Overexpression of Toll-Like Receptors 2, 3, 4, and 8 Is Correlated to the Vascular Atherosclerotic Process in the Hyperlipidemic Rabbit Model: The Effect of Statin Treatment.

BACKGROUND: Atherosclerosis is the major cause of cardiovascular disease; hypercholesterolemia is a major risk factor. We hypothesized that specific TLR members (TLR2, TLR3, TLR4, TLR8) may play a role in atherosclerosis progression and its accompanying inflammatory response. We determined the association of atherosclerotic lesions and TLR mRNA expression in different aortic sites. We also assessed the effects of fluvastatin (Flu) treatment on TLR expression and plaque characteristics. METHODS: Male rabbits, fed with an atherogenic diet for a duration of 3 months, were screened for advanced atherosclerotic lesions in the aorta. Additional animals received normal diet or normal diet plus Flu for 1 additional month. TLR mRNA expression in various thoracic and abdominal aortic segments was assessed, together with atherosclerotic changes. RESULTS: After high lipid diet, the atherosclerotic burden increased more in the abdominal than in the thoracic aorta; TLR2, 3, 4, and 8 also increased significantly. Flu decreased atherosclerotic plaque, calcium deposition, lipid cores, intraplaque hemorrhage, erythrocyte membranes, endothelial cells, and macrophage infiltration, while increasing smooth muscle cells in plaques of both aortic segments; it also lowered TLR2, 3, 4, and 8 expression in all aortic segments to a stronger degree than resumption of normal diet. There was a strong association between blood and tissue parameters during experimental period and finally a strong correlation found between these parameters with mRNA of TLR2, 3, 4, and 8 in various stages. CONCLUSION: For the first time TLR2, 3, 4, and 8 mRNA expression is prospectively explored after hypercholesterolemic diet in the rabbit model. TLR2, 3, 4, and 8 mRNA expression is strongly upregulated and correlates with the progression of atherosclerosis in the aorta. Flu significantly inhibited this progress and reduced inflammation via TLR downregulation which was strongly associated with regression of plaque morphology and atherosclerosis promoting factors.

Toll-Like Receptors -2, -3, -4 and -7 Expression Patterns in the Liver of a CLP-Induced Sepsis Mouse Model.

Objective: To investigate the expression of toll-like receptors (TLRs) in the liver of septic mouse model. Materials and methods: For this study seventy-two C57BL/6J mice were utilized. Sepsis was induced by cecal ligation and puncture (CLP) in the mice of the three septic (S) groups (euthanized at 24 hours, 48 hours and 72 hours). Sham (laparotomy)- operated mice constituted the control (C) groups (euthanized at 24, 48 and 72 hours). Blood samples were drawn and liver tissues were extracted and examined histologically. The expression of TLRs 2, 3, 4 and 7 was assessed via immunohistochemistry (IHC) and qrt-PCR (quantitative- Polymerase Chain Reaction). Results: Liver function tests were elevated in all S-groups in contrast to their time-equivalent control groups (S24 versus C24, S48 versus C48 and S72 versus C72) (p < 0.05). Liver histology displayed progressive deterioration in the septic groups. IHC and qrt-PCR both showed an increased expression of all TLRs in the septic mice in comparison to their analogous control ones (p < 0.05). Analysis of livers and intestines of the septic animals proved that all TLRs were significantly expressed in higher levels in the intestinal tissues at 24h and 48h (p < 0.05) except for TLR 3 in S48 (p > 0.05); whereas at 72 hours only TLR 4 levels were significantly elevated in the intestine (p < 0.05). Conclusion: TLRs seem to be expressed in significant levels in the livers of septic rodents, indicating that they have a possible role in the pathophysiology of liver damage in septic conditions.

Increased expression of Toll-like receptors 2, 3, 4 and 7 mRNA in the kidney and intestine of a septic mouse model.

