Publicação
Monitoring the interplay between ER stress and STING signaling in the plasmacytoid dendritic cell line CAL-1
| Resumo: | Innate immune responses are fundamental to defend against bacterial and viral infections. Plasmacytoid Dendritic Cells (pDCs) are a subset of Dendritic Cells (DCs) crucial for anti-viral responses that quickly produce and secrete large amounts of type I interferon (IFN-I) upon the activation of either Toll-like Receptor (TLR) 7 or TLR9. Additionally, pDCs are also activated through the Cyclic GMP-AMP synthase (cGAS) – Stimulator of Interferon Genes (STING) pathway, by recognizing and responding to double-stranded DNA (dsDNA). STING is located in the Endoplasmic Reticulum (ER), an organelle that is prominent in highly secretory cells, including pDCs. Alterations in ER homeostasis trigger ER stress, characterized by accumulation of misfolded and unfolded proteins and subsequent activation of the Unfolded Protein Response (UPR), a key mechanism for pDC development. Since the discovery of STING, its relationship with ER stress has been explored, but with conflicting conclusions and no studies on pDCs. Therefore, the main objective of this thesis is to clarify the interplay between STING and ER stress in pDCs. For this, we characterized the response of a pDC cell line, CAL-1, to STING activation and ER stress induction, and investigated the ER stress - STING activation interplay. Upon stimulation with the STING ligands cGAMP and diABZI, CAL-1 cells exhibited a rapid STING response, but not PERK activation. On the other hand, treatment of CAL-1 with thapsigargin rapidly decreased protein synthesis and induced phosphorylation of PERK and eIF2a, followed by a recovery within two hours. Importantly, STING stimulation under ER stress conditions resulted in increased STING signaling and expression of IFN-I mRNA, in a PERK- dependent manner. To further investigate the molecular mechanisms underlying this effect, a proximity-based labelling assay was conducted with CAL-1-STING-TurboID cells, followed by mass spectrometry analysis to identify molecules interacting with STING. We explored the potential role of the identified candidates TAX1BP1, p62 and MAVS and found that deletion of MAVS potentiated even further the ER stress-promoted increase on STING signaling. In conclusion, the insights gained from this study, along with the developed tools and methodologies, contribute to a deeper understanding of STING and ER stress interactions. These findings could help elucidate how different cell types respond to these stimuli and how the pathways intersect, ultimately providing valuable knowledge for the development of potential therapeutic strategies targeting innate immune responses. |
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| Autores principais: | Pinheiro, Fátima Cátia Leite |
| Assunto: | Plasmacytoid dendritic cells STING Interferon-β Endoplasmic reticulum Stress Unfolded protein response |
| Ano: | 2025 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Aveiro |
| Idioma: | inglês |
| Origem: | RIA - Repositório Institucional da Universidade de Aveiro |
| Resumo: | Innate immune responses are fundamental to defend against bacterial and viral infections. Plasmacytoid Dendritic Cells (pDCs) are a subset of Dendritic Cells (DCs) crucial for anti-viral responses that quickly produce and secrete large amounts of type I interferon (IFN-I) upon the activation of either Toll-like Receptor (TLR) 7 or TLR9. Additionally, pDCs are also activated through the Cyclic GMP-AMP synthase (cGAS) – Stimulator of Interferon Genes (STING) pathway, by recognizing and responding to double-stranded DNA (dsDNA). STING is located in the Endoplasmic Reticulum (ER), an organelle that is prominent in highly secretory cells, including pDCs. Alterations in ER homeostasis trigger ER stress, characterized by accumulation of misfolded and unfolded proteins and subsequent activation of the Unfolded Protein Response (UPR), a key mechanism for pDC development. Since the discovery of STING, its relationship with ER stress has been explored, but with conflicting conclusions and no studies on pDCs. Therefore, the main objective of this thesis is to clarify the interplay between STING and ER stress in pDCs. For this, we characterized the response of a pDC cell line, CAL-1, to STING activation and ER stress induction, and investigated the ER stress - STING activation interplay. Upon stimulation with the STING ligands cGAMP and diABZI, CAL-1 cells exhibited a rapid STING response, but not PERK activation. On the other hand, treatment of CAL-1 with thapsigargin rapidly decreased protein synthesis and induced phosphorylation of PERK and eIF2a, followed by a recovery within two hours. Importantly, STING stimulation under ER stress conditions resulted in increased STING signaling and expression of IFN-I mRNA, in a PERK- dependent manner. To further investigate the molecular mechanisms underlying this effect, a proximity-based labelling assay was conducted with CAL-1-STING-TurboID cells, followed by mass spectrometry analysis to identify molecules interacting with STING. We explored the potential role of the identified candidates TAX1BP1, p62 and MAVS and found that deletion of MAVS potentiated even further the ER stress-promoted increase on STING signaling. In conclusion, the insights gained from this study, along with the developed tools and methodologies, contribute to a deeper understanding of STING and ER stress interactions. These findings could help elucidate how different cell types respond to these stimuli and how the pathways intersect, ultimately providing valuable knowledge for the development of potential therapeutic strategies targeting innate immune responses. |
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