| Abstract | Izvanstanični Hsp70 protein (eHsp70) pripada skupini molekularnih obrazaca oštećenja (DAMP), a može
aktivirati stanice imunosnog sustava vezujući se za TLR2 i TLR4 receptore te tako poticati sintezu upalnih
citokina. U bolesnika s kroničnom opstrukcijskom plućnom bolešću (KOPB), koja je karakterizirana kroničnom
upalom dišnih putova, izmjerene su povišene koncentracije eHsp70 u krvi. Glavna hipoteza ovog istraživanja je
da sam eHsp70 može aktivirati imunosni i upalni odgovor, ali da može i modulirati upalni odgovor izazvan
patogenim bakterijama ili dimom cigareta u staničnim modelima KOPB-a (NCI-H292, NHBE, PBEC (od
zdravih ispitanika i bolesnika s KOPB-om) i THP-1 stanice te monociti).
Istražen je utjecaj rekombinantnog humanog (rh) Hsp70 kada je prisutan sam ili zajedno s bakterijskim
komponentama LPS-om i LTA te ekstraktom dima cigareta (CSE) na upalni odgovor određivanjem
koncentracije upalnih citokina IL-1α, IL-1β, IL-6, IL-8 i TNF-α, kao i njihov utjecaj na vijabilnost i način
umiranja stanica MTS testom, određivanjem aktivnosti LDH te aktivnosti kaspaza-3/7, -8 i -9. Mehanizmi
djelovanja ovih molekula ispitani su određivanjem aktivacije TLR2 i TLR4, MAPK i NF-κB signalnih putova te
korištenjem specifičnih inhibitora MAPK, NF-κB, Hsp70, transkripcije i translacije.
rhHsp70 značajno potiče upalni odgovor u NCI-H292, PBEC i THP-1 stanicama te monocitima (pojačano
izlučivanje upalnih citokina). Može modulirati upalni odgovor na LPS, LTA i CSE tako da s LPS-om uglavnom
pokazuje sinergistički, a s LTA i CSE-om antagonistički tip interakcija. Sam rhHsp70 nema citotoksično
djelovanje, a uglavnom djeluje zaštitno naspram citotoksičnosti LPS-a i CSE-a. Ovisno o vrsti stanica rhHsp70
može potisnuti aktivnost izvršnih kaspaza-3/7. rhHsp70 može modulirati ekspresiju TLR2, TLR4 i
unutarstaničnog Hsp70. rhHsp70 djeluje preko MAPK i NF-κB signalnih putova koje aktivira ovisno o vrsti
stanica, koncentraciji i vremenu tretiranja.
Rezultati ovog istraživanja potvrdili su upalno djelovanje rhHsp70 te pokazali da on može modulirati upalni
odgovor na bakterijske komponente i CSE. Istovremena prisutnost eHsp70, LPS-a, LTA i/ili CSE može dovesti
do smanjenog djelovanja ili do pojačane aktivacije TLR2 i TLR4 receptora, što dovodi do pogoršanja upale i
nepovoljnih posljedica za bolesnike s KOPB-om. |
| Abstract (english) | Extracellular Hsp70 (eHsp70) can act as damage-associated molecular pattern (DAMP), activate immune cells
via Toll-like receptors (TLR) 2 and 4, and induce cytokine synthesis. Elevated levels of eHsp70 have been
measured in blood of patients with chronic obstructive pulmonary disease (COPD), which is characterised by
chronic airway inflammation. The main hypothesis of this research is that eHsp70 alone can cause immune and
inflammatory responses, but can also modulate inflammatory responses caused by pathogens or cigarette smoke
in cellular models of COPD (NCI-H292, NHBE, PBEC (isolated from healthy subjects and COPD patients) and
THP-1 cells and monocytes).
We explored the effects of recombinant human (rh) Hsp70 when present alone or together with bacterial
components LPS and LTA as well as cigarette smoke extract (CSE) on inflammatory response determined by the
levels of secreted pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8 and TNF-α). We also assessed cell
viability and mode of cell death by MTS assay, and by measurements of LDH activity secreted in cell
supernatants and cellular activities of caspases-3/7, -8 and -9. Mechanisms of action of aforementioned
molecules were explored by assessing TLR2, TLR4, MAPK and NF-κB signalling pathways activation, and by
using specific inhibitors of MAPK, NF-κB, Hsp70, transcription and translation.
rhHsp70 significantly induced inflammatory responses in NCI-H292, PBEC, THP-1cells and monocytes
(increased secretion of pro-inflammatory cytokines). It can modulate inflammatory response to LPS, LTA and
CSE, and has shown mainly synergistic type of interaction with LPS, and mainly antagonistic type of interaction
with LTA and CSE. rhHsp70 alone is not cytotoxic and mainly has protective effects against LPS and CSE
induced cytotoxicity. Depending on the cell type it can also reduce caspase-3/7 activities. rhHsp70 can modulate
the expression of TLR2, TLR4 and intracellular Hsp70. It exhibits its effects via MAPK and NF-κB signalling
pathways, but their activation is dependent on the type of cells, concentration and time.
Our results confirmed pro-inflammatory function of eHsp70 and showed that it can modulate inflammatory
response to bacterial components and CSE. Combined presence of eHsp70, LPS, LTA and/or CSE could lead to
desensitization or inappropriate activation of TLR2 and TLR4 receptors, which might contribute to the
aggravation of chronic inflammation and lead to worsening of COPD patients’ condition. |
