Abstract
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis and thrombosis, and bone marrow failure. Affected cells harbor somatic mutation in X-linked PIGA gene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis. Loss of GPI biosynthesis results in defective cell-surface expression of GPI-anchored complement regulators CD59 and DAF. The affected stem cells generate many abnormal blood cells after clonal expansion, which occurs under bone marrow failure. Here, we report the mechanistic basis of a disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH), caused by germline mutation plus somatic loss of PIGT on chromosome 20q. A region containing maternally imprinted genes implicated in clonal expansion in 20q-myeloproliferative syndromes was lost together with normal PIGT from paternal chromosome 20. Taking these findings together with a lack of bone marrow failure, the mechanisms of clonal expansion in AIF-PNH appear to differ from those in PNH. AIF-PNH is characterized by intravascular hemolysis and recurrent autoinflammation, such as urticaria, arthralgia, fever and aseptic meningitis. Consistent with PIGT’s essential role in synthesized GPI’s attachment to precursor proteins, non-protein-linked free GPIs appeared on the surface of PIGT-defective cells. PIGT-defective THP-1 cells accumulated higher levels of C3 fragments and C5b-9 complexes, and secreted more IL-1β than PIGA-defective cells after activation of the complement alternative pathway. IL-1β secretion was dependent upon C5b-9 complexes, accounting for the effectiveness of the anti-C5 drug eculizumab for both intravascular hemolysis and autoinflammation. These results suggest that free GPIs enhance complement activation and inflammasome-mediated IL-1β secretion.
Introduction
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell (HSC) disorder characterized by complement-mediated hemolysis, thrombosis and bone marrow failure (1, 2). Affected cells harbor a somatic mutation in the PIGA gene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis that occurs in the endoplasmic reticulum (ER)(Figure 1A(a)) (3). Loss of GPI biosynthesis results in the defective expression of GPI-anchored proteins (GPI-APs) including complement inhibitors CD59 and DAF/CD55 (Figure 1A(b)). The affected stem cells generate large numbers of abnormal blood cells after clonal expansion that occurs under bone marrow failure. The affected erythrocytes are defective in complement regulation and destroyed by the membrane attack complex (MAC or C5b-9) upon complement activation (1). Eculizumab, an anti-complement component 5 (C5) monoclonal antibody (mAb), has been used to prevent intravascular hemolysis and thrombosis (4, 5). Eculizumab binds to C5 and inhibits its activation and subsequent generation of C5b-9 complexes.
Among more than 20 genes involved in GPI biosynthesis and transfer to proteins, PIGA is X-linked whereas all others are autosomal (6). Because of X-linkage, one somatic mutation in PIGA causes GPI deficiency in both males and females (3). In contrast, two mutations are required for an autosomal gene, but the probability of somatic mutations in both alleles at the same locus is extremely low, which explains why GPI deficiency in most patients with PNH is caused by PIGA somatic mutations. Recently, we reported two patients with PNH whose GPI-AP deficiency was caused by germline and somatic mutations in the PIGT gene localized on chromosome 20q (7, 8). Both patients had a heterozygous germline loss-of-function mutation in PIGT, along with loss of the normal allele of PIGT by a deletion of 8 or 18 Mb occurring in HSCs (7, 8). PIGT, forming a GPI transamidase complex with PIGK, PIGS, PIGU and GPAA1, acts in the transfer of preassembled GPI to proteins in the ER (Figure 1A(a)) (9). In PIGT-defective cells, GPI is synthesized but is not transferred to precursor proteins, resulting in GPI-AP deficiency on the cell surface (Figure 1A(c)). We showed recently that non-protein-linked, free GPI remaining in the ER of PIGT-defective Chinese hamster ovary (CHO) cells is transported to and displayed on the cell surface (Figure 1A(c)) (10).
Two reported PNH patients with PIGT defect suffered from recurrent inflammatory symptoms that are unusual in patients with PNH (7, 8). Here, we report two more patients with PNH who lost PIGT function via a similar genetic mechanism, and present insights into the expansion of PIGT-defective clones common among four patients. We also present integrated clinical characteristics of these four patients and show that PIGT-defective mononuclear leukocytes, but not PIGA-defective mononuclear leukocytes, secreted IL1β in response to inflammasome activators. Using a PIGT-knockout THP-1 cell model, we show that complement activation is enhanced on the surface of PIGT-defective cells leading to MAC-dependent elevated secretion of IL1β. Against this background, we propose a distinct disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH).
