Određivanje aktivnosti upalnih i apoptoznih kaspaza u THP-1 stanicama i monocitima

master's thesis

• Title (english): Quantification of activity of inflammatory and apoptotic caspases in THP-1 cells and monocytes
• Author: Lucija Franin
• Mentor: Lada Rumora (mentor)
• Committee member: Lada Rumora (predsjednik povjerenstva)
• Committee member: Andrea Hulina (član povjerenstva)
• Committee member: Sandra Šupraha Goreta (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2018-06-06, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Izvanstanični protein Hsp70 djeluje kao molekularni obrazac oštećenja (DAMP, engl. damage-associated molecular pattern) preko receptora sličnog Tollu TLR2 i TLR4 te može uzrokovati upalni odgovor. Povišen je u krvi bolesnika s kroničnom opstrukcijskom plućnom bolesti. Patogeneza te bolesti uključuje kroničnu upalu i oštećenje tkiva, no nije potpuno razjašnjena. Cilj rada bio je ispitati djelovanje različitih koncentracija izvanstaničnog proteina Hsp70 te lipopolisaharida (LPS) i lipoteikoične kiseline (LTA) preko receptora TLR2 i TLR4 na diferencirane monocite iz periferne krvi zdravih donora i na THP-1 stanice, te utvrditi uzrokuju li aktivaciju inflamasoma i programirane stanične smrti (apoptoze). Prisutnost upale i aktivacije inflamasoma ispitani su mjerenjem aktivnosti kaspaze-1 u stanicama, a prisutnost apoptoze mjerenjem aktivnosti kaspaza-3/7, -8 i -9. Istraživanje smo proveli na THP-1 stanicama koje smo prethodno diferencirali u makrofage pomoću forbol 12-miristat-13-acetata (PMA) te na monocitima iz periferne krvi zdravih donora koje smo diferencirali pomoću čimbenika koji stimulira rast makrofagnih kolonija (M-CSF). Stanice su tretirane rekombinantnim humanim (rh) Hsp70 proteinom u koncentracijama od 0,3, 1, 3 i 10 μg/mL, s 0,1 μg/mL LPS-a te 1 μg/mL LTA tijekom 2, 4, 6 i 8 sati. Aktivnosti kaspaza u stanicama izmjerene su luminometrijskim testom. Rezultati su pokazali da tretiranje LPS-om i LTA u THP-1 stanicama dovodi do aktivacije kaspaze-1. LPS potiče i aktivaciju kaspaza-3/7 te kaspaze-8 u THP-1 stanicama. Kod monocita, LPS i LTA potiskuju aktivnost kaspaze-1, a LTA uzrokuje i smanjenje aktivnosti kaspaze-9. rhHsp70 primijenjen u nižim koncentracijama i nakon kraćeg vremena inkubacije potiskuje aktivnost kaspaze-1 u THP-1 stanicama, a pri višim koncentracijama i nakon duljeg vremena inkubacije stimulira njenu aktivnost. Nakon 8 sati inkubacije rhHsp70 uzrokuje i smanjenje aktivnosti kaspaza-3/7 u ovim stanicama. Kod monocita, tretiranje s rhHsp70 potiče aktivnost kaspaze-1, dok visoke koncentracije rhHsp70 snižavaju aktivnost kaspaze-9. Zaključno, LPS i LTA uzrokuju aktivaciju inflamasoma, a LPS aktivira i apoptozne kaspaze-3/7 i -8 u THP-1 stanicama, dok u monocitima LPS i LTA ne dovode do aktivacije inflamasoma i apoptoze. rhHsp70 djeluje protu-upalno u nižim koncentracijama, a u višim koncentracijama uzrokuje aktivaciju inflamasoma u THP-1 stanicama, dok na monocite djeluje upalno i protu-apoptozno.

Abstract (english)

Extracellular Hsp70 protein acts as a damage-associated molecular pattern (DAMP) and can activate Toll-like receptors 2 and 4 (TLR2 and TLR4) and cause an immune response. It is elevated in blood of patients with chronic obstructive pulmonary disease. Its pathogenesis involves chronic inflammation and tissue damage, but it is not yet completely understood. Aim of this study was to examine the effect of different concentrations of extracellular Hsp70, lipopolysaccharide (LPS) and lipoteichoic acid (LTA) via TLR2 and TLR4 receptors on differentiated monocytes from peripheral blood of healthy donors and on THP-1 cells, and to determine if they cause activation of inflammasome and programmed cell death (apoptosis). Presence of inflammation and inflammasome activation is examined by measuring caspase-1 activity in cells, and presence of apoptosis by measuring caspase-3/7, -8 and -9 activities. We did our study on THP-1 cells which we had previously differentiated to macrophages with phorbol 12-myristate-13-acetate (PMA) and on monocytes from peripheral blood of healthy donors which we had differentiated with macrophage-colony stimulating factor (M-CSF). Cells were treated with recombinant human (rh) Hsp70 protein in concentrations of 0,3, 1, 3 and 10 μg/mL, with 0,1 μg/mL of LPS and with 1 μg/mL of LTA during 2, 4, 6 and 8 hours. Caspase activities in cells were measured with luminometric test. Results showed that treatments with LPS and LTA in THP-1 cells stimulate caspase-1 activation. LPS also causes activation of caspases-3/7 and caspase-8 in THP-1 cells. In monocytes LPS and LTA reduce caspase-1 activity and LTA additionaly causes reduction of caspase-9 activity. Low concentrations of rhHsp70 applied for a short period of time decrease caspase-1 activity in THP-1 cells, while high concentrations and longer incubation time increase activity. After 8 hours of incubation, rhHsp70 also decreases caspases-3/7 activity. In monocytes, treatment with rhHsp70 induces caspase-1 activity, while high concetrations of rhHsp70 lower caspase-9 activity. In conclusion, LPS and LTA trigger inflammasome activation, and LPS also induces activity of caspase-3/7 and -8 in THP-1 cells. However, LPS and LTA don't cause activation of inflammasome and apoptosis in monocytes. rhHsp70 has anti-inflammatory effect in low concentrations, but in high concetrations it causes inflammasome activation in THP-1 cells, while in monocytes it has pro-inflammatory and anti-apoptotic effect.

• Keywords: izvanstanični Hsp70; LPS; LTA; kaspaze; THP-1 stanice; monociti
• Keywords (english): extracellular Hsp70; LPS; LTA; caspases; THP-1 cells; monocytes
• Language: croatian
• URN:NBN: urn:nbn:hr:163:102213
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije (magistar/magistra medicinske biokemije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2019-03-06 13:25:39