In-vitro safety assessment of meropenem on human retinal pigment epithelium (RPE)

Abstract

Purpose: Endophthalmitis is a severe infection accompanied by inflammation that affects the anterior and posterior parts of the eye. It is typically treated with a combination of antibiotics that cover various microorganisms. However, retinal pigment epithelium (RPE) cells are highly susceptible to damage from intravitreal injection therapy. This study aimed to investigate the impact of clinically relevant concentrations of meropenem (alone or in combination with vancomycin) on the viability and inflammation of RPE cells.

Design: In-vitro Study.

Methods: RPE cells from passages 5-7 were treated with different concentrations of meropenem (1/4x, x, and 4x; [x = 16 mg/L]), vancomycin (30 mg/L), and meropenem (x) plus vancomycin for 24 h. The morphology assessment and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay were performed to evaluate cytotoxicity due to drug treatment. Real-time PCR was used to measure the relative expression of apoptotic genes (BCL-2 and BAX) and inflammation biomarkers (IL-1b and IL-6).

Results: Meropenem (alone or in combination with vancomycin) did not have any significant effect on RPE cell morphology, density, and viability. Gene expression analysis confirmed these results, showing no significant changes in the BCL-2/BAX ratio in drug-treated RPE cells compared to controls. Treatment with meropenem significantly induced the expression of IL-1b at all studied concentrations. Additionally, at concentrations of x and 4x, it also significantly increased the expression of IL-6, which was dose-dependent. However, this effect was not observed with vancomycin alone or in combination with meropenem.

Conclusions: The results of this study suggest that meropenem, either alone or in combination with vancomycin, does not induce RPE cytotoxicity. There was an upregulation of IL-1b and IL-6 in meropenem monotherapy, the clinical implication of which should be elucidated in future in-vivo or clinical studies.

Keywords: Apoptosis; Endophthalmitis; Inflammation; Meropenem; RPE.

Fig. 1

RPE characterization; (A) Representative image showing proliferating RPE cells with highly pigmented morphology at early passages, observed approximately one week after the initiation of culture. (B) Representative image demonstrating changes in cell morphology as pigmentation gradually diminishes and cells adopt a spindle-shaped appearance with repeated passages. (C) Immunocytochemistry (ICC) analysis confirming expression of the RPE65 marker in primary cultures of RPE cells. (D) & (E) fields show the secondary antibody control confirming the lack of fluorescence in cultures without the secondary antibody of RPE65.

Fig. 2

RPE cell morphology under varying treatments of meropenem, vancomycin, and their combination (invert microscopy, Mag: 100x). The data showed consistent RPE cell distribution and density across treatment with meropenem (1/4x, x, and 4x; [x = 16 mg/L]), vancomycin (30 mg/L), and meropenem (x) plus vancomycin, in comparison with controls, indicating the safety of meropenem alone or in combination with vancomycin. The incubation time with the various concentrations of meropenem (either alone or in combination with vancomycin) was 24 h.

Fig. 3

Top left: MTT assay; Top right: BCL-2/BAX ratio; Bottom left: Relative gene expression of BCL-2 and BAX; Bottom right: Relative gene expression of inflammatory genes (IL-1b, IL-6) in RPE cells treated with meropenem (1/4x, x, and 4x; [x = 16 mg/L]), vancomycin (30 mg/L), and meropenem (x) plus vancomycin, in comparison with controls (the dashed line). The incubation time with the various concentrations of meropenem (either alone or in combination with vancomycin) was 24 h. The mean value of relative expression ± standard error of mean (SEM) is represented in the graph. All experiments were performed in triplicate. The individual data points of independent biological repetitions are provided on the figure. Asterisk signs (*) bold the statistically significant differences (P < 0.05).