Results
Case Report
Japanese patient J1 (8) and German patients G1 (7), G2, and G3 were diagnosed with PNH at the ages of 68, 49, 65, and 66, respectively. The changes in PNH clone sizes in J1, G1 and G3 after PNH diagnosis are shown in Figure 1B. They were treated with eculizumab, which effectively prevented intravascular hemolysis. We reported that in G1, direct Coombs test positive erythrocytes appeared after commencement of eculizumab treatment, suggesting extravascular hemolysis (7). Blood cell counts for G1 and G3 are shown in Figure S1A. Before the diagnosis of PNH, J1, G1 and G3 had suffered inflammatory symptoms including urticaria, arthralgia and fever from the ages of 30, 26, and 48, respectively (Table 1). Urticaria in J1 was associated with neutrophil infiltration (8) and that in G3 with a mixed inflammatory infiltrate (Figure 1C). J1 (8) and G3 suffered from recurrent aseptic meningitis characterized by an abundance of neutrophils in cerebrospinal fluid. Following the initiation of eculizumab treatment for hemolysis 3–5 years previously, J1 and G3 had not suffered any episodes of meningitis (Figure 1D). Urticaria and arthralgia were also ameliorated in all three by eculizumab treatment. G2 had severe arteriosclerosis, which might be related to autoinflammation (Table 1), however, whether G2 had autoinflammatory symptoms is unclear and could not be confirmed because the patient passed away.
Genetic basis of GPI-AP deficiency
Four patients did not have PIGA somatic mutations but had a germline mutation in one allele of PIGT located on chromosome 20q: J1, NM_015937 (8): c.250G>T; G1, c.1401-2A>G (7); G2, c.761_764delGAAA; and G3, c.197delA (Figure S2A). These cause E84X, exon 11 skipping, frameshift after G254, and frameshift after Y66, respectively. The functional activities of variant PIGT found in J1 and G1 were reported to be very low (7, 11). Variants in G2 and G3 causing frameshifts should also be severely deleterious to PIGT function. In addition to the germline PIGT mutation, all four had in the other allele a somatic deletion of 8–18 Mb, which includes the entire PIGT gene (Figure S2B) (7, 8). Therefore, in contrast to GPI-AP deficiency caused by a single PIGA somatic mutation in PNH, GPI-AP deficiency in all four is caused by a combination of germline loss-of-function PIGT mutation and somatic loss of the whole of normal PIGT in hematopoietic stem cells (Figure 2A).
A possible mechanism of clonal expansion in AIF-PNH
Deletion of chromosome 20q represents the most common chromosomal abnormality associated with myeloproliferative disorders. The deleted region of 8–18 Mb included a “myeloid common deleted region” (CDR) (12) (Figure 2A). A fraction (approximately 10%) of patients with myeloproliferative neoplasm (MPN) such as polycythemia vera commonly have a deletion of 2.7 Mb in chromosome 20q (12). Moreover, a fraction (approximately 4%) of patients with myelodysplastic syndrome (MDS) also have a deletion of 2.6 Mb in this region (12). The CDR region shared by MPN and MDS spanning approximately 1.9 Mb has been called “myeloid CDR” (Figure 2B) and its loss was shown to be causally related to clonal expansion of the affected myeloid cells in these 20q− syndromes (13). In contrast to previous cytogenetic analysis on classical PNH cases that showed no aberrations in 20q (14, 15), one allele of the myeloid CDR was lost in PNH cells of all four patients (Figure S2B) (7, 8).
The tumor suppressor-like gene L3MBTL1 and the kinase gene SGK2 located within the myeloid CDR (Figure 2A,B) are expressed only in the paternal allele due to gene imprinting (16). It was shown that losses of active paternal alleles of these two genes had a causal relationship with clonal expansion of these 20q− myeloid cells (13). L3MBTL1 and SGK2 transcripts were undetectable in GPI-AP-defective granulocytes from J1 and extremely low in whole blood cells from G1, whereas they were found in granulocytes from healthy individuals (Figure 2C). The transcripts of two unimprinted genes, IFT52 and MYBL2, were detected in both GPI-AP-defective granulocytes from J1 and normal granulocytes (Figure 2C, top). The results therefore indicate that the expression of L3MBTL1 and SGK2 is lost in GPI-AP-defective cells in J1 and G1.
