTg-iRBC and then subdivided into groups that received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb on d0 p.i. (early depletion). On day 6 p.i., tissue sections from the brain parenchyma of individual mice were assessed for pathological changes. (A) Standard H&E staining is depicted in the upper panels whereas immunohistochemical staining for anti-Mac3 and anti-CD3 are shown in the middle and lower panels, respectively. Representative sections from naïve mice are shown on the left whereas those from PbTg infected mice are displayed on the right. (B, C) Quantification of Mac3+ cells (B) and CD3+ cells T cells (C) in brain sections from the meninges and frontal cortex of individual mice. Scale bars indicate 100μm in the magnifications. Bars show mean ± SEM from n = 8 mice per group from 1 out of 3 independent depletion-infection experiments counted in 10 defined fields (High power fields, HPF) in the frontal cortex. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test and significant differences are indicated by the stars in brackets between the groups (* p<0.05).
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pone.0124080.g003: Histological analysis of brains of PbTg infected mice upon early depletion.C57BL/6 mice were infected i.v. with 5*104 PbTg-iRBC and then subdivided into groups that received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb on d0 p.i. (early depletion). On day 6 p.i., tissue sections from the brain parenchyma of individual mice were assessed for pathological changes. (A) Standard H&E staining is depicted in the upper panels whereas immunohistochemical staining for anti-Mac3 and anti-CD3 are shown in the middle and lower panels, respectively. Representative sections from naïve mice are shown on the left whereas those from PbTg infected mice are displayed on the right. (B, C) Quantification of Mac3+ cells (B) and CD3+ cells T cells (C) in brain sections from the meninges and frontal cortex of individual mice. Scale bars indicate 100μm in the magnifications. Bars show mean ± SEM from n = 8 mice per group from 1 out of 3 independent depletion-infection experiments counted in 10 defined fields (High power fields, HPF) in the frontal cortex. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test and significant differences are indicated by the stars in brackets between the groups (* p<0.05).

Mentions: Next, we determined whether the selective depletion of these immune cell subsets using mAb also altered the amount of lymphocyte infiltration into the CNS since this has also been shown to play a decisive role in the outcome of murine malaria [6, 8]. At the peak of ECM incidence in PbTg infected C57BL/6 mice (day 6 p.i.), tissue sections from the brains of naïve and infected mice were analysed for the number of infiltrating mononuclear phagocytes (Mac3 staining) and T cells (CD3 staining). Fig 3A shows representative stainings from brain frontal cortex prepared from naïve and PbTg-infected mice, respectively. As expected, we detected a strong increase in infiltrating cells into the CNS parenchyma of infected mice compared to naïve controls (representative images in Fig 3A, upper right). These CNS infiltrates consisted of increased numbers of CD11b+ mononuclear cells and CD3+ T cells (Fig 3A, 3B and 3C). Early depletion of inflammatory monocytes in the blood by using anti-Gr1 or anti-CCR2 mAb resulted in significantly reduced the numbers of both infiltrating mononuclear cells and T cells in the frontal cortex (Fig 3B and 3Cc.f. bars 2 with 3 and 5). No differences in the number of infiltrating lymphocytes were observed when infected mice were subjected to treatment with anti-Ly6G mAb (Fig 3B and 3Cc.f. bars 2 with 4). Depletion using anti-Gr1 and anti-CCR2 but not anti-Ly6G during ongoing PbTg infection on days 3 and 5 also reduced the influx of immune cells as shown by immunohistochemistry, but to a lesser extent (S2 Fig). Therefore, these data showing a massive reduction in lymphocyte influx into the brains of anti-Gr1 or anti-CCR2 mAb-injected mice strongly support our observations described above, that using anti-Gr1 or anti-CCR2 mAb at the time point of infection prolonged the survival of Plasmodium infected mice (Fig 2C and 2G).


Specific depletion of Ly6C(hi) inflammatory monocytes prevents immunopathology in experimental cerebral malaria.

Schumak B, Klocke K, Kuepper JM, Biswas A, Djie-Maletz A, Limmer A, van Rooijen N, Mack M, Hoerauf A, Dunay IR - PLoS ONE (2015)

