Amelioration of endotoxin-induced uveitis treated with an IκB kinase β inhibitor in rats

Abstract

Purpose: Endotoxin-induced uveitis (EIU) is an animal model for acute ocular inflammation. Several substances play major roles in the development of inflammatory changes in EIU, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. These inflammatory cytokines trigger the degradation of IκB by activating IκB kinases (IKKs). Released nuclear factor kappaB (NFκB) subsequently translocates to the nucleus, where NFκB expresses its proinflammatory function. IMD-0354, N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide, selectively inhibits IKKβ, particularly when induced by proinflammatory cytokines, such as TNF-α and IL-1β. In the present study, we examined whether IKKβ inhibition has therapeutic effects on EIU by using IMD-0354 and its prodrug IMD-1041.

Methods: Six-week-old male Lewis rats were used. EIU was induced with subcutaneous injections of 200 μg of lipopolysaccharide (LPS) from Escherichia coli that had been diluted in 0.1 ml of phosphate-buffered saline. IMD-0354 was administered intraperitoneally at 30, 10, 3, or 0 mg/kg, suspended in 1.0 ml of 0.5% carboxymethyl cellulose sodium. The prodrug IMD-1041 (100 mg/kg) was also administered orally. The rats were euthanized 24 h after LPS injection, and EIU severity was evaluated histologically. The number of infiltrating cells and the protein, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) concentrations in the aqueous humor were determined. TNF-α and MCP-1 concentrations were quantified with enzyme-linked immunosorbent assay. Eye sections were also stained with anti-NFκB and phosphorylated I-κBα antibodies.

Results: The number of infiltrating cells in aqueous humor was 53.6±9.8×10(5), 72.5±17.0×10(5), 127.25±32.0×10(5), and 132.0±25.0×10(5) cells/ml in rats treated with 30, 10, 3, or 0 mg/kg of IMD-0354, respectively. The total protein concentrations of aqueous humor were 92.6±3.1 mg/ml, 101.5±6.8 mg/ml, 112.6±1.9 mg/ml, and 117.33±1.8 mg/ml in rats treated with 30, 10, 3, and 0 mg/kg of IMD-0354, respectively. Infiltrating cells and protein concentrations were significantly decreased by treatment with IMD-0354 (p<0.01). IMD-0354 treatment significantly reduced the concentration of TNF-α (p<0.05) and MCP-1 (p<0.01) in aqueous humor. The number of NFκB positive nuclei was reduced when treated with IMD-0354. Furthermore, IMD-0354-treated EIU rats showed only background levels of phosphorylated I-κBα; however, it was strongly expressed in the iris-ciliary body cell cytoplasm of the IMD-0354 untreated EIU rats. Oral administration of IMD-1041 also decreased the cell number (p<0.01) and protein concentration (p<0.05) of aqueous humor in EIU.

Conclusions: Acute uveitis was ameliorated by inhibition of IKKβ in rats. IMD-0354 and its prodrug IMD-1041 seem to be promising candidates for treating intraocular inflammation/uveitis.

Figure 1

Histological changes in the iris-ciliary body, vitreous cavity, and retina 24 h after lipopolysaccharide injection. Photographs on the left side show the iris-ciliary body (ICB) region, and those on the right side show the vitreous and retina in rats. Control animals E, J: were not injected with lipopolysaccharide (LPS); no inflammation was observed. Severe inflammatory cell infiltration was observed in endotoxin-induced uveitis (EIU) rats D: I: In the group of EIU rats treated with IMD-0354 (30 mg/kg; A, F: 10 mg/kg; B, G: reductions in cell infiltration were observed compared to untreated EIU rats. No noticeable reduction in cell infiltration was observed in EIU rats treated with IMD-0354 (3 mg/kg: C: H: Infiltrating cells in the ICB of sections K: and in the posterior part of the eye (l) were counted and averaged. Data are shown as mean±standard error of mean (SEM; n=8). *p<0.05, **p<0.01, significantly different from the LPS group.

Figure 2

Effect of IMD-0354 on cellular infiltration A: and protein concentration B: in aqueous humor collected 24 h after lipopolysaccharide (LPS) treatment. Data are shown as mean±standard error of mean (SEM; n=8). *p<0.05, **p<0.01, significantly different from the LPS group.

Figure 3

Effect of IMD-0354 (30 mg/kg) on tumor necrosis factor (TNF)-α A: and monocyte chemoattractant protein (MCP)-1 B: in aqueous humor collected 24 h after lipopolysaccharide (LPS) treatment. Data are shown as mean±standard error of mean (SEM; n=8). *p<0.05, **p<0.01, significantly different from the LPS group.

Figure 4

Effect of IMD-0354 on nuclear factor (NK) κB p65 (red) activation in the iris-ciliary body 3 h after lipopolysaccharide injection. Dual-immunofluorescence labeling showed the NFκB co-localization (yellow) in nuclei (green). Control animals A: were not injected with lipopolysaccharide (LPS); only weak NFκB signal detected in cytoplasm area of the cells, no nuclear co-localization of NF NFκB was detected. In the group of endotoxin-induced uveitis (EIU) rats treated with IMD-0354 30 mg/kg B: reductions of NFκB co-localization were observed compared to untreated EIU rats C: Quantitative analysis of NF-κB-positive cells in the iris-ciliary body (ICB) presented in graph D: Data are shown as mean±standard error of mean (n=4). *Significantly different from LPS group (p<0.05).

Figure 5

Effect of IMD-0354 on phosphorylated inhibitors of κB (green) in the iris-ciliary body 3 h after lipopolysaccharide injection. No phosphorylated inhibitor of κB (p-IκB signal was detected in negative control A: where no p-IκB antibodies were applied. The p-IκB signals were similarly expressed in cytoplasm of naïve controls B: and IMD-0354-treated (30 mg/kg) LPS injected rats C: Intensive p-IκB expression was observed in cytoplasm of iris-ciliary body (ICB) cells in untreated endotoxin-induced uveitis (EIU) rats D: Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) as blue.

Figure 6

Effect of 100 mg/kg of IMD-1041 oral application on cellular infiltration A: and protein concentration B: in aqueous humor collected 24 h after lipopolysaccharide (LPS) treatment. Data are shown as mean±standard error of mean (SEM; n=4). *p<0.05, **p<0.01, significantly different from the LPS group.