Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements.

Neutrophils are important effector cells in the host defense against invading microorganisms. One of the mechanisms they use to eliminate pathogens is the release of neutrophil extracellular traps (NETs). Although NET release and subsequent cell death known as NETosis have been intensively studied, the cellular components and factors determining or facilitating the formation of NETs remain incompletely understood. Using various actin polymerization and myosin II modulators on neutrophils from healthy individuals, we show that intact F-actin dynamics and myosin II function are essential for NET formation when induced by different stimuli; that is, phorbol 12-myristate 13-acetate, monosodium urate crystals, and Candida albicans. The role of actin polymerization in NET formation could not be explained by the lack of reactive oxygen species production or granule release, which were normal or enhanced under the given conditions. Neutrophils from patients with very rare inherited actin polymerization defects by either actin-related protein 2/3 complex subunit 1B or megakaryoblastic leukemia 1 deficiency also failed to show NETosis. We found that upon inhibition of actin dynamics, there is a lack of translocation of neutrophil elastase to the nucleus, which may explain the impaired NET formation. Collectively, our data show the essential requirement of an intact and active actin polymerization process, as well as active myosin II to enable the release of nuclear DNA by neutrophils during NET formation.

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.

Defective neutrophil development and specific granule deficiency caused by a homozygous splice-site mutation in SMARCD2.

BACKGROUND: SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2. OBJECTIVE: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells. METHODS: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34+ cells were then isolated, phenotyped, and assessed functionally. RESULTS: Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34+ cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later. CONCLUSIONS: This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency.

Red blood cell transfusion results in adhesion of neutrophils in human endotoxemia and in critically ill patients with sepsis.

BACKGROUND: Red blood cell (RBC) transfusion is associated with adverse effects, which may involve activation of the host immune response. The effect of RBC transfusion on neutrophil Reactive Oxygen Species (ROS) production and adhesion ex vivo was investigated in endotoxemic volunteers and in critically ill patients that received a RBC transfusion. We hypothesized that RBC transfusion would cause neutrophil activation, the extent of which depends on the storage time and the inflammatory status of the recipient. STUDY DESIGN AND METHODS: Volunteers were injected with lipopolysaccharide (LPS) and transfused with either saline, fresh, or stored autologous RBCs. In addition, 47 critically ill patients with and without sepsis receiving either fresh (<8 days) or standard stored RBC (2-35 days) were included. Neutrophils from healthy volunteers were incubated with the plasma samples from the endotoxemic volunteers and from the critically ill patients, after which priming of neutrophil ROS production and adhesion were assessed. RESULTS: In the endotoxemia model, ex vivo neutrophil adhesion, but not ROS production, was increased after transfusion, which was not affected by RBC storage duration. In the critically ill, ex vivo neutrophil ROS production was already increased prior to transfusion and was not increased following transfusion. Neutrophil adhesion was increased following transfusion, which was more notable in the septic patients than in non-septic patients. Transfusion of fresh RBCs, but not standard issued RBCs, resulted in enhanced ROS production in neutrophils. CONCLUSION: RBC transfusion was associated with increased neutrophil adhesion in a model of human endotoxemia as well as in critically ill patients with sepsis.

A false-carrier state for the c.579G>A mutation in the NCF1 gene in Ashkenazi Jews.

BACKGROUND: Mutations in the NCF1 gene that encodes p47phox, a subunit of the NADPH oxidase complex, cause chronic granulomatous disease (CGD). In Kavkazi Jews, a c.579G>A (p.Trp193Ter) mutation in NCF1 is frequently found, leading to CGD. The same mutation is found in about 1% of Ashkenazi Jews, although Ashkenazi CGD patients with this mutation have never been described. METHODS: We used Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), gene scan analysis and Ion Torrent Next Generation Sequencing for genetic analysis, and measured NADPH oxidase activity and p47phox expression. RESULTS: In an Ashkenazi couple expecting a baby, both parents were found to be heterozygotes for this mutation, as was the fetus. However, segregation analysis in the extended family was consistent with the fetus inheriting both carrier alleles from the parents. MLPA indicated four complete NCF1 genes in the fetus and three in each parent. Gene sequencing confirmed these results. Analysis of fetal leucocytes obtained by cordocentesis revealed substantial oxidase activity with three different assays, which was confirmed after birth. In six additional Ashkenazi carriers of the NCF1 c.579G>A mutation, we found five individuals with three complete NCF1 genes of which one was mutated (like the parents), and one individual with in addition a fusion gene of NCF1 with a pseudogene. CONCLUSION: These results point to the existence of a 'false-carrier' state in Ashkenazi Jews and have wide implications regarding pre-pregnancy screening in this and other population groups.

