Deficiency in astrocyte CCL2 production reduces neuroimmune control of Toxoplasma gondii infection.

Toxoplasma gondii is an obligate intracellular parasite that infects one-third of the world's human population and establishes infection in the brain. Cerebral immune cell infiltration is critical for controlling the parasite, but little is known about the molecular cues guiding immune cells to the brain during infection. Activated astrocytes produce CCL2, a chemokine that mediates inflammatory monocyte recruitment to tissues by binding to the CCR2 receptor. We detected elevated CCL2 production in the brains of C57BL/6J mice by 15 days after T. gondii infection. Utilizing confocal microscopy and intracellular flow cytometry, we identified microglia and brain-infiltrating myeloid cells as the main producers of CCL2 during acute infection, and CCL2 was specifically produced in regions of parasite infection in the brain. In contrast, astrocytes became the dominant CCL2 producer during chronic T. gondii infection. To determine the role of astrocyte-derived CCL2 in mobilizing immune cells to the brain and controlling T. gondii infection, we generated GFAP-Cre x CCL2fl/fl mice, in which astrocytes are deficient in CCL2 production. We observed significantly decreased immune cell recruitment and increased parasite burden in the brain during chronic, but not acute, infection of mice deficient in astrocyte CCL2 production, without an effect on peripheral immune responses. To investigate potential mechanisms explaining the reduced control of T. gondii infection, we analyzed key antimicrobial and immune players in host defense against T. gondii and detected a reduction in iNOS+ myeloid cells, and T. gondii-specific CD4+ T cells in the knockout mice. These data uncover a critical role for astrocyte-derived CCL2 in immune cell recruitment and parasite control in the brain during chronic, but not acute, T. gondii infection

CCL2 production by myeloid cells in the brain during acute <i>T</i>. <i>gondii</i> infection.

CCL2-RFP mice were injected with PBS as a control or infected with PRU strain T. gondii and examined at 15 DPI. (A) Percentage of immune cells out of CD45+ cells in the brains of PBS-injected (open circles) or T. gondii-infected (closed circles) mice. Cells were identified as infiltrating myeloid cells (CD45hiCD11b+), microglia (CD45int CD11b+), inflammatory monocytes (CD45+CD11b+Ly6Chi), patrolling monocytes (CD45+CD11b+Ly6Clo), neutrophils (CD45+Ly6G+), or T cells (CD45+CD3+). (B) Immune cell numbers in the meninges of PBS-injected (open circles) or T. gondii-infected (closed circles) mice. Cell types were identified as in (A) with the addition of meningeal macrophages (CD45+CD11b+CD206+F4/80+). (C) Frequencies of CCL2+ cells within each immune cell population in the brains of PBS-injected (open circles) or T. gondii-infected (closed circles) mice. n = 8–9 mice per group from three independent experiments. Statistical significance was determined by a randomized block ANOVA. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.</p

Frequencies of myeloid cells in the brains of GFAP-Cre CCL2^fl/fl and CCL2^fl/fl mice during chronic <i>T</i>. <i>gondii</i> infection.

CCL2fl/fl or GFAP-cre CCL2fl/fl mice were infected with T. gondii (ME49 strain), and the brains were harvested at 28 DPI. The frequencies of myeloid immune cells in the brain were determined by flow cytometry. n = 7–8 mice per group from two experiments. Statistical significance was determined by randomized block ANOVA. *p**p (TIF)</p

Gating strategies for immune cells in the brain and meninges.

Gates were drawn based on the fluorescence minus one (FMO) controls for the brains and meninges. (A) Representative flow cytometry gating scheme of brain cells from PBS-injected (top) or PRU strain T. gondii-infected (bottom) CCL2-RFP mice at 15 DPI. (B) Representative flow cytometry gating scheme of meningeal cells isolated from PBS-injected (top) or T. gondii-infected (bottom) CCL2-RFP mice at 15 DPI. (TIF)</p

Astrocyte-derived CCL2 drives immune cell recruitment to the brain during chronic <i>T</i>. <i>gondii</i> infection.

CCL2fl/fl and GFAP-cre CCL2fl/fl mice were infected with T. gondii and examined at 28 DPI. (A) Representative confocal microscopy of GFAP+ astrocytes (green), CCL2-RFP (red), and Iba1+ myeloid cells (blue) in CCL2fl/fl and GFAP-cre CCL2fl/fl mice at 28 DPI with PRU strain. (B) Quantification of CCL2+ GFAP+ cells of total GFAP+ cells per FOV. (C and D) qPCR for ccl2 (C) or ccr2 (D) transcripts from brains of CCL2fl/fl and GFAP-cre CCL2fl/fl mice at 28 DPI with ME49 strain. Transcripts are normalized to gapdh and shown relative to the mean transcript level of the CCL2fl/fl mice. (E) Quantification of brain myeloid immune cells by flow cytometry of CCL2fl/fl and GFAP-cre CCL2fl/fl mice at 28 DPI with ME49 strain. (F) Quantification of brain T cells by flow cytometry at 28 DPI with ME49 strain. In (B) n = 5–7 mice per group from three experiments. In (C and D) n = 3–4 mice per group, and in (E and F) n = 7–8 mice per group from two experiments. Statistical significance was determined by Student’s t-test (B-D) or randomized block ANOVA (E-F). *p**p<0.01, ***p<0.001, ****p<0.0001, ns: not significant.</p

Identity of CCR2-RFP⁺ cells in the brain during <i>T</i>. <i>gondii</i> infection.

