The mechanisms of NLRP3 inflammasome activation and inhibition

Vnetni odziv v okviru prirojene imunosti je ključnega pomena za uspešno spopadanje z vdorom patogenih organizmov in za regeneracijo poškodovanega tkiva. V ta namen so imunske celice opremljene z receptorji za zaznavanje molekul mikrobnega izvora in različnih nemikrobnih endogenih molekul. Eden najpomembnejših regulatorjev nemikrobnega vnetja v imunskih celicah je proteinski kompleks inflamasom NLRP3, ki se aktivira ob zaznavi molekul, kot sta ATP in mitohondrijska DNA, pa tudi raznovrstnih patoloških delcev, kot so amiloidni plaki pri nevrodegenerativnih boleznih, holesterolni kristali pri aterosklerozi ali azbestna vlakna pri azbestozi. Inflamasomski kompleks NLRP3 je sestavljen iz oligomera aktiviranih receptorjev NLRP3, okoli katerega se preko medproteinskih interakcij v domenah PYD in CARD nanizajo molekule adapterskega proteina ASC in molekule pro-kaspaze-1, ki po avtokatalitskem procesiranju v aktivno kaspazo-1 aktivirajo citokina IL-1ß in IL-18 ter piroptotično smrt. Mehanizem aktivacije senzorskega proteina NLRP3 ostaja neznan, kar otežuje razvoj selektivnih terapevtikov za številne bolezni, ki so povezane z njegovo prekomerno aktivacijo. V pričujočem doktorskem delu smo osvetlili nekatera odprta vprašanja v zvezi z začetnimi stopnjami aktivacije inflamasoma NLRP3. Na podlagi celičnih linij, ki so izražale himerne konstrukte NLRP3, pri katerih smo njegovo interakcijsko domeno PYD zamenjali z drugimi interakcijskimi domenami, smo ugotovili, da je domena NLRP3PYD pomembno vpletena v vzdrževanje neaktivne oblike senzorskega proteina NLRP3, saj se je njena zamenjava z različnimi domenami CARD odražala v konstitutivni aktivnosti inflamasoma. S pomočjo konstruktov, pri katerih smo interakcijsko domeno PYD povezali z različnimi oligomerizacijskimi domenami, smo raziskali tudi stehiometrijo senzorskega proteina NLRP3 in ugotovili, da je že povezava domen NLRP3PYD v trimer zadostna za aktivacijo inflamasoma NLRP3. V nadaljevanju nas je zanimala tudi struktura in razporeditev proteinov znotraj inflamasomskega kompleksa NLRP3. S superresolucijsko mikroskopijo 3D SIM fluorescenčno označenih proteinov znotraj kompleksa v živih celicah smo ugotovili, da oligomerizirani proteini ASC-GFP tvorijo visoko zamreženo strukturo z gosto sredico, v kateri se nahaja NLRP3. Spremljanje dinamike njegove tvorbe v realnem času pa je pokazalo, da se filamenti ASC-GFP v značilno kompaktno obliko kondenzirajo preko intenzivnega razvejanja filamentov, na kar pomembno vpliva domena ASCCARD. Na podlagi strukturnih in molekularno- bioloških študij proteinov, udeleženih v inflamasom NLRP3, ki so identificirale njihove interakcijske površine, smo načrtovali tudi peptidne inhibitorje inflamasoma NLRP3. Identificirali smo pet načrtovanih inhibitorjev, ki so inhibirali sproščanje IL-1ß, aktivacijo kaspaze-1 in oligomerizacijo ASC. Peptid iz segmenta ASCPYD/H2-H3, ki je inflamasom NLRP3 inhibiral selektivno, smo za morebitne terapevtske aplikacije na področju nevrodegenerativnih bolezni opremili s peptidnim zaporedjem Angiopep-2 in pokazali, da je tako opremljena različica inhibitorja ASCPYD/H2-H3 uspešno prehajala krvno-možgansko pregrado, obenem pa ni imela vpliva na njegov inhibitorni potencial. V tem doktorskem delu smo prispevali novo znanje o molekularnem mehanizmu aktivacije inflamasoma NLRP3 in načrtovali set peptidnih inhibitorjev, ki bi lahko predstavljali podlago za nove pristope k terapiji z inflamasomom NLRP3 povezanih bolezni.Innate immunity associated inflammatory responses are crucial for the efficient elimination of the infectious agents and tissue repair. For this purpose immune cells are equipped with receptors capable of sensing molecules of both microbial and endogenous origin. One of the key sterile inflammation regulators in imune cells is the NLRP3 inflammasome. Formation of this multiprotein complex is triggered by the endogenous molecules such as extracellular ATP and mitochondrial DNA as well as various pathological deposits including amyloid plaques in neurodegenerative diseases, cholesterol crystals in atherosclerosis and asbestos fibres in asbestosis. NLRP3 inflammasome assembly is initiated by an oligomer of activated NLRP3 receptors that provides a nucleus for the oligomerisation of the adaptor protein ASC and molecules of pro-caspase-1 via interactions in their PYD and CARD domains. Autocatalytical cleavage of pro-caspase-1 results in the formation of active caspase-1 which in turn cleaves cytokine IL-1β and IL-18 precursors into their active forms as well as initiates pyroptotic cell death. The molecular mechanism of the NLRP3 inflammasome activation remains largely elusive which hinders the development of selective therapeutic strategies for numerous diseases associated with disregulation of NLRP3. In this doctoral dissertation we addressed some of the open questions regarding the early stages of the NLRP3 inflammasome activation. Establishing cell lines that expressed chimeric NLRP3 variants where PYD domain was replaced by other interaction domains we found that NLRP3PYD domain is critically involved in the maintenance of inactive NLRP3 conformation as its replacement with various CARD domains resulted in constitutive inflammasome activation. Using the constructs where NLRP3PYD domain was coupled to various oligomerisation domains we also investigated the minimal active stoichiometry of NLRP3 oligomer and found that a trimer of NLRP3PYD domains was sufficient for the inflammasome activation. In addition, we were interested in the structure and protein arrangement within the complex. 3D SIM superresolution microscopy of fluorescently labelled proteins within the complex revealed that ASC-GFP forms a highly branched network with a dense core containing the NLRP3 oligomer. Monitoring the dynamics of the complex formation in real time further revealed that ASC-GFP filaments are being condensed into a speck through intense branching of filaments, a process supported by ASCCARD domains. Moreover, based on the available protein crystal structures and mutagenesis studies that identified interaction surfaces of inflammasome proteins we also designed peptide inhibitors of the NLRP3 inflammasome. Five of the designed peptides inhibited IL-1β release, caspase-1 activation and ASC oligomerisation. Peptide comprising the ASCPYD/H2-H3 segment selectively inhibited the inflamasom NLRP3. In order to underline its relevance in a neurological setting, it was equipped with Angiopep-2 peptide sequence which enabled its transfer through the brain-blood barrier in the absence of any negative effects on its inhibitory potential. This doctoral thesis provides new mechanistic clues on NLRP3 inflammasome activation as well as a toolbox of designed peptide inhibitors that could represent the basis for the novel therapeutic approaches to treating NLRP3 inflammasome associated diseases

