Study of anti-inflammatory functions of HDL in experimental mouse models

• Κύριος συγγραφέας: Πετροπούλου, Περιστέρα-Ιωάννα
• Άλλοι συγγραφείς: Κυπραίος, Κυριάκος
• Μορφή: Thesis
• Γλώσσα: Greek
• Έκδοση: 2016
• Θέματα: Sepsis; HDL; Macrophages; Σήψη; 572.68

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. Τhe purpose of this study was to investigate the potential correlation between HDL shape and composition and its anti-inflammatory functions and properties in LPS-induced inflammation. As lecithin-cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat-/-) mice that lack mature HDL present an increased LPS-induced inflammatory response. The effects in Lcat-/- mice were compared to Apolipoprotein A-I-deficient (Apoa1-/-) mice, which lack classical HDL and are known to have an increased response to LPS, and control wild-type (WT) mice. Characterization of apolipoprotein composition of HDL, revealed that LCAT-deficient HDL is primarily composed of ApoE, while HDL from Apoa1-/- mice is highly enriched in ApoE and ApoA-II. Further analyses, showed clear differences in the lipid composition of HDL among the three groups. As expected based on the structural data of HDL, LPS (100 μg/kg body weight)-induced cytokine response in both Lcat-/- and Apoa1-/- mice was markedly enhanced and prolonged compared to WT mice. Ex vivo stimulation of whole blood with LPS (1-100 ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons and VLDL (triglyceride-rich lipoproteins; TRL fraction) or the lipid-free protein fraction of Lcat-/- mice, had a reduced capacity to attenuate the LPS-induced TNFα response. ApoA-Ι-deficiency did not affect the capacity of HDL to neutralize LPS. Additional immunophenotyping showed that Lcat-/-, but not Apoa1-/- mice, have markedly increased circulating monocyte numbers as a result of an increase in ‘mildly pro-inflammatory’ Cd11b+LyCmid monocytes, whereas ‘highly pro-inflammatory’ Cd11b+LyChi monocytes were reduced. In line with this observation, Kuppfer cells in the Lcat-/- liver had a rather anti-inflammatory, regulatory phenotype, while peritoneal macrophages of Lcat-/- mice also showed a markedly dampened LPS-induced TNFα response. However, fluorescent microscopy studies of membrane cholesterol content and fluidity, were not able to provide a correlation between membrane cholesterol and rigidity, and macrophage responsiveness to LPS. Importantly, reintroducing LCAT by adenovirus-mediated gene transfer (AdLCAT) to Lcat-/- mice reverted their lipid profile and Ly6Chi/Ly6Cmid monocyte ratio back to that of WT mice. Consequently, AdLCAT-treated Lcat-/- mice, presented significant decrease in TNFα levels when stimulated with LPS, compared to the Lcat-/- group treated with the control adenovirus AdGFP. Based on the above, we conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL, as well as increased monocyte number despite the disturbed monocyte/macrophage phenotype.