Toll-like receptors (TLRs) are the key regulators of innate and adaptive immunity and are highly expressed during sepsis. Thus, studying the expression of TLRs in an animal septic model might indicate their possible association with acute kidney injury in sepsis. Seventy-two male C57BL/6J mice were used for this study. Randomly, these animals were divided into 6 groups (N = 12/group): 3 control and 3 septic groups depending on the euthanasia time (24 h, 48 h, 72 h). Septic groups underwent cecal ligation and puncture (CLP) to induce peritonitis, while control groups had a sham operation. Hematological tests were performed in serum for immune biomarkers; immunohistochemistry, morphometry and qRT-PCR analysis were used on both kidney and intestine tissues to evaluate the expression of TLR 2, 3, 4 and 7 in a septic process. At the end of each experimental period, we found that TLRs 2, 3, 4 and 7 were expressed in both tissues but there were differences between those at various time points. Also, we found that mRNA levels were significantly higher in qRT-PCR evaluation in septic groups than control groups in both kidney and intestinal tissues (p < 0.05); showing a steady increase in the septic groups as the time to euthanasia was prolonged (p < 0.05). Overall, our study provides a suggestion that TLRs 2, 3, 4 and 7 are highly expressed in the kidneys of septic mice and especially that these TLRs are sensitive and specific markers for sepsis. Finally, our study supports the diagnostic importance of TLRs in AKI and provides an insight on the contribution of septic mice models in the study of multi organ dysfunction syndrome in general.

Expression of Toll-like receptors (TLRs) in the lungs of an experimental sepsis mouse model.

BACKGROUND: Sepsis is a condition characterized by high mortality rates and often accompanied by multiple-organ dysfunction. During sepsis, respiratory system may be affected and possibly result in acute respiratory distress syndrome (ARDS). Toll-like receptors (TLRs), as a first line defense against invading pathogens, seem to be highly expressed in septic states. Therefore, expression of TLRs in the lungs of a sepsis animal model could indicate the involvement of the respiratory system and appear as a severity index of the clinical course. MATERIALS AND METHODS: A total of 72 C57BL/6J mice, aged 12-14 weeks, were studied. The animals were divided into 3 sepsis (S) groups (24h, 48h and 72h) and 3 control (C) groups (24h, 48h and 72h), each consisting of 12 mice. The S-groups were subjected to cecal ligation and puncture (CLP) while the C-groups had a sham operation performed. Blood samples were drawn from all groups. Total blood count analysis was performed along with the measurement of certain biochemical markers. Additionally, lung tissues were harvested and the expression of TLRs, namely TLR 2, TLR 3, TLR 4 and TLR 7 were evaluated by means of immunofluorescence (IF) and qRT-PCR (quantitative-Polymerase Chain Reaction). Statistical analysis was performed by using one-way ANOVA followed by student t-test. Results were considered statistically significant when p<0.05. RESULTS: WBCs and lymphocytes were decreased in all S-groups compared to the corresponding C-groups (p<0.05), while RBCs showed a gradual decline in S-groups with the lowest levels appearing in the S72 group. Only, monocytes were higher in S-groups, especially between S48-C48 (p<0.05) and S72-C72 (p<0.05). Creatinine, IL-10 and IL-6 levels were significantly increased in the S-groups compared to the corresponding C-groups (S24 vs C24, S48 vs C48 and S72 vs C72, p<0.05). IF showed that expression of TLRs 2, 3, 4 and 7 was increased in all S-groups compared to the time-adjusted C-groups (p<0.05). Similarly, qRT-PCR revealed that expression of all TLRs was higher in all S-groups compared to their respective C-groups in both lungs and intestine (p<0.05). Comparing lung and intestinal tissues from S-groups, TLRs 2 and 4 were found increased in the lung at 24, 48 and 72 hours (p<0.05), whereas TLR 3 was higher in the intestine at all time points examined (p<0.05). Finally, TLR 7 levels were significantly higher in the intestinal tissues at 24 hours (p<0.0001), while lungs predominated at 48 hours (p<0.0001). CONCLUSION: TLRs seem to be highly expressed in the lungs of septic mice, therefore suggesting a potential role in the pathogenesis of ARDS during sepsis. While more studies need to be conducted in order to completely understand the underlying mechanisms, TLRs may represent a promising target for establishing novel therapeutic strategies in the treatment of sepsis.