Loss-of-function nuclear factor κB subunit 1 (NFKB1) variants are the most common monogenic cause of common variable immunodeficiency in Europeans.

BACKGROUND: The genetic cause of primary immunodeficiency disease (PID) carries prognostic information. OBJECTIVE: We conducted a whole-genome sequencing study assessing a large proportion of the NIHR BioResource-Rare Diseases cohort. METHODS: In the predominantly European study population of principally sporadic unrelated PID cases (n = 846), a novel Bayesian method identified nuclear factor κB subunit 1 (NFKB1) as one of the genes most strongly associated with PID, and the association was explained by 16 novel heterozygous truncating, missense, and gene deletion variants. This accounted for 4% of common variable immunodeficiency (CVID) cases (n = 390) in the cohort. Amino acid substitutions predicted to be pathogenic were assessed by means of analysis of structural protein data. Immunophenotyping, immunoblotting, and ex vivo stimulation of lymphocytes determined the functional effects of these variants. Detailed clinical and pedigree information was collected for genotype-phenotype cosegregation analyses. RESULTS: Both sporadic and familial cases demonstrated evidence of the noninfective complications of CVID, including massive lymphadenopathy (24%), unexplained splenomegaly (48%), and autoimmune disease (48%), features prior studies correlated with worse clinical prognosis. Although partial penetrance of clinical symptoms was noted in certain pedigrees, all carriers have a deficiency in B-lymphocyte differentiation. Detailed assessment of B-lymphocyte numbers, phenotype, and function identifies the presence of an increased CD21low B-cell population. Combined with identification of the disease-causing variant, this distinguishes between healthy subjects, asymptomatic carriers, and clinically affected cases. CONCLUSION: We show that heterozygous loss-of-function variants in NFKB1 are the most common known monogenic cause of CVID, which results in a temporally progressive defect in the formation of immunoglobulin-producing B cells.

Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects.

Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.

Impaired killing of Candida albicans by granulocytes mobilized for transfusion purposes: a role for granule components.

Granulocyte transfusions are used to treat neutropenic patients with life-threatening bacterial or fungal infections that do not respond to anti-microbial drugs. Donor neutrophils that have been mobilized with granulocyte-colony stimulating factor (G-CSF) and dexamethasone are functional in terms of antibacterial activity, but less is known about their fungal killing capacity. We investigated the neutrophil-mediated cytotoxic response against C. albicans and A. fumigatus in detail. Whereas G-CSF/dexamethasone-mobilized neutrophils appeared less mature as compared to neutrophils from untreated controls, these cells exhibited normal ROS production by the NADPH oxidase system and an unaltered granule mobilization capacity upon stimulation. G-CSF/dexamethasone-mobilized neutrophils efficiently inhibited A. fumigatus germination and killed Aspergillus and Candida hyphae, but the killing of C. albicans yeasts was distinctly impaired. Following normal Candida phagocytosis, analysis by mass spectrometry of purified phagosomes after fusion with granules demonstrated that major constituents of the antimicrobial granule components, including major basic protein (MBP), were reduced. Purified MBP showed candidacidal activity, and neutrophil-like Crisp-Cas9 NB4-KO-MBP differentiated into phagocytes were impaired in Candida killing. Together, these findings indicate that G-CSF/dexamethasone-mobilized neutrophils for transfusion purposes have a selectively impaired capacity to kill Candida yeasts, as a consequence of an altered neutrophil granular content.

Invasive fungal infection and impaired neutrophil killing in human CARD9 deficiency.

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule in the cytosol of myeloid cells, required for induction of T-helper cells producing interleukin-17 (Th17 cells) and important in antifungal immunity. In a patient suffering from Candida dubliniensis meningoencephalitis, mutations in the CARD9 gene were found to result in the loss of protein expression. Apart from the reduced numbers of CD4(+) Th17 lymphocytes, we identified a lack of monocyte-derived cytokines in response to Candida strains. Importantly, CARD9-deficient neutrophils showed a selective Candida albicans killing defect with abnormal ultrastructural phagolysosomes and outgrowth of hyphae. The neutrophil killing defect was independent of the generation of reactive oxygen species by the reduced NAD phosphate oxidase system. Taken together, this demonstrates that human CARD9 deficiency results in selective defect in the host defense against invasive fungal infection, caused by an impaired phagocyte killing.

Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.

Macrophages migrate in an activation-dependent manner to chemokines involved in neuroinflammation.

BACKGROUND: In neuroinflammatory diseases, macrophages can play a dual role in the process of tissue damage, depending on their activation status (M1 / M2). M1 macrophages are considered to exert damaging effects to neurons, whereas M2 macrophages are reported to aid regeneration and repair of neurons. Their migration within the central nervous system may be of critical importance in the final outcome of neurodegeneration in neuroinflammatory diseases e.g. multiple sclerosis (MS). To provide insight into this process, we examined the migratory capacity of human monocyte-derived M1 and M2 polarised macrophages towards chemoattractants, relevant for neuroinflammatory diseases like MS. METHODS: Primary cultures of human monocyte-derived macrophages were exposed to interferon gamma and lipopolysaccharide (LPS) to evoke proinflammatory (M1) activation or IL-4 to evoke anti-inflammatory (M2) activation. In a TAXIScan assay, migration of M0, M1 and M2 towards chemoattractants was measured and quantified. Furthermore the adhesion capacity and the expression levels of integrins as well as chemokine receptors of M0, M1 and M2 were assessed. Alterations in cell morphology were analysed using fluorescent labelling of the cytoskeleton. RESULTS: Significant differences were observed between M1 and M2 macrophages in the migration towards chemoattractants. We show that M2 macrophages migrated over longer distances towards CCL2, CCL5, CXCL10, CXCL12 and C1q compared to non-activated (M0) and M1 macrophages. No differences were observed in the adhesion of M0, M1 and M2 macrophages to multiple matrix components, nor in the expression of integrins and chemokine receptors. Significant changes were observed in the cytoskeleton organization upon stimulation with CCL2, M0, M1 and M2 macrophages adopt a spherical morphology and the cytoskeleton is rapidly rearranged. M0 and M2 macrophages are able to form filopodia, whereas M1 macrophages only adapt a spherical morphology. CONCLUSIONS: Together our results indicate that the alternative activation status of macrophages promotes their migratory properties to chemoattractants relevant for neuroinflammatory diseases like MS. Conversely, classically activated, proinflammatory macrophages have reduced migratory properties. Based on our results, we postulate that the activation status of the macrophage influences the capacity of the macrophages to rearrange their cytoskeleton. This is the first step in understanding how modulation of macrophage activation affects macrophage migration in neuroinflammatory diseases like MS.

Reduced myeloid commitment and increased uptake by macrophages of stem cell-derived HPS2 neutrophils.

Hermansky-Pudlak syndrome type 2 (HPS2) is a rare autosomal recessive disorder, caused by mutations in the AP3B1 gene, encoding the ß3A subunit of the adapter protein complex 3. This results in mis-sorting of proteins within the cell. A clinical feature of HPS2 is severe neutropenia. Current HPS2 animal models do not recapitulate the human disease. Hence, we used induced pluripotent stem cells (iPSCs) of an HPS2 patient to study granulopoiesis. Development into CD15POS cells was reduced, but HPS2-derived CD15POS cells differentiated into segmented CD11b+CD16hi neutrophils. These HPS2 neutrophils phenocopied their circulating counterparts showing increased CD63 expression, impaired degranulation capacity, and intact NADPH oxidase activity. Most noticeable was the decrease in neutrophil yield during the final days of HPS2 iPSC cultures. Although neutrophil viability was normal, CD15NEG macrophages were readily phagocytosing neutrophils, contributing to the limited neutrophil output in HPS2. In this iPSC model, HPS2 neutrophil development is affected by a slower rate of development and by macrophage-mediated clearance during neutrophil maturation.

Platelet proteomic profiling in sitosterolemia suggests thrombocytopenia is driven by lipid disorder and not platelet aberrations.

