Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial to Control Cerebral Infection with Toxoplasma gondii

Abstract

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations including LPS. However, the physiological role of IFN-γ production by brain-resident cells including glial cells in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b⁺ cells suggested that IFN-γ production by microglia, which is the only CD11b⁺ cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.

Introduction

T. gondii, an obligate intracellular protozoan parasite, is one of the pathogens that can establish infection in the brain. During the acute stage of infection, IFN-γ-mediated immune responses and, to a lesser degree humoral immunity, control proliferation of tachyzoites, but the parasite establishes a chronic infection by forming cysts preferentially in the brain. It is estimated that 500 million to 2 billion people worldwide are chronically infected with this parasite. The requirement of host immunity to maintain the latency of this chronic infection is evident by an occurrence of reactivation of the infection that can cause life-threatening toxoplasmic encephalitis in immunocompromised individuals such as those with AIDS and organ transplants (1).

Murine models of reactivation of cerebral T. gondii infection demonstrated that IFN-γ is essential for the protective immunity to control the chronic infection. In addition to T cells, cells other than T and NK cells need to produce this cytokine to prevent reactivation of the infection in the brain (2). Microglia and astrocytes from the brain have been shown to produce IFN-γ in vitro in response to various stimulations including LPS (3, 4). However, it is unknown whether IFN-γ produced by brain-resident cells including glial cells plays any roles in resistance to cerebral infections with microorganisms including T. gondii. The present study revealed that IFN-γ production by brain-resident cells including microglia is crucial for upregulating IFN-γ-mediated cerebral protective innate immunity and recruiting T cells into the brain to control the parasite.

Materials and Methods

Mice

Female BALB/c, RAG1^−/−, IFN-γ^−/−, SCID, and (C57BL/6 × SJL)F1 hybrid mice were from the Jackson Laboratories and Swiss-Webster mice were from Taconic. RAG1^−/−IFN-γ^−/− mice were generated by mating RAG1^−/− with IFN-γ^−/− animals. For generation of a transgenic mouse strain that produces IFN-γ only in CD11b-expressing cells, the pCD11b-IFN-γ transgene (Fig. 2B) was constructed by placing IFN-γ coding sequence under control of the CD11b promoter by modifying a CD11b-Thy1.1 construct (5) kindly provided by Dr. Daniel Tenen of Harvard Medical School. After confirming the activity of pCD11b-IFN-γ to induce production of IFN-γ only in CD11b⁺ cells by transfecting CD11b⁺ (J774) and CD11b⁻ (COS7) cells in vitro (Supplemental Table I), pCD11b-IFN-γ transgene was microinjected into zygotes from (C57BL/6 × SJL)F1 hybrid animals. Pups carrying the transgene identified by PCR (Fig. 2C) were mated to (C57BL/6 × SJL)F1, backcrossed to BALB/c mice 6 times, and then mated with IFN-γ^−/− mice to generate animals that express this cytokine only by CD11b⁺ cells (CD11b only-IFN-γ mice). Experimental procedures were performed in accordance with approved protocols from the Institutional Animal Care and Use Committee.

FIGURE 2.

Microglia produce IFN-γ in response to T. gondii tachyzoite antigens and their IFN-γ production can limit cerebral tachyzoite growth during reactivation of the infection. (A) IFN-γ production by EOC20 following stimulation with TLA. (B) pCD11b-IFN-γ gene construct. (C) Genotyping to determine CD11b-IFN-γ mice. Upper band: β-globin. Lower band: CD11b-IFN-γ transgene. (D) IFN-γ expression in PBMC of CD11b-IFN-γ mice in the gated area for monocytes and neutrophils. “Reactivation index” (E) and IFN-γ mRNA (F) in the brains of RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ and RAG1^−/−IFN-γ^−/−→IFN-γ^−/− mice. Mean ± SEM, n=4 or 8 (A) n=3 or 4 (E and F). *P<0.05, **P<0.01, and ***P<0.001.

