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. 2021 Sep 30:12:711876.
doi: 10.3389/fimmu.2021.711876. eCollection 2021.

Eosinophils Suppress the Migration of T Cells Into the Brain of Plasmodium berghei-Infected Ifnar1-/- Mice and Protect Them From Experimental Cerebral Malaria

Johanna F Scheunemann  1 , Julia J Reichwald  1 , Patricia Jebett Korir  1 , Janina M Kuehlwein  1 , Lea-Marie Jenster  1 , Christiane Hammerschmidt-Kamper  2 , Matthew D Lewis  2 , Katrin Klocke  1 , Max Borsche  1 , Kim E Schwendt  1 , Camille Soun  3 , Stephanie Thiebes  3 , Andreas Limmer  4 , Daniel R Engel  3 , Ann-Kristin Mueller  2   5 , Achim Hoerauf  1   6 , Marc P Hübner  1   6 , Beatrix Schumak  1
Affiliations

Eosinophils Suppress the Migration of T Cells Into the Brain of Plasmodium berghei-Infected Ifnar1-/- Mice and Protect Them From Experimental Cerebral Malaria

Johanna F Scheunemann et al. Front Immunol. .

Abstract

Cerebral malaria is a potentially lethal disease, which is caused by excessive inflammatory responses to Plasmodium parasites. Here we use a newly developed transgenic Plasmodium berghei ANKA (PbAAma1OVA) parasite that can be used to study parasite-specific T cell responses. Our present study demonstrates that Ifnar1-/- mice, which lack type I interferon receptor-dependent signaling, are protected from experimental cerebral malaria (ECM) when infected with this novel parasite. Although CD8+ T cell responses generated in the spleen are essential for the development of ECM, we measured comparable parasite-specific cytotoxic T cell responses in ECM-protected Ifnar1-/- mice and wild type mice suffering from ECM. Importantly, CD8+ T cells were increased in the spleens of ECM-protected Ifnar1-/- mice and the blood-brain-barrier remained intact. This was associated with elevated splenic levels of CCL5, a T cell and eosinophil chemotactic chemokine, which was mainly produced by eosinophils, and an increase in eosinophil numbers. Depletion of eosinophils enhanced CD8+ T cell infiltration into the brain and increased ECM induction in PbAAma1OVA-infected Ifnar1-/- mice. However, eosinophil-depletion did not reduce the CD8+ T cell population in the spleen or reduce splenic CCL5 concentrations. Our study demonstrates that eosinophils impact CD8+ T cell migration and proliferation during PbAAma1OVA-infection in Ifnar1-/- mice and thereby are contributing to the protection from ECM.

