Complement and inflammasome overactivation mediates paroxysmal nocturnal hemoglobinuria with autoinflammation
Britta Höchsmann, … , Peter M. Krawitz, Taroh Kinoshita
Britta Höchsmann, … , Peter M. Krawitz, Taroh Kinoshita
Published August 20, 2019
Citation Information: J Clin Invest. 2019;129(12):5123-5136. https://doi.org/10.1172/JCI123501.
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Research Article Hematology Inflammation

Complement and inflammasome overactivation mediates paroxysmal nocturnal hemoglobinuria with autoinflammation

Abstract

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent autoinflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients’ leukocytes and of PIGT-KO THP-1 cells we show that, through increased IL-1β secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of autoinflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also autoinflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.

Authors

Britta Höchsmann, Yoshiko Murakami, Makiko Osato, Alexej Knaus, Michi Kawamoto, Norimitsu Inoue, Tetsuya Hirata, Shogo Murata, Markus Anliker, Thomas Eggermann, Marten Jäger, Ricarda Floettmann, Alexander Höllein, Sho Murase, Yasutaka Ueda, Jun-ichi Nishimura, Yuzuru Kanakura, Nobuo Kohara, Hubert Schrezenmeier, Peter M. Krawitz, Taroh Kinoshita

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Figure 4

IL-1β secretion from PIGT-PNH and PIGA-PNH cells.

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IL-1β secretion from PIGT-PNH and PIGA-PNH cells. (A) The peripheral blo...
(A) The peripheral blood mononuclear cells from JI, PIGA-PNH4, and a healthy individual were incubated with 10–100 ng/mL Pam3CSK4 (Pam3) at 37°C for 4 hours, and then were incubated with 1.5 mM ATP for 30 minutes. IL-1β in the supernatants was measured by ELISA (top) and Western blotting (bottom). (Left) J1 (red bars) and a healthy individual (blue bars). (Right) PIGA-PNH4 (green bars) and a healthy individual (blue bars). P10, P50, and P100, 10, 50, and 100 ng/mL Pam3CSK4, respectively; H, healthy donors; T, PIGT-PNH; A, PIGA-PNH. (B) The peripheral blood mononuclear cells from J1, 2 or 3 patients with PIGA-PNH, and 2 or 5 healthy controls were stimulated with 200 ng/mL of Pam3 or 1 μg/mL of lipoteichoic acid (LTA) from Staphylococcus aureus for 4 hours at 37°C, and after washing were incubated with 3 mM ATP or 200 μg/mL monosodium ureate (MSU) for 4 hours at 37°C. IL-1β secreted into the medium was determined by ELISA. Data for healthy donors and PIGA-PNH were shown as mean ± SD. Cells from 3 patients with PIGA-PNH (PIGA-PNH4-6) secreted very low levels of IL-1β (403 ± 326 pg/mL) after stimulation of NLRP3-inflammasomes with Pam3CSK4 and ATP under these strong conditions. In contrast, cells from PIGT-PNH J1 secreted IL-1β at a high level (25,000 pg/mL), a level that is higher than those from 3 healthy donors (6693 ± 1711 pg/mL). Similar results were obtained by stimulation with Pam3CSK4 and MSU instead of ATP. Moreover, similar results were obtained by stimulation with LTA plus ATP or MSU.