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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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Depletion of Ly6C+F4/80+ cells, which are present in brains of ECM positive mice, prevents ECM.(A) Survival of PbTg-infected C57BL/6 mice. 24 hr before inoculation with iRBC (5*104) of PbTg, indicated C57BL/6 mice received 200μl CloLip i.v.. Graph shows survival rates of mice p.i. (n = 10 mice per group) and represents 1 of 2 independent infection studies. Statistical analysis was performed using log-rank test. (B) Flow cytometric analysis of Ly6C+F4/80+ cells in spleens of non-depleted mice (upper row) and CloLip depleted mice (lower row), which were either naïve (uninfected) (left panel) or PbTg infected (right panel; 24 hours p.i.). Dot plots show representative stainings from 1 of 3 independent depletion studies in 3 mice per group. (C) Emergence of peripheral lymphocytes and mononuclear cells in brains of PbTg infected mice. Flow cytometric analyses show representative plots of naïve and PbTg infected mice, day 6 p.i.. Expression of CD45 and CD11b were used to discriminate lymphocytes (CD45hi CD11b-) and mononuclear cells (CD45+CD11b+) from brain-resident microglia (CD45-CD11b+). (D) Scheme for discrimination of cell populations and gating of Ly6ChiLy6G- inflammatory monocytes [M] versus neutrophils (Ly6CintLy6G+) [N] out of mononuclear cells as shown in data set of (B). CD45 hi cells were identified as CD3+ T cells (lower panel). (E) Ly6ChiLy6Gneg inflammatory monocytes but not Ly6G+ neutrophils from the brain of PbTg infected mice express CCR2, F4/80, CD54 and I-Ab. Flow cytometric data of brains isolated from PbTg infected animals, which were gated as depicted in (C). FMO = fluorescence minus one are shown as control. Representative data from 1 of 3 independent experiments are shown.
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pone.0124080.g001: Depletion of Ly6C+F4/80+ cells, which are present in brains of ECM positive mice, prevents ECM.(A) Survival of PbTg-infected C57BL/6 mice. 24 hr before inoculation with iRBC (5*104) of PbTg, indicated C57BL/6 mice received 200μl CloLip i.v.. Graph shows survival rates of mice p.i. (n = 10 mice per group) and represents 1 of 2 independent infection studies. Statistical analysis was performed using log-rank test. (B) Flow cytometric analysis of Ly6C+F4/80+ cells in spleens of non-depleted mice (upper row) and CloLip depleted mice (lower row), which were either naïve (uninfected) (left panel) or PbTg infected (right panel; 24 hours p.i.). Dot plots show representative stainings from 1 of 3 independent depletion studies in 3 mice per group. (C) Emergence of peripheral lymphocytes and mononuclear cells in brains of PbTg infected mice. Flow cytometric analyses show representative plots of naïve and PbTg infected mice, day 6 p.i.. Expression of CD45 and CD11b were used to discriminate lymphocytes (CD45hi CD11b-) and mononuclear cells (CD45+CD11b+) from brain-resident microglia (CD45-CD11b+). (D) Scheme for discrimination of cell populations and gating of Ly6ChiLy6G- inflammatory monocytes [M] versus neutrophils (Ly6CintLy6G+) [N] out of mononuclear cells as shown in data set of (B). CD45 hi cells were identified as CD3+ T cells (lower panel). (E) Ly6ChiLy6Gneg inflammatory monocytes but not Ly6G+ neutrophils from the brain of PbTg infected mice express CCR2, F4/80, CD54 and I-Ab. Flow cytometric data of brains isolated from PbTg infected animals, which were gated as depicted in (C). FMO = fluorescence minus one are shown as control. Representative data from 1 of 3 independent experiments are shown.

