Low Dose of Lipopolysaccharide Pretreatment Preventing Subsequent Endotoxin-Induced Uveitis Is Associated with PI3K/AKT Pathway

Purpose To explore the effects of LPS pretreatment on endotoxin-induced uveitis and PI3K/AKT pathway. Methods Endotoxin-induced uveitis was induced by a single subcutaneous injection of 200 μg LPS. For the endotoxin tolerance group, induction of EIU was preceded by daily subcutaneous injection of 0.1 mg/kg LPS for five days. Clinical scores were graded at 24 h after EIU under a slit lamp microscope. HE stain was performed to observe the histopathology. Aqueous humor TNF-α concentration was quantified with enzyme-linked immunosorbent assay. The expressions of PI3K and AKT were detected through Western blot analyses, and the activation of AKT was detected through immunofluorescence study. Results Endotoxin tolerance produced suppressive effects by significantly reducing the inflammatory reaction of anterior segment of the rats as measured by slit lamp and histopathology. Low dose of LPS pretreatment significantly reduced TNF-α concentrations and the expressions of PI3K and AKT. Furthermore, the activation of AKT was also inhibited. Conclusions LPS pretreatment can ameliorate endotoxin-induced uveitis in rats. This protection of endotoxin tolerance against EIU is associated with PI3K/AKT pathway by reducing level of TNF-α in the aqueous humor.


Introduction
Uveitis is one of the world's major sight-threatening diseases, which mainly affects the iris, ciliary body, and choroid [1]. Lipopolysaccharide (LPS), which is a kind of pathogenassociated molecular patterns (PAMPs), activates the innate immune system through toll-like receptor 4 (TLR4) which is a member of innate immune recognition receptors (PRRs) [2]. Substantial clinical and experimental evidences support the role of LPS in the pathogenesis of immunemediated, noninfectious acute anterior uveitis (AAU), particularly human leukocyte antigen-(HLA-) B27-associated AAU [3]. In terms of LPS-induced inflammatory response, there is an important protective mechanism, which is called endotoxin tolerance that the body or cells preexposed to low dose of LPS are refractory to subsequent LPS challenge [4]. Elucidating the effect of endotoxin tolerance on ocular inflammation and the underlying mechanism are of great importance for the prevention and treatment of uveitis.
Endotoxin-induced uveitis (EIU) with the injection of LPS into certain susceptible strains of rodents inducing an acute and preferential inflammation of the iris and ciliary body is a well-established animal model that closely resembles AAU in humans [5]. The inflammation of the cellular infiltration and protein leakage into the anterior chamber of the eye reaches a maximum 24 h after LPS injection [6]. Elevated expression of cytokines such as TNF-α and IL-6 has been observed to be concomitant with maximum EIU [6,7]. Cytokine levels, especially those of TNF-α, can be used as a marker for monitoring the inflammation and disease progression.
As is known to us, LPS could activate the PI3K/AKT pathway, which regulates the expressions of cytokine and chemokine, such as TNF-α [8][9][10], moreover, which is involved in endotoxin tolerance [11]. Furthermore, a study of gene expression microarray analysis revealed that PI3K and AKT play critical roles in HLA-B27-associated AA [12]. We sought to explore the effect of endotoxin tolerance on endotoxin-induced uveitis and evaluate whether endotoxin tolerance could modulate the PI3K/AKT pathway.

Animal Model.
Endotoxin-induced uveitis (EIU) was induced by a single subcutaneous injection of 200 μg LPS dissolved in 0.1 ml sterile saline (1 mg/kg) as previously described [13]. All animals were randomly divided into three groups: normal control (NC) group, endotoxin tolerance (ET) group, and endotoxin-induced uveitis (EIU) group. In the ET group, endotoxin tolerance was induced by daily subcutaneous injection of 0.1 mg/kg LPS for five days [14]. The other two groups of rats were treated with sterilized saline in the same manner as pretreatment. On day 6, the animals in the ET and EIU groups received a single subcutaneous injection of 200 μg LPS to induce EIU. The NC group received subcutaneous injection of 0.1 ml sterile saline.