Bone marrow (BM) chimeric mice and infection

RAG1^−/−, IFN-γ^−/−, and CD11b only-IFN-γ mice received whole body irradiation (950 rads) and were injected intravenously with 2.4 × 10⁷ BM cells from RAG1^−/− or RAG1^−/−IFN-γ^−/− mice. BM chimera were infected with 10 cysts of the ME49 strain orally by gavage (6) and treated with sulfadiazine beginning at 4-6 days after infection for 2-3 weeks to establish a chronic infection in their brains (6). For T cell transfer, immune T cells were purified from the spleens of chronically infected BALB/c mice, and 1 × 10⁷ immune T cells were injected intravenously into sulfadiazine-treated BM chimeric mice at 2-3 weeks after infection (6).

Real time RT-PCR, ELISA, flow cytometry, and immunohistochemistry

RNA was isolated from a half brain of each of infected BM chimeric mice and real-time PCR was performed (6). A half brain were homogenized and sonicated, and amounts of IFN-γ and CXCL9 in the sonicates were measured by ELISA (7). Mononuclear cells were purified from brains and stained with PE-anti-CD3ε, FITC-anti-CD4, and PE-Cy5-anti-CD8 α mAbs (BD Biosciences) (6). The staining was triplicated in each group using pooled cells from mice in the same group. Immunhistochemistry for T. gondii was performed as described (6).

Stimulation of a microglial cell line (EOC20) with Toxoplasma tachyzoite lysate antigens (TLA)

EOC20 (ATCC) were maintained in DMEM with 10% FBS and 20% LADMAC-conditioned medium as a source of CSF-1. To examine their IFN-γ production in response to TLA, the cells (4 × 10⁴ cells/well) were cultured in a 96-well tissue culture plate in 10% FBS-DMEM with TLA for 96 hrs.

Statistical Analysis

Levels of significance between experimental groups were determined by One-way ANOVA or Student's t or Mann-Whitney test using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA). Differences which provided P<0.05 were considered significant.

Results and Discussion

IFN-γ production by brain-resident cells is required for upregulating cerebral innate IFN-γ-mediated protective immunity and suppressing tachyzoite growth

We first confirmed the production of IFN-γ by non-T, non-NK cells in the brain following reactivation of T. gondii infection. SCID mice infected and treated with sulfadiazine were treated with anti-asialo GM1 antibody to deplete NK cells, and sulfadiazine treatment was discontinued to initiate reactivation of the infection, which is initiated by rupture of the cysts followed by conversion of released bradyzoites to tachyzoites and proliferation of tachyzoites. Increased amounts of IFN-γ proteins were detected in brains of both NK-depleted and NK-sufficient SCID mice at 5 days after initiation of reactivation of the infection (Day 5) when compared to the last day of sulfadiazine treatment (Day 0) (Supplemental Fig. 1A). Neutralization of IFN-γ by anti-IFN-γ mAb enhanced cerebral tachyzoite growth (Supplemental Fig. 1B), whereas the NK cell-depletion did not (Supplemental Fig. 1C), suggesting that cerebral innate IFN-γ production by non-T, non-NK cells restricts proliferation of tachyzoites in the brain during the reactivation of T. gondii infection.

To address a possibility that brain-resident cells are important non-T, non-NK cell populations that produce IFN-γ against cerebral T. gondii infection, we generated BM chimeras by transferring BM cells from RAG1^−/− mice to irradiated RAG1^−/− (RAG1^−/−→RAG1^−/−) and IFN-γ^−/− mice (RAG1^−/−→IFN-γ^−/−). Hematopoietic cells are irradiation-sensitive, and therefore both groups of the chimeras have hematopoietic cells derived only from the BM donor RAG1^−/− mice, which lack T and B cells but have innate immune cells such as NK cells and neutrophils that can produce IFN-γ. The only difference in the brains of these two groups of animals is the presence (RAG1^−/−→RAG1^−/−) or absence (RAG1^−/−→IFN-γ^−/−) of IFN-γ production by brain-resident cells, which are irradiation-resistant.

