Interferon-gamma, macrophages, and virus spread after HSV-1 injection

Abstract

Purpose: After uniocular anterior chamber (AC) injection of HSV-1, the anterior segment of BALB/c mice becomes inflamed and infected; however, virus does not spread from the anterior segment to cause retinitis in the injected eye. The purpose of these studies was to determine whether interferon (IFN-)-γ and Mac-1(+) cells play a role in preventing direct anterior-to-posterior spread of HSV-1 in the injected eye.

Methods: One AC of adult female BALB/c mice was injected with HSV-1 (KOS). The location of IFN-α, IFN-β, and IFN-γ in the injected eye was determined by immunofluorescence, and mRNA expression was quantified by qPCR. Injected eyes of IFN-γ knockout or clodronate-treated macrophage-depleted mice were examined to determine whether the absence of IFN-γ or Mac-1(+) macrophages affected the sites or timing of virus spread.

Results: IFN-α, IFN-β, and IFN-γ were observed in the anterior segment of injected eyes through 72 hours and mRNA levels of IFN-β and IFN-γ were increased in virus-infected eyes 48 to 120 hours after infection. However, the absence of IFN-γ or macrophages did not affect either the sites or the timing of HSV-1 infection in injected eyes.

Conclusions: Protection of the retina of the injected eye does not depend on a single cell type or cytokine. In addition, in the eye, as in other sites of the body, there are redundancies in the innate response to virus infection.

Figure 1.

Representative photomicrographs of the ciliary body (CB) of the injected eye showing the location (arrowheads) of IFN-α⁺ cells in normal control animals (A–C) and in virus-injected animals 24 (D–F), 48 (G–I), and 72 (J–L) hours p.i. The cornea (C), iris (I), and retina (R) are indicated. Original magnification (A–L), ×200.

Figure 2.

Representative photomicrographs of the ciliary body (CB) of the injected eye showing the location (arrowheads) of IFN-β⁺ cells in normal control animals (A–C) and in virus-injected animals 24 (D–F), 48 (G–I), and 72 (J–L) hours p.i. The cornea (C), iris (I), and retina (R) are indicated. Original magnification, (A–L), ×200.

Figure 3.

Representative photomicrographs of the ciliary body (CB) of the injected eye showing the location (arrowheads) of IFN-γ⁺ cells in normal control animals (A–C) and in virus-injected animals 24 (D–F), 48 (G–I), and 72 (J–L) hours p.i. The cornea (C), iris (I), and retina (R) are indicated. Original magnification, (A–L), ×200.

Figure 4.

IFN gene expression (fold upregulation) in mock-injected eyes 48 hours p.i. and virus-injected eyes 24, 48, 72, and 120 hours p.i. compared with normal control eyes (average of two independent experiments shown). To determine the fold change in gene expression, the normalized expression of the gene of interest (GOI) in the experimental sample was divided by the normalized expression of the same GOI in the control sample (normal control): 2^{−ΔCt(experimental)}/2^{−ΔCt(control)} = 2^−ΔΔCt.

Figure 5.

Representative photomicrographs of the ciliary body (CB) in the injected eye showing HSV-1⁺ staining in IFN-γ⁺/⁺ (A–C) or IFN-γ^−/− (D–F) normal control (A, D) and virus-infected mice at 72 (B, E) and 120 (C, F) hours p.i. Average percentage area of HSV-1⁺ staining in the CB (IFN-γ⁺/⁺, n = 18; IFN-γ^−/−, n = 16). The cornea (C), and iris (I) are also shown. *P = 0.03, significantly different from IFN-γ⁺/⁺ mice (G). Statistical significance was evaluated using a mixed-model ANOVA. Data represent the average of two independent experiments. Error bars represent 95% confidence intervals. Original magnification, ×200.

Figure 6.

Flow cytometric histograms of Mac-1⁺ cells isolated from the eyes of normal control mice, HSV-1–injected mice, mock-treated/HSV-1–injected mice, and Cl₂MBP-treated/HSV-1-injected mice (n = 3) 48 hours p.i. Cells were stained with FITC-conjugated anti-mouse Mac-1 (green line) or FITC-conjugated rat IgG2b,κ (solid black). Percentages are those of isotype-matched control staining subtracted from those of cell marker staining. Data are representative of two independent experiments.

Figure 7.

Flow cytometric histograms of Mac-1⁺ cells isolated from the eyes of normal control mice, HSV-1–injected mice, mock-treated/HSV-1–injected mice, and Cl₂MBP-treated/HSV-1–injected mice (n = 3) 72 hours p.i. Cells were stained with FITC-conjugated anti-mouse Mac-1 (green line) or FITC-conjugated rat IgG2b,κ (solid black). Percentages are those of isotype-matched control staining subtracted from those of cell marker staining. Data are representative of two independent experiments.

Figure 8.

Representative photomicrographs of the ciliary body in the injected eye showing HSV-1⁺ staining in mock-treated/HSV-1–infected (A, B) or Cl₂MBP-treated/HSV-1–infected mice (C, D) at 48 and 72 hours p.i. Average percentage area of HSV-1⁺ staining in the ciliary body (mock treated, n = 21; Cl₂MBP treated, n = 19) (E). *P = 0.005, significantly different from mock treated. Statistical significance was evaluated using a mixed-model ANOVA. Data represent the average of two independent experiments. Error bars represent 95% confidence intervals. Original magnification, ×200.