Vrednotenje celičnih modelov za določanje sproščanja proinflamatornih citokinov

Kuzma, Jure (author), Mlinarič-Raščan, Irena (mentor), Markovič, Tijana (comentor)

S pojavom pandemije bolezni covid-19 je postalo izjemno aktualno raziskovanje zaviranja sindroma sproščanja citokinov oziroma citokinske nevihte. V magistrski nalogi smo se osredotočili na vzpostavitev in vrednotenje celičnih modelov prekomernega sproščanja citokinov. Ti omogočajo vrednotenje novih učinkovin in terapevtskih pristopov, s katerimi bi lahko zavirali ali znižali sproščanje vnetnih citokinov. Želeli smo vzpostaviti ustrezne in vitro celične modele prekomernega sproščanja citokinov, ki bi čim bolje posnemali pogoje in vivo. S tem namenom smo uporabili celične linije imunskih celic limfocitov T Jurkat, monocitov THP-1 in limfoblastoidne celične linije (LCL), ki so pridobljene iz limfocitov B zdravih darovalcev. Celične linije smo tretirali z izbranimi spojinami, ki bi lahko povišale sproščanje citokinov, in po 24 urah pomerili koncentracije izločenih klinično relevantnih citokinov IL-8, IL-1β, IL-6, IL-10, TNFα in IL-12p70 s pretočno citometrijo. Ugotovili smo, da je v celični liniji Jurkat aktivacija s kombinacijo spojin ionomicin/PMA povišala izločanje citokinov IL-8 in TNFα. Tretiranje celic THP-1 s kombinacijo spojin ionomicin/PMA/LPS je aktiviralo najvišje zvišanje izločanja citokinov IL-8, IL-6, IL-1β, TNFα ter IL-10. Tretiranje celic LCL s kombinacijo spojin ionomicin/PMA je aktiviralo povišano sproščanje citokinov IL-8, IL-6, TNFα in IL-12p70. Pred-tretiranje aktiviranih celic LCL 1823 z deksametazonom in ciklosporinom A je statistično značilno znižalo koncentracije vnetnih citokinov TNFα, IL-6, IL-8, s čimer smo potrdili ustreznost in vitro celičnega modela za vredntotenje imunomodulatornih učinkov novih spojin. V nadaljevanju smo in vitro model celic LCL uporabili za vrednotenje imunomodulatornih lastnosti zaviralcev konstititutivnega proteasoma (cP) in imunoproteasoma (iP). Proteasom ima pomembno vlogo pri regulaciji transkripcijskega dejavnika NF-κB, ki nadzira ekspresijo vnetnih citokinov. Zaviralci cP so delovali citotoksično pri nižjih koncentracijah kot zaviralci iP. Ugotovili smo, da je imelo šest od enajstih zaviralcev cP in iP statistično značilen vpliv na sproščanje citokinov. Na poročevalskih celicah Ramos-Blue smo potrdili, da zaviralci cP in iP zavirajo s TNFα aktivirano signalno pot NF-κB. Na podlagi rezultatov lahko zaključimo, da so celice LCL ustrezno vzpostavljen in vitro model prekomernega sproščanja citokinov. Ugotovili smo, da zaviralci iP delujejo manj citotoksično kot zaviralci cP. Za dokončno ovrednotenje imunomodulatornih lastnosti zaviralcev cP in iP bi bile smiselne nadaljne raziskave.

• Language: Slovenian
• Keywords: celični modeli, konstitutivni proteasom, imunoproteasom, citokinska nevihta, citokini, NF-κB, zaviralci proteasoma in imunoproteasoma
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-150945
• Publication date in RUL: 26.09.2023

Secondary language

• Language: English
• Title: Evaluation of cellular models for determining pro-inflammatory cytokines release

Abstract

With the emergence of the covid-19 pandemic, research into inhibiting the cytokine release syndrome or cytokine storm, has become extremely relevant. In the master's thesis, we focused on the establishment and evaluation of cellular models of excessive cytokine release. These enable the evaluation of new drugs and therapeutic approaches that could inhibit or reduce the release of proinflammatory cytokines. We aimed to establish suitable in vitro models of excessive cytokine release that would mimic in vivo conditions as closely as possible. For this purpose, we used cell lines representing immune cells, such as Jurkat for lymphocytes T, THP-1 for monocytes and lymphoblastoid cell lines (LCL), which are obtained from lymphocytes B of healthy donors. The cell lines were treated with selected compounds that could potentially increase the release of cytokines. We measured the concentrations of secreted clinically relevant cytokines IL-8, IL-1β, IL-6, IL-10, TNFα and IL-12p70 after 24 hours by flow cytometry. We found that activation of Jurkat cells with a combination of ionomycin/PMA increased the release of cytokines IL-8 and TNFα. Treatment of THP-1 cells with the combination of ionomycin/PMA/LPS activated the highest release of cytokines IL-8, IL-6, IL-1β, TNFα and IL-10. Treatment of LCL cells with the combination of ionomycin/PMA increased the release of IL-8, IL-6, TNFα and IL-12p70. Pre-treatment of activated LCL 1823 cells with dexamethasone and cyclosporin A statistically significantly reduced the concentrations of proinflammatory cytokines TNFα, IL-6, IL-8, thus confirming the suitability of the in vitro cell model for evaluation of the immunomodulatory effects of novel compounds. Next, LCL cells were used to evaluate the immunomodulatory properties of constitutive proteasome (cP) and immunoproteasome (iP) inhibitors. The proteasome has an important role in the regulation of the transcription factor NF-κB, which controls the expression of proinflammatory cytokines. We idetified cP inhibitors as more cytotoxic compared to iP inhibitors in tested immune cells. Moreover, six out of eleven cP and iP inhibitors statistically significantly decreasedthe release of cytokines. We confirmed that cP and iP inhibitors inhibit the TNFα-activated NF-κB signaling pathway on Ramos-Blue reporter cells. Based on the results, we can conclude that LCL cells are an suitable in vitro model of excessive cytokine release. We found that iP inhibitors are less cytotoxic than cP inhibitors. Further research would be worthwhile to evaluate the immunomodulatory properties of cP and iP inhibitors.

• Keywords: cell models, constitutive proteasome, immunoproteasome, cytokine storm, cytokines, NF-κB, proteasome and immunoproteasome inhibitors