Study of Inflammasome Activation in Autoinflammatory Diseases and Tendinopathies

Resumen

For the present Thesis the following specific objectives were proposed: 1. Determine the effect of autoinflammatory-associated NLRC4 mutations on the structure of NLRC4. 2. Evaluate the activation of the NLRC4 and NLRP3 inflammasome by fluorescence microscopy. 3. Characterize the effect of galvanic current application in the activation of the NLRP3 inflammasome in macrophages. 4. Study the implication of the NLRP3 inflammasome in the inflammation and regeneration responses in the Achilles tendon of mice after percutaneous electrolysis application. 5. Elucidate the role of NLRP3 inflammasome in a mouse model of sterile tissue damage. During this project, in vitro studies were performed using cell cultures of wild-type macrophages and macrophages deficient in different components of the inflammasome. After activation of these macrophages with LPS and galvanic current, the release of different pro-inflammatory cytokines such as IL-1β or IL-18 was determined, as well as the release of LDH and the uptake of YoPro-1. In addition, fluorescence microscopy was also used, as well as the BRET technique to study the oligomerization and conformation, respectively, of the inflammasome. On the other hand, ex vivo studies were carried out using blood from patients with autoinflammatory diseases to isolate peripheral blood mononuclear cells, specifically activate NLRP3 with LPS+ATP and NLRC4 with LPS+FlaTox, and measure the release of IL-1β, IL-18, IL-6 and TNF-α by ELISA, and the formation of ASC oligomers by cytometry. In vivo studies were also performed using wild-type, Nlrp3-/- and Pycard-/- mice to measure the expression of different pro-inflammatory cytokines, and the type, orientation and strength of collagen fibers by picrosirius red staining, second harmonic microscopy and biomechanical tests, after applying percutaneous electrolysis or dry needling. In addition, measurement of the expression and production of IL-1β, IL-18, TNF-α and IL-6 by qPCR and ELISA, respectively; as well as quantification of polymorphonuclear cells by hematoxylin-eosin staining, after inducing tendon damage following application of collagenase enzyme, was performed. Finally, percutaneous electrolysis or dry needling were performed in collagenase-treated tendons. After this Thesis, we obtained the following conclusions: 1. Mutations affecting the WHD domain of NLRC4 induce significant changes in the conformation of NLRC4. 2. Different NLRC4 mutants described in patients with autoinflammatory syndromes induce a puncta distribution of NLRC4 in the cells. 3. The p.Ser171Phe NLRC4 variant present a gain-of-function behavior in assembly an inflammasome. 4. The postzygotic p.Ser171Phe NLRC4 variant is a plausible cause of the autoinflammatory disease in the enrolled patient. 5. Galvanic current applicated in HEK293T cells expressing NLRP3 results in a puncta distribution of NLRP3 in the cells. 6. Galvanic current applicated in LPS-primed macrophages is able to activate the NLRP3 inflammasome. 7. Galvanic current applicated in LPS-primed macrophages induce the release of IL-1β and IL-18 cytokines. 8. Galvanic current applicated in LPS-primed macrophages do not induce pyroptotic cell death. 9. Increasing the time and the number of pulses in galvanic current applicated to in LPS-primed macrophages is related with an increase in IL-1β release and also in current-dependent cell death. 10. Percutaneous needle electrolysis applicated in the Achilles tendon of mice induces an increase in type I collagen and tendon stiffness, improving tendon resistance. 11. Tendon resistance after percutaneous needle electrolysis is dependent on the NLRP3 inflammasome. 12. Collagenase administration in the Achilles tendon of mice induces a sterile tissue damage and an inflammatory response partially dependent on NLRP3. 13. Collagenase damage of the Achilles tendon of mice is not affected by the application of percutaneous needle electrolysis by delivering 3 times every 3 days with 3 impacts of 3 mA for 3 seconds.