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.
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-1β 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.
Abstract
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.
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.
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.
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.
•Patients with p.Cys89Tyr mutation in CD59 have recurrent peripheral neuropathy and strokes.•Three additional mutations in CD59 were described.•Overall, 12/12 of patients with any mutation presented ...with neurological symptoms.•CD59 is related to demyelination and thrombophilic state.
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.
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Background. CD59 encodes a 77-amino acid glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein synthesized as a 128-amino acid protein that includes a signal sequence and the ...sequence for a GPI anchor replacement. The CD59 protein inhibits the final and most important step of membrane attack complex (MAC) formation. A novel primary homozygous Cys89Tyr CD59 deficiency was recently reported by us in 5 children. The mutation resulted in amino acid substitution of p.Cys89Tyr and function failure to of CD59 protein on the cell surface. In this study, we characterized an additional 10 children, and summarized outcomes from a clinical study of eculizumab in these patients (NCT01579838).
Methods. Participants were recruited from our new patient registry for homozygosity of the p.Cys89Tyr mutation on CD59. 15 homozygous and 42 heterozygous patients were identified and characterized using clinical history, lab results, medical treatment, hospitalizations, biopsies, and in one case post mortem analysis. 4 patients were recruited for a clinical study where participants received repeated treatment with 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 IV IgG, and ameliorates neurological deficits compared to those observed before treatment. Treatment response was evaluated every 2-4 weeks over 34 weeks, including examination with gross motor scoring, ASIA score, INCAT disability score, laboratory examination, and SF-12 fulfillment. Neurological relapses and the cumulative dose of IVIG and/or corticosteroids before and after treatment initiation were documented. RBCs and neutrophils were stained to evaluate C5b-9 deposition.
Results. All 15 homozygous patients suffered from chronic Coombs’ negative hemolysis with hemoglobin levels of 6.5-12 gr%. All patients had post infectious peripheral neuropathy resembling recurrent Gillian Barre syndrome (GBS) and 2 patients suffered from recurrent strokes. A dramatic and significant neurological amelioration in the upper limbs and trunk, with a more modest amelioration in the lower limbs was observed in all patients. Corticosteroid and immunoglobulin treatment, which had been used extensively prior to eculizumab initiation, was completely stopped. No patient suffered a relapse during the treatment period despite infections, and there were no hospital admissions. The SF-12 health questionnaires indicated significant improvement (p<0.003). Decreased deposition of C3bi and C5b-9 on RBCs and neutrophils was documented (p<0.0001).
Conclusions. The natural history of patients with homozygous Cys89Tyr CD59 deficiency includes chronic hemolysis and recurrent peripheral neuropathy resembling GBS. Recurrent strokes were observed in 2/15 patients. Eculizumab was safely given to 4 patients. The clinical improvement suggests this is a lifesaving treatment for patients with primary CD59 deficiency. Cys89Tyr CD59 homozygous deficiency is similar to PNH with regard to chronic hemolysis and the risk for stroke, but differs from recurrent GBS episodes that are eliminated once eculizumab was used. Heterozygous patients seem to have normal life expectancy and no hemolysis or any clinical syndromes.
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