Analysis of NLRP3 activation in human conjunctival myofibroblasts cultivated under in vitro dry eye conditions

Abstract

First of all, I would like to thank my two directors, Amalia and Alfredo, for warmly welcoming me into their group more than a year and a half ago. It has been a pleasure working with you, because you have always made everything easier for me, showing constant dedication, kindness, and a willingness to help whenever I needed it. Your guidance has been indispensable throughout this journey. With you, I have learned not only new techniques and skills in the laboratory, but also how a research group works, with all its changes and unexpected challenges. Thank you for trusting me from the very beginning. I would also like to thank my academic tutor, Teresa, for her continuous support and for guiding me through all the administrative aspects during this process. Her attentiveness made everything significantly easier. I also want to thank my family for being by my side during the ups and downs of this project. Your support and understanding gave me the strength to complete this work, even when things got difficult. Your support has kept me motivated and helped me keep moving forward. Thank you for always teaching me to give my best. Lastly, I want to thank my friends for always listening, offering their best advice, and helping me organise my ideas and overcome doubts. You were an important part of this process too, and I truly appreciate everything you have done for me during these past four years. University has been one of the best stages of my life, just as everyone says, and it would not have been the same without you by my side. Without all of you, this would not have been possible. Dry eye disease (DED) is a prevalent and multifactorial disorder of the ocular surface, characterised by tear film instability and chronic inflammation. Recent studies have implicated inflammasomes, particularly the NOD-like receptor family, pyrin domain containing 3 (NLRP3), in the pathogenesis of DED. In this study, we investigated the effects of hyperosmolar (500 mOsM) and inflammatory (25 ng/mL TNF-) conditions on NLRP3 gene expression in conjunctival epithelial cells and conjunctival fibroblasts, exposed to stimulated conjunctival epithelial cells secretome, using an in vitro model. NLRP3 gene expression was analysed by RT-qPCR with relative mRNA levels assessed using the 2(−ΔΔCt) method and statistical significance determined via parametric, unpaired Student’s t-test with equal variance and a two-tailed distribution. Our results demonstrated that epithelial cells exposed to these stressors significantly increased NLRP3 expression, suggesting activation of inflammatory signalling pathways. However, the secretome from stressed epithelial cells did not induce a comparable response in conjunctival fibroblasts, indicating that paracrine signalling alone may not be sufficient to activate NLRP3 in these cells under the tested conditions. These findings highlight the need for further research into the epithelial–mesenchymal communication in DED, including protein-level analysis of the secretome and investigation of additional inflammasome components, to better understand the contribution of fibroblasts to chronic ocular surface inflammation.

La enfermedad de ojo seco (EOS) es un trastorno prevalente y multifactorial de la superficie ocular, caracterizado por inestabilidad de la película lagrimal e inflamación crónica. Estudios recientes han implicado a los inflamasomas, en particular la familia de receptores tipo NOD con dominio de pirina, miembro 3 (NLRP3), en la fisiopatología de la EOS. En este estudio, investigamos los efectos de las condiciones hiperosmolares (500 mOsM) e inflamatorias (25 ng/mL TNF-) sobre la expresión del gen NLRP3 en células epiteliales conjuntivales y fibroblastos conjuntivales, expuestos al secretoma de las células epiteliales conjuntivales estimuladas, utilizando un modelo in vitro. La expresión del gen NLRP3 fue analizada mediante RT-qPCR, evaluando los niveles relativos de ARNm mediante el método 2(−ΔΔCt) y determinando la significancia estadística mediante una prueba t de Student paramétrica, no pareada, con varianzas iguales y distribución bilateral. Nuestros resultados mostraron que las células epiteliales expuestas a estos estímulos incrementan significativamente la expresión de NLRP3, lo que sugiere la activación de vías de señalización inflamatorias. Sin embargo, el secretoma procedente de células epiteliales sometidas a estrés no indujo una respuesta comparable en los fibroblastos conjuntivales, lo que indica que la señalización paracrina por sí sola podría no ser suficiente para activar el NLRP3 en estas células bajo las condiciones evaluadas. Estos hallazgos subrayan la necesidad de profundizar en el estudio de la comunicación epitelio-mesenquimal en la EOS, incluyendo el análisis proteico del secretoma y la investigación de otros componentes del inflamasoma, para comprender mejor la contribución de los fibroblastos a la inflamación crónica de la superficie ocular.