Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans. METHODS: We examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. RESULTS: We demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack. CONCLUSION: The Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.

Eculizumab Is Safe and Effective as a Long-term Treatment for Protein-losing Enteropathy Due to CD55 Deficiency.

OBJECTIVES: Loss of the complement inhibitor CD55 leads to a syndrome of early-onset protein-losing enteropathy (PLE), associated with intestinal lymphangiectasia and susceptibility to large-vein thrombosis. The in vitro and short-term treatment benefits of eculizumab (C5-inhibitor) therapy for CD55-deficiency have been previously demonstrated. Here we present the 18-months treatment outcomes for 3 CD55-deficiency patients with sustained therapeutic response. METHODS: Three CD55-deficiency patients received off-label eculizumab treatment. Clinical and laboratory treatment outcomes included frequency and consistency of bowl movements, weight, patient/parent reports of overall well-being, and serum albumin and total protein levels. Membrane attack complex deposition on leukocytes was tested by flow cytometry, before and during eculizumab treatment. RESULTS: Marked clinical improvement was noted in all 3 patients with resolution of PLE manifestations, that is, diarrhea, edema, malabsorption, overall well-being, growth, and quality of life. In correlation with the clinical observations, we observed progress in all laboratory outcome parameters, including increase in albumin and total protein levels, and up to 80% reduction in membrane attack complex deposition on leukocytes (P < 0.001). The progress persisted over 18 months of treatment without any severe adverse events. CONCLUSIONS: CD55-deficiency patients present with early-onset diarrhea, edema, severe hypoalbuminemia, abdominal pain, and malnutrition. Targeted therapy with the terminal complement inhibitor eculizumab has positive clinical and laboratory outcomes in PLE related to CD55 loss-of-function mutations, previously a life-threatening condition. Our results demonstrate the potential of genetic diagnosis to guide tailored treatment, and underscore the significant role of the complement system in the intestine.

Molecular pathogenesis of human CD59 deficiency.

OBJECTIVE: To characterize all 4 mutations described for CD59 congenital deficiency. METHODS: The 4 mutations, p.Cys64Tyr, p.Asp24Val, p.Asp24Valfs*, and p.Ala16Alafs*, were described in 13 individuals with CD59 malfunction. All 13 presented with recurrent Guillain-Barré syndrome or chronic inflammatory demyelinating polyneuropathy, recurrent strokes, and chronic hemolysis. Here, we track the molecular consequences of the 4 mutations and their effects on CD59 expression, localization, glycosylation, degradation, secretion, and function. Mutants were cloned and inserted into plasmids to analyze their expression, localization, and functionality. RESULTS: Immunolabeling of myc-tagged wild-type (WT) and mutant CD59 proteins revealed cell surface expression of p.Cys64Tyr and p.Asp24Val detected with the myc antibody, but no labeling by anti-CD59 antibodies. In contrast, frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* were detected only intracellularly and did not reach the cell surface. Western blot analysis showed normal glycosylation but mutant-specific secretion patterns. All mutants significantly increased MAC-dependent cell lysis compared with WT. In contrast to CD59 knockout mice previously used to characterize phenotypic effects of CD59 perturbation, all 4 hCD59 mutations generate CD59 proteins that are expressed and may function intracellularly (4) or on the cell membrane (2). None of the 4 CD59 mutants are detected by known anti-CD59 antibodies, including the 2 variants present on the cell membrane. None of the 4 inhibits membrane attack complex (MAC) formation. CONCLUSIONS: All 4 mutants generate nonfunctional CD59, 2 are expressed as cell surface proteins that may function in non-MAC-related interactions and 2 are expressed only intracellularly. Distinct secretion of soluble CD59 may have also a role in disease pathogenesis.

Prothrombotic mechanisms in patients with congenital p.Cys89Tyr mutation in CD59.

BACKGROUND: Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome. METHODS: Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5-0.9 µm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight. FINDINGS: 2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2-2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients. INTERPRETATION: Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

Demyelination, strokes, and eculizumab: Lessons from the congenital CD59 gene mutations.

