Imunska modulacija vnetne celične komponente v eksperimentalnem modelu postopka zunajtelesne fototerapije

Cajhen, Anja (author), Švajger, Urban (mentor)

Zunajtelesna fototerapija je celična imunoterapija, ki na imunski sistem deluje tako zaviralno kot spodbujevalno, kar omogoča njeno uporabo pri zdravljenju bolezni presadka proti gostitelju in kožnega T-celičnega limfoma. Postopek ECP se izvede v treh stopnjah: odvzem mononuklearnih celic z aferezo, obdelava zbranih celic z 8-metoksipsoralenom in UVA svetlobo, ki sproži apoptozo celic ter ponovno injiciranje obdelanih celic v pacientov krvni obtok. Tako obdelane celice v postopku zunajtelesne fototerapije predstavljajo zdravilo, natančneje napredno celično zdravilo. Med pripravo celičnega zdravila oz. celičnega produkta se ustvarijo imunozaviralne in imunospodbujevalne oz. vnetne celične komponente. Slednje predstavljajo med postopkom zunajtelesne fototerapije aktivirani monociti, ki v telesu kot zrele dendritične celice inducirajo nastanek efektorskih imunskih odzivov. Naš namen je bil razviti standardiziran protokol za in vitro tretiranje celic s spojinami, ki inhibirajo aktivacijo monocitov, in s tem narediti prvi korak k optimizaciji zunajtelesne fototerapije za zdravljenje bolezni presadka proti gostitelju. Vrednotili smo učinek deksametazona na diferenciacijo in zorenje monocitov v kulturi MNC, izoliranih iz krvi prostovoljnih zdravih krvodajalcev. Mononuklearne celice smo z 28 do 558 µg/ml deksametazona tretirali 24 oz. 48 ur. Nato smo jih 24 ur aktivirali z različnimi stimulansi: LPS (1 µl/ml), LPS (1 µl/ml) + IFNγ (0,5 µl/ml), mCD40L (2 µl/ml), mCD40L (2 µl/ml) + IFNγ (0,5 µl/ml) in R848 (1 µl/ml). Kot ustrezna stimulansa za uporabo v našem eksperimentalnem modelu sta se izkazala le LPS+IFNγ in R848, saj z ostalimi nismo pridobili ustrezne pozitivne kontrole. Celice, tretirane z deksametazonom, so izražale nižjo raven ko-stimulacijskih molekul CD40, CD80, CD83 in CD86 kot netretirane celice. Do upada izražanja ko-stimulacijskih molekul na tretiranih monocitih v primerjavi z netretiranimi monociti je prišlo tudi po aktivaciji z LPS+IFNγ. Učinka deksametazona na izražanje inhibitornih molekul ILT-3, ILT-4 in PD-L1 zaradi slabe ponovljivosti nismo uspeli ugotoviti. Po aktivaciji z LPS+IFNγ ali R848 se je zmanjšala kapaciteta izločanja vnetnih citokinov IL-8, IL-1β, IL-6, TNFα, IL-12p70 in protivnetnega citokina IL-10 iz monocitov tretiranih z deksametazonom. Rezultati naših poskusov torej kažejo, da dekametazon delno inhibira diferenciacijo in zorenje monocitov.

• Language: Slovenian
• Keywords: deksametazon, monociti, ko-stimulacijske molekule, zunajtelesna fototerapija, bolezen presadka proti gostitelju
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-106186
• Publication date in RUL: 07.02.2019

Secondary language

• Language: English
• Title: Immune modulation of proinflammatory cell component in experimental model of extracorporeal phototherapy

Abstract

Extracorporeal phototherapy is a cell immunotherapy that has inhibitory and stimulatory effects which enables its use in the treatment of graft versus host disease as well as cutaneous T-cell lymphoma. The process is performed in three stages: leukapheresis, photoactivation of collected cells with 8‐methoxypsoralen (8‐MOP)/UVA which later triggers apoptosis of cells and re‐infusion of treated cells into the patients' bloodstream. The cells thus treated therefore constitute a medicinal product or, more precisely advanced therapy medicinal product. During the preparation of the cell product imunosupresive and imunostimulatory cell components are generated. Monocytes activated during the extracorporeal phototherapy induce efector immune responses in the patient’s organism and therefore represent the immunostimulatory component of the cell product. Our purpose was to develop a standardized protocol for the in vitro treatment of cells with compounds that inhibit monocyte activation and thereby to make the first step towards the optimization of the extracorporeal phototherapy for treatment of graft versus host disease. We studied the effect of dexamethason on differentiation and maturation of human monocytes in a culture of mononuclear cells isolated from the blood of healty blood donors. We treated mononuclear cells with 28 to 558 µg/ml dexamethason for 24 or 48 hours. Then we activated them with stimulants: LPS (1 µl/ml) + IFNγ (0,5 µl/ml), mCD40L (2 µl/ml), mCD40L (2 µl/ml) + IFNγ (0,5 µl/ml) and R848 (1 µl/ml). Only LPS+IFNγ and R848 proved to be appropriate stimulants for use in our experimental model, as with the rest we haven't acquired the relevant positive control. Cells treated with dexamethason expressed lower levels of co-stimulatory molecules CD40, CD80, CD83 and CD86 as untreated cells. Treated cells expressed lower levels of co-stimulatory molecules than untreated cells even after exposure to LPS+IFNγ. We were unable to evaluate the effect of dexamethason on the expression of inhibitory molecules ILT-3, ILT-4 and PD-L1 due to the lack of repeatability. Dex decreased the capacity of treated monocytes for excretion of inflammatory cytokines (IL-8, IL-1β, IL-6, TNFα, IL-12p70) as well as anti-inflammatory cytokine IL-10 after activation with LPS+IFNy or R848. In conclusion, our data suggest that dexamethason partially inhibits differentiation and maturation of monocytes.

• Keywords: dexamethason, monocytes, co-stimulatory molecules, extracorporeal phototherapy, graft versus host disease