Limits...
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.

Show MeSH

Related in: MedlinePlus

Early monocyte depletion prevents IFNγ producing T cell infiltration into the brain but does not affect peripheral PbTg-specific CTL responses.C57BL/6 mice were infected with 5*10e4 PbTg iRBC i.v.. Simultaneously, groups of mice received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb. Six days later, T cell subsets in the brain and spleen were analysed and cellular immune responses were evaluated. (A) Frequencies of CD8+ cells and (B) CD4+ cells among brain infiltrates were determined by flow cytometry. Each group contained 4–5 mice. (C) Leukocyte preparations from the brains of individual mice were re-stimulated ex vivo and intracellular stained to determine the frequency of IFNγ producing CD8+ T cells. Representative images of brain derived IFNγ+CD8+ T cells by flow cytometry are shown. Calculated frequencies of CD8+ IFNγ+ T cells and CD8+ IFNγ+ T cells in the brains as analysed from (A) are shown in Table 1. (D) Fold increase of IFNγ mRNA levels relative to GAPDH in the brains of PbTg-infected mice ± d0 depletion on day 6 p.i. n = 6–8 per group, Kruskal Wallis test with Dunn’s Post test was performed. (E) Total cell count of splenocytes from all d0 depletion groups and controls at day 6 p.i. (F) Frequency of CD3+CD8+ splenocytes in percent from all d0 mAb depletion groups and controls at day 6 p.i. (G) Calculated total amount of CD8+ splenocytes according to data from F and G. (H) In vivo cytotoxicity assay analysing PbTg-specific T cells. Recipient PbTg-infected mice, that were or were not additionally treated with depletion antibodies, received an adoptive transfer of CSFE-labelled T cells, that were loaded with ovalbumin-derived MHC class I peptide SIINFEKL and unloaded control cells i.v. (1x107/mouse) on day 5 p.i.. 18 hours later the level of lytic activity was measured by flow cytometry in the spleen. (I) In addition, splenocytes from the same animals as in (H) were re-stimulated with SIINFEKL ex vivo for 24 hours and IFNγ production was quantified by sandwich ELISA. (A-I) 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, ** p<0.01).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4401438&req=5

pone.0124080.g005: Early monocyte depletion prevents IFNγ producing T cell infiltration into the brain but does not affect peripheral PbTg-specific CTL responses.C57BL/6 mice were infected with 5*10e4 PbTg iRBC i.v.. Simultaneously, groups of mice received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb. Six days later, T cell subsets in the brain and spleen were analysed and cellular immune responses were evaluated. (A) Frequencies of CD8+ cells and (B) CD4+ cells among brain infiltrates were determined by flow cytometry. Each group contained 4–5 mice. (C) Leukocyte preparations from the brains of individual mice were re-stimulated ex vivo and intracellular stained to determine the frequency of IFNγ producing CD8+ T cells. Representative images of brain derived IFNγ+CD8+ T cells by flow cytometry are shown. Calculated frequencies of CD8+ IFNγ+ T cells and CD8+ IFNγ+ T cells in the brains as analysed from (A) are shown in Table 1. (D) Fold increase of IFNγ mRNA levels relative to GAPDH in the brains of PbTg-infected mice ± d0 depletion on day 6 p.i. n = 6–8 per group, Kruskal Wallis test with Dunn’s Post test was performed. (E) Total cell count of splenocytes from all d0 depletion groups and controls at day 6 p.i. (F) Frequency of CD3+CD8+ splenocytes in percent from all d0 mAb depletion groups and controls at day 6 p.i. (G) Calculated total amount of CD8+ splenocytes according to data from F and G. (H) In vivo cytotoxicity assay analysing PbTg-specific T cells. Recipient PbTg-infected mice, that were or were not additionally treated with depletion antibodies, received an adoptive transfer of CSFE-labelled T cells, that were loaded with ovalbumin-derived MHC class I peptide SIINFEKL and unloaded control cells i.v. (1x107/mouse) on day 5 p.i.. 18 hours later the level of lytic activity was measured by flow cytometry in the spleen. (I) In addition, splenocytes from the same animals as in (H) were re-stimulated with SIINFEKL ex vivo for 24 hours and IFNγ production was quantified by sandwich ELISA. (A-I) 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, ** p<0.01).

