Lipopolysaccharide enhances HSV-1 replication and inflammatory factor release in the ARPE-19 cells

Abstract

Purpose: During acute retinal necrosis (ARN), retinal pigment epithelial (RPE) cells could be stimulated by both herpes simplex virus (HSV) and lipopolysaccharide (LPS). We aim to investigate the impact of LPS on HSV-1 infection and inflammatory factors in human retinal pigment epithelial cell lines (ARPE-19 cells).

Methods: ARPE-19 cells were infected by HSV-1F strain and HSVg4 strain, a modified HSV strain with GFP genes cloned in, for 1 h. Different concentrations of LPS were added. Green fluorescence protein (GFP) of HSVg4 and the infected cell protein 4 (ICP4) expression were observed. Cell culture supernatants were collected to detect 34 kinds of related cytokines and chemokines by multiplex immunoassay assay.

Results: Under LPS treatment, the cytopathic effect displayed as enlarged multinucleated cells, and the GFP fluorescence intensity and ICP4 expression increased in the HSV-1-infected ARPE-19 cells. HSV-1 infection stimulated cytokines IL-1α, IL-1β, IL-1RA, IL-2, IL-4, IL-6, IL-9, IL-12P70, IL-15, IL-18, IL-21, IL-27, TNF-α, IFN-γ and chemokines CXCL1, CXCL8, CXCL10, CXCL12, CCL2, CCL3, CCL4, CCL5, CCL11 while LPS further enhanced their expression.

Conclusion: LPS promoted HSV-1 infection and inflammatory factor release in ARPE-19 cells, indicating that ARN could deteriorate when complicated with endotoxemia.

Keywords: ARPE-19 cells; Chemokines; Cytokines; Herpes simplex virus type 1; Lipopolysaccharide.

Figure 1

Lipopolysaccharide (LPS) enhanced herpes simplex virus 1 H129-G4 strain (HSVg4) infection in human retinal pigment epithelial cell lines ARPE-19 cells. The cells were infected with HSVg4 at MOIs of 0.01, 0.1 and 1 for 1 h and then treated with LPS at 0, 10, 50 and 100 μg/ml for 24 h. The control cells were not infected with HSVg4. A. The cytopathic effects (CPE) of ARPE-19 cells under different treatments. The CPE is presented as enlarged multinucleated cells with diaphanous margins. Typical CPE was magnified in Figure 1A. Scale bar = 100 μm. B. The correlated green fluorescent protein (GFP) image of HSVg4 in ARPE-19 cells. GFP fluorescence represents HSV-1 replication in ARPE-19 cells. C. The relative fluorescence values in ARPE-19 cells under different conditions. Compared to the group of LPS 0 μg/ml in different HSV-1 concentrations, ∗∗p < 0.01. n = 5.

Figure 2

Infected cell protein 4 (ICP4) expression was promoted by LPS in HSV-1-infected ARPE-19 cells. A. ICP4 expression in ARPE-19 cells at the multiplicity of infection (MOI) of 0.01. The uncropped images of blots were displayed in S1. Fig. B. The ratio of ICP4/β-actin band intensities in different groups in A. C. ICP4 expression at the MOI of 0.1. The uncropped images of blots were displayed in S1. Fig. D. The ratio of ICP4/β-actin band intensities in different groups in C. n = 3. The unedited images of blots in the second and third repeated experiments were shown in S2. Fig.

Figure 3

Cytokine secretion in ARPE-19 cells under LPS treatment and HSV-1 infection. ARPE-19 cells were infected with HSV-1 F for 1 h and incubated with different concentrations of LPS for 12 h. The supernatant of cell culture medium was collected and analyzed at 6 and 12 h post-infection. Statistical analysis was conducted using Kruskal-Wallis test. Compared to the control group under the same LPS levels and at the same time point, ## < 0.01; Compared to the LPS 0 μg/ml group at the same MOI and time point, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Each data point is the average value of 3 independent assays.

Figure 4

Chemokine secretion in ARPE-19 cells under LPS treatment and HSV-1 infection. Kruskal-Wallis test was used in statistical analysis. Compared to the LPS 0 μg/ml group at the same MOI and time point, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Each data point is the average value of 3 independent assays.