Ovrednotenje vpliva aktivacije z AMP aktivirane protein kinaze na inflamasom NLRP3

Miklavčič, Rok (avtor), Hafner Bratkovič, Iva (mentor), Gunčar, Gregor (komentor)

Inflamasom NLRP3 je večproteinski kompleks prirojene imunosti in služi za aktivacijo kaspaze 1, ki proteolitično aktivira IL-1β, IL-18 in gasdermin D. Po aktivaciji NLRP3 se zreli obliki interlevkinov sprostita v okolico celic preko por gasdermina D ali zaradi nekroze in sprožita vnetje. Sestavljanje kompleksa sprožijo mnogi PAMP-i in DAMP-i, ki delujejo po kanonični, nekanonični ali alternativni poti aktivacije NLRP3. Delovanje inflamasoma NLRP3 je povezano tudi s sterilnim kroničnim vnetjem v starosti in spremlja starostne bolezni, kot so diabetes tipa 2, Alzheimerjeva bolezen ter ateroskleroza. Trenutno še ni jasno, katera od aktivacijskih poti inflamasoma sodeluje pri tem učinku, slabo pa je razjasnjena tudi sama regulacija delovanja inflamasoma NLRP3. Namen našega dela je bil sistematično ovrednotiti učinek spojin, ki podaljšujejo življenje, na kanonično, nekanonično in alternativno aktivacijo inflamasoma NLPR3, da bi pridobili informacije o regulaciji delovanja NLRP3 pri staranju. Za delo smo izbrali spojine, ki so preko aktivacije AMPK ali avtofagije podaljšale življenjsko dobo modelnih organizmov. Pri celicah iBMDM smo uspešno vzpostavili protokol za nekanonično aktivacijo NLRP3 s transfekcijo LPS in protokol za alternativno aktivacijo NLRP3 s peptidoglikanom. Pokazali smo, da rapamicin, spermidin, spermin, resveratrol, kurkumin, metformin, AICAR in PT-1 inhibirajo zorenje IL-1β pri nekanonični aktivaciji NLRP3. Večina teh spojin je inhibirala tudi zorenje IL-1β pri alternativni aktivaciji NLRP3, vendar smo predvsem za spermin in spermidin pokazali, da ob daljši inkubaciji lahko delujeta citotoksično. Pri kanonični aktivaciji NLRP3 smo spremljali zorenje IL-1β le za metformin, spermidin in spermin, kjer smo sicer za spermin in spermidin opazili znižanje procesiranja IL-1β, vendar smo hkrati opazili tudi citotoksičnost pri danih pogojih. Zanimivo je posredni aktivator AMPK metformin nasprotno celo povečal celični odziv pri kanonični aktivaciji NLRP3. Drugi namen naloge je bil pripraviti celično linijo iBMDM, ki inducibilno izraža konstitutivno aktivno AMPK, pri kateri bi lahko preučevali učinek AMPK na NLRP3. Z molekulskim kloniranjem in retrovirusno transdukcijo smo uspešno pripravili celični liniji CA/iBMDM in nCA/iBMDM. Obe liniji izražata konstitutivno aktivno AMPKα1 (1-312) T183D, ki pa se razlikujeta na N-koncu. Pri obeh linijah smo s prenosom western potrdili, da lahko z doksiciklinom induciramo sintezo encimov CA-AMPK oziroma nCA-AMPK. Fiziološke aktivnosti encimov še nismo uspeli enolično potrditi, sta pa oba encima nakazala učinek na fosforilacijo ACC po indukciji z doksiciklinom. Pri delu smo določili dobre kandidate za nadaljnje testiranje inhibicije NLRP3 in preučevanje njegove regulacije pri vnetju, ki spremlja staranje. Poleg tega smo pripravili celični liniji, pri katerih bi lahko po optimizaciji indukcije preučevali učinek AMPK na delovanje inflamasoma NLRP3.

• Jezik: Slovenski jezik
• Ključne besede: NLRP3, inflamasom, staranje, AMPK, avtofagija
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2020
• PID: 20.500.12556/RUL-117445
• COBISS.SI-ID: 23501827
• Datum objave v RUL: 10.07.2020

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Characterization of the effect of AMP-activated protein kinase activation on the NLRP3 inflammasome

Izvleček

NLRP3 inflammasome is a multiprotein complex of innate immunity that is used to activate caspase 1, which proteolytically activates IL-1β, IL-18 and gasdermin D. After the activation of NLRP3 the interleukins are released through gasdermin D pores or by necrosis and trigger inflammation. Inflammasome assembly is triggered by many PAMPs and DAMPs, which act via the canonical, non-canonical or alternative NLRP3 activation pathway. NLRP3 inflammasome activity is also connected with inflammaging and accompanies geriatric diseases such as type 2 diabetes, Alzheimer's disease and atherosclerosis. It is not yet clear which of the activation pathways is involved in this effect. The regulation of NLRP3 activity is also poorly understood. The purpose of our work was to systematically evaluate the effect of lifespan-enhancing compounds on canonical, non-canonical and alternative NLRP3 inflammasome activation to obtain information on the regulation of NLRP3 activity in aging. We selected compound that extended the lifespan of model organisms through activation of AMPK or autophagy. On iBMDM cells, we successfully established a protocol for non-canonical NLRP3 activation by LPS transfection and a protocol for alternative NLRP3 activation by peptidoglycan. We have shown that rapamycin, spermidine, spermine, resveratrol, curcumin, metformin, AICAR and PT-1 inhibited IL-1β maturation by the non-canonical inflammasome. The majority of tested compounds also inhibited IL-1β release by the alternative inflammasome, however, particularly spermine and spermidine exposed some cytotoxic activity at prolonged incubation. Metformin, spermidine and spermine were also tested in canonical NLRP3 activation setup, where spermine and spermidine inhibited IL-1β release, yet some cytotoxicity was observed at the same time. Interestingly, the indirect AMPK activator metformin even increased the cellular response to canonical activation of NLRP3. The second aim of our work was to prepare an iBMDM cell line that inducibly expresses constitutively active AMPK to study the effect of AMPK on NLRP3 response. By molecular cloning and retroviral transduction, we have successfully prepared CA/iBMDM and nCA/iBMDM cell lines, each expressing a variant of constitutively active AMPKα1 (1-312) T183D. We confirmed by western blot that doxycycline induces the synthesis of CA-AMPK and nCA-AMPK enzymes in CA/iBMDM and nCA/iBMDM cell lines, respectively. We have not yet uniformly confirmed the physiological activity of the enzymes, but both enzymes have indicated an effect on ACC phosphorylation after doxycycline induction. Our work provided good candidates for further research on NLRP3 inhibition and its regulation in aging. In addition, we prepared two cell lines where we could study the effect of AMPK on NLRP3 inflammasome activity after we optimize the induction.

• Ključne besede: NLRP3, inflammasome, aging, AMPK, autophagy