Mentions: To determine whether the reduced number of CD3+ T cells, which we observed in immunohistochemistry in the frontal cortex of anti-Gr1 or CCR2 mAb PbTg-infected mice also affected different T cell populations that cross the blood brain barrier, we analysed the frequencies of CD8+ and CD4+ T cells in the preparations from the whole brains by flow cytometry. In addition, we analysed the capacity of CD8+ T cells to produce IFNγ. On day 6 p.i., PbTg infected mice presented elevated frequencies of CD8+ T cells and CD4+ T cells in the brain when compared to naïve mice (Fig 5A and 5Bc.f. bars 1 and 2). More than half of the CD8+ T cells that had infiltrated the brains of PbTg-infected mice, produced IFNγ (Table 1, Fig 5C). Administration of anti-Gr1 at the start of PbTg infection resulted in a significant reduction of CD8+ T cells, including IFNγ producing and non-producing CD8+ T cells (Table 1) but did not change the influx of CD4+ T cells within the brain (Fig 5A and 5Bc.f. bars 2 with 3). These results were also observed when we administered antibodies on days 3 and 5 of infection (Table 1). Importantly, administration of anti-CCR2 mAb led to significantly reduced frequencies of IFNγ producing CD8+ T cells in the brains of the mice, whereas the frequencies of CD4+ T cells were not changed (Table 1). The depletion of neutrophils however, using anti-Ly6G mAb, did neither result in reduced frequencies of CD8+ T or CD4+ T cells nor in the changed proportion of IFNγ producing CD8+ T cells in the brain compared to non-depleted infected mice (Fig 5A and 5Bc.f. bars 2 and 4, Fig 5C, Table 1). Thus, we conclude that the protection of PbTg-infected mice from ECM upon depletion of Ly6Chi monocytes by anti-Gr-1 or anti-CCR2 mAb was due to abrogated infiltration of lymphocytes into the CNS, mainly of IFN-γ-producing CD8+ T cells. Next, we analysed the expression of IFNγ in the brains of PbTg-infected and depleted mice. We detected elevated mRNA levels of IFNγ in the brains of PbTg-infected mice (Fig 5Dc.f. bars 1 and 2), which were 10-fold reduced in the brains of anti-Gr1 mAb treated infected mice and five-fold reduced in the brains of anti-CCR2 treated infected mice (Fig 5Dc.f. bars 2 with 3 and 5). Administration of anti-CCR2 or anti-Gr-1 mAb during an ongoing PbTg infection also resulted in reduced IFNγ levels in the brains, which was significant in the case of anti-Gr-1, demonstrating again a strong effect of this clone (S4 Fig). Interestingly, the d0 application of anti-Ly6G mAb to infected mice resulted in a two-fold decrease of IFNγ levels in the brain, but remained a trend (Fig 5Dc.f. bars 2 and 4), similar to day 3 and 5 application of anti-Ly6G (S4 Fig 4). Taken together, we observed the strongest effects concerning influx of T cells into the brain and block of IFNγ production in anti-Gr-1 mAb injected mice, but importantly, comparable effects were achieved with the selective depletion of Ly6Chi inflammatory monocytes with the help of anti-CCR2-injection.


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 IFNγ producing T cell infiltration into the brain but does not affect peripheral PbTg-specific CTL responses.C57BL/6 mice were infected with 5*10e4 PbTg iRBC i.v.. Simultaneously, groups of mice received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb. Six days later, T cell subsets in the brain and spleen were analysed and cellular immune responses were evaluated. (A) Frequencies of CD8+ cells and (B) CD4+ cells among brain infiltrates were determined by flow cytometry. Each group contained 4–5 mice. (C) Leukocyte preparations from the brains of individual mice were re-stimulated ex vivo and intracellular stained to determine the frequency of IFNγ producing CD8+ T cells. Representative images of brain derived IFNγ+CD8+ T cells by flow cytometry are shown. Calculated frequencies of CD8+ IFNγ+ T cells and CD8+ IFNγ+ T cells in the brains as analysed from (A) are shown in Table 1. (D) Fold increase of IFNγ mRNA levels relative to GAPDH in the brains of PbTg-infected mice ± d0 depletion on day 6 p.i. n = 6–8 per group, Kruskal Wallis test with Dunn’s Post test was performed. (E) Total cell count of splenocytes from all d0 depletion groups and controls at day 6 p.i. (F) Frequency of CD3+CD8+ splenocytes in percent from all d0 mAb depletion groups and controls at day 6 p.i. (G) Calculated total amount of CD8+ splenocytes according to data from F and G. (H) In vivo cytotoxicity assay analysing PbTg-specific T cells. Recipient PbTg-infected mice, that were or were not additionally treated with depletion antibodies, received an adoptive transfer of CSFE-labelled T cells, that were loaded with ovalbumin-derived MHC class I peptide SIINFEKL and unloaded control cells i.v. (1x107/mouse) on day 5 p.i.. 18 hours later the level of lytic activity was measured by flow cytometry in the spleen. (I) In addition, splenocytes from the same animals as in (H) were re-stimulated with SIINFEKL ex vivo for 24 hours and IFNγ production was quantified by sandwich ELISA. (A-I) 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, ** p<0.01).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401438&req=5

