ErbB4 Signaling Restricts Pro‐Inflammatory Macrophage Numbers through an ADAM17/γ‐secretase Dependent Mechanism
Authors: Schumacher Michael A , Bernard Jessica K , Hsieh Jonathan J , Punit Shivesh , Frey Mark R 
Published: November 2023

BACKGROUNDThe ErbB4‐specific ligand neuregulin‐4 (NRG4) is protective when administered in rodent colitis models. The cell populations and mechanisms mediating this protection are not well defined, but ErbB family receptor expression has recently been demonstrated on immune cells. In particular, ErbB4 has been detected on circulating human monocytes and neuronal macrophages (Mø), but its expression on intestinal Mø and role in Mø biology are unknown. In some cell types, a metalloproteinase/secretase‐mediated cleavage of ErbB4 generating an intracellular domain (4ICD) is required for ErbB4 signaling, but whether 4ICD is present or functional in Mø is not defined.HYPOTHESISErbB4 is induced on Mø during colitis, and NRG4 regulates Mø number or activity during inflammation through ErbB4 activation and generation of 4ICD.METHODSTo study the protective effect of neuregulin‐4, colitis was induced in mice (C57Bl/6) by administering 3% dextran sodium sulfate (DSS) in drinking water for 4 days followed by daily NRG4 injections (100 μg/kg) on days 4–7. Colonic Mø populations were identified by flow cytometry (F4/80HI/CD11bHI). Murine bone marrow‐derived Mø (BMMø) were generated and classically (IFN‐γ/LPS) or alternatively (IL‐4) activated for in vitro studies. Bone marrow‐derived dendritic cells (BMDC) were also generated for TLR‐4 dependent (IFN‐γ/LPS) activation analysis. Some cells were exposed to NRG4 (100 ng/ml) to activate ErbB4, with or without ErbB4 blocking antibody. Inhibitors of ADAM17 (GM6001) and γ‐secretase (DAPT) were used to test the requirement for ligand‐stimulated ErbB4 proteolysis and 4ICD generation in response to NRG4.RESULTSDuring DSS colitis, the proportion of ErbB4+ Mø significantly increased (4.2‐fold, p<0.05). These cells predominantly expressed Ly6C, suggesting ErbB4 is present on recruited pro‐inflammatory Mø. However, colonic expression of the ErbB4 ligand NRG4 was reduced during colitis, coincident with elevated pro‐inflammatory cytokine levels. Exogenous NRG4 injection during colitis ameliorated disease parameters, reduced IFNγ, IL‐6, and TNF levels, and attenuated colonic Mø numbers by 35%. In vitro, classical activation (IFNγ/LPS) induced ErbB4 expression on cultured BMMø, whereas alternative activation (IL‐4) inhibited expression. ErbB4 expression was undetectable in resting and IFNγ/LPS‐stimulated BMDCs. Treatment of classically M1‐polarized Mø with NRG4 induced apoptosis as measured by caspase‐3 and annexin V analysis, leading to a significant reduction in viable cell number after 48 hours. This reduction was blocked by ErbB4 blocking antibody or by pre‐treatment with ADAM17 and γ‐secretase inhibitors. This suggests proteolytic cleavage of ErbB4 and liberation of 4ICD mediates the effect of NRG4 on Mø death.CONCLUSIONSColitis induces ErbB4 on colonic Mø. NRG4 binding to ErbB4 stimulates ADAM17/γ‐secretase dependent cleavage of ErbB4 which signals for Mø apoptosis. NRG4‐stimulated ErbB4 signaling in these cells is an inhibitory feedback mechanism restricting Mø numbers, and thus is a potential therapeutic target to reduce inflammation in IBD.Support or Funding InformationNIH R01 DK095004 (MRF); Senior Research Award (MRF) and Research Fellowship Award (MAS) from the Crohn's & Colitis Foundation of America