Imunoreakcije uzrokovane infekcijom virusima Hantaan i Andes u MRC-5 i HEK 293 stanicama

Cebalo, Ljiljana (2007) Imunoreakcije uzrokovane infekcijom virusima Hantaan i Andes u MRC-5 i HEK 293 stanicama. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Hantaviruses (HTV), genus Hantavirus, family Bunyaviridae are rodent-borne viruses that are associated with two main clinical syndromes: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Little is known about the immunopathogenesis in HTV infections, and especially why some HTV cause mainly kidney disorders, while others mostly affect the lungs. The aim of this study was to analyze some non-specific imunoreactions regulated by two viruses; Hantaan virus (HFRS) and Andes virus (HPS) in two different cell types: human lung fibroblasts (MRC-5 cells) and human epithelial kidney cells (HEK 293 cells). In lung fibroblasts we analyzed the gene expression for different cytokines/chemokines; extracellular matrix and adhesion molecules (ECM) and molecules involved in NF-kB signal transductional pathway in the early phase of infection (six hours). In HEK 293 cells we analyzed the genes for different ECM molecules and molecules that take part in different apoptosis pathways in two time points during infection (3 and 5 days). GEArray method was used to compare gene expression profiles of different RNA samples (GEArrays, SuperArray Bioscience, Frederick, MD, USA). Selected genes identified by GEArray analysis were also analyzed by RT-PCR to verify transcriptional responses. Both viruses increased gene expression for the members of Rel/NFkB/IkB family, NF-kB responsive genes, transcription factors and adaptor proteins. These changes suggest a possible role for NF-kB pathway in the early immune response against Hantaan and Andes viral infections. We shown for the first time that pathogenic hantaviruses induce changes in the levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). As MMPs and TIMPs are critical modulators of ECM remodeling, the deteriorated homeostasis found could have a role during immunoreactions to hantaviral infections. We confirmed the considerable interactions of tested viruses and integrins, and their effect to different cytokines. Furthermore, we identified the influence of both tested viruses on different caspases, molecules that belong to Bcl-2 family and other factors which participate in death program associated with endoplasmic reticulum and mitochondria. Taken together, these results indicate that inner apoptotic pathway have an important role in hantaviral infections. Our findings may direct future studies which should explain the mechanisms behind the immunopathogenesis of kidney and lung disorders during HFRS/HPS.

Abstract in Croatian

Hantavirusi (HTV), rod Hantavirus, obitelj Bunyaviridae prenose se s mišolikih glodavaca na ljude te u čovjeka uzrokuju dvije različite bolesti: hemoragijsku vrućicu s bubrežnim sindromom (HVBS) te hantavirusni plućni sindrom (HPS). Vrlo se malo zna o imunopatogenezi hantavirusnih infekcija, te osobito o tome zašto određeni hantavirusi uzrokuju pretežito bubrežne promjene, dok drugi poglavito zahvaćaju pluća. Cilj ovoga rada bio je istražiti određene nespecifične imunološke reakcije uzrokovane infekcijom virusom Hantaan (uzročnik HVBS-a) i virusom Andes (uzročnik HPS-a). Promjene su praćene u dvije različite vrste stanica: ljudskim plućnim fibroblastima (MRC-5 stanice) i ljudskim bubrežnim epitelnim stanicama (HEK 293 stanice). Kod infekcije plućnih fibroblasta analizirali smo ekspresiju gena za različite citokine/kemokine, adhezijske i molekule izvanstaničnog matriksa te gene uključene u NF-kB transkripcijski put u ranoj fazi infekcije (šest sati). Kod infekcije HEK 293 stanica analizirali smo gene uključene u različite puteve apoptoze te promjene u adhezijskim i molekulama izvanstaničnog matriksa u dvije vremenske točke tijekom infekcije (3 i 5 dana). Kako bi odredili gensku ekspresiju testiranih uzoraka RNK koristili smo GEArray metodu (GEArrays, SuperArray Bioscience, Frederick, MD, SAD). Određeni geni ili obitelji gena kod kojih smo našli značajnu promjenu GEArray metodom također smo testirali metodom RT-PCR. Oba virusa utjecala su na gensku ekspresiju kod članova Rel/NFkB/IkB obitelji, molekula koje su modulirane preko NF-kB puta, transkripcijskih čimbenika te adaptornih proteina. Ove promjene mogu govoriti u prilog našoj hipotezi da tijekom rane faze hantavirusnih infekcija dolazi do aktivacije NF-kB transkripcijskog puta. Po prvi puta smo pokazali utjecaj virusa Hantaan i Andes na ekspresiju matriks metaloproteinaza i njihovih tkivnih inhibitora. Budući da ove proteaze imaju glavnu ulogu u razgradnji izvanstaničnog matriksa, njihova neravnoteža može znatno doprinijeti razvoju patoloških promjena. Nadalje, potvrdili smo značajnu interakciju hantavirusa i integrina te utjecaj na ekspresiju različitih citokina. Značajne promjene tijekom infekcije virusima Hantaan i Andes primijetili smo kod pripadnika Bcl-2 obitelji te molekula koje mogu sudjelovati u procesima apoptoze pokrenutim putem endoplazmatskog retikuluma i mitohondrija. Ovi rezultati upućuju da unutarnji put apoptoze ima ključnu ulogu pri infekciji patogenim hantavirusima. Ovi rezultati mogli bi usmjeriti buduća istraživanja koja bi trebala objasniti imunopatogenetske mehanizme koji stoje u pozadini bubrežnih i plućnih promjena koje se javljaju tijekom hantavirusnih infekcija.

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