Mentions: Although ECM in PbA infected C57BL/6 mice is predominantly mediated by CD8+ T cells and IFNγ [6–8], the exact contribution of responding phagocytic cell subpopulations in developing such Th1 responses remains insufficiently defined. Therefore, we examined the participation of phagocytic cells in the development of ECM using a transgenic strain of Plasmodium berghei ANKA that expresses ovalbumin (PbTg) [22]. Initially, groups of C57BL/6 mice were intravenously injected with clodronate liposomes (CloLip) that result in the depletion of all cells possessing phagocytic activity [23] and then infected with PbTg. CloLip administration protected mice from developing lethal ECM and resulted in an 80% survival rate (Fig 1A). Parasitemia levels did not differ significantly between the infected groups (Supplementary information S1 Fig). When compared to naïve mice, flow cytometric analysis of splenocytes from PbTg-infected C57BL/6 mice revealed increased frequencies of phagocytic cells, in particular more Ly6ChiF4/80+ monocytes (6.42% vs. 1.12%), Ly6CintF4/80+ monocytes (8.25% vs. 3.08%) and Ly6C+F4/80- neutrophils (12% vs 5.84%) (Fig 1B, upper row). As expected, F4/80+ cells were depleted upon injection of CloLip, in both PbTg infected C57BL/6 mice and uninfected control mice (Fig 1B lower panel), in comparison to non-depleted mice, that received PBS-loaded liposomes (Fig 1B, upper panel). Monocytes express high levels of both Ly6C and F4/80 and were depleted upon protective CloLip administration. On the other hand, there are data for a detrimental role of neutrophils in P. berghei ANKA induced ECM [21]. Therefore, we investigated the role of monocytes versus neutrophils in the ECM model. First, we evaluated the composition of cellular infiltrates in brains of PbTg-infected mice at day 6 post infection (p.i.) and sought for the presence of lymphocytes, inflammatory monocytes (defined by Ly6ChiLy6G-, box M) and neutrophils (defined by Ly6CintLy6G+, box N) (see gating scheme in Fig 1C and 1D). We observed that in the brains of PbTg-infected C57BL/6 mice with ECM, compared to naïve animals, there was a strong influx of CD45hi leukocytes, which were positive for CD3 (Fig 1D, lower panel), and CD45+CD11b+ mononuclear cells from the periphery that could be discriminated from CD45-CD11b+ microglia. In brains of PbTg-infected mice, but not in brains of naïve mice were both Ly6ChiLy6G- cells (inflammatory monocytes) and CD11b+Ly6G+Ly6Cint cells (neutrophils) among the CD45+CD11b+ mononuclear cells present (Fig 1C lower panel right). The inflammatory monocytes from brains of PbTg infected mice also showed strong expression of CCR2 and F4/80, MHC class II, and CD54, in contrast to Ly6G+ neutrophils, which showed either intermediate expression of those surface molecules or were devoid of these surface molecules (Fig 1E). Both mononuclear cell populations showed intermediate expression of CD11c, but were negative for CD3 (Fig 1E).


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)

Depletion of Ly6C+F4/80+ cells, which are present in brains of ECM positive mice, prevents ECM.(A) Survival of PbTg-infected C57BL/6 mice. 24 hr before inoculation with iRBC (5*104) of PbTg, indicated C57BL/6 mice received 200μl CloLip i.v.. Graph shows survival rates of mice p.i. (n = 10 mice per group) and represents 1 of 2 independent infection studies. Statistical analysis was performed using log-rank test. (B) Flow cytometric analysis of Ly6C+F4/80+ cells in spleens of non-depleted mice (upper row) and CloLip depleted mice (lower row), which were either naïve (uninfected) (left panel) or PbTg infected (right panel; 24 hours p.i.). Dot plots show representative stainings from 1 of 3 independent depletion studies in 3 mice per group. (C) Emergence of peripheral lymphocytes and mononuclear cells in brains of PbTg infected mice. Flow cytometric analyses show representative plots of naïve and PbTg infected mice, day 6 p.i.. Expression of CD45 and CD11b were used to discriminate lymphocytes (CD45hi CD11b-) and mononuclear cells (CD45+CD11b+) from brain-resident microglia (CD45-CD11b+). (D) Scheme for discrimination of cell populations and gating of Ly6ChiLy6G- inflammatory monocytes [M] versus neutrophils (Ly6CintLy6G+) [N] out of mononuclear cells as shown in data set of (B). CD45 hi cells were identified as CD3+ T cells (lower panel). (E) Ly6ChiLy6Gneg inflammatory monocytes but not Ly6G+ neutrophils from the brain of PbTg infected mice express CCR2, F4/80, CD54 and I-Ab. Flow cytometric data of brains isolated from PbTg infected animals, which were gated as depicted in (C). FMO = fluorescence minus one are shown as control. Representative data from 1 of 3 independent experiments are shown.
