Utjecaj izvanstaničnog proteina Hsp70 i cigaretnog dima na aktivaciju inflamasoma, nekroptoze i piroptoze u staničnim modelima kronične opstrukcijske plućne bolesti

master's thesis

• Title (english): Effect of extracellular protein Hsp70 and cigarette smoke on the inflammasome, necroptosis and pyroptosis activation in cellular models of chronic obstructive pulmonary disease
• Author: Marija Fabijanec
• Mentor: Lada Rumora (mentor)
• Committee member: Lada Rumora (predsjednik povjerenstva)
• Committee member: Marija Grdić Rajković (član povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of medical biochemistry and haematology); Zagreb
• Defense date and country: 2020-09-04, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Kronična opstrukcijska plućna bolest (KOPB) složena je bolest čija patogeneza uključuje kroničnu upalu, a povezana je s dugotrajnom izloženošću štetnim česticama, posebice cigaretnom dimu. U krvi bolesnika s KOPB-om izmjerene su povišene koncentracije izvanstaničnog Hsp70 (eHsp70) koji djeluje imunomodulacijski. Cilj ovog diplomskog rada bio je ispitati potiču li ekstrakt cigaretnog dima (CSE), eHsp70 te njihove kombinacije aktivaciju NLRP3 inflamasoma te izazivaju li piroptozu i nekroptozu. Oponašanje imunomodulacijskog djelovanja eHsp70 postignuto je korištenjem rekombinantnog humanog Hsp70 proteina (rhHsp70). Provodila su se tretiranja in vitro modela KOPB-a, a to su NCI-H292, THP-1, NHBE i MDM stanice, s rhHsp70 u koncentracijama 0,3, 1 i 3 μg/mL, 2,5, 5 i 15 %-tnim CSE-om te njihovim kombinacijama tijekom 24 sata. Aktivacija NLRP3 inflamasoma i upalni odgovor ispitani su određivanjem ekspresije mRNA za NLRP3 i IL-1β qPCR-om te određivanjem koncentracije izlučenog IL-1β. Vijabilnost stanica ispitana je određivanjem aktivnosti laktat-dehidrogenaze (LDH) izlučene u medij iznad stanica, a njihov način umiranja ispitan je određivanjem aktivacije i ekspresije RIPK3 i MLKL-a (nekroptoza) te ekspresije GSDMD-a (piroptoza) Western blot analizom nakon tretiranja tijekom 2 i 8 h te određivanjem koncentracije IL-1β (piroptoza). Dokazano je da je ekspresija gena za NLRP3 i IL-1β, kao i izlučivanje IL-1ß, u svim stanicama značajno povećana nakon primijenjenih pojedinačnih tretiranja, što upućuje na aktivaciju inflamasoma i upalu. Kombinirana tretiranja bila su stimulacijska u MDM stanicama, inhibicijski učinak imala su na NCI-H292 i NHBE stanice, a u THP-1 stanicama nisu imala učinak u odnosu na pojedinačna tretiranja. Litička stanična smrt dokazana je u staničnim linijama (NCI-H292 i THP-1), ali ne i u primarnim stanicama (NHBE i MDM). Promjena u ekspresiji GSDMD-a ili aktivacija RIPK3 i MLKL-a nije dokazana u MDM stanicama. Ekspresija GSDMD-a smanjena je u NCI-H292 i THP-1 stanicama, ali nije detektiran GSDMD-NT. U NHBE stanicama detektirani su aktivirani, odnosno fosforilirani oblici RIPK3 i MLKL-a što ukazuje na nekroptozu ili alternativni put aktivacije NLRP3 inflamasoma za koji se pretpostavlja da bi mogao biti uključen u mnoga sterilna upalna stanja, primjerice kod stabilne faze KOPB-a.

Abstract (english)

Chronic obstructive pulmonary disease (COPD) is a complex disease whose pathogenesis includes chronic inflammation and is associated with prolonged exposure to harmful particles, especially cigarette smoke. Elevated concentrations of extracellular Hsp70 (eHsp70), which has an immunomodulatory effect, were measured in the blood of patients with COPD. The aim of this study was to examine whether cigarette smoke extract (CSE), eHsp70 and their combinations stimulate the activation of NLRP3 inflammasome and whether they cause pyroptosis and necroptosis, thus contributing to the propagation of inflammation. rhHsp70 protein was used to mimic immunomodulatory action of eHsp70. In vitro models of COPD, and those are NCI-H292, THP-1, NHBE, and MDM cells, were treated for 24 hours with rhHsp70 at concentrations of 0.3, 1, and 3 μg/mL, 2.5, 5 and 15 % CSE and their combinations. Activation of NLRP3 inflammasome and inflammatory response were examined by determining mRNA expression for NLRP3 and IL-1β by quantitative PCR, and by determining the concentration of secreted IL-1β. Cell viability was determined by measuring LDH activity in the media above the cells and their mode of dying was explored by determining the activation and expression of RIPK3 and MLKL (necroptosis) and GSDMD expression (pyroptosis) by Western blot analysis after 2 and 8 h treatments, as well as the already mentioned IL-1β concentration (pyroptosis). It has been shown that gene expression for NLRP3 and IL-1β, as well as IL-1β secretion, were significantly increased in all cells after individual treatments, indicating inflammasome activation and inflammation. Combination treatments were stimulatory in MDM cells, had an inhibitory effect on NCI-H292 and NHBE cells, and had no effect on THP-1 cells in comparison to individual treatments. Lytic cell death has been observed in cell lines (NCI-H292 and THP-1) but not in primary cells (NHBE and MDM). A change in GSDMD expression or activation of RIPK3 and MLKL has not been demonstrated in MDM cells. GSDMD expression was reduced in NCI-H292 and THP-1 cells, but GSDMD-NT was not detected. Activated or phosphorylated forms of RIPK3 and MLKL were detected in NHBE cells, indicating necroptosis or an alternative activation of NLRP3 inflammasome that is thought to be involved in many sterile inflammatory conditions, such as the stable phase of COPD.

• Keywords: kronična opstrukcijska plućna bolest; KOPB; eHsp70; cigaretni dim; inflamasom; nekroptoza; piroptoza
• Keywords (english): chronic obstructive pulmonary disease; COPD; eHsp70; cigarette smoke; inflammasome; necroptosis; pyroptosis
• Language: croatian
• URN:NBN: urn:nbn:hr:163:603840
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije i laboratorijske medicine (magistar/magistra medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-04-27 15:07:03