RAG1^−/−→RAG1^−/− and RAG1^−/−→IFN-γ^−/−mice were infected and treated with sulfadiazine, and a degree of reactivation of the infection in their brains after discontinuation of sulfadiazine was indicated by the “Reactivation index”, which is a ratio of the amounts of tachyzoite-specific SAG1 mRNA at Day 5 versus the amounts of bradyzoite-specific BAG1 mRNA at Day 0. Although RAG1^−/−→RAG1^−/− mice had greater levels of BAG1 mRNA levels on Day 0, they limited reactivation of infection in their brains more efficiently than did RAG1^−/−→IFN-γ^−/− mice (Fig. 1A). Notably, marked increases in cerebral expression of IFN-γ mRNA (Fig. 1B) and IFN-γ protein (Fig. 1C) after initiation of reactivation of the infection were observed only in the RAG1^−/−→RAG1^−/− mice. These results indicate that the presence of hematopoietic innate immune cells alone in the periphery is not sufficient to increase cerebral innate expression of IFN-γ following reactivation of the infection, and that IFN-γ production by brain-resident cells is required for upregulating the innate IFN-γ expression, the molecule essential for controlling cerebral tachyzoite growth. However, these results do not necessarily mean that the IFN-γ detected in the brains of RAG1^−/−→RAG1^−/− mice were all produced by brain-resident cells. It is possible that IFN-γ produced by brain-resident cells induces migration of hematopoietic innate immune cells from the periphery and these infiltrated cells also contribute to produce IFN-γ to inhibit the parasite growth.

FIGURE 1.

IFN-γ production by brain-resident cells is required for upregulating cerebral innate protective immunity to limit reactivation of T. gondii infection. Sulfadiazine treatment on infected RAG1^−/−→RAG1^−/− and RAG1^−/−→IFN-γ^−/− mice was discontinued to initiate reactivation of the infection. (A) “Reactivation index”, which is a ratio of the amounts of tachyzoite-specific SAG1 mRNA at Day 5 versus the amounts of bradyzoite-specific BAG1 mRNA at Day 0. (B) Cerebral IFN-γ mRNA. (C) Cerebral IFN-γ protein. (D) Cerebral mRNA for Irgm3, Gbp1, IDO1, NOS2, and Arg1. Mean ± SEM, n=7 or 8 (A, B, and D) and n=4 (C). *P<0.05, **P<0.01, ***P<0.001.

IFN-γ increases expression of the immunity-related GTPases, such as Irgm3, and guanylate-binding protein 1 (Gbp1), and an accumulation of these proteins on the parasitophorous vacuole containing tachyzoites is associated with killing of the parasite (8, 9). The depletion of intracellular L-tryptophan pools by IDO is another important mechanism by which IFN-γ controls the intracellular tachyzoite replication (10). Amounts of mRNA for these three effector molecules increased 7-46 times in the brains of RAG1^−/−→RAG1^−/− mice, but not RAG1^−/−→IFN-γ^−/− animals, during the first 5 days of reactivation of cerebral T. gondii infection (Fig. 1D).

Production of NO from L-arginine by inducible NO synthase (NOS2) is important for the activity of murine microglia activated by IFN-γ against tachyzoites in vitro (11). Arginase 1 (Arg1) competes with NOS2 for L-arginine. In the brains of RAG1^−/−→RAG1^−/− and RAG1^−/−→IFN-γ^−/− mice, amounts of NOS2 mRNA were greater in the former than the latter at Day 5 of reactivation of the infection (Fig. 1D), whereas amounts of Arg1 mRNA were 9 times less in the former than the latter (Fig. 1D). The combination of these effects on NOS2 and Arg1 expressions likely induces production of greater amounts of NO by NOS2 in the brains of RAG1^−/−→RAG1^−/− than RAG1^−/−→IFN-γ^−/− mice and thus possibly has contributed to suppress cerebral tachyzoite growth in the former. These results together indicate that IFN-γ production of brain-resident cells plays a critical role in increasing cerebral innate expression of Irgm3, Gbp1, and IDO1 and production of NO, and these molecules have most likely contributed to inhibiting cerebral tachyzoite growth in RAG1^−/−→RAG1^−/− mice following reactivation of the infection.

