Ovrednotenje signalne poti JAK-STAT in učinkov zaviralca JAK tofacitiniba v sinovijskih celičnih in tkivnih modelih revmatoidnega artritisa z analizo sekvenciranja RNA na nivoju posamičnih celic : magistrski študijski program Laboratorijska biomedicina

Ižanc, Nadja (avtor), Čučnik, Saša (mentor), Frank Bertoncelj, Mojca (komentor)

Revmatoidni artritis, kronična sistemska vnetna bolezen sklepov, vodi v invalidnost, zmanjšano kakovost življenja in prezgodnjo umrljivost. Tarčno bolezensko tkivo pri revmatoidnem artritisu je sinovijska membrana sklepov, h kroničnemu sinovijskemu vnetju in poškodbi sklepa pa ključno prispevata infiltracija imunskih celic ter aktivacija lokalnih stromalnih celic (npr. sinovijskih fibroblastov). Kljub obstoju učinkovitih zdravil (npr. zaviralec signalne poti JAK-STAT tofacitinib) številni bolniki ne dosežejo zadostnega odgovora na zdravljenje. Odkrivanje heterogenosti tarčnih tkiv in mehanizmov, s katerimi se za bolezen ključne celične populacije odzivajo na zdravila, lahko pomembno prispeva k razumevanju terapevtske neodzivnosti. Namen magistrske naloge je bil proučiti celično sestavo sinovijskega tkiva, identificirati sinovijske celične populacije, ki so ključne za signalizacijo JAK-STAT in poglobiti znanje učinkov tofacitiniba pri revmatoidnem artritisu. Pri tem smo uporabljali tri eksperimentalne sisteme: z ultrazvočno biopsijo pridobljeno sveže sinovijsko tkivo, v kulturi gojeno sinovijsko tkivo in iz sinovijskega tkiva izolirane gojene fibroblaste bolnikov z revmatoidnim artritisom. Celično sestavo sinovije, izražanje glavnih komponent poti JAK-STAT v celičnih populacijah in učinke tofacitiniba na sinovijske celice smo analizirali z uporabo merjenja izražanja RNA na nivoju posameznih celic. S tofacitinibom sprožene spremembe genskega in proteinskega izražanja v sinovijskih fibroblastih (stimuliranih z vnetnimi dejavniki, ki aktivirajo signalizacijo JAK-STAT) smo raziskali z encimsko imunsko metodo in kvantitativno verižno reakcijo s polimerazo. Analiza celične sestave sinovijskega tkiva 16 vzorcev je pokazala razporeditev 66.878 celic v glavne celične tipe (fibroblasti, endotelijske celice, makrofagi, dendritične celice, limfociti B in T, naravne celice ubijalke, celice žilne stene, periciti, mastociti in do sedaj z metodo sekvenciranja RNA na nivoju posameznih celic nedetektirane nevtrofilce). Pokazali smo, da pod našimi eksperimentalnimi pogoji tofacitinib ni bistveno vplival na celično sestavo in gensko ekspresijo 19.584 treh ex vivo gojenih sinovijskih tkiv. Sinovijski fibroblasti so močno izražali komponente signalne poti JAK-STAT, zlasti JAK1 in STAT3, kar kaže, da so te celice ena izmed glavnih tarč zaviralcev JAK. V in vitro celičnih kulturah sinovijskih fibroblastov smo dokazali, da je stimulacija z vnetnimi citokini vzpodbudila izražanje genov za IL-6, IL-8, ISG15, TNFAIP3, JUNB in SOCS3. Tofacitinib je zavrl od vnetja odvisno izražanje IL-6, ISG15, JUNB, SOCS3, ne pa tudi IL-8 in TNFAIP3. Izločanje IL-6 in IL-8 na proteinski ravni je bilo skladno z izsledki genske ekspresije.

• Jezik: Slovenski jezik
• Ključne besede: signalna pot JAK-STAT, tofacitinib, analiza sekvenciranja RNA na nivoju posameznih celic, sinovijska ovojnica, signalne poti JAK-STAT
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [N. Ižanc]
• Leto izida: 2021
• Št. strani: 75 f.
• PID: 20.500.12556/RUL-139444
• UDK: 616.72-002.77-085(043.3)
• COBISS.SI-ID: 80171523
• Datum objave v RUL: 02.09.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Single-cell RNA sequencing analysis of the JAK-STAT signaling pathway and its therapeutic inhibition with tofacitinib in synovial cell- and tissue-based models of rheumatoid arthritis

Izvleček

Rheumatoid arthritis, a chronic systemic inflammatory disease of the joints, leads to disability, reduced quality of life and premature mortality. The target tissue in rheumatoid arthritis is the synovial membrane of the joints, where immune cell infiltration and activation of local stromal cells (e.g. synovial fibroblasts) are key contributors to chronic synovial inflammation and joint damage. Despite the existence of powerful drugs (e.g. tofacitinib, an inhibitor of the JAK-STAT signaling), many patients do not achieve a sufficient response to treatment. Uncovering the heterogeneity of target tissues and the mechanisms by which disease-essential cell populations respond to drugs can make an important contribution to understanding the therapeutic non-responsiveness. The aim of this master thesis was to investigate the cellular composition of synovial tissue, to identify synovial cell populations critical for JAK-STAT signaling and to increase knowledge of the effects of tofacitinib in rheumatoid arthritis. We used three experimental systems: fresh synovial tissue obtained by ultrasound biopsy, cultured synovial tissue, and cultured fibroblasts isolated from synovial tissue from patients with rheumatoid arthritis. The cellular composition of the synovium, the expression of major components of the JAK-STAT pathway in synovial cell populations and the effects of tofacitinib on synovial cells were analyzed using single-cell RNA sequencing. Tofacitinib-induced changes in gene and protein expression in synovial fibroblasts (stimulated with inflammatory factors activating JAK-STAT signaling) were investigated with enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Cellular composition of 16 synovial tissues showed a distribution of 66,878 cells into major cell types (fibroblasts, endothelial cells, macrophages, dendritic and natural killer cells, B and T cells, mural and mast cells, pericytes and to date -with scRNA-sequencing - undetected neutrophils). We demonstrated that under our experimental conditions tofacitinib did not significantly affect the synovial cell composition and gene expression of 19,584 cells in 3 ex vivo cultured synovial tissue explants. Synovial fibroblasts strongly expressed components of the JAK-STAT signaling pathway, in particular JAK1 and STAT3, demonstrating that these cells represent important targets of JAK inhibitors. Inflammatory cytokine stimulation in vitro increased expression of IL-6, IL-8, ISG15, TNFAIP3, JUNB and SOCS3 in synovial fibroblasts. Inflammation-driven expression of IL-6, ISG15, JUNB and SOCS3 was tofacitib-dependent, whereas IL-8 and TNFAIP3 were not repressed with tofacitinib. IL-6 and IL-8 protein secretion was consistent with the gene expression changes in synovial fibroblasts.