Quantification of NLRP3–caspase-1 complex assembly reveals a non-linear association with 30-day mortality in sepsis: a proof-of-concept pilot study

BACKGROUND: Direct, cell-resolved quantification of NLRP3 inflammasome activation in clinical patients has not been feasible. Previous studies have relied on indirect surrogates such as downstream cytokines, which are influenced by inflammasome-independent pathways and systemic clearance kinetics, potentially explaining the inconsistent prognostic findings reported in the literature. We applied a proximity ligation assay (PLA) to directly visualise NLRP3-caspase-1 complex formation in peripheral blood mononuclear cells of septic patients, providing a cell-specific, in situ readout of inflammasome assembly at the single-cell level. METHODS: In this prospective, multicentre proof-of-concept pilot study, 78 patients fulfilling Sepsis-3 criteria were enrolled across three university hospitals. The PLA was validated in THP-1-derived macrophages against established readouts of canonical inflammasome activation prior to clinical application. Patients were stratified into low (n = 28), intermediate (n = 23), and high (n = 27) NLRP3 inflammasome activity groups. RESULTS: Median age was 64 years (IQR: 56-78), 63% were male, and median Sequential Organ Failure Assessment score was 8 (IQR: 4-12). Baseline characteristics, disease severity scores, and routine inflammatory biomarkers did not differ across activity groups. Kaplan-Meier analysis revealed a non-linear pattern of 30-day survival, with the most favourable outcomes in the intermediate activity group. Both low and high activity were associated with increased mortality (log-rank p = 0.003). In adjusted Cox regression, low inflammasome activity (HR 7.88, 95% CI 1.79-34.65; p = 0.006) and high inflammasome activity (HR 4.35, 95% CI 1.01-19.88; p = 0.048) remained associated with 30-day mortality. Modelling PLA activity as a continuous variable using restricted cubic splines confirmed a significant non-linear association with 30-day mortality, independent of the tertile-based categorisation (p for non-linearity = 0.015). CONCLUSIONS: This proof-of-concept pilot study demonstrates that direct in situ quantification of NLRP3-caspase-1 complex assembly is feasible in septic patients and reveals a non-linear association with 30-day survival. These findings indicate that PLA-based profiling captures immune heterogeneity beyond routine inflammatory biomarkers. Larger independent cohorts are needed to validate these findings before clinical application or patient stratification can be considered.