A20 and the non-canonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny

Background Newborns are at high risk to develop neonatal sepsis, especially when born prematurely, which has been linked to their immature and functionally limited innate immune response.

Aims The ontogenetically regulated maturation of the immune system during fetal life is still elusive. To shed new light on this process, we set out to investigate the molecular mechanisms regulating neutrophil function during fetal life in mice and humans.

Methods We performed a transcriptomic analysis of human fetal and adult neutrophils and found 128 differentially regulated genes, with a prominent focus on genes being involved in the NF-κB signaling pathway. In a second step we evaluated interesting targets from the transcriptomic analysis using in vitro and in vivo functional assays in mice and humans.

Results Among those differentially regulated genes we could identify 40 targets that were transcriptionally upregulated in fetal human neutrophils and were additionally postulated to be regulated by RelB, a transcription factor activated by non-canonical NF-κB signaling. In addition, RelB also localized to the nucleus of fetal but not adult neutrophils, indicating higher baseline activity of non-canonical NF-κB signaling in fetal neutrophils. In contrast, we observed down-regulation of the classical NF-κB pathway, as fetal neutrophils displayed reduced phosphorylation of IκB, which is required for the nuclear translocation of p65 and thus initiation of an inflammatory response. Testing these observations under in vivo conditions in the mouse, we found that intrauterine application of LPS as well as TNF-α, and therefore activation of canonical NF-κB signaling, led to neutrophil adhesion in inflamed murine yolk sac vessels only at later time points during gestation (E17.5) while no response was observed at early time points (E14.5). Among the genes upregulated in fetal neutrophils and modulated by RelB, we identified the ubiquitin-modifying enzyme A20 (Tnfaip3), a strong negative regulator of the canonical NF-κB signaling pathway. By generating A20 overexpressing Hoxb8 cells and thereby mimicking fetal neutrophils, we were indeed able to show reduced neutrophil adhesion to the inflammatory substrate in a microflow chamber assay. In contrast, mice with a neutrophil specific A20 deletion displayed increased inflammation in an in vivo model of TNF-α-stimulated cremaster muscle venules.

Conclusions Taken together, our results indicate a constitutive activation of the non-canonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils compared to adults, identifying non-canonical NF-κB signaling and A20 as master regulators of neutrophil function during fetal ontogeny.

Interessenkonflikt

All authors have declared that no conflict of interest exists.

Publication History

Article published online:
06 June 2023