Pathways regulating lipopolysaccharide‐induced neutrophil survival revealed by lentiviral transduction of primary human neutrophils

Abstract

Summary Human neutrophils express Toll‐like receptor 4 (TLR4) at low levels, and the role of this receptor in neutrophil responses to microbial stimuli has been questioned. Genetic manipulation of these cells to enable the study of the role of proteins such as TLR4 in their function is challenging. Here, we show that primary human neutrophils rapidly express novel proteins such as enhanced green fluorescent protein (eGFP) after transduction with lentivirus. Stimulation of transduced neutrophils with lipopolysaccharide (LPS) resulted in increased cell survival, which was inhibited when neutrophils were transduced with a lentivirus encoding a dominant negative (dn) TLR4 protein. LPS‐induced survival was also inhibited by lentiviruses encoding dnMyD88 or a truncated TRIF (Toll/interleukin‐1R homologous domain‐containing adapter protein inducing interferon‐β) molecule, whilst, in contrast, neutrophil survival was enhanced by overexpression of kinase‐mutated interleukin‐1 receptor‐associated kinase 1 (kmIRAK‐1), which activated nuclear factor (NF)‐κB. These studies provide proof of the role of TLR4 in human neutrophil biology, have begun to elucidate TLR‐dependent pathways regulating neutrophil survival, and demonstrate that neutrophils can be genetically manipulated to enhance or inhibit survival.

The effects of <i>n</i>-3 polyunsaturated fatty acid-rich total parenteral nutrition on neutrophil apoptosis in a rat endotoxemia

“…Prolongation of neutrophil survival by LPS stimuli is mediated by Toll-like receptor 4-MyD88 pathway, which activated nuclear factor κB. (16) Mitogen-activated protein kinase (MAPK) also plays critical roles for neutrophil survival. In the presence of LPS, the signals generated via activated extracellular signal-regulated protein kinases (ERK) may inhibit the regulation of neutrophil apoptosis by p38MAPK, resulting delayed neutrophil apoptosis.…”

Association of polymorphisms in the TLR4 gene with the risk of developing neutropenia in children with leukemia

“…Several studies previously demonstrated an association of the TLR4 gene with enhanced neutrophil survival by inhibiting neutrophil apoptosis. 9,11-13 Dick et al 10 showed that neutrophil survival is inhibited when neutrophils are transduced with a lentivirus encoding a dominant-negative TLR4 protein, and that neutrophils can be genetically manipulated to enhance or inhibit survival. OR is calculated by logistic regression (95% CI) in their best-fitting model (D ¼ minor allele is dominant and R ¼ minor allele is recessive).…”

“…A candidate gene that might be associated with the risk of developing chemotherapy-induced neutropenia is Toll-like receptor 4 (TLR4), as TLR4 has been shown to have a role in inhibition of neutrophil apoptosis, and extension of the functional lifespan of neutrophils during stimulation with lipopolysaccharide. [9][10][11][12][13] TLR4 is 1 of 11 known mammalian Toll-like membrane receptors that have a key role in innate immunity by detecting and eliminating invading pathogens. Expression of TLR4 has been detected on neutrophils.…”

Regulation of wound healing and organ fibrosis by toll-like receptors

“…Except for intracellular TLR3, human neutrophils express all other TLRs, highlighting their importance in the induction of an immune response [121]. TLR stimulation on neutrophils contributes to a prolonged lifespan of this otherwise short-lived cell population [122]. It also modulates cell surface receptor expression and initiates degranulation processes as well as ROS production and phagocytosis - all of which add to their potent microbicidal and proinflammatory effects [123].…”