A20 and the noncanonical NF-κB pathway are key regulators of neutrophil recruitment during fetal ontogeny
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
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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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 4

A20 upregulation in fetal neutrophils results in diminished adhesion.

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A20 upregulation in fetal neutrophils results in diminished adhesion. (A...
(A) mRNA expression of TNFAIP3 in fetal human neutrophils was compared to neutrophils isolated from peripheral blood of adult healthy donors by quantitative RT-PCR. Expression is shown relative to the housekeeping gene (HKG) GAPDH. (**P < 0.005; n = 3.) Unpaired Student’s t test. (B) Western blot and respective quantitative analysis of A20 expression in neutrophils isolated from human fetal cord blood samples and neutrophils from adult peripheral blood. Band intensity was normalized to Gapdh protein. All data are presented as mean ± SEM. (*P < 0.05, n = 8–10.) Unpaired Student’s t test. (C) Expression of Tnfaip3 mRNA relative to the HKGs B2m and Gyk in murine neutrophils was investigated by quantitative RT-PCR in isolated neutrophils of E14.5 and E17.5 embryos and from the peripheral blood of adult mice. (**P < 0.005; n = 3–4.) Ordinary 1-way ANOVA with Dunnett’s multiple comparisons test. (D) Workflow of Hoxb8 experiments. Hoxb8 precursor cells overexpressing A20 and Hoxb8 control cells were differentiated into Hoxb8 neutrophils, and subsequently flow chamber experiments were performed in addition to Western blot verification of A20 overexpression. (E) Representative Western blot image of A20 expression in control and A20-overexpressing Hoxb8 cells. Gapdh expression is displayed to ensure equal loading. (F) Flow chamber analysis of differentiated Hoxb8 control and A20-overexpressing cell adhesion in microflow chambers coated with rmE selectin/rmICAM-1 and rmCXCL1. One representative field was recorded for 10 minutes using an Olympus BX51WI microscope with a CCD camera (model CF8/1, Kappa) and a water immersion objective (×40/0.8 NA, Olympus). All data are presented as mean ± SEM. (***P < 0.001; n = 3.) Unpaired Student’s t test.