Internalized components of membrane attack complexes disrupt proteostasis and acquire alarmin-like properties

Area of Science:

• Immunology
• Cell Biology
• Molecular Biology

Background:

• Membrane attack complexes (MACs) are known for inducing cell death.
• The pro-inflammatory roles of MAC components, particularly C9, are not fully understood.
• Endothelial cell (EC) activation is a key feature of inflammatory processes.

Purpose of the Study:

• To investigate the non-cytolytic functions of C9.
• To elucidate the mechanisms by which C9 contributes to inflammation.
• To identify the cellular pathways involved in C9-mediated endothelial cell activation.

Main Methods:

• Detection of intracellular C9 aggregates in patient tissues.
• Identification of NUMBL as a C9-binding protein using Rab35 effector pathway.
• Analysis of C9 aggrephagy involving ZFYVE21, RNF34, and LC3B.
• In vivo studies using three mouse models to assess the role of the ZFYVE21-RNF34 axis.
• Conditional knockout of ZFYVE21 in ECs to evaluate its impact on inflammation and tissue injury.

Main Results:

• C9 forms non-cytolytic intracellular aggregates in inflamed tissues, associated with EC activation.
• NUMBL mediates C9 internalization and endolysosomal trafficking.
• C9 aggregates undergo aggrephagy, activating NF-κB signaling.
• The ZFYVE21-RNF34 axis is essential for C9 aggrephagy and NF-κB-dependent EC activation in vivo.
• Loss of ZFYVE21 in ECs reduces C9 aggrephagy, systemic inflammation, and skin transplant injury.

Conclusions:

• C9, a MAC component, forms intracellular aggregates with alarmin-like properties.
• These C9 aggregates promote inflammation through NF-κB activation and EC activation, independent of cell death.
• The endolysosomal pathway and aggrephagy machinery, involving NUMBL, ZFYVE21, and RNF34, are critical for regulating C9-mediated inflammation.