Improvement of herpetic stromal keratitis with fumaric acid derivate is associated with systemic induction of T helper 2 cytokines

Abstract

Fumaric acid derivates have been shown to stimulate T helper-2-cytokines (interleukin (IL)-4, -5) without affecting the T-helper-1-cytokine (IL-2, interferon (IFN)-gamma)-response. Herein, the influence of systemic treatment with the fumaric acid derivate dimethylfumarate (DMF) on the secretion of T helper-cytokines and the development of HSV-1 stromal keratitis (HSK) was studied in mice. The corneas from BALB/c mice were infected with 10(5) PFU of HSV-1 (KOS strain). While one group of mice was treated intraperitoneally with PBS, another group of mice received DMF at 15 mg/kg of body weight. Expression of IL-2, -4, -10 and IFN-gamma was analysed in HSV-1 activated lymphocytes by ELISA. The severity of epithelial and stromal herpetic keratitis was investigated clinically. Corneas were studied for the inflammatory cell infiltration, and the CD3-, CD4- and CD8-positive cells were analysed by immunohistochemistry. The IL-2, -4, 10 and IFN-gamma content was measured in the corneas. Virus replication in the eyes was analysed by a plaque-assay. The DTH-response, the HSV-specific T cell proliferation and the serum neutralizing antibody-titres were investigated. DMF increased IL-4 and IL-10, but not IL-2 and IFN-gamma, secretion in activated lymphocytes from the spleen. Incidence and severity of stromal HSV-1 keratitis was reduced in the DMF group (P < 0.01). In the corneas from DMF-treated mice, the numbers of CD3+ and CD4+ cells were decreased and IL-4 was increased. Severity of epithelial disease and the virus-clearance from the eyes did not differ between the PBS and DMF group of mice. DTH, HSV-specific T cell proliferation and the neutralizing antibody-titres were not impaired. DMF increased the T helper-2-cytokine secretion in activated lymphocytes. After corneal HSV-1 infection, corneas from DMF treated mice had increased IL-4 content. This is associated with an improvement of herpetic stromal keratitis and reduced corneal T cell infiltration. DMF did not impair the systemic antiviral response.

Fig. 1

Influence of dimethylfumarate (DMF) on the secretion of T helper cytokines in splenic cells. ELISA data from cell culture supernatants of cells harvested at day 14 p.i. from the spleen. Up-regulation of IL-4 and IL-10 but not IL-2 and IFN-γ secretion after 24 h incubation with DMF. P < 0·05.

Fig. 2

Influence of dimethylfumarate (DMF) on the development of herpetic stromal keratitis mice. Groups of mice were treated daily intraperitoneally with DMF or PBS 28 days before and 14 days after corneal HSV-1 infection. The severity of stromal keratitis on day 14 after infection was graded as: 0 clear cornea; +1 mild corneal haze; +2 moderate corneal opacity or scarring; +3 severe corneal opacity, iris visible; +4 opaque cornea, iris not visible, necrotizing stromal keratitis with ulceration. Mean score ± SD of all mice in each group. DMF reduced the severity of keratitis (P < 0·01).

Fig. 3

Influence of dimethylfumarate (DMF) on the histopathological appearance of the cornea after HSV-1 infection. Groups of mice were treated daily intraperitoneally with DMF or PBS 28 days before and 14 days after corneal HSV-1 infection. Corneal sections representative for (a) PBS- or (b) DMF-treated mice; haematoxylin-eosin staining. Magnification ×120. Scale bar indicates 100 µm.

Fig. 4

Influence of dimethylfumarate (DMF) on the secretion of certain T helper cytokines in the HSV-1 infected corneas. Groups of mice were treated daily intraperitoneally with DMF or PBS 28 days before and 14 days after corneal HSV-1 infection. ELISA data are shown. Up-regulation of IL-4 but not IL-2 and IFN-γ after 24 h incubation with DMF (P < 0·05).

Fig. 5

Influence of dimethylfumarate (DMF) on the virus replication in eyes after corneal HSV-1 infection in mice. Groups of mice were treated daily intraperitoneally with DMF or PBS 28 days before and 14 days after corneal HSV-1 infection. DMF treatment did not alter the clearance of infectious virus particles from the infected eyes (n = 5, each time point and group).