The results shown in Figure 2C also indicate that the somatically deleted region in J1 and G1 included active L3MBTL1 and SGK2, so it was in the paternal chromosome. Owing to mRNA from patients G2 and G3 not being available, we determined the methylation status of the L3MBTL1 gene using DNA from blood leukocytes, among which the large majority of cells were of the PNH phenotype. L3MBTL1 in G1 and G3 samples was hypermethylated (Figure 2D and Figure S3A), indicating that the myeloid CDR allele remaining in their PNH clones was imprinted. In contrast, the G2 sample was hypomethylated. It was reported that, in some MPN patients with myeloid CDR deletion, the remaining allele was hypomethylated; nevertheless, its transcription was suppressed (13). G2 might be in a similar situation, although it was not possible to draw a definitive conclusion on this by RT-PCR analysis as the patient had passed away. These results indicate that the loss of expressed myeloid CDR allele is associated with clonal expansion of PIGT-defective cells similar to 20q− MPN and MDS.
Appearance of free GPI on the surface of PIGT-defective cells
PIGA is required for the first step in GPI biosynthesis (17); therefore, no GPI intermediate is generated in PIGA-defective cells (Figure 1A(b)). PIGT is involved in the attachment of GPI to proteins. GPI is synthesized in the ER, but is not used as a protein anchor in PIGT-defective cells (Figure 1A(c)). We used T5 mAb that recognizes free GPI, but not protein-bound GPI, as a probe to characterize free GPI (Figure 3A: see Methods for epitope and other characteristics of this antibody)(18, 19). Using T5 mAb in western blotting and flow cytometry, we first compared PIGT-defective CHO cells with PIGL-defective CHO cells, in which an early GPI biosynthetic step is defective, like in PIGA-defective cells. T5 mAb revealed a strong band of free GPI at a position corresponding to approximately 10 kDa in lysates of PIGT-defective cells but not of PIGL-defective cells (Figure 3B). DAF and CD59 were not detected in either mutant cell, confirming that un-GPI-anchored precursor proteins were degraded (Figure 3B) (20). T5 mAb stained the surface of PIGT-defective CHO cells but not PIGL-defective cells, confirming that free GPI transported to the cell surface is detectable by T5 mAb (Figure 3C).
We then analyzed blood cells from J1, G1, and G3, and from patients with PIGA-PNH by flow cytometry. All four patients had PNH-type blood cells defective in various GPI-APs (Figures 3D–F and S4A, B). Erythrocytes from J1, G1, and G3 contained 3%, 84%, and 60% PNH cells, respectively, and a sizable fraction of them (36%, 87%, and 87%, respectively) were stained by T5 mAb (Figure 3D). J1 had PNH cells in granulocytes (81%), monocytes (87%), and B-lymphocytes (54%), but not T-lymphocytes (<2%), as revealed by anti-CD59 and GPI-binding probe fluorescence-labeled nonlytic aerolysin (FLAER). Affected monocytes and B-lymphocytes were strongly stained by T5 mAb, whereas affected granulocytes were weakly but clearly stained (Figures 3E and S4B). Normal populations in granulocytes, monocytes and B-lymphocytes were not stained by T5 mAb (Figure 3E). Similar results, showing strong T5 staining of affected monocytes and granulocytes, were obtained with leukocytes from G1 and G3 (Figure 3F). In contrast, PNH cells from PIGA-PNH patients and cells from healthy individuals were not positively stained by T5 mAb (Figure 3E, F). Small fractions of wild-type erythrocytes from J1, G1, and G3 (0.13%, 9.7%, and 9.5%, respectively) were positively stained by T5 mAb (Figure 3D). Free GPI might be transferred from PNH cells to wild-type erythrocytes in vivo (21, 22), although the exact mechanism involved needs to be clarified. Thus, the surface expression of the T5 mAb epitope is specific for PIGT-defective cells and T5 mAb is useful to diagnose AIF-PNH.
We next investigated whether GPI-AP-defective clone was present in the stage only with autoinflammation. After determining the break points causing the deletion of 18 Mb in J1 (Figure S5), we quantitatively analyzed blood DNA samples for the presence of the break. It was estimated that approximately 3% of total leukocytes obtained 4 months before the onset of recurrent hemolysis had the break, that is, were GPI-AP-defective cells (Figure 3G).