Keywords: CCL5; CD8 T cells; IFNAR1; NK cells; Plasmodium berghei; eosinophils; experimental cerebral malaria (ECM); malaria.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
PbAAma1OVA-infected Ifnar1-/- mice are protected from ECM. (A–E) C57BL/6 WT and Ifnar1-/- mice were infected i.v. with 5x104 PbAAma1OVA-infected red blood cells and monitored for ECM development and parasitemia. (A) Days until onset of ECM, stated as survival of infected mice. Due to ethical reasons animals were sacrificed with a score of six or less. (B) Neurological symptoms of PbAAma1OVA-infected (PbA inf.) mice according to the rapid murine coma and behavior scale (RMCBS) on day 6 p.i. compared to naïve WT controls. (C) Blood-stage parasitemia levels on day 6 p.i. (D, E) The stability of the blood brain-barrier was analyzed with an Evans Blue assay on day 6 p.i. in PbA inf. mice and naïve controls. (D) Photos from brains isolated from representative animals one hour after injection of Evans Blue. (E) Colorimetric quantification of dye extravasation from isolated brains of naïve and PbA inf. mice on day 6 p.i. (A) n=10 per group; Log-rank (Mantel-Cox) test. (B) n=14-16 per group, data are pooled from 4 individual experiments; Kruskal-Wallis with Dunn’s post-test. (C) n=6 per group, representative for 3 individual experiments; Mann-Whitney-U-test. (E) n=3-4 per group. Kruskal-Wallis with Dunn’s post-test. (B, C, E) Data show median with IQR. p.i., post infection; PbA inf., PbAAma1OVA-infected, RMCBS, rapid murine coma and behavior scale.
Figure 2
Figure 2
Cellular infiltration and brain inflammation are reduced in PbAAma1OVA-infected Ifnar1-/- mice. (A–L) C57BL/6 WT mice and Ifnar1-/- mice were infected i.v. with 5x104 PbAAma1OVA-infected red blood cells and analyzed ex vivo on day 6 p.i. regarding cellular brain infiltration via flow cytometry and ELISA. (A) Enriched brain leukocytes from naïve and infected (PbA inf.) mice were analyzed according to the scheme in (B) via flow cytometry to distinguish CD11bloCD45hi-infiltrated leukocytes (gated in P1) and CD11bintCD45hi mononuclear cells (gated in P2) from CD11bhiCD45int-brain resident immune cells (gate P3). (C) Dotplots according to the gating in (B) from brain-enriched leukocytes of representative samples. (D) Total count of CD45hi-infiltrated leukocytes (P1+P2). (E) Total count of CD8+ T cells (CD45+CD3+CD8+). (F) Total count of lineage positive NK cells (CD45+Lineage+CD49b+NKp46+T-bet+). (G) Expression of CCR5 on CD8+ T cells. (H) Dotplots of Granzyme B (GrzB) expression in CD8+ T cells of representative samples. (I) Expression of GrzB in CD8+ T cells in PbA-inf. mice. (J–L) ELISA analysis of enriched brain leukocyte culture supernatants after 24h for (J) TNF, (K) CCL3 and (L) CCL5. (A–L) n=4-11, pooled data from 2 individual experiments. (A, D-G, I–L) Data show median with IQR. When comparing two groups Mann-Whitney-U-test, for more groups Kruskal-Wallis with Dunn’s post-test. (A, D) Mann-Whitney-U-test for comparing the PbA-infected groups. GrzB, Granzyme B; p.i., post infection; PbA inf., PbAAma1OVA-infected.
Figure 3
Figure 3
Accumulation of CD8+T cell T cells in spleens of PbAAmaOVA1-infected Ifnar1-/- mice. (A–K) C57BL/6 WT mice and Ifnar1-/- mice were infected i.v. with 5x104 PbAAma1OVA-infected red blood cells and analyzed ex vivo on day 6 p.i. regarding cellular composition and immune activation in the spleen by flow cytometry (B–I) and ELISA (J, K). (A) Total splenocyte count. (B) Frequency of T cells (CD3+) among all splenocytes. (C) Frequency of CD8+ T cells (CD3+CD8+) and (D) frequency of CD4+ T cells (CD3+CD4+) of all T cells. (E) Ratio of CD4+ T cells to CD8+ T cells in the spleen based on their frequency. (F) Frequency of lineage positive NK cells (CD45+Lineage+CD49b+NKp46+T-bet+) among all splenocytes. (G) Frequency of IFN-y positive CD8+ T cells among all CD8+ T cells after in vitro stimulation with PMA and ionomycin. (H) CXCR3 on CD8+ T cells. (I) SIINFEKL (S8L)-specific lysis. (J, K) Cytokine quantification in total splenocyte culture supernatants after 24h for (J) IFN-γ, and (K) Granzyme B (GrzB). (A–E, H, J, K) n=4-6, representative for 3 individual experiments (F) n= 5-10, WT naïve and PbA-infected data pooled from two individual experiments. (G, I) n=4-6, data from 1 experiment. (A–K) Data shown as median with IQR; Kruskal-Wallis with Dunn’s post-test. GrzB, Granzyme B; ns, non-significant; p.i., post infection; PbA inf., PbAAma1OVA-infected; PMA, phorbol myristate acetate.