Clinical Manifestations Scoring.
Clinical features of ocular inflammation were evaluated in both eyes at 24 h after injection of 200 μg LPS or vehicle under a slit lamp microscope. Inflammatory signs were recorded. The severity of uveitis was graded according to the scoring system in Table 1 by two observers who were blinded to the treatment groups [15,16].
2.5. Enzyme-Linked Immunosorbent Assay (ELISA). After clinical evaluation, an aqueous humor sample was collected from both eyes of all rats using a 30-gauge needle attached to a 1 mL syringe under a microscope. TNF-α levels in the aqueous humor were assessed with rat TNF-α ELISA kit. All measurements were conducted according to the manufacturer's instructions.
2.6. Histopathological Examination. For histopathologic analysis, the rats were killed 24 hours after the last injection of LPS or vehicle; their intact eyes were enucleated and placed in 10% neutral buffered formalin solution for 24 h. The eye specimens were dehydrated in a graded ethanol series and embedded in paraffin. Sagittal sections (4-5 μm thick), cut near the optic nerve head, were stained with hematoxylin and eosin. As previously described [16,17], anterior chamber tissues were scored for severity of inflammation as follows: grade 0 = normal tissue; grade 1+ = dilated iris vessels and thickened iris stroma with exudate, protein, and/or a few scattered inflammatory cells in the anterior chamber; grade 2+ = infiltration of inflammatory cells into the stroma of the iris and/or ciliary body with a moderate number of inflammatory cells within the anterior chamber; grade 3+ = heavy infiltration of inflammatory cells within the iris stroma, ciliary body, and the anterior chamber; and grade 4+ = heavy exudation of cells, dense protein aggregation in the anterior chamber, and in inflammatory cell deposits on the corneal endothelium. Histopathologic analyses were performed by a single pathologist blinded to treatment history.

Western
Blot. Each ICB sample was harvested, rinsed with PBS, and lysed. A total of 40 μg of protein per sample was separated by SDS-polyacrylamide gel electrophoresis, incubated with anti-PI3-kinase p85α (Santa Cruz Biotechnology) at a dilution of 1 : 1000 or anti-AKT1/2/3 (Santa Cruz Biotechnology) at a dilution of 1 : 500. Signal was visualized after secondary antibody staining (donkey antirabbit IgG antibody, 1 : 4000). All values were normalized with β-actin as loading control. Each sample was collected from two rats (4 eyes).
2.8. Immunofluorescence. Eye specimens were fixed in 4% paraformaldehyde for 1-2 h after being enucleated. The ICB was dissected into four segments; each prepared tissue was permeabilized with 0.3% Triton X-100 for 30 min at room temperature and then washed three times with PBS. After blocking nonspecific binding with 3% bovine serum albumin (BSA)/PBS, the tissues were incubated with rabbit polyclonal PI3-kinase p85α antibody (

Endotoxin Tolerance Attenuates EIU.
At 24 h after injection of 200 μg LPS or vehicle, the EIU group showed typical signs mimicking human uveitis, including ciliary congestion, iris blood vessel dilatation, pupil occlusion, and fibrinous membrane formation (Figure 1(a)). Low dose of LPS pretreatment (ET group) significantly attenuated iris blood vessel dilatation, pupil occlusion, and fibrinous membrane formation; just slight iris blood vessel dilatation could be found in the ET group, small amounts of exudate occasionally (Figure 1(b)). No ocular inflammatory signs were observed in rats of the blank control group (Figure 1(c)). The clinical score of the ET group was 1.65 ± 0.49, significantly lower than that of the EIU group (6.65 ± 0.59, p < 0 05) (Figure 1(d)).

Histopathological
Changes. HE staining results were in accordance with clinical features of ocular inflammation. In the EIU group, heavy infiltration of inflammatory cells within the iris stroma, ciliary body, and the anterior chamber and inflammatory cells depositing on the corneal endothelium were observed (Figure 2(a)). Inflammatory cellular infiltration was significantly reduced by low dose of LPS pretreatment in the ET group (Figure 2(b)). The histopathologic score of the ET group was 0.6 ± 0.5, significantly lower than that of the EIU group (3.8 ± 0.4, p < 0 05) (Figure 1(d)).

The Levels of Cytokines in the Aqueous Humor during
Endotoxin-Induced Uveitis in Wistar Rats. As shown in Figure 3, the aqueous TNF-α concentration in the EIU group (30.96 ± 1.55 pg/ml) was significantly higher than that of the NC group (10.32 ± 0.61 pg/ml). However, the level of TNFα was significantly reduced in the ET group when dose of LPS pretreatment (10.58 ± 0.67 pg/ml) is low.

Effects of Endotoxin Tolerance on the Expression of PI3K
and AKT in the Iris-Ciliary Body. Western blot was performed to detect the expression of PI3K and AKT. As shown in Figure 4, the protein levels of PI3K and AKT in the ET group decreased significantly compared with the EIU group. Moreover, the levels also decreased significantly compared with the NC group.

