NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival

Abstract

Key Points• We demonstrate an important role for NR4A receptors in regulating neutrophil lifespan and homeostasis in vitro and in vivo.• These findings may define targets for therapies for diseases driven by defects in neutrophil number and/or survival.The lifespan of neutrophils is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis-related genes, the most highly regulated of these being NR4A2 and NR4A3. Direct PKA activation by the siteselective PKA agonist pair N6/8-AHA (8-AHA-cAMP and N6-MB-cAMP) and treatment with endogenous activators of PKA, including adenosine and prostaglandin E2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA-dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and small interfering RNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologs in zebrafish larvae significantly reduces the absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis.

A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models

“…Blood was taken from healthy volunteers (pool of 9 donors, males and females, aged between 18 and 40) with written informed consent and ethical approval from the South Sheffield Research Ethics Committee (STH18729). Human PBMCs were collected after routine neutrophil preparation by dextran sedimentation followed by plasma-Percoll gradient centrifugation from whole blood ( Prince et al , 2017 ). PBMCs were further purified by positive selection using the CD34 MicroBead kit (Miltenyl Biotech, Bergisch Gladbach, Germany).…”

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“…Neutrophils are relatively short‐lived cells which originate and mature in the bone marrow, then circulate in the blood for several hours before either being removed or engaged in the inflammatory response and recruited into tissues. Typically, neutrophils undergo cell death within 24 h of their release into circulation (McCracken & Allen, ), although perturbations in neutrophil cell death pathways have been reported to increase neutrophil lifespan in some infectious and inflammatory disease states (McCracken & Allen, ; Kinkead, Fayram & Allen, ; Prince et al , ). Like AC‐DCs, neutrophil numbers are also continuously renewed by haemopoiesis from myeloid precursors in the bone marrow, with a production rate of 1 × 10 10 –1 × 10 11 neutrophils/day in humans.…”

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“…involved in cellular function and inflammation reaction. 11,12 Although NR4A3 is expressed in various cell types, the function of this molecule has poorly been studied. The first study on NR4A3 focused on neurons; NR4A3-deficient mice showed defects in their inner ear development.…”