CXCL10 is required to maintain T-cell populations and to control parasite replication during chronic ocular toxoplasmosis

Abstract

Purpose: Toxoplasma gondii is a major cause of ocular disease, which can lead to permanent vision loss in humans. T cells are critically involved in parasite control, but little is known about the molecules that promote T-cell trafficking and migration in the retina. Thus, the aim of this study was to image and dissect the T-cell response during chronic toxoplasmic retinochoroiditis.

Methods: C57BL/6 mice were infected with the Me49 strain of T. gondii, and T cells that infiltrated the eye were analyzed by flow cytometry and imaged using multiphoton microscopy. IFN-γ, CXCL9, CXCL10, and CXCR3 mRNA levels were measured by real-time PCR. To investigate the role of CXCL10, mice were treated with anti-CXCL10 antibodies, and histopathology and immunohistochemistry were performed to monitor changes in pathology, cellular infiltration, and parasite burden in the eye.

Results: Infection with T. gondii leads to the infiltration of highly activated motile T cells into the eye. These cells express CXCR3 and are capable of producing IFN-γ and TNF-α, and CD8+ T cells express granzyme B. The expression of CXCL9 and CXCL10 in the retina was significantly upregulated during chronic infection. Treatment of chronically infected mice with anti-CXCL10 antibodies led to decreases in the numbers of CD3+, CD4+, and CD8+ T cells and the amount of IFN-γ mRNA expression in the retina and an increase in replicating parasites and ocular pathology.

Conclusions: The maintenance of the T-cell response and the control of T. gondii in the eye during chronic infection is dependent on CXCL10.

Figure 1.

T cells isolated from the eye during ocular toxoplasmosis are highly activated. Cells were isolated from infected eyes and analyzed by flow cytometry. CD45 expression was analyzed on live cells isolated from the eye (A). CD45⁺ cells were analyzed for CD3 expression (B) and subsequently gated on CD4 and CD8 expression (C). The total number of live cells (total) and CD4⁺ and CD8⁺ T cells are presented as mean ± SE from four independent experiments (D). CD44 and CD62L (E) and CD69 expression (F) on T cells isolated from infected eyes are shown. Shaded histogram represents fluorescence minus one control. All plots are representative of three independent experiments with a minimum of three mice per group.

Figure 2.

T cells isolated from the eye during ocular toxoplasmosis produce effector cytokines and lytic granules. Ex vivo cytokine production by T cells from the spleen and eye was assessed by flow cytometry. TNF-α and IFN-γ production was assessed in CD4⁺ cells (A) and CD8⁺ cells (B). Granzyme and IFN-γ production was measured in CD8⁺ T cells (C). Graphs are representative of three independent experiments with a minimum of three mice per group.

Figure 3.

MP imaging of T cells in the eye reveals heterogeneity in behavior. (A) GFP expression in the eye was evaluated by flow cytometry. (B) GFP-expressing cells were analyzed for CD4 and CD8 expression. (C) Immunohistochemistry revealed that GFP⁺ T cells (green) were found in proximity to a T. gondii cyst (red). Scale bar, 10 μm. GFP⁺ T-cell behavior in the eye was imaged using MP microscopy. (D) GFP⁺ cells (green), secondary harmonic emitting structures (blue), and example cells tracks (multicolored lines) are shown from the retina (left) and iris (right). Images were collected to generate four-dimensional (x-, y-, and z- planes over time) analysis of T-cell migration. Scale bars, 100 μm. Individual cells were analyzed for (E) mean velocity and (F) meandering index. Retina, n = 192 cells; iris, n = 60 cells. Graphs are representative of three independent experiments.

Figure 4.

IFN-γ–dependent, T cell-associated chemokines and chemokine receptors are expressed in the retina during chronic T. gondii infection. Retinas were isolated from chronically infected mice, and gene expression was analyzed by real-time PCR. (A) IFN-γ, CXCL9, CXCL10, and CXCR3 expression was measured by real-time PCR. Levels were normalized to β-actin, and the results are depicted as fold increase over retinas from uninfected mice. **P < 0.01. (B) CXCR3 expression on T cells isolated from the spleen and eye was determined by flow cytometry. Shaded histograms represent fluorescence minus one control. Plots are representative of three independent experiments.

Figure 5.

CXCL10 neutralization during chronic ocular toxoplasmosis causes increased pathology in the eye. (A) Representative images from hematoxylin and eosin–stained retinas from naive, (1) infected control mice, (2–4) and infected mice treated with anti–CXCL10 antibodies. (5–7) Magnified regions from image (5) are indicated by black boxes and are shown in images (6) and (7). Examples of vasculitis (arrows), disorganization of the normal retinal structure (asterisks), inflammatory cell infiltration in the vitreous (arrowhead), and retinal pigment epithelium (#) were noted. Scale bars: 50 μm (1, 3, 6); 500 μm (2, 5); 10 μm (4, 7). (B) A pathology score was determined for control and anti–CXCL10-treated mice with the following parameters: cell infiltration in retinal perivascular region (vasculitis), disorganization of the retinal architecture (disorganization), retinal pigment epithelium migration or proliferation (pigment), and cell infiltration in the choroid (choroiditis), vitreous (vitreous cells), anterior chamber (AC cells), and retina (retinal cells). *P < 0.05. Mean pathology score ± SE from three independent experiments is shown (n = 13 for control and n = 11 for anti–CXCL10 treated mice).

Figure 6.

CXCL10 is required to maintain T-cell populations, IFN-γ mRNA expression, and parasite control in the retina during chronic T. gondii infection. (A) Images from immunohistochemical analysis of retinas from control treated T. gondii–infected mice (A1–3) and anti–CXCL10 treatment (A4–6) are shown. Sections were stained for CD3⁺ (green) and B220⁺ (red; A1, A4) cells, CD4⁺ cells (red; A2, A5), and CD8⁺ cells (red; A3, A6), and nuclei were stained with DAPI (blue). Scale bars, 10 μm. Numbers of various cell types in the retina in control and anti–CXCL10 treated mice were compiled from the immunohistochemical analysis, and the data are presented as the mean ± SE (n = 5) from three independent experiments. N.D., not detected (B). IFN-γ expression was measured by real-time PCR from the retinas of control and anti–CXCL10-treated mice (C). T. gondii parasites in the retina were examined by immunohistochemistry using anti–T. gondii antibodies (green) and Dolichos-binding lectin (red; D). All parasites in control mice were found in intact cysts (D1). Parasites in anti–CXCL10-treated mice were found in cyst form (D2) and tachyzoite form (arrows; D3). Scale bars, 10 μm. (E) Regions of T. gondii parasites (cysts or free parasites) in the retinas of control and anti–CXCL10-treated mice were enumerated from sections totaling 180 to 200 μm in depth. Data are expressed as the average number of parasite regions per tissue depth (μm) from eight control and six anti–CXCL10-treated mice. Data are presented as mean ± SE. *P < 0.05.