Differential Effect of Extracellular Acidic Environment on IL-1β Released from Human and Mouse Phagocytes

Areas of locally decreased pH are characteristic for many chronic inflammatory diseases such as atherosclerosis and rheumatoid arthritis, acute pathologies such as ischemia reperfusion, and tumor microenvironment. The data on the effects of extracellular acidic pH on inflammation are conflicting with respect to interleukin 1 beta (IL-1β) as one of the most potent proinflammatory cytokines. In this study, we used various mouse- and human-derived cells in order to identify potential species-specific differences in IL-1β secretion pattern in response to extracellular acidification. We found that a short incubation in mild acidic medium caused significant IL-1β release from human macrophages, however, the same effect was not observed in mouse macrophages. Rather, a marked IL-1β suppression was observed when mouse cells were stimulated with a combination of various inflammasome instigators and low pH. Upon activation of cells under acidic conditions, the cytosolic pH was reduced while metabolic activity and the expression of the main inflammasome proteins were not affected by low pH. We show that IL-1β secretion in mouse macrophages is reversible upon restoration of physiological pH. pH sensitivity of NLRP3, NLRC4 and AIM2 inflammasomes appeared to be conferred by the processes upstream of the apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization and most likely contributed by the cell background rather than species-specific amino acid sequences of the sensor proteins

Supramolecular organizing centers at the interface of inflammation and neurodegeneration

