Expression Of Toll-Like Receptor 2, 4 And Its Downstream Adaptor Protein Tollip On Mouse With Endotoxin Tolerance

Objective: To determine the dosage and the duration of LPS stimulation for establishing model of endotoxin tolerant mouse and investigate the correlation of TLR4 expression in spleen tissues with endotoxin tolerance.Methods: Age- and weight-matched C57BL/6J mouse were applied to the study. 1) Determination of LPS dosage: mouse were injected with 0.1, 0.5, 1.0, 1.5 or 2.0mg/kg/day LPS for one week, and control animals were treated with normal saline. 2) Definition of LPS duration: mouse were injected with 0.5mg/kg/day LPS by peritoneum, lasting for one week, two weeks and three weeks. The physiological states of mouse were observed. TLR4 in spleen tissues was quantified by Western blot.Results: After LPS stimulation, mouse were weak. Animals in the 0.1mg/kg and the 0.5mg/kg group were more active than others, and they recovered at fifth day. The animals of other groups suffered from less activity and weight loss till seventh day. Expression of TLR4 in all LPS groups was decreased compared with that in the control subjects (P<0.05). And TLR4 in the 0.1mg/kg group was increased compared with those in other LPS groups (all P<0.05). There was no difference of TLR4 expression in the one week group, the two weeks group and the three weeks group for definition of LPS duration.Conclusions: Endotoxin tolerance is associated with TLR4 downregulation in spleen tissues. Protocol, 0.5mg/kg/day and duration of two weeks of LPS stimulation, is suitable for establishing mice model with endotoxin tolerance.Objective: To evaluate expression of Toll-like receptor 2, 4 and Tollip by LPS challenge on mice model with endotoxin tolerance.Methods: Forty-eight C57BL/6J mouse were randomized into endotoxin tolerance animals (ET group) and normal control animals (NC group). Endotoxin tolerance was induced by two weeks daily intraperitoneal injections of 0.5mg/kg LPS. NC group received the same volume saline. On the fifteenth day, mouse were administrated with intravenous injections of 10mg/kg LPS to induce tissue damage. Spleen, liver, heart tissues were collected 0h (before injection), 1h, 6h, 20h after the high dose of LPS (six mouse for each time point, altogether eight subgroups). TLR2, 4 and Tollip protein were quantified by western blot, the tissue location of TLR2, 4 and Tollip protein was measured by immunohistochemistry.Results:(1) Spleen: TLR4 protein in the ET 0h was significantly depressed compared with that in the NC 0h (P<0.05). LPS stimulation did not change the expression of TLR4 in all the ET animals; whereas, TLR4 in the NC animals was increased during LPS challenge, and its peak value was at 20h.TLR2 protein in the ET 0h was significantly depressed compared with that in the NC 0h (P<0.05). LPS stimulation did not change the expression of TLR2 in all the ET animals; whereas, TLR2 in the NC animals was increased at 1h and reached its peak at 20h.Tollip protein in the ET 0h was depressed compared with that in the NC 0h (P<0.05). After LPS challenge, Tollip protein in the ET and the NC animals was all significantly downregulated.TLR2, TLR4 and Tollip protein were all located in medulla of spleen by immunohistochemistry.(2) Liver: There was no difference of TLR2 protein in the ET 0h compared with that in the NC 0h. LPS stimulation did not change the expression of TLR2 in all the ET animals; whereas, TLR2 in the NC animals quickly reached its peak at 1h.TLR4 protein was not expressed in liver tissues.There was no difference of Tollip protein in the ET 0h compared with that in the NC 0h. After LPS challenge, Tollip protein in the ET and the NC animals was all upregulated at 1h(P<0.05); yet, the elevated range in the ET animals was shorter than in the NC animals.Tollip protein was located in sinus of liver by immunohistochemistry.(3) Heart: TLR4 protein in the ET 0h was significantly depressed compared with that in the NC 0h (P<0.05). LPS stimulation did not change the expression of TLR4 in all the ET animals; whereas, TLR4 in the NC animals was increased and its peak value was at 6h.TLR2 protein was not expressed in heart tissues.There were no difference of Tollip protein in the ET 0h compared with that in theNC 0h. After LPS challenge, Tollip protein in the ET and the NC animals was all upregulated during experiment(P<0.05); yet, the elevated range in the ET animals was shorter than in the NC animals.TLR4 was located in membrane of cardiac myocytes by immunohistochemistry, and Tollip protein in cytoplasm of cardiac myocytes.Conclusions: Endotoxin tolerance is associated with a profoundly low level of TLR2, 4 expression in spleen, liver, heart tissues. TLR2, 4 deactivation and Tollip activation in tissues after high dose LPS challenge may result in LPS hyporesponsiveness on endotoxin tolerant mouse.