A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny
Ina Rohwedder, … , Matthias Heinig, Markus Sperandio
Ina Rohwedder, … , Matthias Heinig, Markus Sperandio
Published January 12, 2023
Citation Information: JCI Insight. 2023;8(4):e155968. https://doi.org/10.1172/jci.insight.155968.
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Research Article Inflammation

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

Abstract

Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.

Authors

Ina Rohwedder, Lou Martha Wackerbarth, Kristina Heinig, Annamaria Ballweg, Johannes Altstätter, Myriam Ripphahn, Claudia Nussbaum, Melanie Salvermoser, Susanne Bierschenk, Tobias Straub, Matthias Gunzer, Marc Schmidt-Supprian, Thomas Kolben, Christian Schulz, Averil Ma, Barbara Walzog, Matthias Heinig, Markus Sperandio

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Figure 2

The noncanonical NF-κB subunit RelB determines a specific gene expression signature in fetal human neutrophils.

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The noncanonical NF-κB subunit RelB determines a specific gene expressio...
(A) Venn diagram representing RelB-regulated genes within all genes up- and downregulated in adult compared with fetal neutrophils. Data were correlated with GM12878 human lymphoblastoid B cells (published data set) (15). (B) Imaging flow cytometry was performed on cord blood neutrophils from premature (gestational age < 37 weeks) and mature (gestational age > 37 weeks) fetal samples and peripheral blood from adult healthy donors. Representative pictures of fetal and adult neutrophils are shown as bright-field image, DAPI, NF-κB subunit RelB, and a DAPI/RelB overlay. Respective similarity scores are displayed. Scale bar: 7 μm. (C) Quantification of nuclear RelB. Similarity score defines overlap of nuclear DAPI signal and the respective NF-κB subunit. All data are presented as mean ± SEM. (*P < 0.05, **P < 0.005, n = 5–10.) Ordinary 1-way ANOVA with Tukey’s multiple comparisons test. (D) Western blot and respective quantitative analysis of p52 in isolated neutrophils from premature (gestational age < 37 weeks), mature (gestational age > 37 weeks), and adult samples. Band intensity was normalized to Gapdh. All data are presented as mean ± SEM (**P < 0.005, n = 4–7). Ordinary 1-way ANOVA with Dunnett’s multiple comparisons test. (E) Flow cytometry analysis of TNFRII expression on human and murine neutrophils out of whole blood from indicated gestational ages. Median fluorescence intensity normalized to isotype control is displayed. All data are presented as mean ± SEM (*P < 0.05, n = 3–5). Ordinary 1-way ANOVA with Dunnett’s multiple comparisons test. (F) LT-α protein levels in cord blood serum from premature (gestational age < 37 weeks) and mature infants (gestational age >37 weeks) and from whole blood of adult healthy donors by quantitative ELISA. Values are displayed in pg/mL. All data are presented as mean ± SEM. (*P < 0.05, n = 6–9.) Ordinary 1-way ANOVA with Dunnett’s multiple comparisons test.