Eliopoulos, Elias

ObjectiveWe describe a 38‐year‐old woman who presented with a history of inflammatory arthritis, rash, and daily fevers. She was noted to have chronic parvovirus infection with persistently detectable viral titers and a novel mutation in the ELANE gene. ELANE encodes neutrophil elastase, a neutrophil serine protease with important antimicrobial effects, and is found as part of neutrophil extracellular trap (NET) complexes. Pathogenic ELANE mutations have been identified in forms of hereditary neutropenia. However, our patient never had neutropenia. Because of the striking clinical scenario, we investigated this mutation functionally.MethodsNET formation by neutrophils was assessed by scanning electron microscopy. Neutrophil activation and neutrophil elastase production were evaluated by flow cytometry and fluorescent substrate–based functional assay, respectively. A multiplex assay was used to quantitate neutrophil inflammatory cytokine production. PyMOL software was used to generate 3‐dimensional models of mutant elastase.ResultsActivated neutrophils from the patient demonstrated a significantly decreased ability to form NETs on scanning electron microscopy, as well as quantitative defects in neutrophil activation and neutrophil elastase activity. The patient's neutrophils showed altered levels of interleukin‐12 (IL‐12) and IL‐8, which are key cytokines for antiviral immunity and neutrophil chemotaxis. Three‐dimensional mapping revealed that the mutation could alter protein folding and surface charge distribution, potentially perturbing protein trafficking. Thus, the mutation could affect neutrophil function by decreasing NETosis and altering key antiviral activities of neutrophils.ConclusionThis is the first report of a human pathogenic ELANE mutation associated with a defect in NETosis and a distinct syndrome of recurrent viral infection and chronic inflammation.

ObjectiveSystemic lupus erythematosus (SLE) is frequently characterized by activation of the type I interferon (IFN) pathway. We previously observed that a missense single‐nucleotide polymorphism (rs1049564) in the purine nucleoside phosphorylase (PNP) gene was associated with high levels of IFN in SLE. PNP is a key enzyme involved in purine metabolism. In this study, we performed functional follow‐up of this polymorphism in human cells.MethodsType I IFN was measured in patient sera, using a reporter cell assay. Structural modeling of the PNP variant was performed using PyMOL software. PNP messenger RNA (mRNA) and protein levels and type I IFN–induced gene expression were measured in lymphoblastoid cell lines with known PNP rs1049564 genotypes. The cell cycle was assayed using flow cytometry.ResultsStructural modeling indicated no major disruption in folding related to rs1049564. We observed that homozygous rs1049564 TT lymphoblastoid cells had decreased PNP mRNA expression and protein levels, and that cells with the TT genotype had reduced PNP enzymatic activity even when the amount of PNP was controlled. Cells with the TT genotype had a 2‐fold increase in S‐phase block as compared with cells with the homozygous CC phenotype. The S‐phase block could be pharmacologically reversed with hypoxanthine and adenosine, supporting the notion that relative PNP deficiency is the cause of the S‐phase block. Type I IFN–induced transcripts were increased in a dose‐response manner related to the rs1049564 T allele, at both baseline and after type I IFN stimulation.ConclusionThe PNP rs1049564 T allele is a loss‐of‐function variant that induces S‐phase block and IFN pathway activation in lymphocytes. The S‐phase block could be rescued in our in vitro experiments, suggesting the potential for personalized treatment.