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Case Reports
. 2019 Dec 2;129(12):5123-5136.
doi: 10.1172/JCI123501.

Complement and inflammasome overactivation mediates paroxysmal nocturnal hemoglobinuria with autoinflammation

Britta Höchsmann  1   2 , Yoshiko Murakami  3   4 , Makiko Osato  3   5 , Alexej Knaus  6 , Michi Kawamoto  7 , Norimitsu Inoue  8 , Tetsuya Hirata  3 , Shogo Murata  3   9 , Markus Anliker  1 , Thomas Eggermann  10 , Marten Jäger  11 , Ricarda Floettmann  11 , Alexander Höllein  12 , Sho Murase  7 , Yasutaka Ueda  5 , Jun-Ichi Nishimura  5 , Yuzuru Kanakura  5 , Nobuo Kohara  7 , Hubert Schrezenmeier  1 , Peter M Krawitz  6 , Taroh Kinoshita  3   4
Affiliations
Case Reports

Complement and inflammasome overactivation mediates paroxysmal nocturnal hemoglobinuria with autoinflammation

Britta Höchsmann et al. J Clin Invest. .

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.

Keywords: Complement; Glycobiology; Hematology; Inflammation.

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Conflict of interest statement

Conflict of interest: HS receives research support through the University of Ulm from Novartis and Alexion Pharmaceuticals.

