Karakterizacija imunosupresivnega fenotipa mezenhimskih stromalnih matičnih celic ter analiza vpliva njihove aplikacije na izbrane biološke označevalce pri osteoartritisu kolena

Meglen, Lara (author), Švajger, Urban (mentor)

Kolenski osteoartritis (KOA) je večfaktorski proces, za katerega je značilna razgradnja hrustanca, poškodba drugih obsklepnih tkiv, kronično vnetje in v končni fazi pojav simptomov, kot sta bolečina v sklepu in omejena funkcionalnost. Zaradi imunsko posredovanih mehanizmov, vključenih v patofiziologijo KOA, so mezenhimske stromalne matične celice (MSC) s svojimi imunomodulatornimi lastnostmi na tem področju zanimiva terapevtska učinkovina. V ortopediji se MSC uporabljajo že dlje časa in poročila o zmanjšanju bolečine in izboljšanju funkcionalnosti sklepov so spodbudna. Kljub temu pa je zaradi kompleksnosti tako bolezni kot imunomodulatornega delovanja MSC le malo znanega o molekularnih mehanizmih delovanja MSC na KOA. V okviru klinične študije smo raziskovali mehanizme učinkov lokalnega zdravljenja z gojenimi, alogenskimi mezenhimskimi stromalnimi matičnimi celicami pri pacientih z osteoartritisom kolena. Pri MSC, uporabljenih za aplikacijo, smo najprej dodatno karakterizirali njihov imunosupresivni fenotip. Preko stimulacije z IFN-γ pa smo preverjali, če so MSC sposobne aktivacije in vitro. V vzorcih sklepne tekočine, pridobljene s punkcijo kolena, smo analizirali prisotnost citokinov in treh bioloških označevalcev katabolizma povezanih s KOA (agrekan, hrustančni oligomerni matriksni protein (COMP) in hialuronan (HA)) ter opazovali spremembe njihovih koncentracij, ki bi lahko nakazovale na učinek intervencije z MSC. Rezultati naših analiz so pokazali, da so aplicirane MSC izražale imunosupresivni fenotip, prav tako pa so bile sposobne aktivacije in vitro. Ugotovili smo, da je pri določanju agrekana v vzorcih sklepne tekočine najverjetneje prisoten matriks efekt oziroma identificirali pomanjkljivost izbrane metode. Koncentracija HA se ni bistveno spremenila, koncentracija COMP pa je pri večini pacientov narasla, kar je v nasprotju z našo hipotezo. Obe ugotovitvi pripisujemo predvsem majhnemu številu pacientov (n=5), potencialni heterogenosti vzorcev in pa kompleksnosti KOA ter delovanja MSC. Določili in analizirali smo opazne koncentracije IL-8 in IL-6. Koncentracije IL-8 so ostale enake, pri čemer sklepamo na vlogo tega citokina v začetnih, ne pa v poznejših fazah KOA. Koncentracija IL-6 se je znižala, kar pa nakazuje na pričakovano imunosupresivno delovanje MSC.

• Language: Slovenian
• Keywords: mezenhimske stromalne matične celice, osteoartritis kolena, sklepna tekočina, imunomodulacija, akademska klinična študija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-151092
• Publication date in RUL: 29.09.2023

Secondary language

• Language: English
• Title: Characterization of the immunosuppressive phenotype of mesenhymal stromal stem cells and analysis of the influence of their application on selected biological markers in knee osteoarthritis

Abstract

Knee osteoarthritis (KOA) is a multifactorial process characterised by cartilage degradation, damage to surrounding tissues, chronic inflammation, pain and limited functionality of the joint. The immune-mediated mechanisms involved in the pathophysiology of KOA make mesenchymal stromal stem cells (MSCs), with their immunomodulatory properties, an interesting therapeutic agent in this field. MSCs have been used in orthopaedics for a long time and reports of pain reduction and improved joint functionality are encouraging. However, due to the complexity of both the disease and action of MSCs, little is known about the molecular mechanisms of MSC action on KOA. In the context of a clinical study, we investigated the mechanisms of the effects of local treatment with cultured, allogeneic mesenchymal stromal stem cells in patients with knee osteoarthritis. The MSCs used for application were first characterised for their immunosuppressive phenotype. We determined the expression of the intracellular enzyme IDO and the surface protein PD-L1 to determine the potency of MSCs. IFN-γ stimulation was used to verify whether MSCs are capable of in vitro activation. We analysed the presence of cytokines and three catabolism related biomarkers (aggrecan, cartilage oligomeric matrix protein (COMP) and hyaluronan (HA)) in knee synovial fluid (SF) samples and observed changes in their concentrations, which could indicate an effect of MSC intervention. Our results showed that the injected MSCs expressed an immunosuppressive phenotype and were capable of in vitro activation. We concluded that a matrix effect is likely to be present in the determination of aggrecan in SF samples and identified a potential disadvantage of the chosen method. HA concentration did not change significantly, whereas COMP concentration increased in most patients, which is contrary to our hypothesis. We attribute both findings to the small sample size of the patients, the potential heterogeneity of the samples and the complexity of KOA and MSC function. We detected and analysed significant levels of IL-8 and IL-6. IL-8 concentrations remained unchanged, suggesting a role for this cytokine in the early but not in the later stages of KOA. IL-6 concentrations decreased, suggesting an expected immunosuppressive effect of MSCs.

• Keywords: mesenchymal stromal stem cells, knee osteoarthritis, synovial fluid, immunomodulation, academic clinical study