TREM-1-accentuated lung injury via miR-155 is inhibited by LP17 nanomedicine

Abstract

Triggering receptors expressed on myeloid cell-1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells. Synergy between TREM-1 and Toll-like receptor amplifies the inflammatory response; however, the mechanisms by which TREM-1 accentuates inflammation are not fully understood. In this study, we investigated the role of TREM-1 in a model of LPS-induced lung injury and neutrophilic inflammation. We show that TREM-1 is induced in lungs of mice with LPS-induced acute neutrophilic inflammation. TREM-1 knockout mice showed an improved survival after lethal doses of LPS with an attenuated inflammatory response in the lungs. Deletion of TREM-1 gene resulted in significantly reduced neutrophils and proinflammatory cytokines and chemokines, particularly IL-1β, TNF-α, and IL-6. Physiologically deletion of TREM-1 conferred an immunometabolic advantage with low oxygen consumption rate (OCR) sparing the respiratory capacity of macrophages challenged with LPS. Furthermore, we show that TREM-1 deletion results in significant attenuation of expression of miR-155 in macrophages and lungs of mice treated with LPS. Experiments with antagomir-155 confirmed that TREM-1-mediated changes were indeed dependent on miR-155 and are mediated by downregulation of suppressor of cytokine signaling-1 (SOCS-1) a key miR-155 target. These data for the first time show that TREM-1 accentuates inflammatory response by inducing the expression of miR-155 in macrophages and suggest a novel mechanism by which TREM-1 signaling contributes to lung injury. Inhibition of TREM-1 using a nanomicellar approach resulted in ablation of neutrophilic inflammation suggesting that TREM-1 inhibition is a potential therapeutic target for neutrophilic lung inflammation and acute respiratory distress syndrome (ARDS).

Keywords: TREM-1; lung injury; miR-155; nanomedicine.

Fig. 1.

Triggering receptors expressed on myeloid cell-1 (TREM-1) knockout mice demonstrate improved survival with attenuated inflammation in response to LPS. A: wild-type (WT) and TREM-1 knockout mice were given 25 mg/kg of LPS or vehicle via intraperitoneal route. Control mice all survived. Wild-type mice treated with LPS all succumbed within 96 h whereas TREM-1 knockout mice showed an improved survival. The results are represented by Kaplan Meier curve; P < 0.01 log-rank test. B: lung histology from TREM-1 knockout mice shows an attenuation of neutrophilic influx compared with wild-type mice treated with LPS. HE, hematoxylin-eosin. C: bronchoalveolar lavage (BAL) cell count showed significantly lower cell numbers in TREM-1 knockout mice. Myeloperoxidase (MPO) assays from lungs (D), lung wet-to-dry ratio (W/D; E), BAL protein (F), TNF-α (G), IL-6 (H), and IL-1β (I) were lower in lungs of TREM-1 knockout mice. *P < 0.05; n = 5–6.

Fig. 2.

Microarray analysis of miRNA expression from TREM-1 knockout (KO) and wild-type macrophages. Heat map of genes from TREM-1 and wild-type macrophages treated with LPS (100 ng/ml) (A). miR-155-3p and miR-155-5p (B) and miR-32, miR-466l, and miR-665 (C) were significantly downregulated in TREM-1 knockout macrophages. *P < 0.01; n = 4–5.

Fig. 3.

Proinflammatory effects induced by TREM-1 are mediated through expression of miR-155. Bone marrow-derived macrophages (BMDM) from wild-type mice were treated with mTREM-1 (10 ng/ml) with or without antagomir against miR-155 (100 nmol/l). A: mTREM-1 induced the expression of miR-155, which was downregulated in the presence of anti-miR-155. mTREM-1 induced expression of TNF (B), IL-6 (C), and IL-1β (D) mRNA; however, treatment with miR-155 antagomir resulted in abrogation of this induction. Wild-type and TREM-1 knockout mice were treated with aerosolized LPS (10 mg/kg). Lungs from TREM-1 knockout mice showed a significant downregulation of miR-155-3p and -5p (E) after treatment with LPS. *P < 0.05; n = 4–5.