Inflammasome- and complement-mediated autoinflammation, a feature of AIF-PNH
IL18 levels were elevated in serum samples taken from J1 before and after the commencement of eculizumab therapy (Table 2), suggesting a complement-independent phenomenon. Serum amyloid A was also elevated before eculizumab therapy, but was within the normal range after the commencement of eculizumab therapy (Table 2), suggesting that the elevation was complement-dependent. In G3, increased levels of soluble IL2 receptor and thymidine kinase before, but not after, the start of eculizumab therapy suggested autoinflammation (Table 2). Serum amyloid A (up to 10.5 μg/ml; normal range <5 μg/ml) was also elevated. Combination therapies of prednisolone with anakinra, an IL1 receptor antagonist, or canakinumab, a mAb against IL1β, were effective at reducing urticaria (but not arthralgia and meningitis episodes) of G3. In G1, the IL18 level was above the normal range during eculizumab therapy (195 pg/ml; normal range <150 pg/ml). These lines of evidence suggest that autoinflammatory symptoms are associated with inflammasome activation. We also measured IL18, serum amyloid A, and lactate dehydrogenase (LDH) in serum samples from four patients with PIGA-PNH who were not undergoing eculizumab therapy. The levels of IL18 (263–443 pg/ml; normal range <211 pg/ml) and serum amyloid A (5.0–8.3 μg/ml) were within or slightly higher than the normal ranges, whereas LDH levels were markedly elevated (Table 2). These results suggest that autoinflammation is a feature of AIF-PNH.
We next compared mononuclear cells from J1, patients with PIGA-PNH, and healthy donors for IL1β production upon stimulation by NLRP3-inflammasome activators (23). Cells from three PIGA-PNH patients secreted only very low levels of IL1β after stimulation by Pam3CSK4 (TLR2 ligand) and ATP or monosodium urate (MSU) (Figure 4A right and 4B). In contrast, cells from J1 secreted 45–60 times as much IL1β and the levels were even higher than those from healthy control cells (Figure 4A left and 4B). A similar difference between PIGT- and PIGA-defective cells was seen upon stimulation by lipoteichoic acid (LTA: another TLR2 ligand) and ATP or MSU (Figure 4B). Low IL1β response of PIGA-defective cells was predicted because they lack CD14, a GPI-anchored co-receptor of TLRs. However, PIGT-defective cells also lacking CD14 showed a strong IL1β response. These results indicate that NLRP3 inflammasomes are easily activated and support the idea that the presence of non-protein-linked free GPI is associated with efficient activation of NLRP3 inflammasomes, contributing to autoinflammatory symptoms in AIF-PNH.
To investigate the roles of complement in inflammasome activation in AIF-PNH, we switched to a model cell system because patients’ blood cells were easily damaged in vitro under conditions of complement activation. PIGTKO and PIGAKO cells were generated from human monocytic THP-1 cells (Figure S6A) and were differentiated to macrophages. They showed IL1β response comparable to those of authentic inflammasome activators (Figure S6B). To analyze the inflammasome response to activated complement, these THP-1-derived macrophages were stimulated with acidified serum (AS), which causes activation of the alternative complement pathway. PIGTKO and PIGAKO cells but not WT cells secreted IL1β (1221.8±91.6, 568.2±101 and 23.7±2.2 pg/ml for PIGTKO, PIGAKO, and WT cells, respectively) (Figure 5A). This result is consistent with impaired complement regulatory activities on PIGTKO and PIGAKO cells, and normal complement regulatory activity on WT cells. PIGTKO cells secreted approximately twice as much IL1β as PIGAKO cells (p<0.01). However, IL1β production returned to near the WT cell level after the transfection of PIGT and PIGA cDNAs into PIGTKO and PIGAKO cells, respectively (Figure 5B). The levels of IL1β mRNA and protein were comparable in WT, PIGTKO, and PIGAKO cells (Figure S7A and S7B). Therefore, PIGT KO enhanced the secretion but not the generation of IL1β.
