IFN-γ production by brain-resident cells activates cerebral mRNA expression of a wide spectrum of molecules critical for both innate and T cell-mediated protective immunity to control reactivation of chronic infection with Toxoplasma gondii

Abstract

We previously demonstrated that brain-resident cells produce IFN-γ in response to reactivation of cerebral infection with Toxoplasma gondii. To obtain an overall landscape view of the effects of IFN-γ from brain-resident cells on the cerebral protective immunity, in the present study we employed NanoString nCounter assay and quantified mRNA levels for 734 genes in myeloid immunity in the brains of T and B cell-deficient, bone marrow chimeric mice with and without IFN-γ production by brain-resident cells in response to reactivation of cerebral T. gondii infection. Our study revealed that IFN-γ produced by brain-resident cells amplified mRNA expression for the molecules to activate the protective innate immunity including 1) chemokines for recruitment of microglia and macrophages (CCL8 and CXCL12) and 2) the molecules for activating those phagocytes (IL-18, TLRs, NOD1, and CD40) for killing tachyzoites. Importantly, IFN-γ produced by brain-resident cells also upregulated cerebral expression of molecules for facilitating the protective T cell immunity, which include the molecules for 1) recruiting effector T cells (CXCL9, CXCL10, and CXCL11), 2) antigen processing (PA28αβ, LMP2, and LMP7), transporting the processed peptides (TAP1 and TAP2), assembling the transported peptides to the MHC class I molecules (Tapasin), and the MHC class I (H2-K1 and H2-D1) and Ib molecules (H2-Q1, H-2Q2, and H2-M3) for presenting antigens to activate the recruited CD8⁺ T cells, 3) MHC class II molecules (H2-Aa, H2-Ab1, H2-Eb1, H2-Ea-ps, H2-DMa, H2-Ob, and CD74) to present antigens for CD4⁺ T cell activation, 4) co-stimulatory molecules (ICOSL) for T cell activation, and 5) cytokines (IL-12, IL-15, and IL-18) facilitating IFN-γ production by NK and T cells. Notably, the present study also revealed that IFN-γ production by brain-resident cells also upregulates cerebral expressions of mRNA for the downregulatory molecules (IL-10, STAT3, SOCS1, CD274 [PD-L1], IL-27, and CD36), which can prevent overly stimulated IFN-γ-mediated pro-inflammatory responses and tissue damages. Thus, the present study uncovered the previously unrecognized the capability of IFN-γ production by brain-resident cells to upregulate expressions of a wide spectrum of molecules for coordinating both innate and T cell-mediated protective immunity with a fine-tuning regulation system to effectively control cerebral infection with T. gondii.

Keywords: IFN-γ; Toxoplasma gondii; antigen presentation; brain-resident cells; chemokine; cytokine; protective immunity; regulatory molecule.

Figure 1

Cerebral mRNA levels for IFN-γ-mediated signaling pathway are upregulated only in the presence of brain-resident cells capable of producing IFN-γ in response to reactivation of cerebral infection with T. gondii. RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice were infected orally with 10 cysts of the ME49 strain of T. gondii and treated with sulfadiazine in the drinking water (400 mg/L) beginning at 4-6 days after infection for 2-3 weeks to control the proliferation of tachyzoites and establish a chronic infection in their brains. Their brains were collected on the last day of sulfadiazine treatment (Day 0) and Day 5 after discontinuation of sulfadiazine (Day 5), which had initiated reactivation of the cerebral infection with T. gondii. The mRNA levels for (A) type I IFN (IFN-α and IFN-β) and type II IFN (IFN-γ), (B) STAT1, 3, 4, 5 and 6, (C) IRF1, 2, 3, 4, 5, 7, and 8, and (D) ISG15 in the brains of both groups of mice at both Day 0 and Day 5 were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 2

Cerebral mRNA levels for the molecules that recruit microglia, monocytes, macrophages, and T cells are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for (A) 21 CC chemokines and (B) 11 CXC chemokines were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 3

Cerebral mRNA levels for molecules that increase phagocytosis of microglia and macrophages are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for IL-18 and caspase1, which generate active IL-18 molecule from pro-IL-18, were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05 and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 4

Cerebral mRNA levels for the molecules that recognize the pathogen-associated molecular patterns are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for (A) 12 TLRs, (B) CD180 (a member of TLR family), and (C) NOD1 and NOD2 were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 5

Cerebral mRNA levels for CD40, which activates intracellular killing of tachyzoites by microglia and macrophages, are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for CD40 were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 6

Cerebral mRNA levels for the molecule involved in processing, transporting, and presentation of antigens through the MHC class I molecules for activating CD8⁺ T cells are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for the molecules for (A) antigen processing and peptide generation, (B) Peptide transport, (C) assembling peptide-MHC class I complex, and (D, E) presenting the peptides by classical MHC class I molecules (D) and non-classical MHC class I molecules (E) to activate CD8⁺ T cells were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 7

Cerebral mRNA levels for the molecules involved in antigen presentation for activating CD4⁺ T cells are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for 9 MHC class II molecules were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05 and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 8

Cerebral mRNA levels for co-stimulatory molecules are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for (A) ICOS, CD80, CD86 and (B) 4-1BBL were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 9

Cerebral mRNA levels for the cytokines that facilitate IFN-γ production by NK and T cells are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for IL-12, IL-15, and IL-15R were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).

Figure 10

Cerebral mRNA levels for the molecules that down-regulate the IFN-γ-mediated protective immunity to prevent overly stimulated immune responses are upregulated by IFN-γ production by brain-resident cells during reactivation of cerebral T. gondii infection. For the details of experimental methods, see the legend of Figure 1 . The mRNA levels for (A) IL-10, IL-10R, STAT3, (B) SOCS-1, (C) CD274, (D) IL-27, and (E) CD36 were measured at both Day 0 and Day 5 in the brains of both groups of mice were measured using NanoString nCounter Mouse Myeloid Innate Immunity Panel. *P<0.05, **P<0.01, and ***P<0.001. The data were combined from two independent experiments (n=5 for Day 0 and n=7 for Day 5 in both RAG1^-/-→RAG1^-/- and RAG1^-/-→IFN-γ^-/- mice).