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pone.0124080.g001: Depletion of Ly6C+F4/80+ cells, which are present in brains of ECM positive mice, prevents ECM.(A) Survival of PbTg-infected C57BL/6 mice. 24 hr before inoculation with iRBC (5*104) of PbTg, indicated C57BL/6 mice received 200μl CloLip i.v.. Graph shows survival rates of mice p.i. (n = 10 mice per group) and represents 1 of 2 independent infection studies. Statistical analysis was performed using log-rank test. (B) Flow cytometric analysis of Ly6C+F4/80+ cells in spleens of non-depleted mice (upper row) and CloLip depleted mice (lower row), which were either naïve (uninfected) (left panel) or PbTg infected (right panel; 24 hours p.i.). Dot plots show representative stainings from 1 of 3 independent depletion studies in 3 mice per group. (C) Emergence of peripheral lymphocytes and mononuclear cells in brains of PbTg infected mice. Flow cytometric analyses show representative plots of naïve and PbTg infected mice, day 6 p.i.. Expression of CD45 and CD11b were used to discriminate lymphocytes (CD45hi CD11b-) and mononuclear cells (CD45+CD11b+) from brain-resident microglia (CD45-CD11b+). (D) Scheme for discrimination of cell populations and gating of Ly6ChiLy6G- inflammatory monocytes [M] versus neutrophils (Ly6CintLy6G+) [N] out of mononuclear cells as shown in data set of (B). CD45 hi cells were identified as CD3+ T cells (lower panel). (E) Ly6ChiLy6Gneg inflammatory monocytes but not Ly6G+ neutrophils from the brain of PbTg infected mice express CCR2, F4/80, CD54 and I-Ab. Flow cytometric data of brains isolated from PbTg infected animals, which were gated as depicted in (C). FMO = fluorescence minus one are shown as control. Representative data from 1 of 3 independent experiments are shown.
Mentions: Although ECM in PbA infected C57BL/6 mice is predominantly mediated by CD8+ T cells and IFNγ [6–8], the exact contribution of responding phagocytic cell subpopulations in developing such Th1 responses remains insufficiently defined. Therefore, we examined the participation of phagocytic cells in the development of ECM using a transgenic strain of Plasmodium berghei ANKA that expresses ovalbumin (PbTg) [22]. Initially, groups of C57BL/6 mice were intravenously injected with clodronate liposomes (CloLip) that result in the depletion of all cells possessing phagocytic activity [23] and then infected with PbTg. CloLip administration protected mice from developing lethal ECM and resulted in an 80% survival rate (Fig 1A). Parasitemia levels did not differ significantly between the infected groups (Supplementary information S1 Fig). When compared to naïve mice, flow cytometric analysis of splenocytes from PbTg-infected C57BL/6 mice revealed increased frequencies of phagocytic cells, in particular more Ly6ChiF4/80+ monocytes (6.42% vs. 1.12%), Ly6CintF4/80+ monocytes (8.25% vs. 3.08%) and Ly6C+F4/80- neutrophils (12% vs 5.84%) (Fig 1B, upper row). As expected, F4/80+ cells were depleted upon injection of CloLip, in both PbTg infected C57BL/6 mice and uninfected control mice (Fig 1B lower panel), in comparison to non-depleted mice, that received PBS-loaded liposomes (Fig 1B, upper panel). Monocytes express high levels of both Ly6C and F4/80 and were depleted upon protective CloLip administration. On the other hand, there are data for a detrimental role of neutrophils in P. berghei ANKA induced ECM [21]. Therefore, we investigated the role of monocytes versus neutrophils in the ECM model. First, we evaluated the composition of cellular infiltrates in brains of PbTg-infected mice at day 6 post infection (p.i.) and sought for the presence of lymphocytes, inflammatory monocytes (defined by Ly6ChiLy6G-, box M) and neutrophils (defined by Ly6CintLy6G+, box N) (see gating scheme in Fig 1C and 1D). We observed that in the brains of PbTg-infected C57BL/6 mice with ECM, compared to naïve animals, there was a strong influx of CD45hi leukocytes, which were positive for CD3 (Fig 1D, lower panel), and CD45+CD11b+ mononuclear cells from the periphery that could be discriminated from CD45-CD11b+ microglia. In brains of PbTg-infected mice, but not in brains of naïve mice were both Ly6ChiLy6G- cells (inflammatory monocytes) and CD11b+Ly6G+Ly6Cint cells (neutrophils) among the CD45+CD11b+ mononuclear cells present (Fig 1C lower panel right). The inflammatory monocytes from brains of PbTg infected mice also showed strong expression of CCR2 and F4/80, MHC class II, and CD54, in contrast to Ly6G+ neutrophils, which showed either intermediate expression of those surface molecules or were devoid of these surface molecules (Fig 1E). Both mononuclear cell populations showed intermediate expression of CD11c, but were negative for CD3 (Fig 1E).

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