Figures

Figure 1
Figure 1. Clinical features of PIGT-PNH.
(A) Schematics of normal and defective biosynthesis of GPI-APs. (Top) In normal cells, GPI is synthesized in the ER from phosphatidylinositol (PI) by sequential reactions and assembled GPI is attached to proteins (orange oval). PIGA acts in the first step whereas PIGT acts in attachment of GPI to proteins. GPI-APs are transported to the plasma membrane (PM). (Middle) No GPI biosynthesis in PNH caused by PIGA defect. (Bottom) Accumulation of free GPI in PNH cells caused by PIGT defect. Non–protein-linked GPI is transported to the PM. (B) Time course of PNH clone sizes in patients G1, G3, and J1. Percentages of PNH cells in monocytes, granulocytes, erythrocytes, and reticulocytes are plotted as a function of time in days. Arrows, start of eculizumab therapy. (C) Examples of urticaria in G3 before the start of the anakinra treatment are shown on the left (chest) and middle (left upper leg); hemoglobinuria in G3 is shown on the right. Brightness was adjusted in the bottom chest image to more clearly show raised skin in the affected area. The pictures were made available by the patient. (D) Clinical courses of G3 in comparison to J1 (Figure 1 in ref. was modified with additional data) including effective treatments. G3 (top) had meningitis 19 times between 62–65 years of age. Eculizumab therapy started at 66 years of age after a severe hemolysis. J1 (bottom) had meningitis 121 times between 53–69 years of age when eculizumab therapy started. Downward green arrows, onset of urticaria and/or arthralgia; blue middle height bars, meningitis; orange short bars, hemolysis; orange long bars, severe hemolysis; horizontal arrows of various lengths, treatment periods of effective therapies (anakinra and canakinumab were given with prednisolone); upward arrows with number and asterisk, serum samples taken for cytokine and other protein determination.
Figure 2
Figure 2. Genetic abnormalities in patients with PIGT-PNH.
(A) PIGT mutations in GPI-AP–positive (GPI +) and –defective (GPI –) cells from patients with PIGT-PNH. (Top) GPI + cells from patients J1, G1, G2, and G3 had a germline PIGT mutation (triangle) in the maternal (M) allele. Two maternally imprinted genes, L3MBTL1 and SGK2, within myeloid common deleted region (CDR) are expressed from the paternal (P) allele. Solid and broken red double arrows, P and M alleles of myeloid CDR, respectively. (Bottom) GPI blood cells from PIGT-PNH patients had an 8 Mb to 18 Mb deletion spanning myeloid CDR and PIGT and/or PIGU in the P chromosome 20q leading to losses of expression of L3MBTL1 and SGK2 genes (dotted boxes). (B) A 1.9-Mb region in chromosome 20q spanning PTPRT gene to OSER1 gene is termed myeloid common deleted region. PIGT and PIGU genes are approximately 1.2 Mb telomeric and 7.4 Mb centromeric to the myeloid CDR, respectively. L3MBTL1 and SGK2 genes marked # are maternally imprinted. (C) qRT-PCR analysis of genes within myeloid CDR in GPI-AP–defective granulocytes from J1 and granulocytes from a healthy control (top) and whole blood cells from G1 and a healthy control (bottom). L3MBTL1 and SGK2, maternally imprinted genes; IFT52 and MYBL2, nonimprinted genes. Relative expression is determined taking means of ABL levels as 1 (J1) or of GAPDH as 1 (G1). Blue bars, cells from J1 and G1; orange bars, cells from healthy individuals; * indicates below detection limits. Mean of duplicate (JI) and triplicate (G1) samples in 1 of the 2 independent experiments. Mean RQ max values for J1 samples were 0.15 (IFT52) and 0.17 (MYBL2), and for normal control samples were 0.034 (L3MBLT), 0.003 (SGK2), 0.078 (IFT52), and 0.002 (MYBL2). Mean RQ max values for G1 samples were 0.01 (SGK2), 0.004 (L3MBTL), and 0.01 (GAPDH), and for normal control samples were 0.08 (SGK2), 0.22 (L3MBTL), and 0.002 (GAPDH). (D) Methylation status of the CpG islands in L3MBTL1 in G1, G2, and G3. Red, methylated CpG islands; pink, unmethylated CpG islands; gray, unknown CpG islands. Bisulfite sequencing data are shown in Supplemental Figure 3A.
Figure 3
Figure 3. Biochemical abnormalities in PIGT-defective cells.
(A) Schematic representation of binding specificity of T5 mAb. T5 mAb recognizes mammalian free GPI bearing GalNAc-side chain linked to the first mannose (left). T5 mAb does not bind to free GPI when Gal is attached to GalNAc (right). Man, mannose; GlcN, glucosamine; EtNP, ethanolamine phosphate; PI, phosphatidylinositol. (B) Western blotting analysis of PIGT-defective and PIGL-defective CHO cells with T5 mAb for free GPI, anti-CD59, and anti-DAF mAbs, and anti-transferrin receptor (TfR) as loading controls. (C) Flow cytometry of PIGT-defective and PIGL-defective CHO cells with T5 mAb and anti-CD59 mAb. (D) Flow cytometry of erythrocytes from J1, G1, G3, a healthy individual, and 2 patients with PIGA-PNH with T5 mAb and anti-CD59 (top panels) or anti-CD58 (bottom panels). (E) Flow cytometry of blood cells from JI, a healthy individual, and a patient with PIGA-PNH with T5 mAb and FLAER. (F) Granulocytes and monocytes from G1 and G3, and a patient with PIGA-PNH, stained by T5 mAb and FLAER. (G) Determination of the PNH clone size in J1 by qPCR analysis of the break causing 18 Mbp deletion. (Left) Threshold cycle in PCR for the break and exon 3 of PIGL as a reference. #1, DNA from whole blood leukocytes taken in a stage with autoinflammation only (4 months before the onset of recurrent hemolysis); #2, DNA from granulocytes (29% of cells were GPI-AP–defective) taken 1 month after start of eculizumab therapy. (Right) Relative levels of the break in samples #1 and #2 by setting the level in #2 as 1. Data are shown in mean of triplicate samples in 1 experiment. Mean RQ max values for #1 and #2 samples were 0.092 and 1.11, respectively.
Figure 4
Figure 4. IL-1β secretion from PIGT-PNH and PIGA-PNH cells.
(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.
Figure 5
Figure 5. IL-1β secretion from and binding of complement components to PIGT- and PIGA-defective THP-1 cells.
(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.
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