DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR‐activated murine macrophages

Abstract

Delta-like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR-induced expression of key pro-inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1-deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN-β and other pro-inflammatory cytokines, including TNF-α, IL-12, and IL-23. The expression of key proteins involved in IFN-β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL-12 and IL-23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS-induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH-mediated, pro-inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.Keywords: Cytokine r DLK1 r Inflammatory inhibitor r Macrophage r NOTCH signaling r TLR Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMacrophages are key cells in innate and adaptive immune response to pathogens. Toll-like receptors (TLR) recognize conserved microbial structures and induce the activation of macrophages, thus increasing their phagocytic and cytotoxic activities, and leading to the production of inflammatory cytokines, Correspondence: Dr. María J. Ruiz-Hidalgo and Dr. María J. M. Díaz-Guerra e-mail: Maria.RHidalgo@uclm.es; MariaJose.Martinez@uclm.es such as TNF-α and IL-1, and cytokines of the IL-6 and IL-12 families, which regulate T-cell differentiation [1].Macrophages display a remarkable plasticity and can change their functioning in response to environmental signals, which allows a fine-tuning of their activity to adapt to different situations. Unrestrained activation of TLR responses can lead to excessive inflammation and tissue damage and contribute to the pathogenesis of inflammatory disorders, such as septic shock. Multiple mechanisms are implicated in the control of macrophage activation, including inhibitory cytokines, transcriptional repressors, and epigenetic modifications [2,3].C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 2616 María J. González et al. Eur. J. Immunol. 2015. 45: 2615-2627 In this line, a critical role for NOTCH signaling in macrophage activation and polarization has been evidenced [4][5][6]. Ligand binding to the NOTCH receptors triggers a two-step proteolytic cleavage causing the release of the NOTCH intracellular doma...

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“…Top upstream regulators of these pathways were interferon regulatory factors 3 and 7 ( IRF3 and IRF7 , respectively) linked to interferon and macrophage regulation [30], and delta like canonical ligand 3 ( DLL3 ), Jagged 1 ( JAG1 ), and mesogenin 1 ( MSGN1 ), which all are linked to Notch signaling [31, 32] (Additional file 6: Table S4). Top diseases and biofunctions involved development, cellular functions, cancer and tumor morphologies, and inflammatory responses (Additional file 7: Table S5).…”

“…Five top upstream regulators of the 48 DEGs were DLL3 and JAG1 (Notch ligands) [37]; MSGN1 (transcriptional activator of a Notch signaling program) [32]; and IRF3 and IRF7 , which are both key transcriptional regulators of interferons and macrophages [30]. Notch signaling may also regulate IRF activity [38].…”

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“…NF-kB is a key transcription factor in TLR activation whose activity has been described to be enhanced by NOTCH signaling (10,(38)(39)(40). We wondered whether the modulation of proinflammatory gene expression by TSPAN33 observed in macrophages ( Fig.…”

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“…Macrophages are involved in immune and inflammatory responses and play important roles in periodontitis . They change functioning flexibly by sensing the different environments . Periodontitis is one of the most prevalent chronic inflammatory diseases.…”

“…3 They change functioning flexibly by sensing the different environments. 4 Periodontitis is one of the most prevalent chronic inflammatory diseases. The periodontal inflammatory microenvironment, which forms primarily because of LPS stimulation, not only disturbs periodontal matrix metabolism and bone metabolism but also suppresses periodontal tissue regeneration and leads to periodontal tissue destruction.…”