Implication of the NLRP3, NLRC4 and Pyrin inflammasomes in inflammatory diseases and sepsis

Resumen

NLRP3 and NLRC4, along with the Pyrin protein, are present on cells of the innate immune system. When activated in monocytes, they trigger the release of pro-inflammatory cytokines through pyroptosis, a specific type of cell death. The incorrect activation of inflammasomes or mutations in the genes that encode them can alter their structure and lead to the formation of autoactivate complexes. Autoinflammatory diseases can develop as a result of this. Sepsis is one of the leading causes of death in hospital intensive care units. However, clinicians currently lack reliable biomarkers to assess the patient's prognosis at an early stage. Additionally, there is no effective therapy targeting the pathophysiological mechanisms of sepsis. So that, one of the main objectives of this Doctoral Thesis has been to characterize the inflammatory markers and the function of the NLRP3 and the Pyrin inflammasomes in septic patients. Moreover, to compare inflammatory markers between septic and COVID-19 patients. Other objectives pursued by this Doctoral Thesis has been to determine the function of NLRP3, NLRC4 and MEFV with pathogenic variants on inflammasome activation and to analyse the activation of the NLRP3 and Pyrin inflammasomes in CMML and MS patients before and after therapeutical treatments. In this Doctoral Thesis, the function of NLRP3, NLRC4, and Pyrin inflammasomes has been characterised in human blood samples. Specifically, blood samples were collected from sepsis patients within the first 24 hours of sepsis onset and from patients undergoing abdominal surgery without sepsis development. Additionally, patients carrying mutations associated with autoinflammatory diseases were studied. In addition, the use of specific NLRP3 inflammasome inhibitors, such as MCC950, has been evaluated in patients with relapsing-remitting multiple sclerosis and chronic myelomonocytic leukemia. Each study was accompanied by an analysis of healthy donors. Different techniques were used including the ELISA technique (determination of cytokines released by stimulated cells) and flow cytometry (determination of the percentage of monocytes with ASC oligomers). In addition, a battery of vectors carrying pathogenic mutations of NLRP3 and NLRC4 was generated. To study its functionality, HEK293T cells were transfected and its expression, degree of autoactivation (oligomerization), and inflammasome conformation were evaluated using Western Blot analysis, flow cytometry, and BRET technique, respectively. Regarding the study of NLRP3 and Pyrin inflammasomes in intraabdominal origin septic patients of, our study confirmed the previous results of our research group. We identified a group of septic patients with an impaired response of the NLRP3 inflammasome, but not the Pyrin inflammasome. However, none of the parameters analysed, such as biochemical markers, allowed us for the identification of this group of NLRP3 immunocompromised septic patients. Moreover, patients with impaired NLRP3 inflammasome activation who survived and recovered from sepsis improved their NLRP3 inflammasome response upon hospital discharge, indicating that NLRP3 immunosuppression is a transient state. Therefore, we have concluded that the survival of septic patients of intra-abdominal origin at day one is associated with an increase in canonical activation of the NLRP3 inflammasome in monocytes stimulated with LPS and ATP. The study of the COVID-19 patients showed that acute-phase proteins, procalcitonin and C-reactive protein, as well as the immune dysregulation biomarker, ferritin, increased with clinical disease severity. Additionally, plasma concentrations of cytokines IL-6, IL-1Ra, IL-15, and IL-18 were found elevated in most severe COVID-19 patients. Therefore, our findings suggest that IL-1-mediated signalling and its cytokines favour the immune response against bacterial infections such as sepsis. Additionally, during the development of this Doctoral Thesis, blood samples from patients with RRMS undergoing treatment with fingolimod were accessed. The evaluation of NLRP3 inflammasome activation in these patients revealed that fingolimod pharmacological treatment reduced the percentage of monocytes with ASC oligomers in responders after six months of treatment. However, in non-responders’ patients, there was not only an increase in the number of cells with ASC oligomers but also an increase in the pyroptosis cell death. Additionally, after six months of treatment, an increase in the production of TNF-α and IL-6 was observed in the supernatants of unstimulated peripheral blood mononuclear cells from non-responders’ patients. In the case of responder’s patients, the secreted level of these cytokines was even lower. These results suggest that pharmacological treatment with fingolimod not only inhibits the activation of the NLRP3 inflammasome but also decreases the NF-κB signalling pathway. Therefore, the lack of inhibition of NLRP3 inflammasome activation in patients with RRMS treated with fingolimod could identify individual’s refractory to treatment, thus emerging NLRP3 as a biomarker. Finally, this Doctoral Thesis cohort of patients with LMMC with or without mutations in the oncogene KRAS was analysed. In patients with LMMC and KRAS mutation, a high percentage (around 60%) of monocytes with ASC oligomers were observed under basal conditions. Furthermore, no increase in the release of IL-1β was observed after stimulation of the NLRP3 inflammasome with LPS and ATP. The specific NLRP3 inhibitor, MCC950, significantly reduced the levels of secreted IL-1β, but not the formation of ASC oligomers. This basal NLRP3 activation was not associated with an increase in pyroptosis-related death. Furthermore, it has been shown that anakinra treatment in a patient with LMMC and G12D mutation in KRAS not only reduced the number of monocytes with ASC oligomers ex vivo, but also the plasma concentration of ASC and the release of proinflammatory cytokines IL-1β and IL-18. Overall, our study showed that patients with LMMC and KRAS mutation present a basal activation of NLRP3 inflammasome. In addition, treatment with inhibitors of the IL-1 signalling pathway could be considered as a therapeutic