Histological analysis of brains of PbTg infected mice upon early depletion.C57BL/6 mice were infected i.v. with 5*104 PbTg-iRBC and then subdivided into groups that received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb on d0 p.i. (early depletion). On day 6 p.i., tissue sections from the brain parenchyma of individual mice were assessed for pathological changes. (A) Standard H&E staining is depicted in the upper panels whereas immunohistochemical staining for anti-Mac3 and anti-CD3 are shown in the middle and lower panels, respectively. Representative sections from naïve mice are shown on the left whereas those from PbTg infected mice are displayed on the right. (B, C) Quantification of Mac3+ cells (B) and CD3+ cells T cells (C) in brain sections from the meninges and frontal cortex of individual mice. Scale bars indicate 100μm in the magnifications. Bars show mean ± SEM from n = 8 mice per group from 1 out of 3 independent depletion-infection experiments counted in 10 defined fields (High power fields, HPF) in the frontal cortex. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test and significant differences are indicated by the stars in brackets between the groups (* p<0.05).
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pone.0124080.g003: Histological analysis of brains of PbTg infected mice upon early depletion.C57BL/6 mice were infected i.v. with 5*104 PbTg-iRBC and then subdivided into groups that received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb on d0 p.i. (early depletion). On day 6 p.i., tissue sections from the brain parenchyma of individual mice were assessed for pathological changes. (A) Standard H&E staining is depicted in the upper panels whereas immunohistochemical staining for anti-Mac3 and anti-CD3 are shown in the middle and lower panels, respectively. Representative sections from naïve mice are shown on the left whereas those from PbTg infected mice are displayed on the right. (B, C) Quantification of Mac3+ cells (B) and CD3+ cells T cells (C) in brain sections from the meninges and frontal cortex of individual mice. Scale bars indicate 100μm in the magnifications. Bars show mean ± SEM from n = 8 mice per group from 1 out of 3 independent depletion-infection experiments counted in 10 defined fields (High power fields, HPF) in the frontal cortex. Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test and significant differences are indicated by the stars in brackets between the groups (* p<0.05).
Mentions: Next, we determined whether the selective depletion of these immune cell subsets using mAb also altered the amount of lymphocyte infiltration into the CNS since this has also been shown to play a decisive role in the outcome of murine malaria [6, 8]. At the peak of ECM incidence in PbTg infected C57BL/6 mice (day 6 p.i.), tissue sections from the brains of naïve and infected mice were analysed for the number of infiltrating mononuclear phagocytes (Mac3 staining) and T cells (CD3 staining). Fig 3A shows representative stainings from brain frontal cortex prepared from naïve and PbTg-infected mice, respectively. As expected, we detected a strong increase in infiltrating cells into the CNS parenchyma of infected mice compared to naïve controls (representative images in Fig 3A, upper right). These CNS infiltrates consisted of increased numbers of CD11b+ mononuclear cells and CD3+ T cells (Fig 3A, 3B and 3C). Early depletion of inflammatory monocytes in the blood by using anti-Gr1 or anti-CCR2 mAb resulted in significantly reduced the numbers of both infiltrating mononuclear cells and T cells in the frontal cortex (Fig 3B and 3Cc.f. bars 2 with 3 and 5). No differences in the number of infiltrating lymphocytes were observed when infected mice were subjected to treatment with anti-Ly6G mAb (Fig 3B and 3Cc.f. bars 2 with 4). Depletion using anti-Gr1 and anti-CCR2 but not anti-Ly6G during ongoing PbTg infection on days 3 and 5 also reduced the influx of immune cells as shown by immunohistochemistry, but to a lesser extent (S2 Fig). Therefore, these data showing a massive reduction in lymphocyte influx into the brains of anti-Gr1 or anti-CCR2 mAb-injected mice strongly support our observations described above, that using anti-Gr1 or anti-CCR2 mAb at the time point of infection prolonged the survival of Plasmodium infected mice (Fig 2C and 2G).

Bottom Line: Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage.Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development.Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany.

ABSTRACT
Plasmodium berghei ANKA (PbA) infection of C57BL/6 mice leads to experimental cerebral malaria (ECM) that is commonly associated with serious T cell mediated damage. In other parasitic infection models, inflammatory monocytes have been shown to regulate Th1 responses but their role in ECM remains poorly defined, whereas neutrophils are reported to contribute to ECM immune pathology. Making use of the recent development of specific monoclonal antibodies (mAb), we depleted in vivo Ly6C(hi) inflammatory monocytes (by anti-CCR2), Ly6G+ neutrophils (by anti-Ly6G) or both cell types (by anti-Gr1) during infection with Ovalbumin-transgenic PbA parasites (PbTg). Notably, the application of anti-Gr1 or anti-CCR2 but not anti-Ly6G antibodies into PbTg-infected mice prevented ECM development. In addition, depletion of Ly6C(hi) inflammatory monocytes but not neutrophils led to decreased IFNγ levels and IFNγ+CD8+ T effector cells in the brain. Importantly, anti-CCR2 mAb injection did not prevent the generation of PbTg-specific T cell responses in the periphery, whereas anti-Gr1 mAb injection strongly diminished T cell frequencies and CTL responses. In conclusion, the specific depletion of Ly6C(hi) inflammatory monocytes attenuated brain inflammation and immune cell recruitment to the CNS, which prevented ECM following Plasmodium infection, pointing out a substantial role of Ly6C+ monocytes in ECM inflammatory processes.

Show MeSH
Related in: MedlinePlus