Regulation of LOX-1 on adhesion molecules and neutrophil infiltration in mouse Aspergillus fumigatus keratitis

Abstract

Aim: To determine whether lectin-like ox-LDL receptor (LOX-1) regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus (A. fumigatus) keratitis of C57BL/6 mice.

Methods: C57BL/6 mice were pretreated with a neutralizing antibody to LOX-1 (5 µg/5 µL) or control nonspecific IgG (5 µg/5 µL), LOX-1 inhibitor Poly-I (2 µg/5 µL) or PBS by subconjunctival injection. Fungal keratitis (FK) mouse models of C57BL/6 mice were established by scraping corneal central epithelium, smearing A. fumigatus on the corneal surface and covering the eye with contact lenses. The corneal response to infection was assessed via clinical score. The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin were tested in control and infected corneas by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of ICAM-1 were evaluated by immunofluorescence (IF) and Western blot. Neutrophils were extracted from the abdominal cavity of C57BL/6 mice followed by pretreatment using antibody to LOX-1 (10 µg/mL) or control nonspecific IgG (10 µg/mL), the Poly-I (4 µg/mL) or PBS. The cells were then stimulated with A. fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1 (LFA-1) using RT-PCR and Western blot. IF and myeloperoxidase (MPO) assays were used to assess neutrophil infiltration in mice corneas.

Results: Pretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS (both P<0.01). And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1, VCAM-1, P-selectin, E-selectin and LFA-1 expression compared with control groups (all P<0.01). Furthermore, pretreated with LOX-1 antibody or Poly-I, the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups (all P<0.05). Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays (both P<0.01).

Conclusion: Inhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

Keywords: adhesion molecules; fungal keratitis; lectin-like ox-LDL receptor; mice; neutrophils.

Figure 1. Disease response to LOX-1 neutralizing antibody and inhibitor treatment in A. fumigatus stimulated mice corneas

A: Typical corneal image of mouse model of A. fumigatus keratitis after pretreatment of LOX-1 antibody or Poly-I and IgG or PBS respectively at 3d p.i. B: LOX-1 inhibition led to lower clinical scores after pretreated with LOX-1 antibody or Poly-I were euthanized 3d p.i. compared with pretreatment of IgG or PBS. The values are expressed as mean±SEM. ^bP<0.01.

Figure 2. The expression of adhesion molecules in the cornea of C57BL/6 mice

The corneas of C57BL/6 mice which were infected by A. fumigates at 0, 0.5, 1, and 3d p.i., the mRNA levels of ICAM-1 (A), VCAM-1 (B), P-selectin (C) and E-selectin (D) in control and infected corneas of C57BL/6 mice using RT-PCR. The values are expressed as mean±SEM. ^aP<0.05; ^bP<0.01.

Figure 3. Effect of LOX-1 neutralizing antibody treatment on C57BL/6 mice corneas

Before infection, C57BL/6 cornea was pretreated with LOX-1 neutralizing antibody or IgG, and then infected with A. fumigatus for 0, 0.5, 1, or 3d. The mRNA levels of ICAM-1 (A), VCAM-1 (B), P-selectin (C) and E-selectin (D) were measured by PCR. The values are expressed as mean±SEM. ^aP<0.05; ^bP<0.01.

Figure 4. Effects of LOX-1 inhibitor Poly-I treatment on C57BL/6 mice corneas

C57BL/6 corneas were pretreated with Poly-I before infection and then infected with A. fumigatus for 0, 0.5, 1, or 3d. The mRNA levels of ICAM-1 (A), VCAM-1 (B), P-selectin (C) and E-selectin (D) were measured by RT-PCR. The values are expressed as mean±SEM. ^aP<0.05; ^bP<0.01.

Figure 5. Protein Expression of ICAM-1 in the corneas of C57BL/6 mice

Immunofluorescence of 10 µm frozen sections was used to observe the expression of corneal ICAM-1 protein in C57BL/6 mice of different treatment groups after 3d p.i. The protein levels of ICAM-1 expression using immunofluorescent staining (A) and Western blot (B). Green represents ICAM-1 and blue represents DAPI (nucleus). Magnification ×40. The values are expressed as mean±SEM. ^aP<0.05; ^bP<0.01.

Figure 6. The expression of the LFA-1 in PMNs of C57BL/6 mice

Before infection, PMNs were pretreated with LOX-1 neutralizing antibody or inhibitor, and then stimulated with A. fumigatus for 4, 8, 12h. The expression of LFA-1 in PMNs was analyzed by RT-PCR (A) and Western blot (B). The values are expressed as mean±SEM. ^aP<0.05; ^bP<0.01.

Figure 7. Effect of LOX-1 on neutrophil infiltration in mice corneas

Mouse corneas were pretreated with LOX-1 neutralizing antibody or inhibitor and IgG or PBS before infection, then stimulate the corneas with A. fumigatus for 3d. A: Infiltrating neutrophils were directly observed and measured as a count per 10 µm cornea. NIMP-R14 (green) and DAPI staining (blue). Magnification: 40×. B: The levels of MPO expression after pretreatment of the LOX-1 neutralization antibody and inhibitor in A. fumigatus stimulated mice corneas compared with IgG and PBS. The values are expressed as mean±SEM. ^bP<0.01.