Heat inactivation of complement and the addition of anti-C5 mAb to AS almost completely inhibited IL1β secretion (Figure 5A). These results indicate that IL1β secretion requires the activation of C5 on PIGTKO and PIGAKO cells. The activation of C5 leads to two biologically active products, C5a and MAC (24). To address which of these is important for IL1β secretion, cells were treated with the C5aR antagonist W-54011 (25) or anti-C5aR mAb to inhibit the signal transduction through C5aR. WT, PIGTKO, and PIGAKO cells expressed C5aR at similar levels (Figure S7C). The two methods of functional inhibition of C5aR had little effect on IL1β secretion, indicating that the signal through C5aR plays no major role in this cell system (Figure 5C). Next, AS-treated cells were analyzed for surface binding of C3b fragments and MAC. Exposure to AS resulted in the higher binding of C3b fragments and MAC on PIGTKO cells compared with that on PIGAKO cells (Figures 5D and S8A, B). The level of MAC was several times higher on PIGTKO cells than on PIGAKO cells, suggesting that complement activation was enhanced, leading to the enhanced formation of MAC on PIGTKO cells. To confirm the role of MAC in IL1β secretion, PIGTKO cells were treated with acidified C6- and C7-depleted sera, in which C5a generation is intact whereas MAC formation is impaired. IL1β secretion was greatly reduced by C6 or C7 depletion and was restored by the replenishment of C6 or C7 (Figure 5E). These results suggest that MAC but not C5a plays a critical role in the secretion of IL1β. It is also suggested that free GPI plays some role in complement activation, leading to the enhanced binding of C3b fragments and MAC formation.
Finally, to determine whether the structure of free GPI (presence or absence of Gal capping) affects complement activation and subsequent IL1β secretion, we knocked out SLC35A2 in PIGTKO THP-1 cells. PIGT-SLC35A2 double-KO THP-1 cells were strongly stained by T5 mAb as expected (Figure S6C). The binding of both C3b fragments and MAC increased approximately five times after SLC35A2 KO (Figure 5F). Concomitantly, the secretion of IL1β more than doubled (Figure 5G). These results indicate that the structure of free GPI influenced complement activation efficiency and subsequent IL1β secretion.
Discussion
We studied patients with PNH caused by PIGT mutations and propose that they represent a new disease entity, AIF-PNH. AIF-PNH caused by PIGT mutation is distinct from PNH in four regards. First, GPI-AP deficiency in PNH is caused by somatic mutation of the X-linked PIGA gene in hematopoietic stem cells, whereas GPI-AP deficiency in AIF-PNH is caused by a germline heterozygous mutation in the PIGT gene on chromosome 20q in combination with somatic deletion of the normal PIGT gene in hematopoietic stem cells.
Second, PIGA mutations cause a defect in the initial step in GPI biosynthesis, whereas PIGT mutations cause a defect in the transfer of preassembled GPI to proteins. Therefore, free GPI remains in PIGT-defective cells, but not in PIGA-defective ones.
Third, the expansion of PIGA-defective clones in PNH is often caused by selective survival under autoimmune bone marrow failure with or without the acquisition of benign tumor characteristics by additional somatic mutations (26–29). In contrast, none of the patients with AIF-PNH had documented bone marrow failure (Figure S1A) (7, 8). In addition, the myeloid CDR is lost in PIGT-defective clones in AIF-PNH, similar to the case in clonal cells in myeloproliferative 20q− syndromes. The causal relationship between the myeloid CDR loss in AIF-PNH and clonal expansion needs to be proven, particularly because boosted lineages under L3MBTL1 and SGK2 loss differ between in vitro study (13) and AIF-PNH patients. Nevertheless, this unique deletion occurs in AIF-PNH but not in PIGA-PNH. Taking these findings together, it is likely that the mechanism of clonal expansion for AIF-PNH is distinct from that for PIGA-PNH cells (see models in Figure S3B).
Fourth, whereas AIF-PNH shares intravascular hemolysis and thrombosis with PNH, AIF-PNH is characterized by autoinflammatory symptoms including recurrent urticaria, arthralgia and aseptic meningitis. AIF-PNH first manifested with autoinflammatory symptoms alone and symptoms of PNH became apparent many years later. It is possible that different clinical symptoms appear depending on the size of the PIGT-defective clone. When the clone size is small, autoinflammation but not PNH may occur and, when the clone size becomes sufficiently large, PNH may become apparent. The idea that the PIGT-defective clone is small when only autoinflammation is seen was supported by analyzing J1 DNA obtained before the start of recurrent hemolysis, only approximately 3% of total leukocytes being PIGT-defective (Figure 3G).
