Generation of human ILC3 from allogeneic and autologous CD34+ hematopoietic progenitors toward adoptive transfer.

Type 3 innate lymphoid cells (ILC3) are important in tissue homeostasis. In the gut, ILC3 repair damaged epithelium and suppress inflammation. In allogeneic hematopoietic cell transplantation (HCT), ILC3 protect against graft-versus-host disease (GvHD), most likely by restoring tissue damage and preventing inflammation. We hypothesize that supplementing HCT grafts with interleukin-22 (IL-22)-producing ILC3 may prevent acute GvHD. We therefore explored ex vivo generation of human IL-22-producing ILC3 from hematopoietic stem and progenitor cells (HSPC) obtained from adult, neonatal and fetal sources. We established a stroma-free system culturing human cord blood-derived CD34+ HSPC with successive cytokine mixes for 5 weeks. We analyzed the presence of phenotypically defined ILC, their viability, proliferation and IL-22 production (after stimulation) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We found that the addition of recombinant human IL-15 and the enhancer of zeste homolog 1/2 inhibitor UNC1999 promoted ILC3 generation. Similar results were demonstrated when UNC1999 was added to CD34+ HSPC derived from healthy adult granulocyte colony-stimulating factor mobilized peripheral blood and bone marrow, but not fetal liver. UNC1999 did not negatively impact IL-22 production in any of the HSPC sources. Finally, we observed that autologous HSPC mobilized from the blood of adults with hematological malignancies also developed into ILC3, albeit with a significantly lower capacity. Together, we developed a stroma-free protocol to generate large quantities of IL-22-producing ILC3 from healthy adult human HSPC that can be applied for adoptive transfer to prevent GvHD after allogeneic HCT.

Circulating Iron in Patients with Sickle Cell Disease Mediates the Release of Neutrophil Extracellular Traps.

Introduction: Neutrophils promote chronic inflammation and release neutrophil extracellular traps (NETs) that can drive inflammatory responses. Inflammation influences progression of sickle cell disease (SCD), and a role for NETs has been suggested in the onset of vaso-occlusive crisis (VOC). We aimed to identify factors in the circulation of these patients that provoke NET release, with a focus on triggers associated with hemolysis. Methods: Paired serum and plasma samples during VOC and steady state of 18 SCD patients (HbSS/HbSß0-thal and HbSC/HbSß+-thal) were collected. Cell-free heme, hemopexin, and labile plasma iron have been measured in the plasma samples of the SCD patients. NETs formation by human neutrophils from healthy donors induced by serum of SCD patients was studied using confocal microscopy and staining for extracellular DNA using Sytox, followed by quantification of surface coverage using ImageJ. Results: Eighteen patients paired samples obtained during VOC and steady state were available (11 HbSS/HbSß0-thal and 7 HbSC/HbSß+-thal). We observed high levels of systemic heme and iron, concomitant with low levels of the heme-scavenger hemopexin in sera of patients with SCD, both during VOC and in steady state. In our in vitro experiments, neutrophils released NETs when exposed to sera from SCD patients. The release of NETs was associated with high levels of circulating iron in these sera. Although hemin triggered NET formation in vitro, addition of hemopexin to scavenge heme did not suppress NET release in SCD sera. By contrast, the iron scavengers deferoxamine and apotransferrin attenuated NET formation in a significant proportion of SCD sera. Discussion: Our results suggest that redox-active iron in the circulation of non-transfusion-dependent SCD patients activates neutrophils to release NETs, and hence, exerts a direct pro-inflammatory effect. Thus, we propose that chelation of iron requires further investigation as a therapeutic strategy in SCD.

Cell-free DNA levels are increased in acute graft-versus-host disease.

BACKGROUND: Cell-free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. However, the role of cfDNA in acute Graft-versus-Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown. OBJECTIVE: The aim of this study is to investigate the role of cfDNA as a marker of aGvHD. METHODS: We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species-specific polymerase chain reaction. RESULTS: In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno-reactive PBMC and damaged mouse cells. CONCLUSION: We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD.

FSAP-mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE.

Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross-linking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.

Platelet concentrates in platelet additive solutions generate less complement activation products during storage than platelets stored in plasma.

BACKGROUND: Platelet transfusions can be associated with adverse reactions, such as febrile non-haemolytic transfusion reaction (FNHTR). It has been suggested that damage-associated molecular patterns (DAMP) and complement play a role in FNHTR. This study investigated the nature of DAMPs and complement activation products contained in platelet concentrates during storage, with a specific focus on different platelet storage solutions. MATERIALS AND METHODS: Buffy coats (BC) from healthy donors were pooled (15 BC per pool) and divided into three groups of the same volume. After addition of different storage solutions (plasma, platelet additive solutions [PAS]-C or PAS-E; n=6 for each group), BC pools were processed to platelet concentrates (PC). Leukoreduced PCs were stored on a shaking bed at 20-24°C and sampled on days 1, 2, 6 and 8 after collection for selected quality parameters: platelet activation, DAMPs (High Mobility Group Box 1 [HMGB1], nucleosomes), and complement activation products. RESULTS: During storage, equal levels of free nucleosomes and increasing concentrations of HMGB1 were present in all groups. Complement activation was observed in all PC. However, by day 8, the use of PAS had reduced C3b/c levels by approximately 90% and C4b/c levels by approximately 65%. DISCUSSION: Nucleosomes and HMGB1 were present in PCs prepared in plasma and PAS. Complement was activated during storage of platelets in plasma and in PAS. The use of PAS is associated with a lower amount of complement activation products due to the dilution of plasma by PAS . Therefore, PC in PAS have less complement activation products than platelets stored in plasma. These proinflammatory mediators in PC might induce FNHTR.