Fig. 4.

miR-155 is induced by TREM-1 in an NF-κB-dependent manner. Suppressor of cytokine signaling-1 (SOCS-1) is a miR-155 target induced by TREM-1. RAW264.7 cells were treated with mTREM-1 (10 ng/ml) or control IgG or LPS (100 ng/ml) or PBS with or without the NF-κB inhibitor Bay 11–7082(1 μmol/l). Expression of miR-155-5p (A), miR-155-3p (B), and Mir155hg (C) was determined by real-time PCR at 16 h posttreatment. RAW264.7 cells were transfected with miR-155-3p and miR-155-5p antagomir (100 nmol/l) and mirVana miRNA Inhibitor Negative Control (100 nmol/l), 48 h later, cultured in DMEM media with or without mTREM-1 (10 ng/ml) and LPS (100 ng/ml), respectively. D: SOCS-1 mRNA expression was determined at 16 h. Data represent the mean ± SD percentages cells from 3 independent experiments for both untreated cells (open bars) and cells treated with 1 μmol/l Bay 11–7082 (solid bars). *P < 0.05.

Fig. 5.

Mitochondrial bioenergetics in RAW264.7 cells showed lower oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in TREM-1 knockdown cells. RAW264.7 cells were transfected with siTREM-1 or control siRNA, and mitochondrial oxidative phosphorylation reactions and respiratory assays were assessed by a Seahorse analyzer. Basal and spare respiratory OCR (A), ATP turnover and maximal respiration (B), proton leak (C), and ECAR (D) were measured in real time under basal conditions and in response to indicated mitochondrial inhibitors. R/A, rotenone/antimycin. E: basal OCR/ECAR ratio. *P < 0.05; n = 12.

Fig. 6.

TREM-1 knockout macrophages showed lower OCR and maximal respiration after treatment with LPS compared with wild-type macrophages. BMDM from wild-type and TREM-1 knockout mice were treated with LPS (100 ng/ml) for 4 h, and mitochondrial bioenergetics were determined using a Seahorse analyzer. OCR and basal respiration (A), ATP turnover and maximal respiration (B), and proton leak (C). *P < 0.05; n = 12.

Fig. 7.

miR-155-3p mimic increases mitochondrial OCR and ECAR. RAW264.7 cells were transfected with miR-155-3p mimic (100 nmol/l) and mirVana miRNA mimic Negative Control (100 nmol/l). A: miR-155-3p expression was measured in RAW264.7 cells transfected with miR-155-3p mimic and negative control. Mitochondrial bioenergetics were measured in transfected cells using a Seahorse analyzer. B: OCR in real time under basal conditions and in response to indicated mitochondrial inhibitors. C: ATP production and maximal respiration. D: proton leak. E: ECAR. F: basal OCR/ECAR ratio. Data are representative of at least 3 independent experiments and shown as mean ± SE. *P < 0.05; **P < 0.0001.

Fig. 8.

LP17 nanomedicine [LP17- sterically stabilized phospholipid nanomicelles (SSM) SSM] attenuate LPS-induced neutrophilic lung inflammation. Wild-type mice were treated with LP17 or scrambled LP17, LP17 SSM, or scrambled SSM via subcutaneous route before treatment with aerosolized LPS (1 mg/ml). Expression of TREM-1 in the lung was significantly reduced by LP17 SSM (A), and BAL total and neutrophil (B), MPO assays from lung homogenates (C), lung wet-to-dry ratio (D), BAL proteins (E), TNF-α (F), and IL-1β (G) levels were significantly lower in mice that were treated with LP17 SSM compared with naked peptide (LP17). *P < 0.05; n = 6–7.