Elucidating inflammasome activation with alpaca single domain antibodies

• Applicant: Professor Dr. Florian Ingo Schmidt
• Subject Area: Immunology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 322568668

Myeloid cells as well as other sentinel cells integrate cytosolic evidence of infection or cell damage by assembling inflammasomes. To form these macromolecular signaling complexes, activated sensors oligomerize to enable the recruitment and autocatalytic activation of pro-caspase-1, often through the assembly of highly ordered assemblies of the adaptor protein ASC, visible as ASC foci. Caspase-1 activity is necessary for the maturation and secretion of the pro-inflammatory cytokines and/or pro-inflammatory cell death by pyroptosis. Inflammasome signaling is critical to contain infections and respond to cell damage, but has to be tightly controlled to avoid auto-inflammatory diseases.We know little about the molecular changes and features that activate sensors, organize the ASC focus, or control caspase-1 activation. This is mostly explained by the lack of tools to study the process in its native environment: Ablating inflammasome components often leads to the complete loss of the macromolecular structures. To overcome these challenges, I have developed an entirely novel approach to investigate inflammasomes: We will immunize alpacas to obtain single domain antibodies (VHHs or nanobodies), which can be expressed in the cytosol to specifically perturb protein function and visualize inflammasome activation in the relevant cell types. To identify VHHs that activate or inhibit distinct steps of inflammasome assembly, we will use a phenotypic screening approach that I have established. We will analyze the exemplary inflammasome sensor NLRP1 to reveal the domains and conformational changes necessary for ligand binding, sensor oligomerization, and nucleation of ASC. Using VHHs as perturbants and sophisticated microscopy tools, we will visualize the molecular interactions and architecture of ASC foci. We will address how ASC integrates input from multiple signals to recruit and control caspase-1 activity. Finally, we will employ the established VHH toolbox to investigate inflammasome activation in response to virus infection, an understudied physiological trigger. Using a representative panel of viruses encoding biosensors for inflammasome assembly, we will infect human and mouse primary cell types as well as selected cell lines. This will systematically define the repertoire of cells capable of assembling inflammasomes. We will identify the inflammasome sensors involved, the steps of the viral life cycle sensed, and analyze how different cell types and cellular factors collaborate to allow sensitive virus detection.The proposed research plan illuminates one overarching question: How can cells of our innate immune system deliver a perfectly balanced inflammatory response to cytosolic threats such as virus infections? Results from this study will contribute to our understanding of antiviral responses and auto-inflammatory diseases, and thus benefit basic and applied research.

• DFG Programme: Independent Junior Research Groups