20.500.12556/RUL-122145 The role of extracellular vesicles snd their synthetic analogues in the modulation of TLR4 activity Vloga zunajceličnih veziklov in njihovih sintetičnih analogov v modulaciji aktivnosti TLR4 During oxidative stress conditions, endogenous stress-derived EVs (stressEVs) were found to activate Toll-like receptor 4 (TLR4) with a gene profile different from lipopolysaccharide (LPS). Here we show that stressEVs in comparison to LPS activate several different transcription factors resulting in activation of different immune response genes underlying the differences between pathogen-induced and sterile inflammation. Additionally, concerted activity of 15-lipoxygenase (15-LO) and secretory phospholipase A2 (sPLA2) is needed for the formation of TLR4 agonists, which we identified as lysophospholipids (lysoPLs) with oxidized unsaturated fatty acid. Hydroxy, hydroperoxy and keto products of 20:4 lysoPI were determined by mass spectrometry and they activated the same gene pattern as stressEVs. Furthermore, sPLA2 activity in the synovial fluid from rheumatoid arthritis patients promoted formation of the TLR4 agonists. Injection of sPLA2 promoted K/BxN serum induced arthritis in mice and swelling of the ankles was partially TLR4-dependent. Because both enzymes are induced during inflammation, these results confirm the role of stressEVs in sterile inflammation that promotes chronic diseases and opens the opportunity to treat diseases without affecting systemic innate immunity. V času trajanja oksidativnega stresa se sproščajo zunajcelični vezikli (stressEV), ki so sposobni aktivacije Tollu podobnega receptorja 4 (TLR4) z genskim profilom, ki se razlikuje od aktivacije z naravnim ligandom lipopolisaharidom (LPS). V tej doktorski dizertaciji pokažemo, da stressEV v primerjavi z LPS aktivirajo različne dejavnike transkripcije, kar prispeva k razlikam med vnetjem, induciranim s patogeni, in sterilnim vnetjem. Pokazali smo, da je za tvorbo agonistov TLR4 potrebna usklajena aktivnost 15-lipoksigenaze (15-LO) in sekretorne fosfolipaze A2 (sPLA2). Te agoniste smo identificirali kot lizofosfolipide (lysoPL) z oksidirano nenasičeno maščobno kislino. Hidroksi, hidroperoksi in keto produkte 20:4 lysoPI smo določili z masno spektrometrijo in le-ti so aktivirali enak genski vzorec kot stressEV. Poleg tega je aktivnost sPLA2 v sinovialni tekočini bolnikov z revmatoidnim artritisom prispevala k tvorbi agonistov TLR4. Injiciranje sPLA2 je poslabšalo s K/BxN serumom induciran artritis pri miših in otekanje gležnjev je bilo delno odvisno od TLR4. Ker se oba encima aktivirata med vnetjem, ti rezultati potrjujejo vlogo stressEV v sterilnem vnetju in spodbujanju kroničnih bolezni ter odpirajo možnost zdravljenja bolezni, ne da bi to vplivalo na sistemsko prirojeno imunost. Immunology/ sterile inflammation/ TLR4/ extracellular vesicles/ oxidized phospholipid/ 15-lipoxygenase/ secreted phospholipase A2 Imunologija / sterilno vnetje / TLR4 / zunajcelični vezikli / oksidirani fosfolipidi / 15-lipoksigenaza / sekretorna fosfolipaza A2 true false false Angleški jezik Slovenski jezik Doktorsko delo/naloga 2020-11-26 07:15:11 2020-11-26 07:15:43 2023-06-16 11:05:08 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 16280 56449027 714.pdf 714.pdf 1 13ADC418358AE861BAD7C550BE354DB9 cf0043155a2e1a4e3cd3cccafe00c0b8337bb76d2eb09833c605183ec5319e40 10eec698-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=137315 715.pdf 715.pdf 1 5D3061A3CD721378695E3F1FC07E0D90 c85b0d28d293d7ea9d593bf6852c4366f15dd09420b4f5dee217804183884937 10ff89d1-a1bb-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=137316 Medicinska fakulteta 0 0 0