Immunofluorescence Analysis of the AKT Activation.
Without LPS stimulation, AKT distributed over cytoplasm ( Figure 5(a)). After 200 μg LPS stimulation, AKT transferred   Figure 3: Expression of TNF-α in aqueous humor. All data shown as mean ± standard deviation (n = 10). * p < 0 05 the EIU group versus the NC group; * * p < 0 05 the ET group versus the EIU group.
from cytoplasm to cell membrane, representing activation of AKT ( Figure 5(b)). However, low dose of LPS pretreatment inhibited translocation of AKT partially ( Figure 5(c)).

Discussion
As a potent inflammatory stimulant, high doses of LPS lead to systemic inflammatory response syndrome and death [18]. However, if the body was given a small dose of LPS stimulation in advance, the body showed low or no response to the subsequent large dose or lethal dose of LPS stimulation. This important protective mechanism is called endotoxin tolerance [4].
To elucidate the effect of endotoxin tolerance on ocular inflammation, endotoxin-induced uveitis with that a wellestablished animal model of AAU was used in this study. The Wistar rats in the EIU group showed typical signs mimicking human uveitis, including ciliary congestion, iris blood vessel dilatation, pupil occlusion, and fibrinous membrane formation. However, the inflammatory reaction of anterior segment of the rats significantly decreased in the ET group as measured by slit lamp assessment and histopathology. These results demonstrate that endotoxin tolerance which was induced by low dose of LPS pretreatment could successfully suppress the ocular inflammation in the EIU model. Further, these findings suggest that endotoxin tolerance is of great potential in the treatment and prevention of uveitis. However, the molecular mechanisms underlying the induction of endotoxin tolerance are not yet fully understood. Hoekzema et al. [15] demonstrated that repeated injection of endotoxin resulted in no detectable IL-6 in the aqueous humor and the absence of uveitis. Mashimo et al. [19] indicated that continuous high expression of IL-10 in the eye plays significant role in the mechanism of endotoxin tolerance in a rat model of EIU. In this study, TNF-α levels in the aqueous humor during endotoxin-induced uveitis in rats were investigated to further clarify the anti-inflammatory effect of endotoxin tolerance and explore the role of cytokines in this protective mechanism. Consistent with the literatures [6,[20][21][22], elevated TNF-α was observed in the EIU group, and the TNF-α levels were significantly reduced by low dose of LPS pretreatment in ET group. This result strongly confirmed the anti-inflammatory effect of endotoxin tolerance, which is consistent with the slit lamp assessment and histopathology. Furthermore, the result suggests that TNF-α as well as IL-6 and IL-10 is closely involved in the protective mechanism of endotoxin tolerance. However, the upstream mechanism remains unclear. As mentioned earlier, the PI3K/AKT pathway plays an important role in regulating LPS-induced cytokine such as TNF-α expression. Therefore, we next evaluated whether endotoxin tolerance could modulate the PI3K/AKT pathway. Similar to many protein kinases, AKT has a specific AH/ PH domain which mediates the interaction between signaling molecules. PI3K generates phosphoinositide-3,4,5-triphosphate (PIP3) that can bind to the AH/PH domain of AKT in response to upstream signals, resulting in AKT translocating to the plasma membrane and being activated [23]. Thus, both the expressions of PI3K and AKT in ICB of the EIU rats and activation of AKT were detected. In this work, the protein levels of PI3K and AKT in the ET group decreased significantly compared with the EIU group, which were associated with the inflammatory outcome of the rats. Moreover, the levels in the ET group were even lower than those in the NC group, which indicated that the expression of PI3K and AKT was suppressed when dose of LPS pretreatment is low. Furthermore, according to the immunofluorescence, the activation of AKT was also suppressed in the ET group. Taken together, both low expressions of PI3K/AKT and inhibition of AKT activation contribute to the induction of endotoxin tolerance.
In summary, the present study demonstrates that low dose of LPS pretreatment could prevent subsequent endotoxininduced uveitis by reducing the expression of TNF-α in the aqueous humor, and this protective effect of endotoxin tolerance is associated with PI3K/AKT pathway. Furthermore, in virtue of the critical roles of PI3K and AKT in HLA-B27associated acute anterior uveitis, endotoxin tolerance holds promise for the prevention and treatment of this sightthreatening diseases.

Conflicts of Interest
The authors declare that they have no conflicts of interest.