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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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Early monocyte depletion prevents lymphocyte infiltration into the brain.C57BL/6 mice were left either untreated or infected with 5*104 PbTg iRBC (A). In addition, groups of infected mice were treated either with anti-Gr1 (B), anti-Ly6G (C) or anti-CCR2 (D) mAb on the day of PbTg-infection (day 0). Six days later, cellular infiltrates from the brains of individual mice were prepared and analysed for the frequency of (E) infiltrating mononuclear cells (CD45+CD11b+) as well as (F) CD45hi lymphocytes (CD45hiCD11b-). Bars show mean ± SEM from n = 4–5 mice per group from 1 out of 3 independent depletion-infection experiments. 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.g004: Early monocyte depletion prevents lymphocyte infiltration into the brain.C57BL/6 mice were left either untreated or infected with 5*104 PbTg iRBC (A). In addition, groups of infected mice were treated either with anti-Gr1 (B), anti-Ly6G (C) or anti-CCR2 (D) mAb on the day of PbTg-infection (day 0). Six days later, cellular infiltrates from the brains of individual mice were prepared and analysed for the frequency of (E) infiltrating mononuclear cells (CD45+CD11b+) as well as (F) CD45hi lymphocytes (CD45hiCD11b-). Bars show mean ± SEM from n = 4–5 mice per group from 1 out of 3 independent depletion-infection experiments. 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: To evaluate how the depletion of inflammatory cell subpopulations in the periphery could influence cellular infiltration into the CNS in more detail, we further determined on day 6 p.i. the frequency and composition of infiltrating cells within the brains of PbTg-infected mice by flow cytometry. When compared to naïve mice, PbTg infected animals exhibited in the brains strongly elevated frequencies of CD45+CD11b-lymphocytes as well as CD45+CD11b+ mononuclear cells (Fig 4A, upper and lower panel). Within the CD11b+ mononuclear cells in brains of PbTg-infected mice, we found Ly6C+Ly6G- inflammatory monocytes and Ly6CintLy6G+ neutrophils (Fig 4A, compare upper and lower right plots). Upon anti-Gr1 mAb treatment, we detected a decrease in infiltrating cells which included 3 fold less infiltrating CD45+CD11b+ cells and a massive reduction in infiltrating CD11b-CD45hi lymphocytes compared to WT infected control mice (Fig 4B), which correlates to our findings in peripheral depletion (Fig 2A) and the observed protection (Fig 2B and 2C) and previous studies [21]. Interestingly, the frequency of inflammatory monocytes within the parental mononuclear CD45+CD11b+ subset was comparable to control infected animals (Fig 4A and 4B; 63.8% vs 63.9.4%), but compared to WT infected control mice, the overall frequency of mononuclear cells was strongly reduced in brains of mAb treated mice (Fig 4E and 4F). These population changes were even stronger when infected mice were injected with anti-Gr1 mAb on days 3 and 5, demonstrating an enduring depletion effect upon administration of the mAb (S3 Fig).


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)

Early monocyte depletion prevents lymphocyte infiltration into the brain.C57BL/6 mice were left either untreated or infected with 5*104 PbTg iRBC (A). In addition, groups of infected mice were treated either with anti-Gr1 (B), anti-Ly6G (C) or anti-CCR2 (D) mAb on the day of PbTg-infection (day 0). Six days later, cellular infiltrates from the brains of individual mice were prepared and analysed for the frequency of (E) infiltrating mononuclear cells (CD45+CD11b+) as well as (F) CD45hi lymphocytes (CD45hiCD11b-). Bars show mean ± SEM from n = 4–5 mice per group from 1 out of 3 independent depletion-infection experiments. 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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401438&req=5

pone.0124080.g004: Early monocyte depletion prevents lymphocyte infiltration into the brain.C57BL/6 mice were left either untreated or infected with 5*104 PbTg iRBC (A). In addition, groups of infected mice were treated either with anti-Gr1 (B), anti-Ly6G (C) or anti-CCR2 (D) mAb on the day of PbTg-infection (day 0). Six days later, cellular infiltrates from the brains of individual mice were prepared and analysed for the frequency of (E) infiltrating mononuclear cells (CD45+CD11b+) as well as (F) CD45hi lymphocytes (CD45hiCD11b-). Bars show mean ± SEM from n = 4–5 mice per group from 1 out of 3 independent depletion-infection experiments. 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: To evaluate how the depletion of inflammatory cell subpopulations in the periphery could influence cellular infiltration into the CNS in more detail, we further determined on day 6 p.i. the frequency and composition of infiltrating cells within the brains of PbTg-infected mice by flow cytometry. When compared to naïve mice, PbTg infected animals exhibited in the brains strongly elevated frequencies of CD45+CD11b-lymphocytes as well as CD45+CD11b+ mononuclear cells (Fig 4A, upper and lower panel). Within the CD11b+ mononuclear cells in brains of PbTg-infected mice, we found Ly6C+Ly6G- inflammatory monocytes and Ly6CintLy6G+ neutrophils (Fig 4A, compare upper and lower right plots). Upon anti-Gr1 mAb treatment, we detected a decrease in infiltrating cells which included 3 fold less infiltrating CD45+CD11b+ cells and a massive reduction in infiltrating CD11b-CD45hi lymphocytes compared to WT infected control mice (Fig 4B), which correlates to our findings in peripheral depletion (Fig 2A) and the observed protection (Fig 2B and 2C) and previous studies [21]. Interestingly, the frequency of inflammatory monocytes within the parental mononuclear CD45+CD11b+ subset was comparable to control infected animals (Fig 4A and 4B; 63.8% vs 63.9.4%), but compared to WT infected control mice, the overall frequency of mononuclear cells was strongly reduced in brains of mAb treated mice (Fig 4E and 4F). These population changes were even stronger when infected mice were injected with anti-Gr1 mAb on days 3 and 5, demonstrating an enduring depletion effect upon administration of the mAb (S3 Fig).

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