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 5

IL-1β secretion from and binding of complement components to PIGT- and PIGA-defective THP-1 cells.

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IL-1β secretion from and binding of complement components to PIGT- and P...
(A) Complement-mediated IL-1β secretion from THP-1–derived macrophages. WT, PIGT-KO, and PIGA-KO cells were incubated with acidified serum (AS), heat-inactivated AS (H-AS), or AS containing anti-C5 mAb. Supernatant samples were collected after a 5-hour incubation and analyzed for IL-1β by ELISA. Mean ± SD of 3 independent experiments. (B) Reductions of IL-1β secretion by transfection of PIGT and PIGA cDNAs into PIGT-KO and PIGA-KO cells (PIGT-KO+T and PIGA-KO+A, respectively). Cells differentiated by PMA were either left untreated (PMA) or incubated with AS (AS) under similar conditions as described in A, and supernatants analyzed for IL-1β. Mean ± SD of 3 independent experiments. (C) Effect of inhibiting C5aR on IL-1β secretion from THP-1–derived macrophages. Cells were incubated with AS alone (no treat), or AS containing C5aR antagonist (W-54011) or anti-C5aR mAb. Supernatant was collected after 5 hours and analyzed for IL-1β by ELISA. Mean ± SD of duplicate samples from 2 independent experiments. (D) Detection of C3b fragments (left) and MAC (right) by flow cytometry on PMA-differentiated THP-1 macrophages after incubation with AS. Geometric mean fluorescence intensity of medium-treated cells was subtracted from that of AS-treated cells. Mean ± SD of 3 independent experiments. (E) IL-1β secretion from PIGT-KO THP-1 macrophages stimulated with C6- or C7-depleted AS. PIGT-KO THP-1 macrophages were incubated with AS, C6-depleted AS (–/C6de), C6de restored by C6 (+C6/C6de), C7-depleted AS (–/C7de), or C7de restored by C7 (+C7/C7de). Supernatant was collected after overnight incubation. Mean ± SD of triplicate samples from 2 independent experiments (normal and C6-depleted sera) and 1 experiment (C7-depleted serum). (F) Binding of C3b fragments (left) and MAC (right) on PIGT-KO and PIGT-SLC35A2 double KO THP-1 macrophages after AS treatments. (G) IL-1β secretion from PIGT-KO and PIGT-SLC35A2 double KO THP-1 macrophages after AS treatments. (H) Binding of C4d (top) and C3b fragments (bottom) on PIGT-KO and PIGA-KO THP-1 macrophages after AS treatments and inhibition by mannose. Two-tailed Student’s t test was used for analysis. Mean ± SD of 3 independent experiments.