Abstract
Neutrophil granulocytes are innate effector cells of the first line of defense against pyogenic bacteria. Neutrophil lifespan is short, is prolonged by pro-inflammatory stimuli, controls functionality of the cells and can determine tissue damage. Experimental analysis of primary neutrophils is difficult because of their short lifespan and lack of possibilities of genetic manipulation. The Hoxb8 system of neutrophil differentiation from immortalized progenitor cells offers the advantage of unlimited production of neutrophils in vitro as well as easy genetic modification. We here use this system to analyze the role of the poorly characterized anti-apoptotic B-cell lymphoma protein 2 (Bcl-2) family member A1/Bfl-1 (Bcl-2-related protein A1) for survival and homeostasis of neutrophils and of neutrophil progenitors. Low constitutive mRNA and protein expression of A1 was detected, while A1 was transiently upregulated early during differentiation. Pro-inflammatory stimuli caused strong, mainly transcriptional, A1 upregulation, in contrast to posttranscriptional regulation of Mcl-1 (induced myeloid leukemia cell differentiation protein). Inhibitor studies showed that phosphoinositide-3 kinase (PI3K)/Akt and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) is required for A1 expression and survival of progenitors and mature neutrophils. ShRNA-mediated constitutive A1 knockdown (KD) impaired maintenance of progenitors. ShRNA experiments further showed that A1 was required early during neutrophil differentiation as well as in mature neutrophils upon pro-inflammatory stimulation. Our data further indicate differential regulation of the two anti-apoptotic proteins A1 and Mcl-1. Relevant findings were confirmed in primary human neutrophils. Our data indicate that A1, in addition to the well-established Mcl-1, substantially contributes to neutrophil survival and homeostasis. A1 may thus be a promising target for anti-inflammatory therapy.
Highlights
Neutrophils have a lifespan of few hours, which is regulated by apoptosis.[4]
We found strong regulation of A1 mRNA and protein expression, modulated mainly by phosphoinositide-3 kinase (PI3K)/Akt and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways, and less by extracellular signal-regulated kinase (ERK)
The results strongly suggest that A1 is an important antiapoptotic factor, which cooperates with Mcl-1 in regulation of neutrophil survival and homeostasis
Summary
Neutrophils have a lifespan of few hours, which is regulated by apoptosis.[4]. Pro-inflammatory stimuli such as cytokines or bacterial components can prolong neutrophil lifespan.[4,5]. Major experimental drawbacks of neutrophil work are lacking possibilities of genetic modification in human cells and limited cell numbers in mice These problems can be overcome using a model system of differentiation of murine myeloid cells from bone marrow progenitors.[20] Conditional estrogen-regulated activation of the transcription factor Hoxb[8] allows the establishment of progenitor lines committed to the neutrophil lineage in the presence of specific cytokines. This system enables large-scale production of mature neutrophils in vitro, but is amenable to genetic modification. When Hoxb[8] is turned off, the cells quickly start differentiation into neutrophils, permitting analysis of gene regulation during this process, and the analysis of cells indistinguishable from mature neutrophils.[20,21]
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