Neurological symptoms of patients with p.Cys89Tyr mutation in the CD59 gene include recurrent peripheral neuropathy resembling Guillain-Barré syndrome, characterized by sensory-motor demyelinating neuropathy with secondary axonal damage and moderate enhancement of the nerve roots on spine MRI, together with recurrent strokes and retinal involvement. Three additional mutations in CD59, leading to loss of function, have been described, and overall, 12/12 (100%) of patients with any mutation presented with neurological symptoms; 11/12 (92%) patients presented with recurrent peripheral neuropathy, 6/12 (50%) with recurrent strokes, and 1/12 (8%) with retinal involvement. We review the possible thrombophilic profile associated with the mutations. In these patients, excessive intravascular hemolysis saturates scavenger mechanisms resulting in free hemoglobin in plasma that irreversibly reacts with nitric oxide to form nitrate and methemoglobin, leading to arterial thrombosis. CD59 loss of function is also one of the major thrombophilic mechanisms in patients with paroxysmal nocturnal hemoglobinuria. We then describe the relationship with demyelination. The lack of CD59 allows uncontrolled complement amplification following low-level spontaneous-, viral-, or post viral-induced complement activation, resulting in severe demyelination in the peripheral nervous system. It is interesting, and certainly encouraging, that after 3 years, following 4 patients with Cys89Tyr mutations who are treated with eculizumab, no strokes occurred and non-permanent neurological insults underwent resolution without any new neurological exacerbations.

Identification and Characterization of Two Human Monocyte-Derived Dendritic Cell Subpopulations with Different Functions in Dying Cell Clearance and Different Patterns of Cell Death.

Human monocyte-derived dendritic cells (mdDCs) are versatile cells that are used widely for research and experimental therapies. Although different culture conditions can affect their characteristics, there are no known subpopulations. Since monocytes differentiate into dendritic cells (DCs) in a variety of tissues and contexts, we asked whether they can give rise to different subpopulations. In this work we set out to characterize two human mdDC subpopulations that we identified and termed small (DC-S) and large (DC-L). Morphologically, DC-L are larger, more granular and have a more complex cell membrane. Phenotypically, DC-L show higher expression of a wide panel of surface molecules and stronger responses to maturation stimuli. Transcriptomic analysis confirmed their separate identities and findings were consistent with the phenotypes observed. Although they show similar apoptotic cell uptake, DC-L have different capabilities for phagocytosis, demonstrate better antigen processing, and have significantly better necrotic cell uptake. These subpopulations also have different patterns of cell death, with DC-L presenting an inflammatory, "dangerous" phenotype while DC-S mostly downregulate their surface markers upon cell death. Apoptotic cells induce an immune-suppressed phenotype, which becomes more pronounced among DC-L, especially after the addition of lipopolysaccharide. We propose that these two subpopulations correspond to inflammatory (DC-L) and steady-state (DC-S) DC classes that have been previously described in mice and humans.

Therapy with eculizumab for patients with CD59 p.Cys89Tyr mutation.

OBJECTIVE: The objective of this work was to report on the outcome of eculizumab treatment in pediatric patients with recurrent acute predominantly motor, demyelinating neuropathy with conduction block, and chronic hemolysis attributed to p.Cys89Tyr mutation in the CD59 gene. METHODS: Four patients were recruited from our new registry of patients with homozygosity for the p.Cys89Tyr mutation on CD59. Participants received repeated intravenous eculizumab. In this 24-month open-label phase IIa study, we aimed to determine whether eculizumab reduces chronic hemolysis, and cumulative doses of steroids and intravenous immunoglobulin (IVIG), and ameliorates neurological deficits, compared to pretreatment status. Treatment response was evaluated every 2 to 4 weeks over 104 weeks and included examination with gross motor scoring by American Spinal Injury Association Impairment Scale and Inflammatory Neuropathy Cause and Treatment disability score, laboratory examination, well-being [12-item Short Form Health Survey; SF-12]). Neurological relapses and cumulative dose of IVIGs and/or corticosteroids before and after treatment were documented. Red blood cells (RBCs) and neutrophils were stained to evaluate C5b-9 deposition. ClinicalTrials.gov: NCT01579838. RESULTS: Dramatic and significant neurological amelioration in the upper limbs and trunk with more-modest amelioration in the lower limbs was observed in all patients. Corticosteroid and IVIG treatment was completely stopped. No patient relapsed during treatment despite infections, and there were no hospital admissions. Decreased C3bi and C5b-9 deposition on RBCs and neutrophils was documented (p < 0.0001). The SF-12 health questionnaires indicated significant improvement (p < 0.003). INTERPRETATION: Eculizumab was safely administered to these patients. Marked clinical improvement suggests that eculizumab may be a life-saving treatment for patients with acute predominantly motor, demyelinating neuropathy with conduction block, and secondary axonal damage attributed to primary p.Cys89Tyr mutation in the CD59 gene. Ann Neurol 2016;80:708-717.