IFN-γ production by microglia suppresses cerebral tachyzoite growth

Our previous studies using flow cytometric analyses showed that CD11b⁺CD45^low microglia (a brain-resident cell population) and CD11b⁺CD45^high blood-derived macrophages are the major non-T and non-NK cell populations that produce IFN-γ in the brain following reactivation of T. gondii infection (12, 13). Therefore, microglia are most likely major IFN-γ-producing cells among brain-resident cells during reactivation of the infection. The capability of microglia to produce IFN-γ in response to T. gondii tachyzoite antigens was confirmed by the presence of greater amounts of IFN-γ in the culture supernatants of a microglial cell line following stimulation with tachyzoite lysate antigens (Fig. 2A). To address the possibility that IFN-γ production by microglia is crucial for the protective innate immunity to this parasite in the brain, we generated a transgenic mouse strain that produces IFN-γ under the control of CD11b promoter (CD11b-IFN-γ mice) (Figs. 2B and 2C) since microglia is the only cell population that expresses CD11b among the brain-resident cells. The majority of IFN-γ-expressing cells were detected in CD11b⁺ population in PBMC of this transgenic strain (Fig. 2D). This transgenic strain was backcrossed to BALB/c and then mated with IFN-γ^−/− mice to generate the mice that express IFN-γ only in CD11b⁺ cell populations (CD11b only-IFN-γ). In their brains, the majority of CD11b⁺ cells were CD45^low microglia along with only small numbers of CD45^high blood-derived macrophages, and IFN-γ expression was detected only in CD11b⁺ cells by flow cytometry (data not shown). In the microglia population, 10% of them were positive for IFN-γ (data not shown). To generate mice that produce IFN-γ only by microglia in the brain without a production of this cytokine by blood-derived macrophages, we generated BM chimeras by transferring BM cells from RAG1^−/−IFN-γ^−/− mice to irradiated CD11b only-IFN-γ animals (RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ). As a control, IFN-γ^−/− mice were used as the recipients of the BM cells (RAG1^−/−IFN-γ^−/−→IFN-γ^−/−). BM cells from RAG1^−/−IFN-γ^−/− mice generate hematopoietic cells that lack T and B cells and the capability to produce IFN-γ, in these chimeras. The only difference in the brains of these two groups is the presence (RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ) or absence (RAG1^−/−IFN-γ^−/−→IFN-γ^−/−) of IFN-γ production by microglia.

After initiation of reactivation of cerebral T. gondii infection by discontinuing sulfadiazine treatment, RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ mice inhibited reactivation of the infection in the brain more efficiently than did RAG1^−/−IFN-γ^−/−→IFN-γ^−/− animals (Fig. 2E), although BAG1 mRNA levels on Day 0 were greater in the former than the latter. IFN-γ mRNA were detected only in the brains of RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ mice on both Day 0 and Day 5 (Fig. 2F). IFN-γ mRNA levels in their brains were within a range between those detected in the brains of RAG1^−/−→RAG1^−/− mice on Day 0 and Day 5 shown in Fig. 1B. Therefore, the IFN-γ levels in the brains of RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ mice mimic physiological levels of this cytokine produced by innate immune cells during reactivation of the infection, although microglial IFN-γ expression in RAG1^−/−IFN-γ^−/−→CD11b only-IFN-γ mice was constitutive and not upregulated by reactivation of the infection (Fig. 2F). These results suggest that IFN-γ production only by microglia is able to limit tachyzoite growth during reactivation of T. gondii infection. Thus, microglia appear to be a key cell population among brain-resident cells that produces IFN-γ by recognizing tachyzoite growth and limit reactivation of T. gondii infection, although these results do not exclude a possibility that brain-resident cells other than microglia also produce IFN-γ in a lesser extent and partially contribute to suppressing cerebral tachyzoite growth.

