Proučevanje prirojenega imunskega odziva po okužbi z virusom Dobrava in virusom Puumala

V Sloveniji hemoragično mrzlico z renalnim sindromom (HMRS) povzročajo hantavirusi Dobrava (DOB), Puumala (PUU) in Dobrava-Kurkino. Potek bolezni je lahko zelo različen, od blage oblike bolezni, do bolezni s težkim potekom, ki se lahko konča tudi s smrtnim izidom. Dosedanje raziskave kažejo na to, da ima pri okužbi s hantavirusi pomembno vlogo odgovor prirojenega imunskega sistema gostitelja. Pokazali so, da patogeni hantavirusi zavirajo zgodnji odziv interferonov tipa I (IFN&#945 in IFN&#946 ), stopnja zaviranja IFN pa je odvisna od stopnje patogenosti hantavirusa. Utišanje prirojenega imunskega odziva posledično privede do hitrega in nenadzorovanega, sistemskega razsoja okužbe. V dostopni literaturi še ni opisanih razlik v prirojenem imunskem odzivu po okužbi z virusom DOB in virusom PUU. V naši raziskavi smo želeli ugotoviti ali obstajajo razlike v prirojenem imunskem odzivu mononuklearnih celic iz periferne krvi (PBMC) pridobljenih iz kliničnih vzorcev bolnikov po okužbi z virusom DOB in virusom PUU ter njihov pomen v patogenezi HMRS. Doktorsko nalogo smo razdelili na tri dele. V prvem delu raziskave smo želeli ugotoviti, ali obstajajo razlike v odzivu IFN tipa I pri PBMC po stimulaciji z virusom DOB in virusom PUU ter ali razlike v odzivu IFN tipa I vplivajo na različen potek bolezni. Z virusom DOB in virusom PUU smo stimulirali PBMC zdravih prostovoljcev in PBMC bolnikov z znanim potekom HMRS. Ugotovili smo, da virus PUU, ne pa virus DOB, stimulira odziv IFN tip I, saj je bilo izražanje genov IFN&#946 , STAT-1 in MxA 48 ur po stimulaciji statistično značilno večje kot pri nestimuliranih celicah. Z rezultati raziskave smo potrdili postavljeno hipotezo, da je stopnja odziva IFN tipa I po stimulaciji PBMC z virusom DOB nižja kot po stimulaciji z virusom PUU, kar posledično lahko pripomore k težji klinični sliki HMRS po okužbi z virusom DOB. S stimulacijo PBMC iz vzorcev bolnikov po preboleli HMRS smo ugotovili, da je izražanje gena MxA pri PBMC bolnikov, ki so imeli blažji potek okužbe z virusom PUU statistično značilno višje, kot pri PBMC bolnikov, ki so imeli težji potek okužbe. Iz rezultatov naše raziskave sklepamo, da večje protivirusno delovanje, ki smo ga potrdili z višjim izražanjem gena MxA, pripomore k blažjemu poteku okužbe z virusom PUU. Odziv PBMC pridobljenih iz akutnih vzorcev bolnikov smo primerjali z odzivom PBMC, ki smo jih pridobili iz vzorcev bolnikov po preboleli HMRS, saj nas je zanimala vloga imunskega spomina. Predpostavljali smo, da bo odziv IFN tipa I je bolj zavrt v PBMC med akutno okužbo kot v PBMC po preboleli okužbi. Statistično značilne razlike so se pokazale med PBMC bolnikov, ki so imeli težji potek okužbe z virusom PUU. Ugotovili smo, da je izražanje gena IFN&#946 statistično značilno nižje, izražanje gena MxA pa statistično značilno višje pri akutnih PBMC, kot pri PBMC po preboleli okužbi. Iz rezultatov sklepamo, da so PBMC pridobljeni v akutni fazi HMRS bolj odzivni na stimulacijo z virusom PUU, kot PBMC pridobljeni po preboleli bolezni. Menimo, da odziv IFN tipa I pri PBMC ne prispeva k zaščiti pred ponovno okužbo z virusom DOB in virusom PUU, saj je del prirojenega imunskega sistema, ki na patogene deluje z nespecifičnimi odgovori. V drugem delu doktorske naloge smo s pretočno citometrijo primerjali profil PBMC bolnikov s HMRS. Želeli smo ugotoviti, ali okužba z virusom DOB in virusom PUU povzročita različen odziv imunskih celic. Zanimalo nas je tudi, ali so prisotne razlike v limfocitnih populacijah povezane s potekom HMRS. Ugotovili smo, da imajo bolniki okuženi z virusom DOB statistično značilno večji delež celic NK, kot bolniki okuženi z virusom PUU. Prav tako smo potrdili večji delež celic NK pri bolnikih okuženih z virusom PUU, ki so imeli težji potek bolezni, kot pri bolnikih z blažjim potekom okužbe. Dodatno smo ugotovili, da imajo bolniki s težjim potekom okužbe z virusom PUU, višjo koncentracijo citotoksičnih celic NK CD56dim. Iz rezultatov sklepamo, da so celice NK povezane s težjim potekom HMRS. Še posebno so s težjim potekom okužbe z virusom PUU povezane citotoksične celice NK CD56dim. Z analizo limfocitov T smo ugotovili, da imajo bolniki okuženi z virusom PUU višjo koncentracijo limfocitov T kot bolniki okuženi z virusom DOB. Prav tako so rezultati pokazali višjo koncentracijo limfocitov T pri bolnikih okuženih z virusom PUU, ki so imeli blažji potek okužbe, kot pri bolnikih s težjim potekom bolezni. Dodatno smo z našo raziskavo ugotovili, da imajo bolniki okuženih z virusom PUU večjo koncentracijo celic Th ter več aktiviranih limfocitov T, ki izražajo aktivacijski marker CD69, ki se pojavi tik pred proliferacijo in aktivacijski marker HLA-DR, ki se