Histone deacetylase inhibitor attenuates experimental fungal keratitis in mice

Abstract

Fungal keratitis is one of the leading causes of blindness of infected corneal diseases, but the pathogenesis of fungal keratitis is not fully understood and therefore the treatment of the disease by medication is still under investigation. In the current study, we sought to study the effect of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on experimental fungal keratitis in mice. SAHA (25 mg/kg) (n = 30) or vehicle (DMSO) (n = 30) was delivered through intraperitoneal injection (IP) 24 hours after the fungal inoculation, and the same amount of SAHA injection or DMSO was followed at day 2. The expression of histone H3 (H3), acetylated histone H3 (AC-H3), histone deacetylase 1 (HDAC)1, tumor necrosis factor-α (TNFα), and Toll-like receptor 4 (TLR4) in surgically excised specimens from the patients and mice with fungal keratitis were detected by immunohistochemistry. The expression of mRNAs for Interleukin-1β (IL-1β), TNFα, and TLR4 were evaluated in the corneas of the mice with fungal infection and the control corneas by real-time PCR. The quantification of IL-1β and TNFα in the corneas of the mice with fungal infection was determined by ELISA. The inhibitory effect of SAHA on mice fungal keratitis was revealed by GMS and H&E staining. We found that the downregulation of histone acetylation and upregulation of HDAC1 expression were associated with the increased inflammation response in fungal keratitis not only in humans but also in experimental animals. SAHA was able to inhibit experimental fungal keratitis in mouse by suppressing TLR4 and inflammatory cytokines such as TNFα and IL-1β; the inhibition of HDAC may be a potential therapeutic approach for the treatment of fungal keratitis.

Figure 1

Immunohistochemical analysis of the expression of H3, AC-H3, HDAC1 in the specimens of patient with fungal keratitis. Red: positive staining; blue: hematoxylin contrast staining. The expression of HDAC1 was strikingly high in fungal infected corneal specimens; in contrast, the acetylated histone H3 was substantially reduced after fungal infection. No obviously difference is seen in the staining of H3 between specimens of fungal infected corneal tissues and control. The red arrows indicate the corneal surface and the black arrows represent focal positive staining. Scale bar: 50 µm. Original magnification, 200x.

Figure 2

Immunohistochemical analysis of the expression of H3, AC-H3, HDAC1 in the specimens of the corneas of mice fungal keratitis. Red: positive staining; blue: hematoxylin contrast staining. The expression of HDAC1 was abundant in fungal infected corneal specimens of mice (f) in contrast, the acetylated histone H3 was substantially reduced after fungal infection. (e) The HDAC1 expression was inhibited after SAHA injection. No obvious difference is seen in the staining of H3 between specimens of fungal infected corneal tissue and control. Note the expression of AC-H3 was increased after SAHA injection. The red arrows indicate the corneal surface and the black arrows represent focal positive staining. Scale bar: 50 µm. Original magnification, 400x.

Figure 3

The expression of TNFα and TLR4 in the specimens of human fungal keratitis and the corneas of mice fungal keratitis. Red: positive staining; blue: hematoxylin contrast staining. Abundant immunoreactivity of TNFα and TLR4 is seen in fungal infected corneas of both human and experimental mice (DMSO injection); the expressions of TNFα and TLR4 in the corneas were inhibited by the application of SAHA in experimental mice. The red arrows indicate the corneal surface and the black arrows represent focal positive staining. Scale bar: 50 µm; Original magnification 300x.

Figure 4

The effects of SAHA on the mRNA expression of IL-1β, TNFα and TLR4 in the corneal specimens of mice with experimental fungal keratitis. The total RNA was isolated from the mice corneas after the mice were sacrificed for the analysis of the mRNA expression of IL-1β, TNFα and TLR4 by real-time PCR. After treatment with SAHA, IL-1β and TNFα mRNA are reduced significantly compared with DMSO injection (*P < 0.05), while the inhibition of TLR4 is even greater (**0.01). The numbers of Y axial indicate the relative fold changes to GAPDH.

Figure 5

The effects of SAHA on the production of TNFα and IL-1β in corneas of mice with experimental fungal keratitis by ELISA. The protein obtained from the mice corneas of experimental fungal keratitis was subjected to ELISA assay, injection of SAHA significantly inhibited TNFα and IL-1β production compared with DMSO application (P < 0.05).

Figure 6

The effects of SAHA injection on the corneal opacity of experimental fungal keratitis in mice. The corneal opacity was considerably reduced in the mice corneas with SAHA injection (a–c) compared with DMSO application. (d–f) The inhibition of the corneal lesions by SAHA injection was significant (g), (P < 0.05), photos are representative.

Figure 7

Injection of SAHA inhibited the fugal growth in the mice cornea. Fungal growth were inhibited in SAHA treated mice cornea as demonstrated by H&E (g,h), GMS staining (i,j) respectively. Dark brown stained fungal. The black arrows indicate the surface of the corneas and the red arrows represent fungal. Scale bar: 50 µm; Original magnification 300x.