NLRP3 inflammasome inhibition fails to prevent red blood cell lysis in PNH

Abstract

Abstract Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated red blood cell (RBC) lysis due to acquired deficiencies in complement regulatory proteins. A recent study in “Cell” (Chen et al., Cell, 2025) proposed a novel form of programmed RBC death in PNH “spectosis” driven by the NLRP3 inflammasome/caspase pathway leading to β-spectrin cleavage. The authors suggested that spectrin cleavage, beyond membrane attack complex (MAC, C5b-9), may represent an additional mechanism of hemolysis, and points to inflammasome signaling as a putative therapeutic target in PNH. However, extensive clinical and experimental evidence supports complement activation as the primary mediator of hemolysis in PNH. We systematically evaluated the pathogenic and therapeutic relevance of the NLRP3/caspase-dependent “spectosis” pathway in RBC lysis using patient-derived models. Methods: Primary PNH RBCs were incubated with ABO-compatible, complement-active serum with or without selective inhibitors. Experimental conditions included heat inactivated serum (56OC for 30 min to inactivate complement proteins), specific complement inhibitors including eculizumab (Ecu, terminal complement inhibitor, 0.34 µM ), factor d inhibitor (Fdi, alternative pathway 1 µM), and sutimlimab (Suti, 0.041µM), pan-caspase inhibitor (Z-VAD-FMK, 5 µM) and Caspase 8 inhibitor (Z-IETD-FMK, 5 µM), and NLRP3 inhibitor (MCC950, 5µM), and NLRP3 inflammasome activator (Nigericin, 1µM). ABO compatible serum was pre-treated with specific inhibitors or heat inactivated for 30 min. Next, RBCs were resuspended in 20% serum + 3mM GVB-MgEGTA, pH 6.4 and incubated for 2 hours at 37OC. After incubation, the degree of hemolysis was measured spectrophotometrically at 414 nm assessing lysis. RBCs incubated with buffer and water were used as a 0% and 100% lysis control respectively. PNH patients were stratified into group A, n=6 (high type III clone, >50%) and group B, n=3 (low/moderate type III clone, <40%). Results: Serum induced marked hemolysis in group A (84.1 ± 7.8% of maximal lysis [water, 100%]), which was significantly reduced by ecu (33.1 ± 11.8%). Fdi provided even greater protection (13.1 ± 6.6%). In contrast, classical pathway inhibition with sutimlimab (79.4 ± 11.7%), as well as caspase inhibition (Z-VAD-FMK: 78.8 ± 13.6%; Z-IETD-FMK: 81.7 ± 7.0%) and NLRP3 inflammasome inhibition (MCC950: 80.4 ± 8.0%), showed no significant reduction in lysis compared to untreated serum. Nigericin (85.8 ± 7.6%) and MCC950 combined with nigericin (86.2 ± 10.4%) similarly had no significant effect. In group B, due to lower type III clone size, serum-induced lysis was reduced (21.5 ± 8.4%). Both eculizumab (8.0 ± 4.3%) and Fdi (3.3 ± 2.9%) reduced hemolysis, with Fdi showing the greatest inhibition. Sutimlimab (16.2 ± 4.5%), Z-VAD-FMK (19.5 ± 6.2%), Z-IETD-FMK (19.5 ± 6.6%), and MCC950 (20.8 ± 8.7%) did not significantly impact lysis. Nigericin (31.2 ± 9.2%) and MCC950 plus nigericin (29.1 ± 8.8%) were associated with slightly increased lysis. Heat-inactivated serum showed consistently minimal lytic activity. Across both groups, only complement blockade was protective. Collectively, these results demonstrate that complement-mediated hemolysis remains the principal mechanism of RBC destruction in PNH, regardless of clone size, with both eculizumab and Fdi providing robust protection. As expected, sutimlimab had no protective effect in calcium-deficient buffer. Inhibition of caspases (pan-caspase or caspase-8) or the NLRP3 inflammasome (MCC950) had minimal impact on hemolysis in both high and low type III clone settings. Nigericin, an inflammasome activator, did not increase RBC lysis, and MCC950, alone or in combination with nigericin had no effect on hemolysis. Ongoing experiments are assessing β-spectrin cleavage in PNH RBCs by western blot and evaluating NLRP3 activation using potassium efflux assays in TF-1 cells with the IPG-2 potassium probe. Conclusion: Our findings reinforce complement inhibition as the primary clinical strategy for PNH, with little evidence supporting inflammasome/caspase targeting for hemolysis. Although inflammasome activation may contribute to PNH-associated inflammation, its role in RBC destruction appears negligible.