[doktorska disertacija]
Tanja Soklič (Avtor), Peter Korošec (Mentor), Irena Hočevar-Boltežar (Komentor)

Povzetek

UVOD: Kronični rinosinuzitis (KRS) je kronično vnetje sluznice nosu in obnosnih votlin, ki traja več kot 12 tednov z občasnimi poslabšanji. KRS razdelimo na polipozni KRS (KRS z nosnimi polipi, KRSwNP) in nepolipozni KRS (KRS brez nosnih polipov, KRSsNP). Vsaj 20% bolnikov ima kljub ustreznemu zdravljenju težek KRS s perzistentnimi motečimi simptomi (ocena ?5 na vizualni analogni lestvici, VAS 0-10) in ponovitvami po endoskopskih operacijah. Raznolikost imunofenotipov - endotipov v sluznici pri KRS še ni dokončno pojasnjena. Podtipi sluzničnih T limfocitov, povezani s težkim KRS, še niso bili raziskani. NAMEN: Namen naše raziskave je bila analiza podtipov sluzničnih T limfocitov pri bolnikih s KRSwNP in bolnikih s KRSsNP, še preden so začeli prejemati nosne ali sistemske glukokortikoide. Za razliko od predhodnih raziskav smo želeli identificirati specifične podtipe sluzničnih T celic, karakteristične za bolnike s težkim KRSwNP in KRSsNP ter za bolnike z urejenim KRSwNP in KRSsNP. Osredotočili smo se na CD8+ citotoksične T limfocite in dvojno negativne CD4-CD8- DN T celice, ki še niso bile opisane pri KRS. Naš dodaten cilj je bila določitev genskih ekspresij glavnih znotrajceličnih transkripcijskih faktorjev podtipov T limfocitov, ki bi lahko kot promotorji in regulatorji vnetnih poti predstavljali potencialne nove tarče topikalnega zdravljenja nosne sluznice z DNAcimi. MATERIALI IN METODE: Prospektivno smo vključili 31 bolnikov z novoodkritim KRS (18 KRSwNP in 13 KRSsNP) s težkimi simptomi, ki do vključitve še niso bili zdravljeni zaradi KRS. Odvzeli smo biopsijo sluznice nosnega polipa v srednjem nosnem hodniku pri bolnikih s KRSwNP ali sluznice s procesus uncinatusa v srednjem nosnem hodniku pri bolnikih s KRSsNP. Vzorce sluznice smo pregledali histopatološko in analizirali s pretočno citometrijo za podtipe T limfocitov (Th1, Th2, Th17, Tc1, Tc17 in DN T celice), aktivacijske markerje (CD69, CD25, HLA-DR) ter označevalce citotoksičnosti (CD28, CD27). Pri 22 bolnikih (14 KRSwNP in 8 KRSsNP) smo izmerili nivoje genskih ekspresij glavnih transkripcijskih faktorjev T-box transkripcijski faktor (T-bet, TBX21), GATA binding protein 3 (GATA3), Retinoic acid-related orphan receptor C (RORC) in Forkhead box P3 (FOXP3) s kvantitativno verižno reakcijo s polimerazo z reverzno transkriptazo (RT-qPCR). Bolnike smo zdravili in jih redno ambulantno spremljali povprečno 24 do 36 mesecev; težek KRS je kljub zavzetemu medikamentoznemu in endoskopskemu kirurškemu zdravljenju imelo 5 od 18 bolnikov s KRSwNP in 5 od 13 bolnikov s KRSsNP. Statistično analizo podatkov smo naredili s programom GraphPad Prism 6.0. REZULTATI: Sluznična eozinofilija z >10 eozinofilci / HPF je bila pogostejša pri bolnikih s KRSwNP kot pri bolnikih s KRSsNP. Bolniki s KRSwNP so imeli v primerjavi z bolniki s KRSsNP več CD4+ T limfocitov in zvišano število aktiviranih CD4+CD25+ T celic, kar kaže na Th-posredovano vnetje. Th1 celice so bile pomnožene v sluznici pri KRSwNP, vendar niso imele vpliva na kontrolo bolezni pri KRSwNP. Bolniki s KRSsNP so imeli zvišano število CD8+ T limfocitov in pomnožene efektorsko citotoksične CD8+CD28-CD27- T celice ob hkrati znižanem številu centralno spominskih CD8+CD28+CD27+ T celic, kar pomeni prevladujoče citotoksično vnetje pri bolnikih s KRSsNP. To potrjuje tudi rezultat, da so imeli bolniki s težkim KRSsNP še dodatno zvišano število CD8+ T celic v sluznici. Pri bolnikih s KRSwNP in bolnikih s KRSsNP smo našli