The pathogenesis of neurodegenerative diseases involves the accumulation of misfolded protein aggregates. These deposits are both directly toxic to neurons, invoking loss of cell connectivity and cell death, and recognized by innate sensors that upon activation release neurotoxic cytokines, chemokines, and various reactive species. This neuroinflammation is propagated through signaling cascades where activated sensors/receptors, adaptors, and effectors associate into multiprotein complexes known as supramolecular organizing centers (SMOCs). This review provides a comprehensive overview of the SMOCs, involved in neuroinflammation and neurotoxicity, such as myddosomes, inflammasomes, and necrosomes, their assembly, and evidence for their involvement in common neurodegenerative diseases. We discuss the multifaceted role of neuroinflammation in the progression of neurodegeneration. Recent progress in the understanding of particular SMOC participation in common neurodegenerative diseases such as Alzheimer’s disease offers novel therapeutic strategies for currently absent disease-modifying treatments

Shikonin Suppresses NLRP3 and AIM2 Inflammasomes by Direct Inhibition of Caspase-1

<div><p>Shikonin is a highly lipophilic naphtoquinone found in the roots of <i>Lithospermum erythrorhizon</i> used for its pleiotropic effects in traditional Chinese medicine. Based on its reported antipyretic and anti-inflammatory properties, we investigated whether shikonin suppresses the activation of NLRP3 inflammasome. Inflammasomes are cytosolic protein complexes that serve as scaffolds for recruitment and activation of caspase-1, which, in turn, results in cleavage and secretion of proinflammatory cytokines IL-1β and IL-18. NLRP3 inflammasome activation involves two steps: priming, i.e. the activation of NF-κB pathway, and inflammasome assembly. While shikonin has previously been reported to suppress the priming step, we demonstrated that shikonin also inhibits the second step of inflammasome activation induced by soluble and particulate NLRP3 instigators in primed immortalized murine bone marrow-derived macrophages. Shikonin decreased NLRP3 inflammasome activation in response to nigericin more potently than acetylshikonin. Our results showed that shikonin also inhibits AIM2 inflammasome activation by double stranded DNA. Shikonin inhibited ASC speck formation and caspase-1 activation in murine macrophages and suppressed the activity of isolated caspase-1, demonstrating that it directly targets caspase-1. Complexing shikonin with β-lactoglobulin reduced its toxicity while preserving the inhibitory effect on NLRP3 inflammasome activation, suggesting that shikonin with improved bioavailability might be interesting for therapeutic applications in inflammasome-mediated conditions.</p></div

Complexing Shikonin with β-lactoglobulin Retains the Inhibitory Activity.

<p>iBMDMs were primed with 100 ng/mL LPS overnight or were left untreated (ctrl). The next day, they were activated with 10 μM nigericin. The complex of shikonin with β-lactoglobulin (molar ratio 1:100) (100–0 μM in shikonin) was applied to cells 30 min before activation with nigericin. (B) Cells exposed to samples of shikonin complexed with β-lactoglobulin showed conserved metabolic activity below 30 μM shikonin. Representative of 2 independent experiments is shown. Error bars represent SD of triplicate wells.</p

Shikonin is More Potent Inhibitor of IL-1β Maturation than Acetylshikonin.

<p>Shikonin (left) or acetylshikonin (right) was applied to cells 30 min before priming with 100 ng/mL LPS (A) or 30 min before activation with 10 μM nigericin (B). Supernatants were collected 1 h after activation with nigericin and assayed using IL-1β ELISA. Representative of three experiments is shown. Error bars represent SD of triplicate wells. Molecular structures of shikonin (left) and acetylshikonin (right) are shown above the corresponding results.</p

Shikonin Inhibits IL-1β Release from Macrophages Treated with Activators of NLRP3 Inflammasome and AIM2 Inflammasome.

<p>iBMDMs were primed with 100 ng/mL LPS for 12 h or left unprimed (ctrl). They were subjected to shikonin (2.5–0 μM) or DMSO (solvent control) 30 min before activation with 20 μg/mL silica (A) for 6 h, 0.5 mg/mL alum (B) for 12 h, 20 μg/mL imiquimod (C) for 24 h, 5 mM ATP (D) for 1 h or 1 μg/mL dAdT (E) for 1 h. Concentrations of mature IL-1β in culture media were determined using IL-1β ELISA. Representative of 3 (A to D) or 2 (E) independent experiments is shown. Error bars represent SD of triplicate wells.</p