Substance P Induces HO-1 Expression in RAW 264.7 Cells Promoting Switch towards M2-Like Macrophages.

Giovanna Montana,Nadia Lampiasi,Partha Mukhopadhyay

doi:10.1371/journal.pone.0167420

Giovanna Montana, Nadia Lampiasi + Show 1 more

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https://doi.org/10.1371/journal.pone.0167420

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Abstract

Substance P (SP) is a neuropeptide that mediates many physiological as well as inflammatory responses. Recently, SP has been implicated in the resolution of inflammation through induction of M2 macrophages phenotype. The shift between M1-like and M2-like, allowing the resolution of inflammatory processes, also takes place by means of hemeoxygenase-1 (HO-1). HO-1 is induced in response to oxidative stress and inflammatory stimuli and modulates the immune response through macrophages polarisation. SP induces HO-1 expression in human periodontal ligament (PDL), the latter potentially plays a role in cytoprotection. We demonstrated that SP promotes M2-like phenotype from resting as well as from M1 macrophages. Indeed, SP triggers the production of interleukine-10 (IL-10), interleukine-4 (IL-4) and arginase-1 (Arg1) without nitric oxide (NO) generation. In addition, SP increases HO-1 expression in a dose- and time-dependent manner. Here we report that SP, without affecting cell viability, significantly reduces the production of pro-inflammatory cytokines and enzymes, such as tumor necrosis factor-alpha (TNF-α), interleukine-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and ameliorates migration and phagocytic properties in LPS-stimulated RAW 264.7 cells. M2-like conversion required retention of NF-κB p65 into the cytoplasm and HO-1 induced expression. Silencing of the HO-1 mRNA expression reversed the induction of pro-inflammatory cytokines in RAW 264.7 stimulated by LPS and down-regulated anti-inflammatory hallmarks of M2 phenotype. In conclusion, our data show that SP treatment might be associated with anti-inflammatory effects in LPS-stimulated RAW 264.7 cells by suppressing NF-κB activation and inducing HO-1 expression.

Highlights

We first investigated whether the neuropeptide Substance P (SP) could stimulate the production of HO-1 in RAW 264.7 macrophages
Cells were challenged with different SP concentrations (1, 10, 100 μM) and different time of exposure (4, 6, 24 h) and the levels of HO-1 mRNA were quantified by RT-qPCR
neurokinin-1 receptor (NK-1R) mRNA expression was detected in cells under baseline conditions

Summary

Introduction

HO-1 is induced in response to oxidative stress and inflammatory stimuli, playing an important role in the suppression of inflammatory reaction and insulin resistance [15] It has been regarded as an adaptive cellular response against NF-κB-mediated inflammation [16] and oxidative cytotoxic conditions, such as excessive production of ROS or TNF-induced apoptosis [17]. The promoter sequences of HO-1 contain two enhancer regions (E1 and E2) providing binding motifs for a variety of transcription factors, such as activator protein (AP)-1, cAMPresponsive element binding protein (CREB), NF-κB or Nuclear factor (erythroid-derived 2)like 2 (Nrf2) [23] Among these transcription factors, Nrf, which is the master regulator of the antioxidant stress response, controls many aspects of cell homoeostasis in response to oxidative and toxic insults. The protective effect of SP on LPS-induced inflammation and the macrophages switching towards M2-like phenotype were confirmed by HO-1 silencing

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