C5 activation must be involved in the autoinflammatory symptoms in AIF-PNH because they were suppressed by eculizumab. It is important to consider GPI-AP deficiency for patients with recurrent autoinflammatory symptoms such as aseptic meningitis even when PNH symptoms are absent because eculizumab may be effective for such cases. Because DAF and CD59 are missing on PIGT-defective monocytes, C5a and MAC might be generated once complement activation has been initiated. It was reported that subarachnoidal application of C5a in rabbits and rats induced acute experimental meningitis (30). Various types of myeloid cells are present in the central nervous system (reviewed in (31)). If C5 activation occurs on some of those cells lacking complement regulatory function and C5a is generated, aseptic meningitis might ensue.
The involvement of complement in inflammasome activation has been shown in various blood cell systems (32–35). Indeed, in the THP-1 cell model system, IL1β secretion was induced by complement in both PIGTKO and PIGAKO cells and more strongly in PIGTKO cells, mainly through MAC formation. It appeared that complement activation is enhanced in PIGTKO cells, although the mechanism involved in this is unclear. AIF-PNH mononuclear cells were activated by conventional stimulators of inflammasomes similar to or even stronger than healthy control cells. Because blood mononuclear cells were easily lysed by acidified serum, the effect of complement on inflammasome activation in mononuclear cells could not be addressed. Taking the obtained findings together with the results for THP-1 cells, we speculate that PIGT-deficient monocytes show an enhanced inflammasome response when complement is activated. How free GPI is involved in inflammasome and complement activation needs to be clarified to fully understand the mechanistic basis of AIF-PNH.
Inflammatory symptoms, recurrent urticaria, arthralgia, fever, and especially meningitis seen in AIF-PNH are shared by children with cryopyrinopathies or cryopyrin-associated periodic syndrome (reviewed by Neven et al (36)). Cryopyrinopathies are caused by gain-of-function mutation in NLRP3 that leads to easy activation of NLRP3 inflammasomes in monocytes and autoinflammatory symptoms (36), further suggesting that inflammasomes are activated in monocytes from AIF-PNH patients. It was reported that autoinflammation occurs in patients having mosaicism with NLRP3-mutant cells even when the mutant clone size is small (frequency of mutant allele in whole blood cells being 4.3% to 6.5%) (37). This is relevant to the symptoms/clone size relationship in AIF-PNH as discussed above.
PIGU is an essential component of GPI transamidase, forming a protein complex with PIGT, PIGS, PIGK, and GPAA1 (Figure 1A) (38). PIGU-defective cells do not express GPI-APs on their surface (38). The PIGU gene is localized at approximately 7.4 Mb centromeric to the myeloid CDR (Figure 2A and 2B), and regions of somatic deletions of 18 and 12 Mb in GPI-AP-defective cells from J1 and G2, respectively, included the entire PIGU gene as well as myeloid CDR and PIGT gene. The levels of PIGU protein in these cells would be around half of the normal levels. It appears unlikely that the 50% reduction in PIGU has a significant impact on these cells. The levels of PIGT protein in the same cells would be zero or very low because mutations in the remaining PIGT gene are a nonsense mutation (E84X) in J1 and a frameshift mutation (frameshift after G254) in G2. For any remaining PIGT protein, half of the normal amounts of PIGU protein would be excessive for making the GPI transamidase complex. However, it is conceivable that, if a similar somatic deletion including PIGU and myeloid CDR occurs in a hematopoietic stem cell of an individual who bears a germline PIGU loss-of-function mutation, AIF-PNH caused by PIGU mutation might occur.
Germline PIGT mutations were reported in patients with IGD (Table S2), which is characterized by developmental delay, seizures, hypotonia, and typical facial dysmorphism (11, 39-43). Inflammatory symptoms and intravascular hemolysis were not reported in IGD patients with PIGT mutations. They had either partial loss-of-function homozygous mutations (families 1, 4, and 6), or combinations of a partial loss-of-function mutation and a null or nearly null mutation (families 2, 3, 5, and 7). Therefore, cells from the patients with IGD have only partially reduced PIGT activities and express only partially reduced levels of CD59 and DAF/CD55, and may have free GPI only to a small extent. In contrast, both germline and somatic mutants in AIF-PNH were functionally null or nearly null (Table 1). Therefore, the affected cells from AIF-PNH patients lost CD59 and DAF/CD55 severely or completely and had high levels of free GPI. Interestingly, the same mutation c.250G>T (p.E84X) was found in J1 and two Japanese patients with IGD (11, 43) who were not related to each other. AIF-PNH patient J1 and mothers of two IGD patients from families 2 and 7 had the same heterozygous non-sense PIGT mutation (Table S2). These mothers were healthy and no inflammatory symptoms were reported for them (11, 43), suggesting that autoinflammation of J1 was not caused by haploinsufficiency but was initiated after the somatic loss of the other PIGT copy occurred. In addition, the reported allele frequency of this variant PIGT in the East Asian population is 0.0002316, suggesting that 55,000 Japanese may have this variant (44). Although inheritance of the germline PIGT mutations in AIF-PNH patients is not formally proven because DNA samples were not available from their families, it is highly likely that J1 inherited the PIGT variant from his mother.