ABSTRACT: Sitosterolemia is a rare autosomal recessive genetic disorder in which patients develop hypercholesterolemia and may exhibit abnormal hematologic and/or liver test results. In this disease, dysfunction of either ABCG5 or ABCG8 results in the intestinal hyperabsorption of all sterols, including cholesterol and, more specifically, plant sterols or xenosterols, as well as in the impaired ability to excrete xenosterols into the bile. It remains unknown how and why some patients develop hematologic abnormalities. Only a few unrelated patients with hematologic abnormalities at the time of diagnosis have been reported. Here, we report on 2 unrelated pedigrees who were believed to have chronic immune thrombocytopenia as their most prominent feature. Both consanguineous families showed recessive gene variants in ABCG5, which were associated with the disease by in silico protein structure analysis and clinical segregation. Hepatosplenomegaly was absent. Thrombopoietin levels and megakaryocyte numbers in the bone marrow were normal. Metabolic analysis confirmed the presence of strongly elevated plasma levels of xenosterols. Potential platelet proteomic aberrations were longitudinally assessed following dietary restrictions combined with administration of the sterol absorption inhibitor ezetimibe. No significant effects on platelet protein content before and after the onset of treatment were demonstrated. Although we cannot exclude that lipotoxicity has a direct and platelet-specific impact in patients with sitosterolemia, our data suggest that thrombocytopenia is neither caused by a lack of megakaryocytes nor driven by proteomic aberrations in the platelets themselves.

Lack of eosinophil extracellular trap formation due to failure of plasma membrane breakdown in the absence of elastase.

Activated eosinophils are described to release eosinophil extracellular traps (EETs), which consist of the cell's DNA covered with granule-derived antimicrobial peptides. Upon stimulation of eosinophils with the known EET-inducers phorbol 12-myristate 13-acetate, monosodium urate crystals, or Candida albicans, we observed that their plasma membrane became compromised, resulting in accessibility of the nuclear DNA for staining with the impermeable DNA dye Sytox Green. However, we did not observe any DNA decondensation or plasma membrane rupture by eosinophils, which sharply contrasts with neutrophil extracellular trap (NET) formation and the subsequent cell death known as NETosis. Neutrophil elastase (NE) activity is thought to be essential for the cleavage of histones and chromatin decondensation during NETosis. We observed that the neutrophils of a patient with a mutation in ELANE, leading to congenital neutropenia and NE deficiency, were unable to undergo NETosis. Taken together, we may suggest that the natural absence of any NE-like proteolytic activity in human eosinophils explains why EET formation is not observed, even when eosinophils become positive for an impermeable DNA dye in response to stimuli that induce NETosis in neutrophils.

Development of an Affinity-Based Probe to Profile Endogenous Human Adenosine A3 Receptor Expression.

The adenosine A3 receptor (A3AR) is a G protein-coupled receptor (GPCR) that exerts immunomodulatory effects in pathophysiological conditions such as inflammation and cancer. Thus far, studies toward the downstream effects of A3AR activation have yielded contradictory results, thereby motivating the need for further investigations. Various chemical and biological tools have been developed for this purpose, ranging from fluorescent ligands to antibodies. Nevertheless, these probes are limited by their reversible mode of binding, relatively large size, and often low specificity. Therefore, in this work, we have developed a clickable and covalent affinity-based probe (AfBP) to target the human A3AR. Herein, we show validation of the synthesized AfBP in radioligand displacement, SDS-PAGE, and confocal microscopy experiments as well as utilization of the AfBP for the detection of endogenous A3AR expression in flow cytometry experiments. Ultimately, this AfBP will aid future studies toward the expression and function of the A3AR in pathologies.

Toll-like receptor-induced reactivity and strongly potentiated IL-8 production in granulocytes mobilized for transfusion purposes.

Transfusion of granulocytes from granulocyte-colony stimulating factor (G-CSF)/dexamethasone (dexa)-treated donors can be beneficial for neutropenic recipients that are refractory to antimicrobial therapy. G-CSF/dexa treatment not only increases the number of circulating neutrophils but also affects their gene expression. Because of the intended transfusion of these granulocytes into patients who are severely ill, it is of importance to establish to what extent mobilization affects the cellular behavior of neutrophils. Here, we studied the effects of mobilization on Toll-like receptor (TLR)-mediated responses. Mobilized granulocytes displayed increased gene and protein expression of TLR2, TLR4, TLR5, and TLR8. Although mobilized granulocytes displayed normal priming of nicotinamide adenine dinucleotide phosphate oxidase activity and a slight increase in adhesion in response to TLR stimulation, these cells produced massive amounts of interleukin-8 (IL-8), in particular to TLR2 and TLR8 stimulation. The increase in IL-8 release occurred despite reduced IL-8 mRNA levels in the donor granulocytes after in vivo G-CSF/dexa treatment, indicating that the enhanced TLR-induced IL-8 production was largely determined by posttranscriptional regulation. In summary, granulocytes mobilized for transfusion purposes show enhanced TLR responsiveness in cytokine production, which is anticipated to be beneficial for the function of these cells on transfusion into patients.