Activation of TREM-1 induces endoplasmic reticulum stress through IRE-1α/XBP-1s pathway in murine macrophages

Highlights

• Activation of TREM-1 induces pro-inflammatory microenvironment in primary macrophages.

• IRE-1α/XBP-1 s is required for the TREM-1-induced pro-inflammatory microenvironment in primary macrophages.

• Blockade of TREM-1 ameliorates lung injury induced by LPS through suppressing ER stress.

Abstract

Increasing evidence suggests that endoplasmic reticulum (ER) stress activates several pro-inflammatory signaling pathways in many diseases, including acute lung injury (ALI). We have reported that blocking triggering receptor expressed on myeloid cells 1 (TREM-1) protects against ALI by suppressing pulmonary inflammation in mice with ALI induced by lipopolysaccharides (LPS). However, the molecular mechanism underlying the TREM-1-induced pro-inflammatory microenvironment in macrophages remains unclearly. Herein, we aimed to determine whether TREM-1 regulates the inflammatory responses induced by LPS associated with ER stress activation. We found that the activation of TREM-1 by a monoclonal agonist antibody (anti-TREM-1) increased the mRNA and protein levels of IL-1β, TNF-α, and IL-6 in primary macrophages. Treatment of the anti-TREM-1 antibody increased the expression of ER stress markers (ATF6, PERK, IRE-1α, and XBP-1s) in primary macrophages. While pretreatment with 4-PBA, an inhibitor of ER stress, significantly inhibited the expression of ER stress markers and pro-inflammatory cytokines and reduced LDH release. Furthermore, inhibiting the activity of the IRE-1α/XBP-1s pathway by STF-083010 significantly mitigated the increased levels of IL-1β, TNF-α, and IL-6 in macrophages treated by the anti-TREM-1 antibody. XBP-1 silencing attenuated pro-inflammatory microenvironment evoked by activation of TREM-1. Besides, we found that blockade of TREM-1 with LR12 ameliorated ER stress induced by LPS in vitro and in vivo. In conclusion, we conclude that TREM-1 activation induces ER stress through the IRE-1α/XBP-1s pathway in macrophages, contributing to the pro-inflammatory microenvironment.

Introduction

Acute lung injury (ALI), a serious respiratory disease worldwide, is recognized as an intense and severe inflammatory process in the lung (Huang et al., 2019; Doycheva et al., 2019). ALI is usually caused by trauma, bacteria, and pneumonia (Sun et al., 2018). ALI is usually related to inflammatory processes, including alveolar epithelium injury, neutrophil accumulation into the alveoli, increased microvascular endothelial permeability, and pulmonary interstitial edema (Yuan et al., 2018; Zhang et al., 2018; Bao et al., 2019). Although considerable progress has been made, the mortality rate remains high (Guo et al., 2018). The mechanisms underlying the lipopolysaccharides (LPS)-mediated ALI are far from completely understood.

The endoplasmic reticulum (ER) is a cell organelle for protein synthesis, processing, secretion, and storing cellular calcium (Ca²⁺) (Joshi et al., 2017; Benham, 2012; Mahanty et al., 2019). Impairment of ER function results in an accumulation of misfolded and unfolded proteins in the ER lumen, inducing ER stress (Chou et al., 2019; Garcia-Gonzalez et al., 2018). Stress-responsive genes, proteins, and pathways provide cellular mechanisms to deal with the ER stress and restore cell homeostasis (Chu et al., 2019). The ER stress is sensed by the ER transmembrane proteins, including activating transcription factor 6 (ATF6), PKR-like ER kinase (PERK), and inositol-requiring enzyme 1α (IRE-1α), which activates the unfolded protein response (UPR), an adaptive response (Chan et al., 2017; Neves et al., 2019). If the UPR fails to cope with the ER stress, the cells will undergo cell dysfunction (Sagar et al., 2019; Huang et al., 2017). It has been reported that LPS-induced ALI and LPS-activated alveolar macrophages show an increase in unfolded protein levels in the ER, which indicates substantial UPR (Liang et al., 2019; Weng et al., 2019).

IRE-1α is the most important regulator of the UPR (Walter and Ron, 2011). IRE-1α converts unspliced XBP-1 mRNA into an active transcription factor XBP-1 (XBP1s) mRNA (Schutt et al., 2018). Then XBP-1s protein, encoded by XBP-1s mRNA, controls the transcription of several targets genes for protein degradation, protein folding, and UPR function (Kroeger et al., 2019). Mutant of IRE-1α and XBP1 has a protective effect against inflammatory diseases, such as inflammatory bowel disease (Negroni et al., 2014). Moreover, in macrophages and stromal cells, XBP1 activation itself is sufficient to drive pro-inflammatory cytokine’s transcription, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (Li et al., 2005).

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a cell-surface-activating receptor, mainly expressing in myeloid cells, such as monocytes, macrophages, and neutrophils (Zhu et al., 2016; Bouchon et al., 2000). TREM-1 exerts an important regulatory role in inflammatory responses. Accumulating studies show that blocking TREM-1 protects against ischemia-reperfusion, sepsis, inflammatory bowel diseases, and pancreatitis (Sigalov, 2014; Wang et al., 2012; Gibot et al., 2008; Dang et al., 2012; Schenk et al., 2007). In our previous study, we found that blocking TREM-1 attenuates ALI by decreasing pulmonary inflammation and improves the survival of mice with ALI induced by LPS (Liu et al., 2016). It has shown that the activation of TREM-1 can increase the production of inflammatory cytokines induced by LPS in human monocytes, such as IL-1β (Dower et al., 2008). However, the current study is very limited in whether TREM-1 regulates the ER stress in inflammatory responses induced by LPS.

Here, we hypothesized that the activation of TREM-1 could enhance the ER stress, contributing to the inflammatory cytokine storm in the lungs of ALI mice. In this study, we investigated whether ER stress was involved in releasing inflammatory cytokines relative to the activation of TREM-1 in vitro, and blockade of TREM-1 could protect against ALI induced by LPS by suppressing ER stress in vivo.