pojavi tik pred klonsko ekspanzijo ter višjo koncentracijo aktiviranih efektorskih celic T (CD25+CD4+) in regulatornih celic T (CD4+CD25+CD127low/-), kot bolniki okuženi z virusom DOB. Iz rezultatov sklepamo, da višja koncentracija limfocitov T in večji specifični T celični odziv pripomoreta k blažjemu poteku HMRS po okužbi z virusom PUU, kot z virusom DOB. Bolniki okuženi z virusom DOB, ki so imeli blažji potek bolezni, so imeli večji delež celic Th, ki so izražale aktivacijski marker HLA-DR, kot bolniki z blažjim potekom okužbe z virusom DOB. Glede na rezultate menimo, da večji delež aktiviranih celic T pomagalk, pripomore k blažji obliki okužbe z virusom DOB. V tretjem delu doktorske naloge smo aktivacijo prirojenega imunskega odziva preverjali na nivoju koncentracije proteina visoko-mobilne skupine B1 (HMGB1). Želeli smo opredeliti vlogo HMGB1 kot napovednega dejavnika pri hantavirusni okužbi. Koncentracijo HMGB1 smo izmerili v supernatantih stimuliranih PBMC zdravih prostovoljcev ter v serumih bolnikov okuženih z virusom DOB in virusom PUU. Ugotovili smo, da je koncentracija HMGB1 višja v supernatantih stimuliranih PBMC z virusom DOB in virusom PUU, kot pri nestimuliranih PBMC, medtem ko razlika v koncentraciji HMGB1 med virusoma ni bila statistično značilna. Rezultate smo potrdili tudi z merjenjem koncentracije HMGB1 v serumih bolnikov s HMRS, pri čemer smo ugotovili, da imajo bolniki okuženi z virusom DOB in virusom PUU povišano koncentracijo HMGB1, med virusoma pa prav tako ni bilo statistično značilnih razlik. Z raziskavo smo prvi pokazali, da ima HMGB1 vlogo tudi v patogenezi HMRS. Pokazali smo, da je HMGB1 uporaben napovedni dejavnik za težji potek okužbe z virusom PUU, saj imajo bolniki s težjim potekom okužbe z virusom PUU višjo koncentracijo HMGB1, kot bolniki z blažjim potekom bolezni. Iz naše raziskave sklepamo, da je odziv prirojenega imunskega sistema bolj aktiviran po okužbi z virusom PUU, kot po okužbi z virusom DOB. Menimo, da zaviranje imunskega odziva gostitelja pripomore k težjemu poteku HMRS po okužbi z virusom DOB, kot po okužbi z virusom PUU. Našo ugotovitev smo v doktorski nalogi potrdili na treh nivojih. Na nivoju proučevanja IFN odziva smo ugotovili, da virus PUU bolj stimulira odziv IFN tipa I, višje izražanje gena MxA pripomore k lažjemu poteku okužbe z virusom PUU. Na nivoju proučevanja limfocitov smo potrdili, da virus PUU bolj aktivira specifični odziv limfocitov T, kot virus DOB. Na biološkem nivoju smo z merjenjem koncentracije beljakovine HMGB1 pokazali, da je HMGB1 vpleten v patogenezo HMRS in da je ustrezen napovedni dejavnik za težji potek bolezni po okužbi z virusom PUU. Zaviranje odziva IFN tipa I z virusom DOB je možen razlog, da HMGB1 ni ustrezen napovedni dejavnik za težji potek okužbe z virusom DOB. Hemorrhagic fever with renal syndrome (HFRS) is an endemic disease in Slovenia and is caused by Dobrava virus (DOBV), Puumala virus (PUUV) and Dobrava-Kurkino virus. The clinical severity of HFRS varies greatly and both mild and severe clinical courses of the disease have been observed, with an overall case fatality rate of 4,5 %. The host innate immune responses play an important role in the infection with hantaviruses. Pathogenic hantaviruses inhibit the early response of type I interferons (IFN&#945 and IFN&#946 ) and the IFN inhibition depends on the hantavirus pathogenicity. Inhibition of innate immune responses consequently leads to a rapid and systemic hantavirus infection. According to available literature, the difference in innate immune response between DOBV and PUUV infection was yet not compared. In our study, differences in activation of the innate immune response in peripheral blood mononuclear cells (PBMCs) from clinical samples of patients infected with DOBV and PUUV were investigated and significance of observed differences in HFRS severity was compared. The doctoral thesis is divided into three sections. In the first part of the study, the difference in the IFN type I-induced antiviral state were investigated in PBMCs in early post-stimulation with DOBV or PUUV. PBMCs from healthy volunteers and HFRS patients were stimulated with DOBV and PUUV. Our results showed that PUUV, but not DOBV, activated the IFN type I-induced antiviral state in stimulated PBMCs, and that IFN&#946 , STAT-1, and MxA were highly up-regulated at 48 h post-stimulation. The results of the study confirmed the hypothesis that DOBV activated lower IFN type I response in comparison to PUUV and the delayed IFN type I response could be a contributor to a more severe clinical outcome of the disease. Furthermore, up-regulation of MxA was statistically significant higher in convalescent-phase PBMCs from patients with mild PUUV infection than in patients with severe disease. Higher