Unbiased Analysis of the Functions and Interactions of Human Inflammasome Mutations

Background: Gain-of-Function (GOF) inflammasome mutations helped define the field of autoinflammation. Genotype/phenotype correlations both found within and between mutated genes that cause inflammasomopathies. We sought to identify features unique to NLRC4 GOF mutations and to develop a system for the unbiased assessment of Protein-Protein Interactions (PPi) made/lost by introduction of NLRC4 or NLRP3 inflammasome mutations in a myelomonocyte cell line. Methods: Part 1: Transduction of cell lines with WT or Mutant NLRC4 alone was preformed, followed by messenger RNA-sequencing and analysis. Part 2: Transduction of cell lines with WT or various of mutated forms of NLRP3 and NLRC4 was preformed to represent a diversity of clinical autoinflammatory phenotypes directly conjugated to the BirA biotinylase. Several experiments were conducted to assess biotinylation and confirm proper transduction. Interactome was established through the use of mass spectrometry. Results: Part 1: The most differentially expressed genes were evaluated. Unbiased GSEA analysis identified a strong IFN-induced gene signature among the most differentially expressed genes. Part 2: Inborn errors of immunity may be an initiating factor in the hyperactivation of the inflammasome. Public Health Relevance: The assessment of various mutations within the same gene or different genes thought to have similar functions may help to diagnose and investigate phenotypic outcomes of disease. Conclusion: NLRC4 may directly induce IFN production, potentially through a non-canonical pathway. Screening for interacting partners may identify the mechanism by which NLRP3 causes most IL-1β mediated disease and NLRC4 drives both Interleukin-1β (IL-1β) and Interleukin-18 (IL-18) production