Title MUNOLOŠKA ULOGA PROTEINA PrPC TIJEKOM
VIRUSNE INFEKCIJE Title (english) IMMUNE ROLE OF PrPC PROTEIN DURING VIRAL
INFECTION Author Dubravka Karner Mentor Tihana Lenac Roviš (mentor)Committee member Gordana Blagojević Zagorac (predsjednik povjerenstva)Committee member Mario Habek (član povjerenstva)Committee member Kristina Pilipović (član povjerenstva)Granter University of Rijeka Defense date and country 2024-05-27, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract Cilj istraživanja: Stanični prionski protein, PrP, mali je glikoprotein na površini stanica
poznat prvenstveno po svojoj ključnoj ulozi u patogenezi neurodegenerativnih poremećaja
poznatih kao prionske bolesti. Sve veći broj istraživanja ukazuje na ulogu proteina PrP u
moduliranju imunosnog sustava, posebno u utišavanju imunosnog odgovora u tkivima poput
mozga, gdje izražena upala može prouzročiti značajna oštećenja. PrP nije samo prisutan na
površini imunosnih stanica, već se može vezati na njih i modulirati njihovu funkciju, što
ukazuje na njegovo prepoznavanje od strane imunosnih receptora. U slučaju prirođene
infekcije citomegalovirusom (CMV-om), pojačana aktivacija imunosnog odgovora može
uzrokovati ozbiljna neurološka oštećenja. Stoga je glavni cilj ovog istraživanja odrediti
imunosnu funkciju proteina PrP u modelu prirođene infekcije mišjim CMV-om (MCMV-om).
Materijali i metode: Za istraživanje utjecaja infekcije MCMV-om na biologiju proteina PrP,
primijenili smo protočnu citometriju, konfokalnu mikroskopiju i metodu imunoblota za analizu
razine PrP-a u inficiranim stanicama te identificirali mehanizam odstranjivanja proteina PrP iz
stanica inficiranih MCMV-om korištenjem inhibitora i detekcije proteina PrP. U modelu
prirođene infekcije MCMV-om kod novookoćenih miševa divljeg tipa i miševa kojima
nedostaje PrP, uklanjanjem populacije limfocita identificirali smo stanice odgovorne za utjecaj
PrP-a na tijek infekcije. Karakterizirali smo te stanice fenotipski i funkcionalno protočnom
citometrijom i sekvenciranjem RNK na razini jedne stanice. Za analizu ispoljavanja topivih
imunosnih medijatora u uzorcima tkiva nakon prirođene infekcije MCMV-om, proveli smo
multipleks analizu citokina i kemokina. Također smo proveli funkcionalne testove proizvodnje
proupalnih citokina kako bismo istražili utjecaj slobodnog proteina PrP na efektorsku funkciju
imunosnih stanica te istražili utjecaj prisustva proteina PrP na ishod imunopatologije mozga.
Rezultati: CMV utječe na ispoljavanje proteina PrP, prvenstveno potičući njegov pojačan
izražaj, pri čemu ujedno dolazi do odcjepljivanja sa stanične membrane pod utjecajem proteaze
ADAM10 i uklanjanja PrP-a iz stanica. Slobodni protein PrP se veže na limfocite CD8 T kod
novookoćenih miševa inficiranih MCMV-om, smanjujući njihovu sposobnost proizvodnje
proupalnih citokina. U miševa kojima nedostaje protein PrP, limfociti CD8 T pokazuju
povećanu aktivnost, uključujući veći broj virus-specifičnih stanica, povećanu citotoksičnost i
izlučivanje citokina. Ovi miševi su pokazali poboljšanu sposobnost uklanjanja virusa bez
izazivanja povećane imunopatologije.
Zaključak: Protein PrP modulira imunosni odgovor tijekom prirođene infekcije CMV-om,
umanjujući aktivnost imunosnih stanica CD8 T i posljedično uklanjanja virusa.
Abstract (english) Objectives: The cellular prion protein, PrP, is a small glycoprotein on the cell surface primarily
known for its critical role in the pathogenesis of neurodegenerative prion diseases. An
increasing body of research points to the role of PrP in modulating the immune system,
particularly in dampening immune responses in tissues such as the brain, where severe
inflammation can cause significant damage. PrP is not only present on the surface of immune
cells but also binds to them and modulates their function, implying its recognition by immune
receptors. In the case of congenital cytomegalovirus (CMV) infection, enhanced activation of
the immune response can lead to serious neurological damage. Therefore, the main goal of this
research is to determine the immune function of PrP protein in a model of congenital infection
with murine CMV (MCMV).
Materials and methods: To investigate the impact of MCMV infection on the biology of the
PrP protein, we used flow cytometry, confocal microscopy and immunoblot to analyze the
levels of PrP in infected cells and identified the mechanism of PrP protein cleavage by detecting
PrP in MCMV infected cells treated by inhibitors. In a model of MCMV congenital infection
in newborn wild-type mice and mice lacking PrP, by depleting the lymphocyte population, we
identified the cells responsible for the influence of PrP on the course of the infection. We
characterized these cells phenotypically and functionally by flow cytometry and single cell
RNA sequencing. To analyze the expression of soluble immune mediators in tissue samples
after congenital MCMV infection, we used a multiplex assay of cytokines and chemokines. We
also conducted functional assay of pro-inflammatory cytokine production to investigate the
influence of cleaved PrP protein on the effector function of immune cells and investigated the
influence of the PrP protein on the outcome of brain immunopathology.
Results: CMV affects the expression of the PrP protein primarily by promoting its increased
expression, while simultaneously causing its removal from the cell membrane through cleavage
by the ADAM10 protease. Cleaved PrP protein binds to CD8 T lymphocytes in neonatal mice
infected with MCMV, reducing their ability to produce pro-inflammatory cytokines. In mice
lacking the PrP protein, CD8 T lymphocytes show increased activity, including a higher
number of virus-specific cells, increased cytotoxicity, and cytokine secretion. These mice
showed an improved ability to clear the virus without causing excessive immunopathology.
Conclusion: The protein PrP modulates the immune response during congenital CMV
infection, reducing the activity of CD8 T immune cells and consequently clearing the virus.
Keywords
Citomegalovirus
mišji
Imunosni odgovor
Prionski protein
Keywords (english)
Cytomegalovirus
Mouse
Immune response
Prion Protein.
Language croatian URN:NBN urn:nbn:hr:184:755747 Promotion 2025 Project Number: IP-2020-02-6617 Project Number: uniri-biomed-18-233 Project Number: KK.01.1.1.01.0006 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2025-04-23 06:30:54
