Inflammatory and fibrotic effects of serum amyloid A and tenascin C on fibroblasts

Nepravilna razrešitev akutnega vnetnega odgovora, dolgotrajna izpostavitev endogenim molekulam, nastalim ob poškodbi, in porušeno ravnovesje med vnetnimi in zaščitnimi molekulami vodijo v kronično vnetje in fibrozo. Med takšni endogeni molekuli spadata tudi serumski amiloid A in tenascin C, ki sodelujeta pri številnih kroničnih vnetnih in fibrotičnih boleznih. Vežeta se na receptor podoben Tollu 4, prvi pa tudi na receptor podoben Tollu 2 in N-formil peptidni receptor 2. Naš namen je bil proučitev možnosti omejevanja vnetnih in fibrotičnih učinkov serumskega amiloida A in tenascina C. Delovanje serumskega amiloida A in tenascina C smo analizirali na mišjih embrionalnih fibroblastih, normalnih človeških pljučnih fibroblastih in mononuklearnih celicah periferne krvi na genskem (z verižno reakcijo s polimerazo v realnem času) in/ali proteinskem nivoju (z encimskoimunskim testom in prenosom western). Ugotovili smo, da so vsi trije geni za receptorje, na katere se vežeta, inducibilni. Pri vseh celicah smo ugotovili razlike v njihovem razmerju izražanja genov za receptorje, zato so se celice različno odzvale na stimulacije. Serumski amiloid A in tenascin C sta povzročila zvišano izražanje gena in proteina vnetnega citokina interlevkina 6 ter zvišano izražanje označevalcev fibrotičnih procesov (kolagena 1A1, aktina α 2), pri čemer je imel najpomembnejšo vlogo receptor podoben Tollu 4. BOC2, antagonist N-formil peptidnega receptorja 2, je zmanjšal vnetne učinke serumskega amiloida A in fosforilacijo signalne poti, preko katere deluje. Vnetne in fibrotične učinke tenascina C sta z znižanjem izražanja genov za interlevkin 6, kolagen 1A1, aktin α 2 in fibronektin zmanjšali molekuli lipoksin A4 in resolvin D1, ki se vežeta na N-formil peptidni receptor 2. Poleg inhibicije serumskega amiloida A in tenascina C z inhibitornimi molekulami in antagonisti receptorjev bi lahko njune vnetne in fibrotične učinke regulirali tudi z uporabo protiteles. Naravna protitelesa proti serumskemu amiloidu A1 so v preteklosti izkazala uspešno vlogo pri omejevanju vnetnih učinkov serumskega amiloida A1 in posledično interlevkina 6. Njihovo uporabo smo testirali na mononuklearnih celicah periferne krvi, kjer so znižala izločanje interlevkina 6, povzročeno s serumskim amiloidom A. Pri zdravljenju vnetnih in fibrotičnih bolezni se tako odpirajo številne možnosti, ki bi jih bilo v bodoče smiselno raziskati, saj bi s tem pripomogli k omejevanju kroničnih bolezni, ki v sodobnem času predstavljajo enega glavnih vzrokov zmanjšanja kvalitete življenja in smrtnosti.An unresolved acute inflammatory response, prolonged exposure to damage-associated molecular patterns and dysbalance between inflammatory and resolution molecules can lead to chronic inflammation and fibrosis. Two such damage-associated molecules, serum amyloid A and tenascin C, are associated with multiple chronic and fibrotic diseases. They both bind to Toll-like receptor 4, while serum amyloid A can additionally bind to Toll-like receptor 2 and N-formyl peptide receptor 2. The aim of our study was to investigate different ways of limiting the inflammatory and fibrotic effects of serum amyloid A and tenascin C. We tested the influence of serum amyloid A and tenascin C on mouse embryonic fibroblasts, normal human lung fibroblasts and human peripheral blood mononuclear cells on gene expression levels (using real time polymerase chain reaction) and/or protein levels (using enzyme-linked immunosorbant assay and Western blot analysis). We confirmed that all three receptor genes are inducible. We observed differences in cells\u27 ratio in expression of receptor genes, which resulted in their different responses to stimulation with serum amyloid A and tenascin C. Serum amyloid A and tenascin C induced the expression and synthesis of the inflammatory cytokine interleukin 6 and expression of markers of fibrosis (collagen 1A1 and actin α 2), in all of which Toll-like receptor 4 played an important role. BOC2, an antagonist of N-formyl peptide receptor 2, reduced the inflammatory effects of serum amyloid A and the phosphorylation of its signal pathway. Two resolution molecules, that bind to N-formyl peptide receptor 2, lipoxin A4 and resolvin D1, attenauted inflammatory and fibrotic effects of tenascin C by reducing expression of genes for interleukin 6, collagen 1A1, actin α 2 and fibronectin. Another possibility for inhibition of serum amyloid A and tenascin C-induced inflammatory and fibrotic effects is inhibition with antibodies. Natural antibodies against serum amyloid A1 had previously been used to limit the inflammatory effects of serum amyloid A1 and consequently interleukin 6. We tested their role on human peripheral blood mononuclear cells and we observed attenuation of serum amyloid A-induced synthesis of interleukin 6. There are many ways for treating inflammatory and fibrotic diseases. However, further investigations are required to limit the amount of chronic diseases, which are nowadays main cause of reduced quality of life and mortality