Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder caused by somatic mutations in PIGA gene (PIGA-PNH). PNH red blood cells are susceptible to complement-mediated intravascular hemolysis because of a loss of the glycosylphosphatidyl-inositol (GPI) anchored proteins including complement regulatory proteins. Recently, a PNH patient caused by the PIGT mutations was reported (PIGT-PNH) and had unusual symptoms similar to autoinflammatory syndrome. Whereas PIGA is essential for the first step in the biosynthesis of GPI anchors, PIGT is involved in transfer of GPI to proteins; thus its defect causes accumulation of free GPI, non-protein linked GPI, in affected cells. It is likely that the free GPI accumulated in affected cells together with activated complement contribute to inflammasome activation. Here, we aimed to investigate whether free GPI together with complement activation can induce inflammasome activation in human monocytic cells in PIGT-PNH.

We generated THP-1 human monocytic cells in which PIGT or PIGA gene was deleted using the CRISPR/Cas9 system. Each knockout cell was rescued with a corresponding wild-type cDNA. Cells were differentiated into macrophages under RPMI-1640 medium containing PMA (100 ng/ml, 3h) in the presence of 10% fetal bovine serum. For induction of inflammasome activation, both of the priming signal and the stimulating signal are necessary. PMA-differentiated cells were primed for 5hr with Pam3CSK4 (200 ng/ml), which specifically activates the Toll-like receptor 2 (TLR 2), followed by ATP stimulation (5 mM, 1h), or cells were stimulated by acidified serum (21:1 of 0.4M HCl) alone, which activates alternative complement pathway. Heat-inactivated (56°C for 30 min) serum was used as a negative control. ELISA kits were used to measure the interleukin (IL) -1β levels in supernatants according to the manufacturers' instructions. Total RNA was extracted from cultured cells and quantitative Real-Time PCR (qRT-PCR) was performed for mRNA level of IL-1β and NLRP3.

Results are expressed as means ± SD of three independent experiments. Priming of PMA-differentiated WT, PIGT deficient (PIGTKO), and PIGA deficient (PIGAKO) THP-1 cells with Pam3CSK4, followed by ATP stimulation induced similar levels of IL-1β secretion (167.9±61.5, 144.3±19.7, 200.3±48.8 pg/1.6x105 cells). In contrast, when cells were stimulated with acidified serum alone, PIGTKO cells significantly enhanced IL-1β secretion compared to WT and PIGAKO cells, in which PIGTKO cells showed about 50- and 2-fold increase, respectively (23.7±2.2, 1221.8±91.6, 568.2±101 pg/1.6x105 cells; P<0.01 PIGTKO compered to PIGAKO). This elevated levels of IL-1β were decreased comparably to WT cells by the treatments with heat-inactivated acidified serum or anti-C5 monoclonal antibody, indicating IL-1β secretion was enhanced due to complement activation in PIGTKO and PIGAKO cells. PIGT and PIGA rescued cells showed similar levels of IL-1β to WT cells. Interestingly, qPCR revealed that PMA treatment alone induced transcription of proIL-1β and NLRP3 and further incubation of PIGTKO cells with acidified serum showed similar elevation in transcription compered to WT and PIGAKO cells.

Thus, our data demonstrated that complement activation works as both a priming signal and a stimulating signal for inflammasome activation in PIGAKO and PIGTKO THP-1 cells. The facts that the PIGT-PNH patients, but not PIGA-PNH patients, showed autoinflammatory symptoms and that PIGTKO THP-1 secreted higher level of L-1β than PIGAKO THP-1 cells upon stimulation with acidified serum, indicate that the free GPI accumulated in PIGTKO cells contribute to inflammasome activation together with complement activation.


Osato: Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura: Alexion Pharma GK: Honoraria, Research Funding. Ueda: Alexion Pharma GK: Honoraria, Research Funding. Kinoshita: Alexion Pharmaceuticals: Honoraria. Kanakura: Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.