Devastating recurrent brain ischemic infarctions and retinal disease in pediatric patients with CD59 deficiency.

Identification of CD59 p.Cys89Tyr mutation in 5 patients from North-African Jewish origin presenting with chronic inflammatory demyelinating polyradiculoneuropathy like disease and chronic hemolysis, led us to reinvestigate an unsolved disease in 2 siblings from the same origin who died 17 years ago. The two patients carried the same CD59 gene mutation previously described by our group. These children had quiet similar disease course but in addition developed devastating recurrent brain infarctions, retinal and optic nerve involvement. Revising the brain autopsy of one of these patients confirmed the finding of multiple brain infarctions of different ages. CD59 protein expression was missing on brain endothelial cells by immunohistochemical staining. This new data expands the clinical spectrum of CD59 mutations and further emphasizes the need for its early detection and treatment.

Apoptotic cells induce NF-κB and inflammasome negative signaling.

As they undergo phagocytosis, most early apoptotic cells negatively regulate proinflammatory signaling and were suggested as a major mechanism in the resolution of inflammation. The dextran sulfate sodium model is generally viewed as an epithelial damage model suited to investigate innate immune responses. Macrophages primed with LPS and subsequently exposed to DSS secrete high levels of IL-1ß in an NLRP3-, ASC-, and caspase-1-dependent manner. The aim of this research was to test the therapeutic effect of a single dose of apoptotic cells in a DSS-colitis model and to explore possible mechanisms. Primary peritoneal macrophages, the DSS mice model, and Nlrp3-deficient mice, were used to assess the effect apoptotic cells on colitis. Immunohistochemistry, flow-cytometer, and western blots helped to explore the effect and mechanisms. Using a variety of NLRP3 triggering mechanisms, we show that apoptotic cells negatively regulate NF-κB and NLRP3 activation in primary peritoneal macrophages, at pre- and post-transcription levels, via inhibition of reactive oxygen species, lysosomal stabilization, and blocking K+ efflux. This property of apoptotic cells is demonstrated in a dramatic clinical, histological, and immunological amelioration of DSS colitis in Balb/c and B6 mice following a single administration of apoptotic cells.

Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack.

Bacteria, such as Fusobacterium nucleatum, are present in the tumor microenvironment. However, the immunological consequences of intra-tumoral bacteria remain unclear. Here, we have shown that natural killer (NK) cell killing of various tumors is inhibited in the presence of various F. nucleatum strains. Our data support that this F. nucleatum-mediated inhibition is mediated by human, but not by mouse TIGIT, an inhibitory receptor present on all human NK cells and on various T cells. Using a library of F. nucleatum mutants, we found that the Fap2 protein of F. nucleatum directly interacted with TIGIT, leading to the inhibition of NK cell cytotoxicity. We have further demonstrated that tumor-infiltrating lymphocytes expressed TIGIT and that T cell activities were also inhibited by F. nucleatum via Fap2. Our results identify a bacterium-dependent, tumor-immune evasion mechanism in which tumors exploit the Fap2 protein of F. nucleatum to inhibit immune cell activity via TIGIT.

The Thr224Asn mutation in the VPS45 gene is associated with the congenital neutropenia and primary myelofibrosis of infancy.