Figure 4
Figure 4
Increased secretion and expression of CCL5/CCR5 in ECM-protected Ifnar1-/- mice. (A–H) C57BL/6 WT mice and Ifnar1-/- mice were infected i.v. with 5x104 PbAAma1OVA-infected red blood cells and spleen cells were analyzed ex vivo on day 6 p.i. by ELISA (A) and flow cytometry (B-H). (A) Cytokine quantification in total splenocyte culture supernatants after 24h for CCL5. (B) Expression of CCR5 on CD8+ T cells. (C) Total count of CCL5 expressing cells in the spleen. (D) Overview of CCL5 expressing cell populations in the spleens of naïve and PbA-inf. WT and Ifnar1-/- mice. (E) Total count of CCL5 expressing CD8+ T cells (CD3+CD8+), (F) CCL5 expressing eosinophils (CD8-CD11b+Ly6Cint-hiLy6G-SiglecF+) and (G) CCL5 expressing M2-like macrophages (CD11b+, Ly6G-, SiglecF-, F4/80+, Relmα+). (H) CCL5 expression in CD8+ T cells, eosinophils and M2-like macrophages. (A, B) n=4-6, representative for 3 individual experiments. (C–H) n=3-5, data from 1 experiment. (A–C, E–H) Data shown as median with IQR; Kruskal-Wallis with Dunn’s post-test. MØ, Macrophage; PbA inf., PbAAma1OVA-infected.
Figure 5
Figure 5
Enhanced type 2-associated immune response in the spleen of PbAAma1OVA-infected Ifnar1-/- mice. (A–I) C57BL/6 WT mice and Ifnar1-/- mice were infected i.v. with 5x104 PbAAma1OVA-infected red blood cells and the spleen was analyzed ex vivo on day 6 p.i.. Cytokine quantification in total splenocyte culture supernatants after 24h for (A) IL-10 and (B) IL-13. (C) Gating of Th2 cells (gated on CD45+Lineage+TCRβ+cells) and (D) total count of Th2 spleen cells. (E) Gating of eosinophils (gated on CD8-CD11b+Ly6G-) and (F) total count of eosinophils in the spleen. (G) Representative immunohistochemical staining of spleen slides of PbA-infected WT and PbA-infected Ifnar1-/- mice for DNA (DAPI), CD8+ T cells (CD8) and eosinophils (SiglecF). Bar in pictures represents 100µm. (H, I) MACS-purified CD11b+ splenocytes from PbAAma1OVA-infected WT versus Ifnar1-/- mice on day 6 p.i. were analyzed for their (H) arginase activity by measuring urea production and expression of (I) YM-1 by PCR. Each sample was normalized to β-actin. The expression level was calculated as fold-increase against naïve controls. (A, B) n=4-6 per group, representative for 3 independent experiments. (D, F) n=5-9, pooled data from 2 individual experiments. (G) n=3, data from 1 experiment. (H) n=4-5, data representative for 2 individual experiments. (I) n=5, data from 1 experiment. (A, B, D, F, H, I) Data shown as median with IQR; Kruskal-Wallis with Dunn’s post-test. ΔCt, delta cycle threshold; PbA inf., PbAAma1OVA-infected.
Figure 6
Figure 6
Eosinophil-mediated protection in PbAAma1OVA-infected Ifnar1-/- mice. (A–M) C57BL/6 WT mice and Ifnar1-/- mice were infected i.v. with 5x104 PbAAma1OVA-infected red blood cells and a subset of Ifnar1-/- mice were depleted for eosinophils. (A) Experimental setup. (B) Neurological symptoms of PbA-infected (PbA inf.) mice according to the rapid murine coma and behavior scale (RMCBS) on day 6 p.i. compared to naïve WT controls. (C) Blood-stage parasitemia levels determined by Giemsa stains on day 6 p.i.. (D) Total count of brain infiltrated CD45hi leukocytes. (E) Total count of CD8+ T cells (CD3+CD8+) among infiltrated CD45hi-infiltrated leukocytes in the brain. (F) Total splenocyte count. (G) Frequency of CD8+ T cells (CD3+CD8+) among all splenocytes. (H) Total count of CD8+ T cells (CD3+CD8+) in the spleen. (I) CCL5 quantification in 24h unstimulated splenocyte culture supernatant. (J) Total count of Th2 cells (CD45+Lineage+TCRβ+CD90.2+GATA-3+) in the spleen. (K) IL-13, (L) IFN-γ and (M) Granzyme B (GrzB) quantification in 24h unstimulated splenocyte culture supernatant. (B, C) n=8-14, data pooled from 2 individual experiments. (D, E, G–I, K–M) n=8-11, data pooled from 2 individual experiments. (F, J) n=4-5, data from one experiment. (M) Data shown as median with IQR; Kruskal-Wallis with Dunn’s post-test. (B, F, G) Mann-Whitney-U-test between PbA-infected Ifnar1-/- vs. eosinophil-depleted Ifnar1 -/-.GrzB, Granzyme B; ns, non-significant; PbA inf., PbAAma1OVA-infected.
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