Notch signaling regulates the responses of lipopolysaccharide-stimulated macrophages in the presence of immune complexes.

Wipawee Wongchana,Patcharavadee Butta,Chutamath Sittplangkoon,Thitiporn Pattarakankul,Pornrat Kongkavitoon,Tanapat Palaga,Supatta Chawalitpong,Barbara A. Osborne,Pattarin Tangtanatakul,Manjula Karpurapu

doi:10.1371/journal.pone.0198609

Wipawee Wongchana, Patcharavadee Butta + Show 8 more

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https://doi.org/10.1371/journal.pone.0198609

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Abstract

Macrophages exhibit diverse effector phenotypes depending on the stimuli and their microenvironment. Classically activated macrophages are primed with interferon (IFN)γ and stimulated with pathogen-associated molecular patterns. They produce inflammatory mediators and inflammatory cytokines, such as IL-12. In the presence of immune complexes (ICs), activated macrophages have decreased IL-12 production and increased IL-10 production and presumably act as regulatory macrophages. Notch signaling has been shown to regulate the effector functions of classically activated macrophages. In this study, we investigated whether Notch signaling is active in lipopolysaccharide (LPS)-stimulated macrophages in the presence of ICs. LPS/IC stimulation increased the level of cleaved Notch1 in murine macrophages, while IC stimulation alone did not. Delta-like 4, but not Jagged1, was responsible for generating cleaved Notch1. The activation of Notch signaling by LPS/ICs depended upon NF-κB and MEK/Erk pathway activation. Macrophages with the targeted deletion of Rbpj, which encodes a DNA-binding protein central to canonical Notch signaling, produced significantly less IL-10 upon LPS/IC stimulation. A similar impact on IL-10 production was observed when Notch signaling was inhibited with a gamma-secretase inhibitor (GSI). Defects in NF-κB p50 nuclear localization were observed in GSI-treated macrophages and in Rbpj-/- macrophages, suggesting cross-regulation between the Notch and NF-κB pathways. Transcriptomic analysis revealed that Notch signaling regulates the transcription of genes involved in the cell cycle, macrophage activation, leukocyte migration and cytokine production in LPS/IC-stimulated macrophages. Taken together, these results suggest that the Notch signaling pathway plays an important role in regulating the functions of macrophages activated by LPS and ICs.

Highlights

Macrophages mediate both innate and adaptive immune responses
Consistent with previous reports, compared with those activated by LPS, macrophages activated by LPS and Immune complexes (ICs) showed significantly higher levels of Il10 mRNA, whereas the levels of Il12b mRNA were significantly lower (Fig 1A) [5]
Notch signaling in LPS/immune complex-stimulated macrophages production was detected in all LPS-stimulated conditions, while priming with IFNγ alone or ICs alone did not result in detectable IL-10 levels

Summary

Introduction

Macrophages mediate both innate and adaptive immune responses. Signaling through lipopolysaccharide (LPS)/TLR4 results in the execution of host defense functions, such as phagocytosis and killing activities, by macrophages [1], and the cascade of downstream signaling molecules that are induced by LPS facilitates the transcriptional activation of inflammatoryassociated cytokines, such as TNFα, IL-1β, IL-6, IL-12, and type I interferon, as well as the production of relatively low amounts of IL-10. The priming of macrophages with IFNγ enhances TLR-induced cytokine gene expression, partly by facilitating the remodeling of chromatin to increase chromatin accessibility and the recruitment of TLR-induced transcription factors to the regulatory promoter regions [2]. These macrophages are well-characterized as classically activated macrophages [3]. This stimulation leads to macrophage activation that yields high levels of IL-10 and low levels of IL-12 while maintaining the levels of other innate cytokines, such as TNFα In addition to these signature cytokines, LPS/IC-activated macrophages express unique gene expression profiles that are different from classically activated macrophages or IL-4-stimulated macrophages, the so-called M2 macrophages [5, 6]. The adoptive transfer of these regulatory macrophages alleviates the severity of autoimmune disease in a mouse model of experimental autoimmune encephalomyelitis (EAE), suggesting that they have a systemic impact in vivo [9]

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