Severe congenital neutropenia as well as primary myelofibrosis are rare in infancy. Elucidation of the underlying mechanism is important because it extends our understanding of the more common adult forms of these disorders. Using homozygosity mapping followed by exome sequencing, we identified a Thr224Asn mutation in the VPS45 gene in infants from consanguineous families who suffered from life-threatening neutropenia, which was refractory to granulocyte CSF, from defective platelet aggregation and myelofibrosis. The mutation segregated in the families, was not present in controls, affected a highly conserved codon, and apparently destabilized the Vps45 protein, which was reduced in the patients' leukocytes. Introduction of the corresponding mutation into yeast resulted in reduced cellular levels of Vps45 and also of the cognate syntaxin Tlg2, which is required for membrane traffic through the endosomal system. A defect in the endosomal-lysosomal pathway, the homologous system in humans, was suggested by the absence of lysosomes in the patients' fibroblasts and by the depletion of α granules in their platelets. Importantly, accelerated apoptosis was observed in the patients' neutrophils and bone marrow. This is the first report of a Vps45-related disease in humans, manifesting by neutropenia, thrombasthenia, myelofibrosis, and progressive bone marrow failure.

CD59 deficiency is associated with chronic hemolysis and childhood relapsing immune-mediated polyneuropathy.

CD59 deficiency is a common finding in RBCs and WBCs in patients with chronic hemolysis suffering from paroxysmal nocturnal hemoglobinuria in which the acquired mutation in the PIGA gene leads to membrane loss of glycosylphosphatidylinositol-anchored membrane proteins, including CD59. The objective of the present study was to elucidate the molecular basis of childhood familial chronic Coombs-negative hemolysis and relapsing polyneuropathy presenting as chronic inflammatory demyelinating polyradiculoneuropathy in infants of North-African Jewish origin from 4 unrelated families. A founder mutation was searched for using homozygosity mapping followed by exome sequencing. The expression of CD59, CD55, and CD14 was examined in blood cells by flow cytometry followed by Western blot of the CD59 protein. A homozygous missense mutation, p.Cys89Tyr in CD59, was identified in all patients. The mutation segregated with the disease in the families and had a carrier rate of 1:66 among Jewish subjects of North-African origin. The mutated protein was present in the patients' cells in reduced amounts and was undetectable on the membrane surface. Based on the results of the present study, we conclude that the Cys89Tyr mutation in CD59 is associated with a failure of proper localization of the CD59 protein in the cell surface. This mutation is manifested clinically in infancy by chronic hemolysis and relapsing peripheral demyelinating disease.

What do we mean when we write "senescence," "apoptosis," "necrosis," or "clearance of dying cells"?

The clearance of dying cells has become an important field of research. Apart from a significant increase in our understanding of the mechanisms for uptake, cell clearance is a basic mechanism in tissue homeostasis, cancer, resolution of inflammation, induction of tolerance, and autoimmunity. Phagocytosis of dying cells is a complex process, involving many interacting molecules on the dying cell and the phagocyte, and in the microenvironment. Although much is known on the subject, there are many questions and unknown variables that remain under investigation. Naturally, different terms were developed, among which some are misused, leading sometimes to pseudoconflicts of understanding. Several receptors were described as "phosphatidylserine receptor: are they all equal?" We will revise terms such as apoptosis, primary and secondary necrosis, lysed cells, senescent cells, clearance of apoptotic cells, efferocytosis, and more. We will try to point out misnomers, misunderstandings, and contradictions, and to define a consensual vocabulary.

Role of class A human thrombospondins in the clearance of dying cells and tolerance induction.

Mammalian thrombospondins (TSPs) are a group of large, secreted, calcium-binding glycoproteins of complex spatial structure that mediate a wide range of intercellular activities and participate in cell-matrix interactions. This family includes five proteins, divided into two subfamilies, that possess different roles and tissue expression. TSPs have complex roles in mediating cellular processes. Apoptotic cell and phagocyte interactions show a dynamic structure with expanding complexity. However, a vast majority of the consequences of these interactions can be mediated by a single protein. One of these signaling molecules is TSP-1, which binds to a wide variety of integrin and nonintegrin cell surface receptors and mediates both engulfment and immune modulation. This mechanism is not only important in homeostasis but may also be a major mechanism for inflammation downregulation and in avoiding autoimmunity.

Thrombospondin-1-N-terminal domain induces a phagocytic state and thrombospondin-1-C-terminal domain induces a tolerizing phenotype in dendritic cells.

In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells.