Complement- and in ammasome-mediated autoin ammation ...
1
Britta Hoechsmann 1* , Yoshiko Murakami 2,3* , Makiko Osato 2,4* , Alexej Knaus 5 , Michi Kawamoto 6 , Norimitsu Inoue 7 , Tetsuya Hirata 2 , Shogo Murata 2,8 , Markus Anliker 1 , Thomas Eggerman 9 , Severin Dicks 9 , Marten Jaeger 10 , Ricarda Floettmann 11 , Alexander Hoellein 12 , Sho Murase 6 , Yasutaka Ueda 4 , Jun-ichi Nishimura 4 , Yuzuru Kanakura 4 , Nobuo Kohara 6 , Hubert Schrezenmeier 1+ , Peter M. Krawitz 5+ , and Taroh Kinoshita 2,3+ ; * shared �rst authors + shared last authors Complement- and in�ammasome-mediated autoin�ammation-paroxysmal nocturnal hemoglobinuria 1 Institute of Transfusion Medicine, University of Ulm, 2 Research Institute for Microbial Diseases, Osaka University, 3 WPI Immunology Frontier Research Center, Osaka University, 4 Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, 5 Institute for Genomic Statistics and Bioinformatics, University Bonn, 6 Department of Neurology, Kobe City Medical Center General, 7 Osaka Medical Center for Cancer, 8 Department of Hematology/Oncology, Wakayama Medical University, 9 Institute for Human Genetics, RWTH, Aachen, 10 BIH Core Genomics Facility, Charité, University Medical Center, Berlin, 11 Institute for Medical Genetics and Human Genetics, Charité Medical Center, Berlin, 12 MLL Muenchner Leukaemielabor GmbH, Munich, Paroxysmal nocturnal hemoglobinuria (PNH) is a complement-mediated hemolytic disease caused by somatic mutations of X- linked PIGA in hematopoietic stem cells. We report autoin�ammation-PNH caused by germline and somatic mutations in PIGT localized in chromosome 20q. Autoin�ammation-PNH is characterized by intravascular hemolysis and in�ammasome-mediated auto-in�am- mation. Eculizumab prevented both hemolysis and autoin�ammation. PIGT-defective cells, but not PIGA- defective cells, accumulated free glycosylphosphatidyl- inositol (GPI), suggesting involvement of complement C5 and accumulated GPI in in�ammasome activation. The paternal “myeloid common deleted region” was always lost together with PIGT, implying a similar clonal expansion mechanism to 20q- myeloproliferative syndromes. We propose a new disease entity, autoin�ammation-PNH (AIF-PNH). Eculizumab Anakinra Canakinumab 70 30 Eculizumab Age J1 Onset (Urticaria, Arthralgia) 65 60 55 Onset (Urticaria) 60 65 55 50 48 G3 Age meningitis hemolysis big hemolysis *number, serum sample Figure 1: A. Clinical courses of patients G3 and J1 B. PIGT mutations in GPI-positive and -defective cells from patients with AIF-PNH: GPI-positive cells from patients with AIF-PNH J1, G1, G2 and G3 had a germline PIGT mutation (triangle) in the maternal (M) allele. Two maternally imprinted genes within myeloid Common Deleted Region (CDR) (red double arrow) are expressed from the paternal (P) allele. GPI-defective blood cells from AIF-PNH patients had an 8 Mb to 18 Mb deletion spanning myeloid CDR and PIGT leading to losses of expression of two maternally imprinted genes (white boxes). C. Methylation status of CpG islands in L3MBTL1 in G1, G2, and G3. D. Models of clonal expansion in PIGA- and AIF-PNH: In PNH, somatic mutation of PIGA gene in a hematopoietic stem cell generates GPI-defective clone (step 1). Under bone marrow failure conditions only GPI-defective clone survives (step 2). The GPI-defective subclone acquires benign-tumor-like growth phenotype and expands greatly (step 3). In addition to a maternal germ line mutation in PIGT a deletion spanning the entire PIGT and myeloid CDR is accuired in the paternal allele in a hematopoietic stem cell, generating GPI-defective clone (step 1). Because of losses of maternally imprinted L3MBL1 and SGK2 genes, the GPI-defective clone obtains a competence to expand and initially contributes to non-erythrocytic myeloid cells causing recurrent autoin�ammation. Years later, GPI-defective clone began to generate GPI-defective erythrocytes, causing PNH phenotype. A. B. C. 40 50Mb 45 35 30 Centro Myeloid CDR 20q M P GPI + M P GPI - 8– 18 Mb J1, G1, G2 and G3 PIGT mutation L3MBTL SGK2 IFT52 MYBL2 ER PIGA PIGT PI (a) Wild type PM (c) AIF-PNH ER PM (b) PIGA-PNH ER PM T5 mAb GalNAc 100bp CpG islands in L3MBTL1 Patient G1 Patient G3 Patient G2 Legend: unmethylated CpG island methylated CpG island unknown CpG status E x o n 5 D. 0 1000 2000 3000 HD PIGT 0 2000 4000 6000 HD PIGA Pam3 0 100 10 25 50 100 ATP 1.5 0 1.5 1.5 1.5 1.5 Pam3 0 100 25 50 100 ATP 1.5 0 1.5 1.5 1.5 15 20 (kDa) P100A H T P50A H T P10A H T P100 H T A H T A H A P100 H A P100A H A IL1β (pg/ml) 0" 400" 800" 1200" IL1β (pg/ml) AS H-AS anti-C5 AS PIGTKO Wild PIGAKO 0" 1000" 2000" 3000" 4000" Fluorescence intensity MAC C3b fragments 0" 100" 200" 300" PIGTKO Wild PIGAKO Figure 2 (left): Normal and defective GPI synthesis: (a) GPI is synthesized in the ER from phosphatidylinositol (PI) by sequential reactions and attached to proteins (orange oval). PIGA acts in the �rst step of GPI biosynthesis whereas PIGT acts in attachment of GPI to proteins. GPI-APs are transported from ER to the plasma membrane (PM). (b) No GPI biosynthesis in PNH cells caused by PIGA defect. (c) Accumulation of free GPI in PNH cells caused by PIGT defect. N-acetylgalactosamine (GalNAc) is exposed and reactive to T5 mAb. Figure 3 (right): Flow cytometry of blood from JI, a healthy individual, and a PIGA-PNH patient with T5 mAb and FLAER. AIF-PNH can be diagnosed using the T5 mAb. Figure 4:A. IL1b production by peripheral blood mononuclear cells from JI, PIGA- PNH4 and a healthy individual. Cells incubated with Pam3CSK4 and ATP. IL1b in the supernatants was measured by ELISA and western blott. (Left) J1 (red bars) and a healthy individual (blue bars). (Right) PIGA-PNH (green bars) and a healthy individual. J1 secreted 45- to 60-times as much IL1b. PIGA defective cells do not respond due to lack of GPI linked CD14, supporting that accumulated free GPI activates NLRP3-in�ammasomes in PIGT de�cient cells. B. Complement-mediated IL1b secretion from THP-1-derived macrophages. WT, PIGTKO and PIGAKO cells incubated with acidi�ed serum (AS), heat- inactivated AS (H-AS), or AS containing anti-C5 mAb. C. Detection of C3b and MAC by �ow cytometry on PMA-differentiated THP-1 macrophages after incubation with AS. A. B. C. Hematopoietic stem cell GPI - cell Somatic mutation of PIGA immunological selection additional abnormality step 1 step 2 step 3 Full expansion of PNH cells PIGA-PNH GPI - cell Somatic mutation (Large deletion in 20q) step 1 step 2 Full expansion of PNH cells Germline mutation of PIGT gene Loss of L3MBTL and SGK2 PIGT-PNH 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 75.4 3.2 10.2 11.2 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 49.6 2.43 44.1 3.8 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0.313 0.481 97.3 1.89 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 13.9 0.747 17.6 67.8 T5 AIF- PNH J1 PIGA- PNH 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0.0226 2.7 96.6 0.685 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 0.249 99.1 0.651 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0.001 0.212 99.5 0.239 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0.0001 0.312 99.6 0.063 Normal Granulocytes Monocytes B cells T cells 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 FLAER
Transcript of Complement- and in ammasome-mediated autoin ammation ...
Britta Hoechsmann1*, Yoshiko Murakami2,3*, Makiko Osato2,4*, Alexej Knaus5, Michi Kawamoto6, Norimitsu Inoue7, Tetsuya Hirata2, Shogo Murata2,8, Markus Anliker1, Thomas Eggerman9, Severin Dicks9, Marten Jaeger10, Ricarda Floettmann11, Alexander Hoellein12, Sho Murase6, Yasutaka Ueda4, Jun-ichi Nishimura4, Yuzuru Kanakura4,
Nobuo Kohara6, Hubert Schrezenmeier1+, Peter M. Krawitz5+, and Taroh Kinoshita2,3+; *shared �rst authors+shared last authors
Complement- and in�ammasome-mediated autoin�ammation-paroxysmal nocturnal hemoglobinuria
1Institute of Transfusion Medicine, University of Ulm, 2Research Institute for Microbial Diseases, Osaka University, 3WPI Immunology Frontier Research Center, Osaka University, 4Department of Hematology and Oncology, Graduate School of Medicine, Osaka
University, 5Institute for Genomic Statistics and Bioinformatics, University Bonn, 6Department of Neurology, Kobe City Medical Center General, 7Osaka Medical Center for Cancer, 8Department of Hematology/Oncology, Wakayama Medical University,
9Institute for Human Genetics, RWTH, Aachen, 10BIH Core Genomics Facility, Charité, University Medical Center, Berlin, 11Institute for Medical Genetics and Human Genetics, Charité Medical Center, Berlin, 12MLL Muenchner Leukaemielabor GmbH, Munich,
Paroxysmal nocturnal hemoglobinuria
(PNH) is a complement-mediated hemolytic
disease caused by somatic mutations of X-
linked PIGA in hematopoietic stem cells. We
report autoin�ammation-PNH caused by
germline and somatic mutations in PIGT
localized in chromosome 20q.
Autoin�ammation-PNH is characterized by intravascular
hemolysis and in�ammasome-mediated auto-in�am-
mation. Eculizumab prevented both hemolysis and
autoin�ammation. PIGT-defective cells, but not PIGA-
defective cells, accumulated free glycosylphosphatidyl-
inositol (GPI), suggesting involvement of complement
C5 and accumulated GPI in in�ammasome activation.
The paternal “myeloid common
deleted region” was always lost
together with PIGT, implying a similar
clonal expansion mechanism to 20q-
myeloproliferative syndromes. We
propose a new disease entity,
autoin�ammation-PNH (AIF-PNH).
Eculizumab
Anakinra
Canakinumab
7030
Eculizumab
Age
J1Onset (Urticaria, Arthralgia)
656055
Onset (Urticaria)
60 65555048
G3
Age
meningitis hemolysis big hemolysis *number, serum sample
Figure 1: A. Clinical courses of patients G3 and J1 B. PIGT mutations in GPI-positive and -defective cells from patients with AIF-PNH: GPI-positive cells from patients with AIF-PNH J1, G1, G2 and G3 had a germline PIGT
mutation (triangle) in the maternal (M) allele. Two maternally imprinted genes within myeloid Common Deleted Region (CDR) (red double arrow) are expressed from the paternal (P) allele. GPI-defective blood cells from AIF-PNH patients
had an 8 Mb to 18 Mb deletion spanning myeloid CDR and PIGT leading to losses of expression of two maternally imprinted genes (white boxes). C. Methylation status of CpG islands in L3MBTL1 in G1, G2, and G3. D. Models of
clonal expansion in PIGA- and AIF-PNH: In PNH, somatic mutation of PIGA gene in a hematopoietic stem cell generates GPI-defective clone (step 1). Under bone marrow failure conditions only GPI-defective clone survives (step 2).
The GPI-defective subclone acquires benign-tumor-like growth phenotype and expands greatly (step 3). In addition to a maternal germ line mutation in PIGT a deletion spanning the entire PIGT and myeloid CDR is accuired in the
paternal allele in a hematopoietic stem cell, generating GPI-defective clone (step 1). Because of losses of maternally imprinted L3MBL1 and SGK2 genes, the GPI-defective clone obtains a competence to expand and initially contributes to
non-erythrocytic myeloid cells causing recurrent autoin�ammation. Years later, GPI-defective clone began to generate GPI-defective erythrocytes, causing PNH phenotype.
A. B.
C.
40 50Mb453530Centro
MyeloidCDR
20q
M
PGPI +
M
PGPI -
8 – 18 Mb
J1, G1, G2 and G3PIGTmutation
L3MBTL SGK2 IFT52 MYBL2
ER
PIGA
PIGT
PI
(a) Wild type
PM
(c) AIF-PNH
ER PM
(b) PIGA-PNH
ER PM
T5 mAb
GalNAc
100bpCpG islands in L3MBTL1
Patient G1
Patient G3
Patient G2
Legend:
unmethylated CpG island
methylated CpG island
unknown CpG status
Exon 5
D.
0
1000
2000
3000
HD PIGT
0
2000
4000
6000
HD PIGA
Pam3 0 100 10 25 50 100ATP 1.5 0 1.5 1.5 1.5 1.5
Pam3 0 100 25 50 100ATP 1.5 0 1.5 1.5 1.5
15
20(kDa)P100A
H TP50AH T
P10AH T
P100H T
AH T
AH A
P100H A
P100AH A
IL1β
(pg
/ml)
0"
400"
800"
1200"
IL1β
(pg
/ml)
AS H-AS anti-C5 AS
PIGTKOWild
PIGAKO
0"
1000"
2000"
3000"
4000"
Flu
ore
sc
en
ce
in
ten
sit
y
MACC3b fragments
0"
100"
200"
300"PIGTKO
Wild
PIGAKO
Figure 2 (left): Normal and defective GPI synthesis: (a) GPI is
synthesized in the ER from phosphatidylinositol (PI) by sequential
reactions and attached to proteins (orange oval). PIGA acts in the
�rst step of GPI biosynthesis whereas PIGT acts in attachment of GPI
to proteins. GPI-APs are transported from ER to the plasma
membrane (PM). (b) No GPI biosynthesis in PNH cells caused by PIGA
defect. (c) Accumulation of free GPI in PNH cells caused by PIGT
defect. N-acetylgalactosamine (GalNAc) is exposed and reactive to T5
mAb. Figure 3 (right): Flow cytometry of blood from JI, a healthy
individual, and a PIGA-PNH patient with T5 mAb and FLAER.
AIF-PNH can be diagnosed using the T5 mAb.
Figure 4:A. IL1b production by peripheral blood mononuclear cells from JI, PIGA-
PNH4 and a healthy individual. Cells incubated with Pam3CSK4 and ATP. IL1b in the
supernatants was measured by ELISA and western blott. (Left) J1 (red bars) and a healthy
individual (blue bars). (Right) PIGA-PNH (green bars) and a healthy individual. J1 secreted
45- to 60-times as much IL1b. PIGA defective cells do not respond due to lack of GPI linked
CD14, supporting that accumulated free GPI activates NLRP3-in�ammasomes in PIGT
de�cient cells. B. Complement-mediated IL1b secretion from THP-1-derived
macrophages. WT, PIGTKO and PIGAKO cells incubated with acidi�ed serum (AS), heat-
inactivated AS (H-AS), or AS containing anti-C5 mAb. C. Detection of C3b and MAC by �ow
cytometry on PMA-differentiated THP-1 macrophages after incubation with AS.
A. B.
C.
Hematopoieticstem cell
GPI- cell
Somatic mutationof PIGA immunological
selectionadditionalabnormality
step 1 step 2 step 3Full expansion
of PNH cells
PIGA-PNH
GPI- cell
Somatic mutation(Large deletion in 20q)
step 1 step 2Full expansionof PNH cells
Germline mutation ofPIGT gene
Loss of L3MBTLand SGK2
PIGT-PNH
0 102 103 104 105
0
102
103
104
105 75.4 3.2
10.211.20 102 103 104 105
0
102
103
104
105 49.6 2.43
44.13.80 102 103 104 105
0
102
103
104
105 0.313 0.481
97.31.890 102 103 104 105
0
102
103
104
105 13.9 0.747
17.667.8
T5
AIF-PNH J1
PIGA-PNH
0 102 103 104 105
0
102
103
104
105 0.0226 2.7
96.60.6850 102 103 104 105
0
102
103
104
105 0 0.249
99.10.6510 102 103 104 105
0
102
103
104
105 0.001 0.212
99.50.2390 102 103 104 105
0
102
103
104
105 0.0001 0.312
99.60.063
Normal
Granulocytes Monocytes B cells T cells
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
0 102 103 104 105
0
102
103
104
105
FLAER
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