Uniform Master's Study Programme Pharmacy
Klara Verbič (Author), Anja Hafner (Mentor), Lara Bolčina (Co-mentor)

Abstract

Celice mikroglije so glialne celice centralnega živčnega sistema (CŽS), podobne makrofagom, ki vzdržujejo homeostazo možganov in so ene izmed najpomembnejših prirojenih celic imunskega sistema v CŽS. Pod vplivom patoloških in imunoloških dražljajev pride do polarizacije mikroglije v pro-vnetni fenotip M1, ki spodbuja procese nevrovnetja ali proti-vnetni fenotip M2, ki izkazuje zaščitno delovanje v možganih. Cisteinski katepsini, ki jih sprošča polarizirana mikroglija M1, so vse bolj prepoznani kot pomembni vnetni dejavniki, ki sprožijo signalne poti, kar vodi v poslabšanje nevrovnetja. Med njimi je cisteinska peptidaza katepsin X, ki je že bila prepoznana kot patogeni dejavnik nevrodegeneracije, povzročene z vnetjem. V okviru magistrske naloge smo želeli ovrednotiti vlogo katepsina X pri polarizaciji celic mikroglije. Najprej smo postavili celični model polarizirane mikroglije z uporabo celične linije BV2, kjer smo pokazali vpliv stimulacije z lipopolisaharidom (LPS), interferonom-γ (IFN-γ), interlevkinom-4 (IL-4) in IL-13 na usmeritev polarizacije mikroglije. Pri tem sta LPS in IFN-γ povzročila polarizacijo mikroglije v fenotip M1, kar smo pokazali s povišano ravnjo pro-vnetnih dejavnikov. Po drugi strani sta citokina IL-4 in IL-13 spodbudila polarizacijo v smeri fenotipa M2, kar smo potrdili z označevalci proti-vnetnega stanja. Nadalje smo opredelili izražanje, aktivnosti in vezikularno lokalizacijo katepsina X v stimuliranih celicah BV2. Stimulacija z LPS in IFN-γ je povišala znotrajcelično izražanje katepsina X, kar smo pokazali s pomočjo prenosa western, in hkrati znižala njegovo aktivnost zaradi sproščanja zrele oblike katepsina X iz celic mikroglije ter zmanjšala njegovo znotrajcelično lokalizacijo v lizosomih, ki smo jo določili s konfokalno mikroskopijo. Pri tem IL-4 in IL-13 na izražanje, aktivnost in lokalizacijo katepsina X nista imela vpliva. V zadnjem delu magistrske naloge smo vrednotili še vpliv ireverzibilnega zaviralca katepsina X, AMS36, na polarizacijo mikroglije. Pokazali smo, da je AMS36 z zaviranjem katepsina X po stimulaciji z LPS in IFN-γ preprečil polarizacijo mikroglije v fenotip M1, kar se je odražalo v upadu ravni pro-vnetnega dejavnika NO, medtem ko je pri stimulaciji celic BV2 z IL-4 in IL-13 spodbudil polarizacijo mikroglije v fenotip M2, kar smo pokazali s povečano aktivnostjo arginaze-1 in izražanjem označevalca CD206, ki sta značilna za polarizirano mikroglijo M2. Pridobljeni rezultati nakazujejo, da ima katepsin X pomembno vlogo pri polarizaciji mikroglije v pro-vnetni fenotip M1 oziroma proti-vnetni fenotip M2 in predstavlja potencialno terapevtsko tarčo za preprečevanje in zdravljenje nevrodegenerativnih bolezni, povezanih s prekomernim vnetjem.

Keywords

mikroglija;polarizacija;vnetje;katepsin X;zaviralec katepsina X;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL FFA - Faculty of Pharmacy
Publisher: [K. Verbič]
UDC: 576.3(043.3)
COBISS: 134865667 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Role of cysteine peptidase cathepsin X in microglia polarization
Secondary abstract: Microglia cells are glial cells of the central nervous system (CNS), similar to macrophages, which maintain brain homeostasis and are among the most important cells of the innate immune system in the CNS. Under the influence of pathological and immunological stimuli, the polarization of microglia occurs, either into the pro-inflammatory phenotype M1, which promotes the processes of neuroinflammation, or the anti-inflammatory phenotype M2, which exhibits a protective function in the brain. Cysteine cathepsins released by M1 polarized microglia are increasingly recognized as important inflammatory factors that trigger signaling pathways, which in turn lead to aggravation of neuroinflammation. Among them is the cysteine peptidase cathepsin X, which has already been recognized as a pathogenic factor in inflammation-induced neurodegeneration. As part of the master's thesis, we evaluated the role of cathepsin X in the polarization of microglia cells. First, we established a cellular model of polarized microglia using the BV2 cell line, where we demonstrated the effect of stimulation with lipopolysaccharide (LPS), interferon-γ (IFN-γ), interleukin-4 (IL-4) and IL-13 on the course of polarization. Here, LPS and IFN-γ induced polarization of microglia to the M1 phenotype, which we demonstrated with increased levels of pro-inflammatory factors. On the other hand, the cytokines IL-4 and IL-13 induced polarization towards the M2 phenotype, which was confirmed by markers of the anti-inflammatory state. We further characterized the expression, activity, and vesicular localization of cathepsin X in stimulated BV2 cells. Stimulation with LPS and IFN-γ increased the intracellular expression of cathepsin X, which was demonstrated by western blotting, and at the same time decreased its activity due to the release of the mature form of cathepsin X from microglial cells and reduced its intracellular localization in lysosomes, which was determined by confocal microscopy. IL-4 and IL-13 had no effect on cathepsin X expression pattern and localization. In the last part of the master's thesis, we also evaluated the influence of the irreversible cathepsin X inhibitor, AMS36, on the polarization of microglia. We showed that AMS36 prevented the polarization of microglia into the M1 phenotype by inhibiting cathepsin X after stimulation with LPS and IFN-γ, as is also indicated by the reduction of the pro-inflammatory factor NO, whereas stimulation of BV2 cells with IL-4 and IL- 13 in the presence of AMS36 enhanced polarization of microglia into the M2 phenotype, which was demonstrated by increased arginase-1 activity and the expression of the marker CD206, which are characteristic of M2 polarized microglia. The obtained results suggest that cathepsin X plays an important role in the polarization of microglia into the pro-inflammatory M1 and the anti-inflammatory M2 phenotype; as such, it represents a potential therapeutic target for the prevention and treatment of neurodegenerative diseases associated with excessive inflammation.
Secondary keywords: Celična biologija;
Type (COBISS): Master's thesis/paper
Thesis comment: Univ. v Ljubljani, Fak. za farmacijo
Pages: XII, 78 str,
ID: 17480030