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.
Toxoplasma 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 were 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 the 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
Female BALB/c, RAG1−/−, IFN-γ−/−, SCID, and (C57BL/6 × SJL)F1 hybrid mice were from The Jackson Laboratory, and Swiss-Webster mice were from Taconic. RAG1−/−IFN-γ−/− mice were generated by mating RAG1−/− mice 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 replacing Thy1.1 coding sequence of a CD11b-Thy1.1 construct (5) (kindly provided by Dr. Daniel Tenen, Harvard Medical School) with the IFN-γ coding sequence. After confirming the ability of pCD11b–IFN-γ to induce the 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 mice, backcrossed to BALB/c mice six 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 (University of Kentucky and Virginia Polytechnic Institute and State University).
Bone marrow chimeric mice and infection
RAG1−/−, IFN-γ−/−, and CD11b only–IFN-γ mice received whole-body irradiation (950 rad) and were injected i.v. with 2.4 × 107 bone marrow (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 d postinfection for 2–3 wk 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 i.v. into sulfadiazine-treated BM chimeric mice at 2–3 wk postinfection (6).
Real time RT-PCR, ELISA, flow cytometry, and immunohistochemistry
RNA was isolated from a half brain of each infected BM chimeric mouse, and real-time PCR was performed (6). The half brain was homogenized and sonicated, and the 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-CD8α mAbs (BD Biosciences) (6). The staining was triplicated in each group using pooled cells from mice in the same group. Immunohistochemistry for T. gondii was performed as described (6).
Stimulation of a microglial cell line (EOC20) with Toxoplasma tachyzoite lysate Ags
EOC20 cells (American Type Culture Collection) 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 Toxoplasma tachyzoite lysate Ag (TLA), the cells (4 × 104 cells/well) were cultured in a 96-well tissue culture plate in 10% FBS-DMEM with TLA for 96 h.
Levels of significance between experimental groups were determined by one-way ANOVA or the Student t or Mann–Whitney test using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA). Differences that 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 Ab 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 d after initiation of reactivation of the infection (day 5) compared with 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 the 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−/− mice (RAG1−/−→RAG1−/−) and IFN-γ−/− mice (RAG1−/−→IFN-γ−/−). Hematopoietic cells are radiation-sensitive; 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 radiation-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 amount of tachyzoite-specific SAG1 mRNA at day 5 to the amount of bradyzoite-specific BAG1 mRNA at day 0. Although RAG1−/−→RAG1−/− mice had greater levels of BAG1 mRNA 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 was 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.
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 intracellular tachyzoite replication (10). Amounts of mRNA for these three effector molecules increased 7–46-fold in the brains of RAG1−/−→RAG1−/− mice, but not RAG1−/−→IFN-γ−/− animals, during the first 5 d of reactivation of cerebral T. gondii infection (Fig. 1D).
Production of NO from l-arginine by 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 in the latter at day 5 of reactivation of the infection (Fig. 1D), whereas the amounts of Arg1 mRNA were nine times less in the former than in the latter (Fig. 1D). The combination of these effects on NOS2 and Arg1 expression likely induces production of greater amounts of NO by NOS2 in the brains of RAG1−/−→RAG1−/− mice than in RAG1−/−→IFN-γ−/− mice and, thus, may have contributed to suppress cerebral tachyzoite growth in the former. Together, these results indicate that IFN-γ production by brain-resident cells plays a critical role in increasing cerebral innate expression of Irgm3, Gbp1, and IDO1 and the production of NO, and these molecules most likely contributed to the inhibition of 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+CD45low microglia (a brain-resident cell population) and CD11b+CD45high 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 Ags 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 Ags (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 the CD11b promoter (CD11b–IFN-γ mice) (Fig. 2B, 2C) because microglia are the only cells that express CD11b among the brain-resident cells. The majority of IFN-γ–expressing cells were detected in the CD11b+ population in PBMCs of this transgenic strain (Fig. 2D). This transgenic strain was backcrossed to BALB/c mice 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 CD45low microglia along with only small numbers of CD45high blood-derived macrophages, and IFN-γ expression was detected only in CD11b+ cells by flow cytometry (data not shown). In the microglia population, 10% were positive for IFN-γ (data not shown). To generate mice that produce IFN-γ only by microglia in the brain without 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 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 ability 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 in the latter. IFN-γ mRNA was detected only in the brains of RAG1−/−IFN-γ−/−→CD11b only–IFN-γ mice on days 0 and 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 days 0 and 5 shown in Fig. 1B. Therefore, the IFN-γ levels in the brains of RAG1−/−IFN-γ−/−→CD11b only–IFN-γ mice mimic the 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 the possibility that brain-resident cells other than microglia also produce IFN-γ to 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 did RAG1−/−→IFN-γ−/− animals in response to reactivation of the infection (Fig. 3A). CXCL9 protein levels also were markedly greater in the brains of RAG1−/−→RAG1−/− mice than in 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 Ags presented by MHC class II and I molecules, respectively, to display their protective activities. The amounts of mRNA for MHC class I (H2-D1) and class II (H2-Aa) molecules increased in the brains of RAG1−/−→RAG1−/− mice but not RAG1−/−→IFN-γ−/− mice following reactivation of cerebral T. gondii infection (Fig. 3C).
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 IFN-γ–producing cells in their brains. At 3 d after the initiation of reactivation of the infection, the numbers of both CD4+ and CD8+ T cells obtained from the brains of RAG1−/−IFN-γ−/−→RAG1−/− mice were 5-fold greater than those from RAG1−/−IFN-γ−/−→IFN-γ−/− mice (Fig. 4A). This is consistent with the presence of 3–5-fold greater amounts of mRNA for the T cell markers CD3δ, CD4, and CD8β in the brains of the former compared with the latter (Fig. 4B).
The recruitment of larger numbers of T cells in RAG1−/−IFN-γ−/−→RAG1−/− mice was associated with 5-fold 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 in the latter compared with control animals of the same strain that received no T cells (Fig. 4D). An accumulation of large numbers of inflammatory cells was observed frequently in the areas of tachyzoite proliferation in the brains of RAG1−/−IFN-γ−/−→RAG1−/− mice (Fig. 4E, left panel) but not RAG1−/−IFN-γ−/−→IFN-γ−/− mice (Fig. 4E, right panel). In addition, fragments of the parasite and Ags most likely released from the destroyed parasites were detected often 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 are effective not only in T cells but also in 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 the RAG1−/−→RAG1−/− mice shown in Fig. 1B. This possibility is supported by the 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 the 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 indicating 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 the 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 the treatment of cerebral infections with those pathogens.
This work was supported in part by National Institutes of Health Grants AI078756 and AI095032 (to Y.S.).
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.