CCR2RFP/+ mice were injected i.p. with 200 T. gondii (PRU strain), and brains were harvested at 15 DPI for flow cytometry of single cells. The percent of CCR2-RFP+ cells from each immune cell population is plotted. n = 7 mice per group. Statistical significance was determined by a one-way ANOVA. ****p (TIF)</p

CCL2 production by NeuN⁺ neurons and Iba1⁺and Mac2⁺ myeloid cells in GFAP-Cre CCL2^fl/fl and CCL2^fl/fl mice.

CCL2fl/fl and GFAP-cre CCL2fl/fl mice were infected with T. gondii (PRU strain) and the brains were harvested and stained with antibodies for analysis at 28 DPI. (A) Representative confocal microscopy of NeuN+ neurons (green), CCL2-RFP (red), and DAPI (blue). (B) Representative confocal microscopy of Mac2+ myeloid cells, (green), CCL2-RFP (red), and Iba1+ myeloid cells (blue). (C) Percent area of CCL2-RFP signal within each cell type. n = 12–23 FOV from 5–7 mice per group from 2 experiments. Statistical significance was calculated using Student’s t-test. ns, not significant. (TIF)</p

Mice deficient in astrocyte-derived CCL2 have reduced parasite control and decreased immune defense during chronic <i>T</i>. <i>gondii</i> infection.

CCL2fl/fl and GFAP-cre CCL2fl/fl mice were infected with ME49 strain T. gondii and examined at 28 DPI. (A) T. gondii cyst counts in brains from infected mice at 28 DPI. (B) Quantification of T. gondii tetramer+ CD4+ T cells in the brains of infected mice by flow cytometry at 28 DPI. (C) Quantification of iNOS+ myeloid cells by flow cytometry of the brains of infected mice at 28 DPI. In (A and C) n = 7–8 mice per group from 2 experiments, and in (B) n = 3–4 mice per group from 1 experiment. Statistical significance was determined by a randomized block ANOVA. *p**p<0.01, ***p<0.001.</p

CCL2 production increases in the brain during acute <i>Toxoplasma gondii</i> infection.

C57BL/6 (A) or CCL2-RFP mice (B-D) were injected with PBS or infected with type II Prugniaud (PRU) strain T. gondii, and brains were harvested and examined at 15 DPI. (A) CCL2 protein levels were measured in whole brain homogenates by ELISA. n = 7 to 10 mice per group from 4 independent experiments. (B) Representative tile scan of T. gondii (white), GFAP+ astrocytes (green), CCL2-RFP (red), and Iba1+ myeloid cells (blue) in brains of infected or PBS control mice. Magnified inset shows a FOV with T. gondii parasites (white arrowheads). (C) Representative confocal microscopy of T. gondii (white), GFAP+ astrocytes (green), CCL2-RFP (red), and Iba1+ myeloid cells (blue) in brains of infected or PBS control mice. (D) Quantification of mean fluorescence intensity (MFI) of CCL2-RFP in FOVs containing CCL2 in infected brains, and in the same brain regions in PBS control mice. n = 23–24 FOV from 6 mice per group from 3–4 independent experiments. Statistical significance was determined by Student’s t-test (A) and (D). ****p<0.0001.</p

Genotyping of mice.

(A) Gel from PCR of genomic DNA isolated from ear punches showing the presence of floxed ccl2 (top band labeled “Mutant CCL2”) in GFAP-Cre CCL2fl/fl, CCL2fl/fl, and GFAP-Cre CCL2fl/+ mice but not in C57BL/6 mouse. Endogenous ccl2 locus without loxP sequences (bottom band) is detected in C57BL/6 mice and GFAP-Cre CCL2fl/+mice, but not in GFAP-Cre CCL2fl/fl nor CCL2fl/fl mice. (B) Gel from PCR showing the presence of cre (top band) in GFAP-Cre CCL2fl/fl and GFAP-Cre CCL2fl/+ mice but not in CCL2fl/fl nor C57BL/6J wildtype mice. To control for the presence of DNA in each sample, primers to detect the T cell receptor (TCR) (bottom band) were used. (TIF)</p