Innate immune responses to Toxoplasma gondii infection

Inflammation is a tightly regulated process necessary to protect the body against infection but, in excess, can be dangerous and lead to damage. Toxoplasma gondii is an obligate intracellular foodborne pathogen with the unique ability to cross the blood-brain barrier and form lifelong parasite cysts in neurons. This infection drives a protective immune response in the brain that can control parasitic growth but not clear the infection. CCR2 chemokine receptor-expressing monocytes play a necessary role in controlling T. gondii infection both in the periphery and the brain. Once in the brain, monocytes are specifically recruited to areas containing clusters of T. gondii. However, the drivers of monocyte recruitment to sites of infection, specifically to areas containing T. gondii, are not well understood. This research aimed to understand the signals that drive protective immunity against T. gondii infection. In the brain, we did determine a critical role for chemokine production in neuroinflammation. In the brain, we determined that the cells producing the potent CCR2-binding chemokine, CCL2, changed over the course of infection: microglia were the main CCL2 producers during acute infection, whereas astrocytes became the dominant CCL2 producers during chronic infection. Interestingly, the ablation of CCL2 production from astrocytes reduced immune cell recruitment to the brain during chronic infection and decreased control of the parasitic infection. We also found that activation of the transcription factor, NF-B, and CCL2 production are increased near parasites in the brain. However, the parasite effector protein GRA15 does not drive immune cell recruitment to parasites. Furthermore, T. gondii replication and host cell lysis play significant roles in driving immune cells to parasites in the brain. Additionally, we found that Piezo1 is upregulated during infection and Piezo1 expression in myeloid cells aids in parasite dissemination to the liver during early acute infection. However, Piezo1 expression in myeloid cells does not affect dissemination to the brain during late acute infection nor control of parasitic burden throughout chronic infection. Collectively, this work highlights the role of host cell responses to T. gondii in driving immune cell recruitment to control parasitic infection

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

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

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

Production of CCL2 by cell type during acute <i>T</i>. <i>gondii</i> infection.

CCL2-RFP mice were injected with PBS or infected with PRU strain T. gondii, and brains were harvested at 15 DPI. (A) Representative confocal microscopy of T. gondii (white), Mac2+ myeloid cells (green), CCL2-RFP (red), and Iba1+ myeloid cells (blue) in brains of PBS control mice or T. gondii-infected mice in FOV with or without parasites. (B) Percent area of each cell type within FOV with or without parasites from infected mice. (C) Percent area of each cell type within CCL2+ area in FOV with or without parasites from infected mice. n = 8–25 FOV from 4–5 mice per group from 4–5 independent experiments. Statistical significance was determined by two-way ANOVA. ****p<0.0001, ns: not significant.</p