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Contains fulltext : 195014.pdf (publisher's version ) (Open Access), Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils.

Item does not contain fulltext, Bloodstream infection is a hallmark of sepsis, a medically emergent condition requiring rapid treatment. However, upregulation of host defense proteins through toll-like receptors and NFkappaB requires hours after endotoxin detection. Using confocal pulmonary intravital microscopy, we identified that the lung provides a TLR4-Myd88-and abl tyrosine kinase-dependent niche for immediate CD11b-dependent neutrophil responses to endotoxin and Gram-negative bloodstream pathogens. In an in vivo model of bacteremia, neutrophils crawled to and rapidly phagocytosed Escherichia coli sequestered to the lung endothelium. Therefore, the lung capillaries provide a vascular defensive niche whereby endothelium and neutrophils cooperate for immediate detection and capture of disseminating pathogens.

Item does not contain fulltext, 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 Fcgamma 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.

Item does not contain fulltext, Retrotransposon-mediated insertion of a long interspersed nuclear element (LINE)-1 or an Alu element into a human gene is a well-known pathogenic mechanism. We report a novel LINE-1-mediated insertion of a transcript from the TMF1 gene on chromosome 3 into the CYBB gene on the X-chromosome. In a Dutch male patient with chronic granulomatous disease, a 5.8-kb, incomplete and partly exonized TMF1 transcript was identified in intron 1 of CYBB, in opposite orientation to the host gene. The sequence of the insertion showed the hallmarks of a retrotransposition event, with an antisense poly(A) tail, target site duplication, and a consensus LINE-1 endonuclease cleavage site. This insertion induced aberrant CYBB mRNA splicing, with inclusion of an extra 117-bp exon between exons 1 and 2 of CYBB. This extra exon contained a premature stop codon. The retrotransposition took place in an early stage of fetal development in the mother of the patient, because she showed a somatic mosaicism for the mutation that was not present in the DNA of her parents. However, the mutated allele was not expressed in the patient's mother because the insertion was found only in the methylated fraction of her DNA.

Contains fulltext : 116529.pdf (publisher's version ) (Closed access), BACKGROUND: Our index case was a patient with common variable immunodeficiency (CVID). She had anaphylactoid reactions on administration of intravenous immunoglobulin (IVIg) associated with the presence of IgG antibodies against IgA. OBJECTIVE: We sought to determine the role of Fcgamma receptor (FcgammaR) IIa in IVIg-induced anaphylactoid reactions. METHODS: Neutrophils and PBMCs were isolated from healthy subjects and IVIg-treated patients. FcgammaRIIa mRNA and DNA were analyzed by using real-time PCR and sequencing. IgG-mediated elastase release and intracellular Ca(2+) mobilization were determined in neutrophils and transfected cell lines, respectively. RESULTS: A novel splice variant of FcgammaRIIa containing an expressed cryptic exon 6* (FcgammaRIIa(exon6 *)) was identified in our index patient. This exon is normally spliced out of all FcgammaRII isoforms, except the inhibitory FcgammaRIIb1. Compared with healthy control subjects, the heterozygous FCGR2A(c.742+871A>G) mutation was more frequent in patients with CVID (n = 53, P < .013). Expression in patients with CVID was associated with anaphylaxis on IVIg infusion (P = .002). On screening of additional IVIg-treated patient cohorts, we identified 6 FCGR2A(c.742+871A>G) allele-positive patients with Kawasaki disease (n = 208) and 1 patient with idiopathic thrombocytopenia (n = 93). None had adverse reactions to IVIg. Moreover, FcgammaRIIa(exon6 *) was also demonstrated in asymptomatic family members. Functional studies in primary cells and transfected murine cells demonstrated enhanced cellular activation by FcgammaRIIa(exon6 *) compared with its native form, as shown by increased elastase release and intracellular calcium mobilization. CONCLUSION: A novel splice variant, FcgammaRIIa(exon6 *), was characterized as a low-frequency allele, coding for a gain-of-function receptor for IgG. In the presence of immune complexes, FcgammaRIIa(exon6 *) can contribute to anaphylaxis in patients with CVID.