Regulación del inflamasoma NLRP3 en enfermedades autoinflamatorias y su implicaciónen el metabolismo

Laburpena

The innate immune system is the body's first defense barrier and is made up of many cells that contain receptors capable of recognizing a wide variety of danger signals. Within these receptors, it is worth highlighting the role of the intracellular receptor NLRP3, which forms a complex called inflammasome, together with the proteins ASC and caspase-1, which is essential for the inflammatory response. This inflammasome is the most characterized to date due to the wide variety of signals it can recognize and its involvement in the development of numerous pathologies, one of them being cryopyrin-associated periodic syndrome (CAPS). This syndrome is a rare autoinflammatory disease that arises from gain-of-function mutations in the NLRP3 gene, which leads to excessive activation of the NLRP3 inflammasome and overproduction of proinflammatory cytokines such as IL-1β. Patients with this syndrome suffer sporadic sterile inflammatory episodes, and lack effective therapies, which focus on IL-1 blockers. This inflammasome often presents as a hypersensitive inflammasome; however, the mechanism of activation and regulation of mutant NLRP3 is not fully described. On the other hand, recent studies describe the importance of metabolism on immune responses, in fact, numerous studies show the relevance of the glycolysis pathway for the correct development of inflammation, however, how the associated pathogenic NLRP3 variants can affect to CAPS to metabolism, is not described. As a main objective, this Thesis aims to elucidate the mechanism of activation of the NLRP3 inflammasome with pathogenic variants associated with CAPS and how this affects metabolism. In addition, the aim is to discover treatments for CAPS syndrome, thus determining the effect of 4-octylitaconate and identifying new NLRP3 inhibitors. In this Thesis, the activation of the NLRP3 inflammasome with variants associated with CAPS has been evaluated in patient samples and in immortalized mouse macrophages that express, through a doxycycline-inducible system, wild-type NLRP3 and different pathogenic variants (p.R260W, p.D303N, p .T348M). These samples have been used in numerous techniques, including flow cytometry, ELISA, Western blot, microscopy, and transcriptomic and metabolomic analysis, among others. The results of this Thesis reveal that the pathogenic variants of NLRP3 associated with CAPS induce a constitutive activation of the inflammasome, causing caspase1 activation, gasdermin processing, IL-18 release and pyroptosis. Furthermore, it is observed that both the induction of NF-kB and deubiquitinases are involved in its regulation. On the other hand, it has been described that the expression of pathogenic variants of NLRP3 produces metabolic reprogramming that affects glycolysis, decreasing the production of IL-1β. This Thesis has also focused on the search for new possible treatments, observing that the use of 4-octylitaconate inhibited the constitutive activation of NLRP3 with pathogenic variants, thus blocking the formation of the NLRP3-ASC complex. In addition, a screening was carried out on 26 molecules that were computationally selected as possible inhibitors of NLRP3. Of these molecules, it was detected that E1 and E3 were capable of inhibiting the wild-type NLRP3 inflammasome and with pathogenic variants associated with CAPS. In conclusion, it should be noted that this Thesis describes to a large extent the mechanism of activation of the mutant NLRP3 inflammasome, observing that the pathogenic variants of NLRP3 associated with CAPS present a constitutive activation of the inflammasome, producing metabolic reprogramming that affects glycolysis, which sheds new light on the possible development of new, more effective therapies.