pomembno visok delež (10-20%) sluzničnih DN T celic, presenetljivo višji od normalnega 1-5% deleža. Med DN T limfociti so prevladovali efektorsko citotoksični CD28-CD27- DN T, le malo je bilo centralno spominskih CD28+CD27+ DN T celic. Po naših podatkih je to prva raziskava, ki je našla tako visoko število DN T limfocitov v sluznici pri KRS. Pri bolnikih s težkim KRSwNP je bilo število vnetnih DN T celic 3-krat višje in manj je bilo delno diferenciranih, efektorsko spominskih DN T kot pri bolnikih z urejenim KRSwNP. Izvor DN T celic pri KRS trenutno ni pojasnjen, lahko pa bi bile pomemben proizvajalec IL-5, glavnega citokina vnetja tipa 2. Pri urejenem KRSwNP je bilo zvišano število Th17 celic, ki verjetno predstavljajo normalen homeostatski imunski odziv zdrave nosne sluznice. Pri urejenem KRSsNP so bile pomnožene DN T celice, ki bi lahko pri teh bolnikih imele regulatorno supresorsko vlogo in bi lahko omejevale škodljivo CD8+ celično aktivacijo ter poškodbe sluznice. Pri bolnikih z eozinofilnim KRSwNP smo s povečano gensko ekspresijo GATA3 mRNA v primerjavi z bolniki z neeozinofilnim KRSwNP potrdili vnetje tipa 2. Vendar verjetno pri vnetju tipa 2 sodelujejo tudi DN T celice, a njihov glavni transkripcijski faktor zaenkrat še ni znan. Pri bolnikih s KRSsNP smo nepričakovano ugotovili simultano zvišane nivoje genskih ekspresij T-bet, GATA3 in RORC v primerjavi z bolniki s KRSwNP; domnevamo, da bi lahko te visoke genske ekspresije vseh treh glavnih transkripcijskih faktorjev za efektorske T celice izvirale iz pomnoženih efektorsko citotoksičnih, aktiviranih CD8+ T celic v sluznici pri KRSsNP. ZAKLJUČKI: Bolniki s KRS so se razlikovali po podtipih sluznični T limfocitov: bolniki s KRSwNP so imeli več CD4+ T celic, bolniki s KRSsNP pa več CD8+ T celic s pomnoženimi efektorsko citotoksičnimi CD8+CD28-CD27- T limfociti. Prvič smo v sluznici KRS našli pomnožene DN T celice, med katerimi so prevladovale efektorsko citotoksične celice. Ta raziskava je prva analizirala podtipe T limfocitov, povezane s kontrolo KRS. Potentne efektorske aktivirane DN T celice, ki jih je bilo v sluznici pri težkem KRSwNP 3-krat več, verjetno igrajo pomembno vlogo v patogenezi težkega KRSwNP, bi morda lahko predstavljale novo tarčo zdravljenja in jih je potrebno še podrobneje raziskati. Obratno pa morda DN T limfociti pri bolnikih s KRSsNP delujejo regulatorno supresorsko in zavirajo škodljivo citotoksično vnetje v sluznici. Nepričakovano visoke genske ekspresije vseh treh glavnih transkripcijskih faktorjev za efektorske T limfocite pri bolnikih s KRSsNP, ki kažejo na pomnožene efektorske aktivirane T celice v sluznici, so trenutno nepojasnjene; najverjetneje izvirajo iz aktiviranih, efektorsko citotoksičnih CD8+ celic. Naši rezultati so tako znanstveno kot tudi klinično aplikativni. V perspektivi razvijanja novih strategij topikalnega nosnega zdravljenja z DNAcimi je potrebno upoštevati dinamično ravnotežje koekspresij dveh različnih glavnih transkripcijskih faktorjev v efektorskih in regulatornih T limfocitih. Prispevajo k razumevanju patogeneze vnetja v sluznici pri bolnikih s KRSwNP in bolnikih s KRSsNP, kar bo v prihodnosti nadgradilo diagnostiko KRS z novimi biomarkerji, modificiralo obstoječe algoritme zdravljenja in omogočilo personalizirano obravnavo bolnikov s KRS. Specifični imunofenotipi - endotipi v sluznici bolnikov s KRS bodo v prihodnosti odločali o vrsti medikamentoznega in obsegu endoskopskega kirurškega zdravljenja, ki postopoma ne bo več enako za vse, ampak bo prilagojeno bolniku, kar bo še posebej pomembno za bolnike s težkim KRS.