IFN-γ production by brain-resident cells is crucial for recruitment of T cells into the brain to inhibit reactivation of T. gondii infection

T cells are required to prevent reactivation of T. gondii infection, although cerebral innate IFN-γ production is able to partially limit tachyzoite growth in the absence of T cells. IFN-γ-mediated expression of CXCL9 is important for recruiting immune T cells into the brain to prevent reactivation of T. gondii infection (6). CXCL10 is also involved in T cell recruitment and controlling cerebral tachyzoite proliferation (14). The RAG1^−/−→RAG1^−/− mice upregulated cerebral CXCL9 and CXCL10 mRNA expression much more efficiently than RAG1^−/−→IFN-γ^−/− animals in response to reactivation of the infection (Fig. 3A). CXCL9 protein levels were also markedly greater in the brains of RAG1^−/−→RAG1^−/− than RAG1^−/−→IFN-γ^−/− mice (Fig. 3B). After T cells migrate to the brain, CD4⁺ and CD8⁺ subsets of the T cells need to recognize T. gondii antigens presented by MHC class II and I molecules, respectively, to display their protective activities. Amounts of mRNA for MHC class I (H2-D1) and class II (H2-Aa) molecules increased in the brains of RAG1^−/−→RAG1^−/− but not of RAG1^−/−→IFN-γ^−/− mice following reactivation of cerebral T. gondii infection (Fig. 3C).

FIGURE 3.

IFN-γ production by brain-resident cells is crucial for cerebral innate protective immunity to upregulate an expression of molecules important for recruiting and activating T cells following reactivation of T. gondii infection. Sulfadiazine treatment on infected RAG1^−/−→RAG1^−/− and RAG1^−/−→IFN-γ^−/− mice was discontinued to initiate reactivation of the infection. (A) Cerebral mRNA for CXCL9 and CXCL10 chemokines. (B) Cerebral CXCL9 protein. (C) Cerebral mRNA for the MHC class I (H2-D1) and II (H2-Aa) molecules. Mean ± SEM, n=7 or 8 (A and C) and n= 4 (B). **P<0.01, and ***P<0.001.

To examine whether IFN-γ production by brain-resident cells is eventually critical for recruiting immune T cells into the brain to prevent reactivation of T. gondii infection, immune T cells from infected BALB/c mice were systemically transferred into infected RAG1^−/−IFN-γ^−/−→RAG1^−/− and RAG1^−/−IFN-γ^−/−→IFN-γ^−/− mice. Before receiving the T cells, the cells that can produce IFN-γ in the brains of RAG1^−/−IFN-γ^−/−→RAG1^−/− mice are only brain-resident cells, and RAG1^−/−IFN-γ^−/−→IFN-γ^−/− animals do not have any of IFN-γ-producing cells in their brains. At three days after initiation of reactivation of the infection, the numbers of both CD4⁺ and CD8⁺ T cells obtained from the brains of RAG1^−/−IFN-γ^−/−→RAG1^−/− were 5 times greater than those of RAG1^−/−IFN-γ^−/−→IFN-γ^−/− mice (Fig. 4A). This is consistent with the presence of 3-5 times greater amounts of mRNA for the T cell markers, CD3δ, CD4, and CD8β, in the brains of the former than the latter (Fig. 4B).

FIGURE 4.

