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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 depletion of inflammatory monocytes protects PbTg infected mice against arising ECM.(A) Effective depletion of inflammatory monocytes (Ly6ChiLy6G-) or neutrophils (Ly6CintLy6G+) in the blood of PbTg-infected C57BL/6 mice upon treatment with anti-Gr1 (middle), anti-Ly6G (right), or anti-CCR2 (far right) monoclonal antibodies, on d+1 post depletion. CD11b+ leukocytes from the blood (upper row) were gated (squares) and analysed for the presence of Ly6C+Ly6G- monocytes and Ly6intLy6G+neutrophils (lower row) The data of the lower row correspond to data of the upper row. n = 4 mice per group. Flow cytometric analyses show a representative data plot for each group from 1 of 3 independent experiments. (B-G) Groups of PbTg-infected C57BL/6 mice were injected i.p. with anti-Gr1 mAb (B and C), anti-Ly6G mAb (D and E) or anti-CCR2 mAb (F and G) on the first day of infection (d0) or on days 3 and 5 p.i. (d3+5). Scores of cerebral pathology were determined on day 6 p.i. and bar graphs in B, D and F depict the mean and SEM of ECM score in individual mice (n = 8 mice per group). Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test (** p<0.01). Survival data shown in C, E and G were analyzed with Mantel-Cox log-rank test. p<0.05 was considered significant (* p<0.05, ** p<0.01, *** p<0.001). n = 10 mice per group.
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pone.0124080.g002: Early depletion of inflammatory monocytes protects PbTg infected mice against arising ECM.(A) Effective depletion of inflammatory monocytes (Ly6ChiLy6G-) or neutrophils (Ly6CintLy6G+) in the blood of PbTg-infected C57BL/6 mice upon treatment with anti-Gr1 (middle), anti-Ly6G (right), or anti-CCR2 (far right) monoclonal antibodies, on d+1 post depletion. CD11b+ leukocytes from the blood (upper row) were gated (squares) and analysed for the presence of Ly6C+Ly6G- monocytes and Ly6intLy6G+neutrophils (lower row) The data of the lower row correspond to data of the upper row. n = 4 mice per group. Flow cytometric analyses show a representative data plot for each group from 1 of 3 independent experiments. (B-G) Groups of PbTg-infected C57BL/6 mice were injected i.p. with anti-Gr1 mAb (B and C), anti-Ly6G mAb (D and E) or anti-CCR2 mAb (F and G) on the first day of infection (d0) or on days 3 and 5 p.i. (d3+5). Scores of cerebral pathology were determined on day 6 p.i. and bar graphs in B, D and F depict the mean and SEM of ECM score in individual mice (n = 8 mice per group). Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test (** p<0.01). Survival data shown in C, E and G were analyzed with Mantel-Cox log-rank test. p<0.05 was considered significant (* p<0.05, ** p<0.01, *** p<0.001). n = 10 mice per group.

Mentions: To analyse the relevance of inflammatory monocytes versus neutrophils in P. berghei ANKA induced ECM in C57BL/6 mice, we used mAb to either deplete (i) Ly6C+ and Ly6G+ cells by anti-Gr-1 mAb as a comparison to previous studies [21], or (ii) to selectively deplete either Ly6G+ neutrophils (anti-Ly6G mAb) [19], or (iii) Ly6Chi inflammatory monocytes with the help of anti-CCR2 mAb [20]. Groups of PbTg-infected mice received the respective depletion antibodies either directly upon PbTg infection (d0) or during infection (days 3 and 5 p.i.). The depletion efficacy of inflammatory monocytes (see Fig 1C for gating, box M, upper left, defined by CD11b+Ly6ChiLy6G-) or neutrophils (see Fig 1C, box N, upper right, defined by CD11b+Ly6Cint Ly6G+) was confirmed in the blood of all groups in comparison to naïve animals and control-infected mice 24h after mAb application (Fig 2A). These injection regimens led to reduction in CD11b+ leukocytes (Fig 2A upper row). We observed an effective elimination of both desired populations in the blood of anti-Gr1-administered mice (Fig 2A, lower row, middle plot). Administration of anti-Ly6G mAb, however, resulted in the depletion of neutrophils, but not of inflammatory monocytes (Fig 2A, lower row, right). In contrast, administration of anti-CCR2 mAb resulted in a strong reduction of Ly6ChiCD11b+ inflammatory monocytes, but not of Ly6G+ neutrophils (Fig 2A, lower row far right).