IFN type I-induced antiviral state could be a contributor to mild HFRS, particularly in PUUV infection. The IFN type I response were compared between acute-phase PBMCs and convalescent-phase cells from the same HFRS patients, and the role of immunological memory was investigated. Higher inhibition of IFN type I response was hypothesized in acute-phase PBMCs. Statistically significant differences were found between acute- and convalescent-phase PBMCs from patients with severe PUUV infection. Our result showed significant lower expression of IFN&#946 and significant higher expression of MxA in acute-phase PBMCs compared to convalescent-phase PBMCs, which indicated higher responsiveness of acute-phase PBMCs to PUUV stimulation. Results indicated no contribution of IFN type I response to the protection against DOBV or PUUV re-infection, as it is part of the innate immune system, with non-specific defense against pathogens. In the second part of the doctoral thesis, the PBMCs profile of patients with HFRS was analyzed by the flow cytometry. The different response of lymphocyte populations was investigated between DOBV and PUUV patients, and connection to HFRS severity was examined. Results showed significantly higher count of NK cells in patients with DOBV infection than in patients with PUUV infection. Higher count of NK cells was detected in patients with severe PUUV infection compared to patients with mild course of infection. Additionally, higher concentration of cytotoxic NK cells CD56dim was detected in patients with severe PUUV infection. Results indicated that NK cells could be associated with severe course of HFRS, especially higher amount of cytotoxic NK cells CD56dim could contribute to the more severe PUUV infection. Analysis showed higher concentration of T lymphocyte, especially higher concentration of Th cells, in PUUV infected patients in comparison to DOBV infected patients. Moreover, results indicated higher concentration of T lymphocyte in PUUV infected patients with mild HFRS compared to those with severe clinical course of the disease. Additionally, PUUV infected patients indicated higher concentration of effector T cells (CD25+CD4+), regulatory T cells (CD4+CD25+CD127low/-) and higher T lymphocyte activation, expressing activation markers CD69 and HLA-DR, in comparison to DOBV infected patients. Higher T lymphocyte concentration and higher specific T cell response contribute to milder HFRS after PUUV infection in comparison to DOBV infection. Results indicated higher amount of Th cell, expressing activation marker HLA-DR in patients with mild DOBV infection in comparison to patients with severe infection. According to results of our study, higher amount of activated Th cell could contribute to milder HFRS after DOBV infection. In the third part of the study, the concentration of protein High mobility group box 1 (HMGB1) was measured and the role of HMGB1 as a prognostic marker for HFRS severity was investigated. The HMGB1 concentration was measured in supernatants of stimulated PBMCs from healthy donors and in serum samples of HFRS patients. Result showed significantly higher concentration of HMGB1 in supernatants of PBMCs stimulated with DOBV and PUUV in comparison to unstimulated PBMCs, but the difference between viruses was not statistically significant. Results of our in vitro experiment was confirmed by measurement of HMGB1 concentration in patients’ serum samples. HMGB1 concentration was significantly elevated in HFRS patients compared to healthy donors, and no statistically significant difference was observed between the viruses. Our study is the first indicating the role of HMGB1 in HFRS pathogenesis. We have demonstrated potential usefulness of HMGB1 as a biomarker for severity in PUUV infection, as patients with a severe PUUV infection had significantly higher concentration of HMGB1 than patients with mild disease. In conclusion, results of our study indicated higher activation of innate immune responses after PUUV infection in comparison to DOBV infection. The inhibition of innate immune response could be one of the contributors to a more severe clinical outcome after DOBV infection. In the doctoral thesis, the differences in innate immunity between DOBV and PUUV infection were confirmed on three different biological levels. Results of PBMCs stimulation indicated high activation of the IFN type I-induced antiviral state after PUUV infection. Flow cytometry analyses showed high specific T lymphocyte activation in patients infected with PUUV. Our study indicated potential usefulness of HMGB1 as a prognostic marker for PUUV severity. Inhibition of IFN&#946 by DOBV could be a possible reason that HMGB1 is not the appropriate biomarker for severity in DOBV infection.