IFN-γ production by brain-resident cells is critical for recruiting immune T cells into the brain and inducing IFN-γ-mediated protective T cell responses to inhibit reactivation of T. gondii infection. Infected, sulfadiazine-treated RAG1^−/−IFN-γ^−/−→RAG1^−/− and RAG1^−/−IFN-γ^−/−→IFN-γ^−/− mice received immune T cells, and 4-5 days later, the sulfadiazine treatment was discontinued to initiate reactivation of the infection. (A) CD4⁺ and CD8⁺ T cells that migrated into the brain in the gated area for lymphocytes. (B) Cerebral mRNA for CD3δ, CD4, and CD8β. (C) Cerebral IFN-γ mRNA. (D) Ratios of tachyzoite-specific SAG1 mRNA in the brains of mice with the T cell transfer vs. those of animals without the T cell transfer. Mean ± SEM, n=4 (A and B) and n=8 (C and D) on Day 3-4. *P<0.05 and ***P<0.001. (E) Immunohistochemical staining for T. gondii on the brain sections on Day 4. Left panel: A representative image from the brains of RAG1^−/−IFN-γ^−/−→RAG1^−/− mice. Arrows indicate representatives of tachyzoites. Arrowheads indicate destroyed tachyzoites and antigens most likely released from the destroyed parasite. Right panel: A representative image from the brain of RAG1^−/−IFN-γ^−/−→IFN-γ^−/− mice. n=3 or 4.

The recruitment of larger numbers of T cells in RAG1^−/−IFN-γ^−/−→RAG1^−/− mice was associated with 5 times greater levels of IFN-γ mRNA in their brains than in RAG1^−/−IFN-γ^−/−→IFN-γ^−/− animals (Fig. 4C). In addition, the transfer of T cells inhibited cerebral tachyzoite growth more efficiently in the former than the latter when compared to control animals of the same strain that had received no T cells (Fig. 4D). An accumulation of large numbers of inflammatory cells was frequently observed in the areas of tachyzoite proliferation in the brains of RAG1^−/−IFN-γ^−/−→RAG1^−/− (Fig. 4E, left panel) but not of RAG1^−/−IFN-γ^−/−→IFN-γ^−/− mice (Fig. 4E, right panel). In addition, fragments of the parasite and antigens most likely released from the destroyed parasites were often detected in the areas associated with inflammatory cells of only the former (Fig. 4E, left panel). These results strongly suggest that T cell recruitment mediated by IFN-γ produced by brain-resident cells is important for inducing IFN-γ-mediated protective activities of T cells in the brain to inhibit tachyzoite growth during the early stage of reactivation of T. gondii infection. It is possible that the regulatory effects of IFN-γ produced by brain-resident cells to facilitate recruitment of immune cells into the brain is not limited to T cells but also be effective to hematopoietic innate immune cells, and that this enhancing effect is involved in the upregulation of cerebral innate IFN-γ production detected in the brains of RAG1^−/−→RAG1^−/− mice shown in Fig. 1B as discussed earlier. This possibility is supported by an evidence that IFN-γ expression levels in the brains of infected RAG1^−/−IFN-γ^−/−→RAG1^−/− mice that had not received T cells were lower than those of RAG1^−/−→RAG1^−/− mice shown in Fig. 1B (data not shown).

The present study revealed that IFN-γ production by brain-resident cells is pivotal for inducing both IFN-γ-mediated protective innate and T cell-mediated immune responses to inhibit reactivation of cerebral infection with T. gondii. To our knowledge, this is the first evidence demonstrating the importance of IFN-γ production by brain-resident cells for facilitating the protective immune responses to control cerebral infection with a pathogen. By considering the fact that IFN-γ plays a critical role in resistance against various microorganisms in addition to T. gondii that can cause cerebral infections (15, 16), IFN-γ production by brain-resident cells appears to be a novel target for developing new methods for treatment of cerebral infections with those pathogens.

Abbreviations used in this article

BM

bone marrow

TLA

Toxoplasma tachyzoite lysate antigens