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 depletion of inflammatory monocytes protects PbTg infected mice against arising ECM.(A) Effective depletion of inflammatory monocytes (Ly6ChiLy6G-) or neutrophils (Ly6CintLy6G+) in the blood of PbTg-infected C57BL/6 mice upon treatment with anti-Gr1 (middle), anti-Ly6G (right), or anti-CCR2 (far right) monoclonal antibodies, on d+1 post depletion. CD11b+ leukocytes from the blood (upper row) were gated (squares) and analysed for the presence of Ly6C+Ly6G- monocytes and Ly6intLy6G+neutrophils (lower row) The data of the lower row correspond to data of the upper row. n = 4 mice per group. Flow cytometric analyses show a representative data plot for each group from 1 of 3 independent experiments. (B-G) Groups of PbTg-infected C57BL/6 mice were injected i.p. with anti-Gr1 mAb (B and C), anti-Ly6G mAb (D and E) or anti-CCR2 mAb (F and G) on the first day of infection (d0) or on days 3 and 5 p.i. (d3+5). Scores of cerebral pathology were determined on day 6 p.i. and bar graphs in B, D and F depict the mean and SEM of ECM score in individual mice (n = 8 mice per group). Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test (** p<0.01). Survival data shown in C, E and G were analyzed with Mantel-Cox log-rank test. p<0.05 was considered significant (* p<0.05, ** p<0.01, *** p<0.001). n = 10 mice per group.
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Related In: Results  -  Collection

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pone.0124080.g002: Early depletion of inflammatory monocytes protects PbTg infected mice against arising ECM.(A) Effective depletion of inflammatory monocytes (Ly6ChiLy6G-) or neutrophils (Ly6CintLy6G+) in the blood of PbTg-infected C57BL/6 mice upon treatment with anti-Gr1 (middle), anti-Ly6G (right), or anti-CCR2 (far right) monoclonal antibodies, on d+1 post depletion. CD11b+ leukocytes from the blood (upper row) were gated (squares) and analysed for the presence of Ly6C+Ly6G- monocytes and Ly6intLy6G+neutrophils (lower row) The data of the lower row correspond to data of the upper row. n = 4 mice per group. Flow cytometric analyses show a representative data plot for each group from 1 of 3 independent experiments. (B-G) Groups of PbTg-infected C57BL/6 mice were injected i.p. with anti-Gr1 mAb (B and C), anti-Ly6G mAb (D and E) or anti-CCR2 mAb (F and G) on the first day of infection (d0) or on days 3 and 5 p.i. (d3+5). Scores of cerebral pathology were determined on day 6 p.i. and bar graphs in B, D and F depict the mean and SEM of ECM score in individual mice (n = 8 mice per group). Statistical analysis was performed using Kruskal-Wallis test and Dunn’s Post test (** p<0.01). Survival data shown in C, E and G were analyzed with Mantel-Cox log-rank test. p<0.05 was considered significant (* p<0.05, ** p<0.01, *** p<0.001). n = 10 mice per group.
Mentions: To analyse the relevance of inflammatory monocytes versus neutrophils in P. berghei ANKA induced ECM in C57BL/6 mice, we used mAb to either deplete (i) Ly6C+ and Ly6G+ cells by anti-Gr-1 mAb as a comparison to previous studies [21], or (ii) to selectively deplete either Ly6G+ neutrophils (anti-Ly6G mAb) [19], or (iii) Ly6Chi inflammatory monocytes with the help of anti-CCR2 mAb [20]. Groups of PbTg-infected mice received the respective depletion antibodies either directly upon PbTg infection (d0) or during infection (days 3 and 5 p.i.). The depletion efficacy of inflammatory monocytes (see Fig 1C for gating, box M, upper left, defined by CD11b+Ly6ChiLy6G-) or neutrophils (see Fig 1C, box N, upper right, defined by CD11b+Ly6Cint Ly6G+) was confirmed in the blood of all groups in comparison to naïve animals and control-infected mice 24h after mAb application (Fig 2A). These injection regimens led to reduction in CD11b+ leukocytes (Fig 2A upper row). We observed an effective elimination of both desired populations in the blood of anti-Gr1-administered mice (Fig 2A, lower row, middle plot). Administration of anti-Ly6G mAb, however, resulted in the depletion of neutrophils, but not of inflammatory monocytes (Fig 2A, lower row, right). In contrast, administration of anti-CCR2 mAb resulted in a strong reduction of Ly6ChiCD11b+ inflammatory monocytes, but not of Ly6G+ neutrophils (Fig 2A, lower row far right).

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