Ključne besede

otorinolaringologija;polipozni kronični rinosinuzitis;nepolipozni kronični rinosinuzitis;limfociti T;podtipi;nosna sluznica;ekspresija genov;topikalno zdravljenje;kirurško zdravljenje;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.08 - Doktorska disertacija
Organizacija: UL MF - Medicinska fakulteta
Založnik: T. Soklič Košak
UDK: 616.214-002-036.1(043.3)
COBISS: 20006659 Povezava se bo odprla v novem oknu
Št. ogledov: 816
Št. prenosov: 216
Ocena: 0 (0 glasov)
Metapodatki: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Immunophenotyping of chronic rhinosinusitis with and without nasal polyps
Sekundarni povzetek: BACKGROUND: Chronic rhinosinusitis (CRS) is a nasal and sinus mucosa chronic inflammation of at least 12 weeks duration with occasional exacerbations. CRS can be further subdivided into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Furthermore, at least 20 % of CRS patients suffer from uncontrolled CRS with persistent bothersome symptoms ( ⡥5 score on the visual analog scale, VAS 0-10) and recurrences after endoscopic surgeries despite appropriate treatment. The CRS mucosal immunophenotypes – endotypes diversity is still not well characterized. Importantly, the uncontrolled CRS - associated mucosal T cells are unknown. OBJECTIVES: We aimed to evaluate the different mucosal T cell subtypes in yet untreated, steroid naïve patients with CRSwNP and patients with CRSsNP. In contrast to previous studies, we sought to identify the characteristic nasal mucosa T cells in patients with uncontrolled CRSwNP and CRSsNP and in patients with well-controlled CRSsNP and CRSsNP. We focused on cytotoxic CD8+ and double-negative (DN) CD4-CD8- T cells, which have not been described in CRS yet. Additionally, we aimed to evaluate the master transcription factors gene expression levels of T cell subtypes in CRSwNP and CRSsNP that could represent new, up-stream targets for DNAzyme topical nasal mucosa treatment to suppress the inflammation. MATERIALS AND METHODS: 31 untreated, steroid-naïve CRS patients (18 CRSwNP, 13 CRSsNP) with severe symptoms were prospectively included. Biopsy was taken from the middle meatal nasal polyp in CRSwNP patients and the uncinate process mucosa in CRSsNP patients. The biopsies were examined histopathologically and analyzed by flow cytometry for T cell subtypes (Th1, Th2, Th17, Tc1, Tc17 and DN T cells), activation markers (CD69, CD25, HLA-DR) and cytotoxicity markers (CD28, CD27). Additionally, in 22 CRS patients (14 CRSwNP and 8 CRSsNP), the gene expression levels of major transcription factors T-box transcription factor (T-bet, TBX21), GATA binding protein 3 (GATA3), Retinoic acid-related orphan receptor C (RORC) and Forkhead box P3 (FOXP3) were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Patients were followed up on average from 24 to 36 months with repeated visits. 5 of 18 CRSwNP and 5 of 13 CRSsNP had uncontrolled CRS despite extensive medical and surgical treatment. The data generated in the study were analyzed using GraphPad Prism 6.0. RESULTS: Significantly more CRSwNP patients compared to CRSsNP patients had tissue eosinophilia >10 per HPF. CRSwNP mucosa was characterized by higher CD4+ cell numbers and more abundant activated CD4+CD25+ cells, suggesting Th-driven inflammation. We also observed increased Th1 cells in CRSwNP; however, we did not find any impact of those cells on the CRSwNP disease control. CRSsNP mucosa was characterized by higher CD8+ T cells and abundant effector cytotoxic CD8+CD28-CD27- cells with simultaneously decreased central memory CD8+CD28+CD27+ cell numbers, speaking for cytotoxic inflammation type in patients with CRSsNP. Furthermore, this was confirmed with even more abundant mucosal CD8+ cells in patients with uncontrolled CRSsNP compared to patients with well-controlled CRSsNP. Importantly, in both CRSwNP and CRSsNP, we found surprisingly high percentages (10-20%) of mucosal DN T cells in contrast to normal 1-5% range. Additionally, higher numbers of effector cytotoxic CD28-CD27- DN T cells and a lower number of central memory CD28+CD27+ DN T cells were observed. According to our knowledge, this is the