Under the supervision of Jérôme Delon et Isabelle Meyts, team Dynamics of the cytoskeleton-dependent responses of immune cells

Abstract

Autoinflammatory syndromes (AIS) are rare genetic disorders characterised by uncontrolled systemic inflammation in the absence of infection. They are caused by innate immune dysregulation, often due to mutations affecting inflammatory pathways such as TNF, NF-ϰB, IL-1β and the inflammasome. Recent discoveries have identified new mutations, including C-terminal CDC42 variants (R186C, C188Y, 192C24), associated with severe phenotypes collectively termed NOCARH syndrome.By contrast, the N-terminal Y64C variant is associated with the Takenouchi-Kosaki syndrome, characterised by dysmorphic features, neurodevelopmental delay, macrothrombocytopenia and mild inflammation. CDC42, a RHO family GTPase, regulates crucial processes for immune function, including cytoskeletal dynamics, adhesion, migration, polarity, and cell cycle progression. Dysregulation of RHO GTPase signalling has been linked to immunodeficiencies and inflammatory diseases, although the underlying mechanisms remain unclear. This study examined CDC42 variants’ roles in autoinflammatory disorders, focusing on cytoskeletal and signalling abnormalities. The Y64C variant exhibited normal subcellular localisation and no alterations in actin polymerisation or NF-ϰB activity. Conversely, all three C-terminal variants showed aberrant Golgi/nuclear accumulation, reduced actin polymerization and increased NF-ϰB phosphorylation and translocation. Interestingly, actin depolymerisation did not cause NF-ϰB hyperactivation, suggesting independent mechanisms. Subsequent analyses investigated ER-Golgi trafficking, ER stress, STING activation, and type I interferon responses in patient-derived and THP-1 monocytic cells expressing CDC42 variants. The recurrent R186C variant disrupted bidirectional ER-Golgi transport, causing ER stress and STING accumulation in the Golgi via defective COPI-mediated retrograde transport. This led to STING overactivation and increased expression of interferon-stimulated genes (ISGs). A similar pattern was observed in 192C24, while Y64C and C188Y, which did not accumulate in the Golgi, failed to activate STING or induce ISGs. A novel heterozygous T43I variant has been identified in the N-terminal region of CDC42. Although it does not affect localisation, trafficking, or IFN-α, it triggers Pyrin inflammasome hyperactivation, likely via enhanced CDC42 binding to the Pyrin’s B30.2 domain, independently of NLRP3 activation. The findings indicate that CDC42 acts as a positive Pyrin inflammasome regulator, suggesting a dual system in which RHOA and CDC42 control Pyrin-mediated inflammation. The results demonstrate that CDC42 variant localisation exerts a significant influence on disease mechanisms, either by disrupting ER-Golgi trafficking and activating STING or by modulating Pyrin inflammasome activity. Furthermore, emphasis is placed on ER-Golgi trafficking defects and ER stress as pivotal immune dysregulation drivers in CDC42 Golgi-trapped variants, aligning with observations in COPI complex subunit mutations such as COPA and COPZ1. Both CDC42 and COPA-related diseases exhibit COPI-dependent retrograde transport defects, vital for cellular homeostasis and immune regulation. In COPA cases, this defect induces ER stress and activates NF-κB and type I interferon pathways. Notably, elevated IFN-α levels in CDC42-R186C carriers correlate with disease severity. These cases show concurrent hyperactivation of NF-κB, IFN-α, and Pyrin inflammasome pathways, suggesting multiple inflammatory cascades converge. Overall, these findings support developing combined therapeutic strategies targeting intracellular trafficking defects, innate immune hyperactivation, and Pyrin inflammasome regulation, potentially offering more effective treatments for AIS driven by CDC42 variants.

Keywords: Autoinflammation, Innate immunity, Cytoskeleton, Intracellular trafficking, COPI, RHO GTPases, CDC42