FLAER is a fluorescent non-lytic variant of aerolysin (45) and is conveniently used to stain cell-surface GPI-APs and to determine the affected cells in patients with PNH (45, 46). Aerolysin specifically binds to the GPI moiety of some but not all GPI-APs, and requires simultaneous association with N-glycan for high-affinity binding (47–49). Our results with AIF-PNH cells (Figure 3E, F) indicate that FLAER binds to protein-bound GPI but not to free GPI.
Materials and Methods
Blood samples and flow cytometry
Peripheral blood samples were obtained from patients J1 (8), G1 (7), G2 and G3 with AIF-PNH, and six patients with PNH after informed consent. Peripheral blood leukocytes (Figure S1B), erythrocytes and reticulocytes were stained for GPI-APs. T5-4E10 mAb (T5 mAb) against free GPI of Toxoplasma gondii was a gift from Dr. J. F. Dubremetz (18). T5 mAb recognizes mammalian free GPI bearing N-acetylgalactosamine (GalNAc) side-chain linked to the first mannose (Figure 3A)(19). T5 mAb does not bind to free GPI when galactose (Gal) is attached to GalNAc. Therefore, reactivity of T5 mAb to free GPI is affected by an expression level of Gal transferase that attaches Gal to the GalNAc. Cells were analyzed by a flow cytometer (MACSQuant Analyzer VYB or FACSCalibur) and FlowJo software.
DNA and RNA analyses
Granulocytes with PNH phenotype were separated from normal granulocytes by cell sorting after staining by FLAER. DNA was analyzed for mutations in genes involved in GPI-AP biosynthesis by target exome sequencing, followed by confirmation by Sanger sequencing (7). DNA was also analyzed by array comparative genomic hybridization for deletion (7). Methylation status of CpG was determined by bisulfite sequencing and a SNuPE assay (13). Total RNA was extracted with the RNeasy Mini Kit (Qiagen) including DNase digestion and DNA cleanup, and reverse transcription was performed with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Levels of L3MBTL1, SGK2, IFT52, MYBL2, ABL and GAPDH mRNAs were analyzed by quantitative real-time PCR (Table S1).
Cell lines
PIGT-defective CHO cells and PIGL-defective CHO cells were reported previously (11, 50). CRISPR/Cas 9 system was used to generate PIGT and PIGA knockout (KO) human monocytic THP-1 cells (ATCC) (Table S1 for guide RNA sequences). KO cells were FACS sorted for GPI-AP negative cells. Each knockout cell was rescued by transfection of a corresponding cDNA.
SLC35A2 gene was knocked out in PIGTKO THP-1 cells by CRISPR/Cas9 system.
Inflammasome activation and IL1β measurements
Toll-like receptor 2 (TLR2) ligands, Pam3CSK4 and Staphylococcus aureus LTA, are from InvivoGen (23, 51). ATP and MSU for activating inflammasomes are from Enzo Life Sciences and InvivoGen (23, 52). Peripheral blood mononuclear cells were stimulated by Pam3CSK4 or LTA for 4 hr at 37℃ and after washing by ATP or MSU for 4 hr at 37℃. IL1β ELISA kit (BioLegend) was used to measure IL1β secreted into the supernatants. Polyclonal rabbit anti-IL1β antibody for western blotting was from Cell Signaling Technology. PIGAKO, PIGTKO and wild-type THP-1 cells were differentiated into adherent macrophages in complete RPMI 1640 medium containing 100 ng/ml phorbol 12-myristate 13-acetate (PMA; InvivoGen) for 3 hr, and then with fresh complete medium for overnight (53). For stimulation, medium was replaced with serum free medium with Pam3CSK4 (200 ng/ml), followed 4hr-later by ATP stimulation for 4 hr (5 mM).