first study to demonstrate such high numbers of DN T cells in CRS mucosa. We demonstrated 3-fold higher numbers of proinflammatory DN T cells and lower count of partly differentiated, effector memory CD28-CD27+ cells in patients with uncontrolled CRSwNP compared to patients with well-controlled CRSwNP. The source of DN T cells in CRS is currently unknown; however, DN T cells might also be an important producer of IL-5, the major type 2 cytokine. Well-controlled CRSwNP was characterized by a higher number of Th17 cells that probably represent a normal homeostatic immune response in healthy nasal mucosa. In patients with well-controlled CRSsNP, we demonstrated higher numbers of DN T cells, which could be suppressive regulatory and could be able to limit harmful CD8+ T cell response and mucosal damage in these patients. In patients with eosinophilic CRSwNP, we confirmed the type 2 inflammation by the higher level of GATA3 gene expression compared to patients with noneosinophilic CRSwNP. However, activated effector DN T cells can also secrete type 2 cytokines, although their master transcription factor is currently unknown. In patients with CRSsNP, we unexpectedly found simultaneous upregulation of T-bet, GATA3 and RORC gene expression levels in comparison to patients with CRSwNP; we might speculate here, that these high gene expression levels of all three effector T cells’ master transcription factors originate from expanded effector cytotoxic, activated CD8+ cells in CRSsNP mucosa. CONCLUSIONS: We demonstrated distinct nasal mucosa T cell patterns for each CRS type: elevated CD4+ T cells in patients with CRSwNP and abundant CD8+ T cells with higher effector cytotoxic CD8+CD28-CD27- type in patients with CRSsNP. Increased mucosal DN T cells were found for the first time in patients with CRS. Moreover, those DN T cells were of elevated effector cytotoxic type. The present study was the first to analyze T cell subtypes in association with CRS disease control. The 3-times higher count of potent effector activated DN T cells in patients with uncontrolled CRSwNP might play a central role in uncontrolled CRSwNP pathogenesis, could represent a new treatment target and therefore, should be further investigated. In contrast, DN T cells might be of the regulatory suppressive type in patients with CRSsNP, limiting the harmful cytotoxic mucosal inflammation. The unexpectedly found simultaneous upregulation of all three effector T cells’ master transcription factors in CRSsNP patients was a sign of expanded activated effector T cells in CRSsNP mucosa and is currently unexplained; however, it might originate from abundant effector cytotoxic CD8+ cells. Our results are scientifically as well as clinically applicable. In the perspective of future transcription factor-targeted topical nasal treatment development, the simultaneous coexpression of two different major transcription factors in effector or regulatory T cells should be taken into account. The understanding of inflammation pathogenesis in the nasal mucosa of patients with CRSwNP and patients with CRSsNP will upgrade the future CRS diagnostics with biomarkers, modify our current treatment algorithms and make the personalized medicine for patients with CRS possible. In the future, specific CRS mucosal immunophenotypes – endotypes will apply for the choice of the medications and tailor the extent of endoscopic sinus surgery that would gradually not follow the same standards for all anymore. Patient-tailored treatment will especially be important for uncontrolled CRS patients.
Sekundarne ključne besede: Kronični rinosinuzitis;Disertacije;Imunofenotipizacija;Immunophenotyping;Otorhinolaryngologic diseases;Pathology;Inflammation;Nasal polyps;Nasal mucosa;T-lymphocytes;Gene expression;STAT transcription factors;Prospective studies;Otorinolaringološke bolezni;Patologija;Vnetje;Nosni polipi;Nosna sluznica;T-limfociti;Gensko izražanje;STAT transkripcijski faktorji;Prospektivne študije;
Vrsta dela (COBISS): Doktorska disertacija
Študijski program: 0
Konec prepovedi (OpenAIRE): 1970-01-01
Komentar na gradivo: Univ. v Ljubljani, Medicinska fak.
Strani: 99 f.
ID: 11448641