pone.0124080.g005: Early monocyte depletion prevents IFNγ producing T cell infiltration into the brain but does not affect peripheral PbTg-specific CTL responses.C57BL/6 mice were infected with 5*10e4 PbTg iRBC i.v.. Simultaneously, groups of mice received either anti-Gr1, anti-Ly6G or anti-CCR2 mAb. Six days later, T cell subsets in the brain and spleen were analysed and cellular immune responses were evaluated. (A) Frequencies of CD8+ cells and (B) CD4+ cells among brain infiltrates were determined by flow cytometry. Each group contained 4–5 mice. (C) Leukocyte preparations from the brains of individual mice were re-stimulated ex vivo and intracellular stained to determine the frequency of IFNγ producing CD8+ T cells. Representative images of brain derived IFNγ+CD8+ T cells by flow cytometry are shown. Calculated frequencies of CD8+ IFNγ+ T cells and CD8+ IFNγ+ T cells in the brains as analysed from (A) are shown in Table 1. (D) Fold increase of IFNγ mRNA levels relative to GAPDH in the brains of PbTg-infected mice ± d0 depletion on day 6 p.i. n = 6–8 per group, Kruskal Wallis test with Dunn’s Post test was performed. (E) Total cell count of splenocytes from all d0 depletion groups and controls at day 6 p.i. (F) Frequency of CD3+CD8+ splenocytes in percent from all d0 mAb depletion groups and controls at day 6 p.i. (G) Calculated total amount of CD8+ splenocytes according to data from F and G. (H) In vivo cytotoxicity assay analysing PbTg-specific T cells. Recipient PbTg-infected mice, that were or were not additionally treated with depletion antibodies, received an adoptive transfer of CSFE-labelled T cells, that were loaded with ovalbumin-derived MHC class I peptide SIINFEKL and unloaded control cells i.v. (1x107/mouse) on day 5 p.i.. 18 hours later the level of lytic activity was measured by flow cytometry in the spleen. (I) In addition, splenocytes from the same animals as in (H) were re-stimulated with SIINFEKL ex vivo for 24 hours and IFNγ production was quantified by sandwich ELISA. (A-I) 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, ** p<0.01).
Mentions: To determine whether the reduced number of CD3+ T cells, which we observed in immunohistochemistry in the frontal cortex of anti-Gr1 or CCR2 mAb PbTg-infected mice also affected different T cell populations that cross the blood brain barrier, we analysed the frequencies of CD8+ and CD4+ T cells in the preparations from the whole brains by flow cytometry. In addition, we analysed the capacity of CD8+ T cells to produce IFNγ. On day 6 p.i., PbTg infected mice presented elevated frequencies of CD8+ T cells and CD4+ T cells in the brain when compared to naïve mice (Fig 5A and 5Bc.f. bars 1 and 2). More than half of the CD8+ T cells that had infiltrated the brains of PbTg-infected mice, produced IFNγ (Table 1, Fig 5C). Administration of anti-Gr1 at the start of PbTg infection resulted in a significant reduction of CD8+ T cells, including IFNγ producing and non-producing CD8+ T cells (Table 1) but did not change the influx of CD4+ T cells within the brain (Fig 5A and 5Bc.f. bars 2 with 3). These results were also observed when we administered antibodies on days 3 and 5 of infection (Table 1). Importantly, administration of anti-CCR2 mAb led to significantly reduced frequencies of IFNγ producing CD8+ T cells in the brains of the mice, whereas the frequencies of CD4+ T cells were not changed (Table 1). The depletion of neutrophils however, using anti-Ly6G mAb, did neither result in reduced frequencies of CD8+ T or CD4+ T cells nor in the changed proportion of IFNγ producing CD8+ T cells in the brain compared to non-depleted infected mice (Fig 5A and 5Bc.f. bars 2 and 4, Fig 5C, Table 1). Thus, we conclude that the protection of PbTg-infected mice from ECM upon depletion of Ly6Chi monocytes by anti-Gr-1 or anti-CCR2 mAb was due to abrogated infiltration of lymphocytes into the CNS, mainly of IFN-γ-producing CD8+ T cells. Next, we analysed the expression of IFNγ in the brains of PbTg-infected and depleted mice. We detected elevated mRNA levels of IFNγ in the brains of PbTg-infected mice (Fig 5Dc.f. bars 1 and 2), which were 10-fold reduced in the brains of anti-Gr1 mAb treated infected mice and five-fold reduced in the brains of anti-CCR2 treated infected mice (Fig 5Dc.f. bars 2 with 3 and 5). Administration of anti-CCR2 or anti-Gr-1 mAb during an ongoing PbTg infection also resulted in reduced IFNγ levels in the brains, which was significant in the case of anti-Gr-1, demonstrating again a strong effect of this clone (S4 Fig). Interestingly, the d0 application of anti-Ly6G mAb to infected mice resulted in a two-fold decrease of IFNγ levels in the brain, but remained a trend (Fig 5Dc.f. bars 2 and 4), similar to day 3 and 5 application of anti-Ly6G (S4 Fig 4). Taken together, we observed the strongest effects concerning influx of T cells into the brain and block of IFNγ production in anti-Gr-1 mAb injected mice, but importantly, comparable effects were achieved with the selective depletion of Ly6Chi inflammatory monocytes with the help of anti-CCR2-injection.

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