Activation of factor VII-activating protease in human inflammation: a sensor for cell death.

INTRODUCTION: Cell death is a central event in the pathogenesis of sepsis and is reflected by circulating nucleosomes. Circulating nucleosomes were suggested to play an important role in inflammation and were demonstrated to correlate with severity and outcome in sepsis patients. We recently showed that plasma can release nucleosomes from late apoptotic cells. Factor VII-activating protease (FSAP) was identified to be the plasma serine protease responsible for nucleosome release. The aim of this study was to investigate FSAP activation in patients suffering from various inflammatory diseases of increasing severity. METHODS: We developed ELISAs to measure FSAP-C1-inhibitor and FSAP-α2-antiplasmin complexes in plasma. FSAP-inhibitor complexes were measured in the plasma of 20 adult patients undergoing transhiatal esophagectomy, 32 adult patients suffering from severe sepsis and 8 from septic shock and 38 children suffering from meningococcal sepsis. RESULTS: We demonstrate plasma FSAP to be activated upon contact with apoptotic and necrotic cells by an assay detecting complexes between FSAP and its target serpins α2-antiplasmin and C1-inhibitor, respectively. By means of that assay we demonstrate FSAP activation in post-surgery patients, patients suffering from severe sepsis, septic shock and meningococcal sepsis. Levels of FSAP-inhibitor complexes correlate with nucleosome levels and correlate with severity and mortality in these patients. CONCLUSIONS: These results suggest FSAP activation to be a sensor for cell death in the circulation and that FSAP activation in sepsis might be involved in nucleosome release, thereby contributing to lethality.

Recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus.

BACKGROUND: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. METHODS: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. RESULTS: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. CONCLUSIONS: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.

Potentiation of Toll-like receptor-induced cytokine production by (1-->3)-beta-D-glucans: implications for the monocyte activation test.

The monocyte activation test (MAT) has been introduced as an alternative for the detection of pyrogens in pharmaceuticals with the rabbit pyrogen test or the Limulus amebocyte lysate (LAL) test. The basis of the MAT is that pyrogens, via Toll-like receptors (TLRs) expressed on monocytes, stimulate cytokine production. Here, we report that, at concentrations that did not induce whole blood cytokine production when tested separately, (1-->3)-beta-D-glucans powerfully co-stimulated cytokine production (IL-6/IL-8) induced by ligands for TLR1/2, TLR2/6, TLR4, and TLR5. Experiments were performed to investigate the involvement of particular (1-->3)-beta-D-glucan receptors such as dectin-1. Spleen tyrosine kinase (Syk) inhibition attenuated the potentiating effects of (1-->3)-beta-D-glucans on TLR-induced cytokine production, suggesting that dectin-1 was involved. However, experiments with low molecular (1-->3)-beta-D-glucans such as laminarin argued against the involvement of dectin-1 in the co-stimulatory effects of (1-->3)-beta-D-glucans. Thus, although the receptors involved in the co-stimulatory actions of (1-->3)-beta-D-glucans on TLR-induced cytokine production are yet to be elucidated, it is clear that (1-->3)-beta-D-glucans may greatly affect MAT results and, when undetected in pharmaceuticals, may give rise to serious side-effects in patients co-exposed to other elicitors of innate immunity, such as during infections.

A plasma nucleosome releasing factor (NRF) with serine protease activity is instrumental in removal of nucleosomes from secondary necrotic cells.

We observed that interaction of secondary necrotic (sn) cells with human serum or plasma leads to loss of DNA staining. The decrease turned out to be a result of nucleosome release and was specific for apoptotic cells as necrotic cells did not show this phenomenon. We named this activity in plasma nucleosome releasing factor (NRF). NRF activity was completely inhibited by trypsin inhibitors suggesting that a serine protease is involved. Upon testing a number of plasma candidate serine proteases we found that plasmin did have NRF activity. However, plasminogen-deficient plasma still had NRF activity indicating that NRF is not plasmin. We conclude that a yet unidentified plasma serine protease is involved in removal of nucleosomes from sn cells.

DNA and factor VII-activating protease protect against the cytotoxicity of histones.

Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes; however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize histones are present in plasma. Importantly, factor VII-activating protease (FSAP) is activated upon contact with histones and subsequently proteolyzes histones. We aimed to determine the effect of FSAP on the cytotoxicity of both histones and nucleosomes. Indeed, FSAP protected against histone-induced cytotoxicity of cultured cells in vitro. Upon incubation of serum with histones, endogenous FSAP was activated and degraded histones, which also prevented cytotoxicity. Notably, histones as part of nucleosome complexes were not cytotoxic, whereas DNA digestion restored cytotoxicity. Histones in nucleosomes were inefficiently cleaved by FSAP, which resulted in limited cleavage of histone H3 and removal of the N-terminal tail. The specific isolation of either circulating nucleosomes or free histones from sera of Escherichia coli challenged baboons or patients with meningococcal sepsis revealed that histone H3 was present in the form of nucleosomes, whereas free histone H3 was not detected. All samples showed signs of FSAP activation. Markedly, we observed that all histone H3 in nucleosomes from the patients with sepsis, and most histone H3 from the baboons, was N-terminally truncated, giving rise to a similarly sized protein fragment as through cleavage by FSAP. Taken together, our results suggest that DNA and FSAP jointly limit histone cytotoxicity and that free histone H3 does not circulate in appreciable concentrations in sepsis.

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos.

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.

Cooperation of factor VII-activating protease and serum DNase I in the release of nucleosomes from necrotic cells.

OBJECTIVE: Removal of dead cells is essential in the maintenance of tissue homeostasis, and efficient removal prevents exposure of intracellular content to the immune system, which could lead to autoimmunity. The plasma protease factor VII-activating protease (FSAP) can release nucleosomes from late apoptotic cells. FSAP circulates as an inactive single-chain protein, which is activated upon contact with either apoptotic cells or necrotic cells. The purpose of this study was to investigate the role of FSAP in the release of nucleosomes from necrotic cells. METHODS: Necrotic Jurkat cells were incubated with serum, purified 2-chain FSAP, and/or DNase I. Nucleosome release was analyzed by flow cytometry, and agarose gel electrophoresis was performed to detect DNA breakdown. RESULTS: Incubation with serum released nucleosomes from necrotic cells. Incubation with FSAP-deficient serum or serum in which FSAP was inhibited by a blocking antibody was unable to release nucleosomes from necrotic cells, confirming that FSAP is indeed the essential serum factor in this process. Together with serum DNase I, FSAP induced the release of DNA from the cells, the appearance of nucleosomes in the supernatant, and the fragmentation of chromatin into eventually mononucleosomes. CONCLUSION: FSAP and DNase I are the essential serum factors that cooperate in necrotic cell DNA degradation and nucleosome release. We propose that this mechanism may be important in the removal of potential autoantigens.

Neutrophil extracellular traps in the host defense against sepsis induced by Burkholderia pseudomallei (melioidosis).

BACKGROUND: Neutrophil extracellular traps (NETs) are a central player in the host response to bacteria: neutrophils release extracellular DNA (nucleosomes) and neutrophil elastase to entrap and kill bacteria. We studied the role of NETs in Burkholderia pseudomallei infection (melioidosis), an important cause of Gram-negative sepsis in Southeast Asia. METHODS: In a prospective observational study, circulating nucleosomes and neutrophil elastase were assayed in 44 patients with Gram-negative sepsis caused by B. pseudomallei (melioidosis) and 82 controls. Functional assays included human neutrophil stimulation and killing assays and a murine model of B. pseudomallei infection in which NET function was compromised using DNase. Specified pathogen-free 8- to 12-week-old C57BL/6 mice were sacrificed post-infection to assess bacterial loads, inflammation, and pathology. RESULTS: Nucleosome and neutrophil elastase levels were markedly elevated in patients compared to controls. NETs killed B. pseudomallei effectively, and neutrophils stimulated with B. pseudomallei showed increased elastase and DNA release in a time- and dose-dependent matter. In mice, NET disruption with intravenous DNase administration resulted in decreased nucleosome levels. Although DNase treatment of mice resulted in diminished liver inflammation, no differences were observed in bacterial dissemination or systemic inflammation. CONCLUSION: B. pseudomallei is a potent inducer of NETosis which was reflected by greatly increased levels of NET-related components in melioidosis patients. Although NETs exhibited antibacterial activity against B. pseudomallei, NET formation did not protect against bacterial dissemination and inflammation during B. pseudomallei-induced sepsis.

Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity But Retained Immunogenicity.

BACKGROUND: Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason. AIM: To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity. METHODS: Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant Fel d 1 (rFel d 1) was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP-inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural Fel d 1 and recombinant unmodified Fel d 1 were used as controls. RESULTS: rFel d 1 demonstrated similar IgE binding and biological activity as its natural counterpart. Upon modification, IgE-binding potency decreased to >1000-fold, which was translated into a >10(6)-fold reduction in the biological activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen. CONCLUSIONS: Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.

Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor.

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.

Lipid transfer proteins from fruit: cloning, expression and quantification.

BACKGROUND: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. METHODS: cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3. RESULTS: The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3. CONCLUSION: rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.