Stimulation of THP-1-derived macrophages with complement
As a source of complement, whole blood was collected from healthy donors after informed consent, and serum separated, aliquoted and stored at −80°C prior to use. Inactivation of complement was carried out by heating serum at 56°C for 30 min. To prepare acidified serum (AS) that allows activation of the alternative pathway on the cell surface, 21 volumes of serum was mixed with 1 volume of 0.4 M HCl to have pH of approximately 6.7. C6- and C7-depleted sera and purified C6 and C7 proteins were purchased from Complement Technology. Differentiated cells were stimulated with acidified normal serum, or acidified C6-depleted and C7-depleted sera, and those reconstituted with C6 and C7, respectively, at 37°C for 5hr and secreted IL1β was measured by ELISA. C5 was inhibited by addition of 35 μg/ml anti-C5 mAb (eculizumab, Alexion Pharmaceuticals).
For ex vivo blockade of human C5aR, anti-human C5aR or nonpeptide C5aR antagonist W-54011 (5 μM, Merck Millipore)(25) was used. Complement C3 fragments and MAC deposited on the cells were measured by flow cytometry. THP-1 cells were suspended in 20 µl FACS buffer (PBS, 1% BSA, 0.05% sodium azide) with 1:20 human TruStain FcXTM (Fc receptor blocking solution) at room temperature for 10 min. Cells were stained with anti-C3/C3b/iC3b/C3d mAb (clone 1H8, BioLegend) or rabbit anti-human SC5b-9 (MAC) polyclonal antibodies (Complement Technology) in FACS buffer. After washing twice, cells were incubated with the PE-conjugated goat anti-mouse IgG (BioLegend) or Alexa Fluor488-conjugated goat anti-rabbit IgG (Thermo Fisher) secondary antibody. The anti-human SC5b-9 polyclonal antibodies positively stained PMA-differentiated THP-1 cells without incubation in AS. The same antibodies did not stain similarly differentiated PIGTKO and PIGAKO THP-1 cells, suggesting that the antibody product contained antibodies reacted with some GPI-AP expressed on THP-1-derived macrophages (Figure S8B, C). Because of this reactivity to non-MAC antigen(s), the anti-SC5b-9 antibodies were used for PIGTKO and PIGAKO cells but not for WT cells in experiments shown in Figure 5D and F.
Statistical analyses
All experiments with THP-1 cells were performed at least three times. All values were expressed as the mean ± SD of individual samples. For two-group comparisons between PIGTKO and PIGAKO cells, Student’s t-test was used. P values below 0.05 were considered statistically significant.
Study approval
This study was approved by institutional review boards of Osaka University (approval number 681), University of Ulm (approval numbers 279/09 and 188/16) and University of Berlin (approval number EA2/077/12).
Data Sharing Statement
All data supporting the findings are available from the corresponding authors.
Authorship Contributions
BH, YM, NI, HS, PMK, and TK designed research. YM, MO, AK, TH, Shogo M, TE, MJ, RF, and AH performed research. BH, MK, MA, Sho M, YU, and NK acquired the data. BH, YM, MO, MK, JN, YK, NK, HS, and PMK analyzed data. BH, YM, MO, HS, PMK, and TK wrote the paper.
Conflict-of-interest statements
BH, YU, TK: honoraria (Alexion Pharma); JN: honoraria (Alexion Pharma), research funding (Japan PNH Study Group); YK: research funding (Chugai Pharmaceutical), honoraria (Alexion Pharma); HS: honoraria and research support (all to University of Ulm from Alexion Pharma).
Acknowledgements
We thank Drs. Morihisa Fujita (Jiangnan University), Tatsutoshi Nakahata (Kyoto University), Hidenori Ohnishi (Gifu University), Tatsuya Saitoh (Tokushima University) and Yusuke Maeda (Osaka University) for discussion, Dr. Jean-Francois Dubremetz (Montpellier University) for T5-4E10 mAb, and Keiko Kinoshita, Kana Miyanagi, Saori Umeshita and Miguel Rodriguez de los Santos for technical help and Dr. med. Lisa A. Gerdes (Munich University) for collaboration regarding patients G3 as well as the patients for providing blood samples and pictures. We thank Edanz (www.edanzediting.co.jp) for editing the English text of a draft of this manuscript. This work was supported by JSPS and MEXT KAKENHI grants (JP16H04753 and JP17H06422) to TK and a grant from the Japan Society of Complement Research to YM.
Footnotes
* equally contributed corresponding authors.