Pravastatin sodium attenuated TREM-1-mediated inflammation in human peripheral blood mononuclear cells

https://doi.org/10.1016/j.bbrc.2018.11.098Get rights and content

Highlights

  • Triggering receptor expressed on myeloid cell-1(TREM-1) mediated inflammation in human peripheral blood mononuclear cells.

  • Pravastatin could inhibit TREM-1-medieted inflammation in human peripheral blood mononuclear cells.

  • NF-κB cellular signaling pathway was involved in the anti-inflammation effect of pravastatin.

Abstract

Pravastatin sodium on triggering receptor expressed on myeloid cell-1 (TREM-1)-mediated inflammation in human peripheral blood mononuclear cells (PBMCs) has been poorly investigated. In this study, we isolated PBMCs from the peripheral blood samples of patients with chronic obstructive pulmonary disease, treated the cells with pravastatin sodium, and determined a concentration at which more than 90% cells could survive. Then we treated cells with 10 ng/ml of lipopolysaccharide, added with 10, 50, 100 μM of pravastatin sodium combined with or without LR-12, a known TREM-1 inhibitor. The expression of TREM-1 was determined by quantitative RT-PCR. The levels of TREM-1, IL-6, and TNF-α in cell culture supernatant were measured with ELISA. Simultaneously, NF-κB signaling pathway-related protein p-p65 and p-IκBα were detected by Western blot assay. Results demonstrated that pravastatin sodium significantly mitigated lipopolysaccharide-stimulated TREM-1 over-expression at mRNA and protein levels dose-dependently. Elevated IL-6 and TNF-α levels changed synchronously. LR-12 inhibited the TREM-1 over-expression and inflammatory factor production but did not show extra synergistic effect to pravastatin. Lipopolysaccharide induced phospho-p65 and -IκBα over-expression was weakened significantly when cells were treated with pravastatin sodium. In conclusion, pravastatin could inhibit TREM-1-medieted inflammation and NF-κB signaling pathway was involved.

Introduction

Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease, characterized by progressive development of the irreversible airway inflammation, airflow limitation, and declined lung function. The pathogenesis of COPD has not been fully elucidated while chronic inflammation in the lower airway was considered to be crucial. Studies showed that neutrophils, monocytes, lymphocytes, and macrophages are involved in the inflammation [1]. These cells release various inflammatory mediators such as interlukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) to attract more inflammatory cells from the circulation, enhance the inflammatory process, and induce structural changes. Peripheral blood mononuclear cells (PBMCs) consist of monocytes and lymphocytes which act as critical components in the immune system to cause the chronic inflammation and lung tissue damage. Obviously, treatment aiming at these inflammation processes may hinder the disease progress potentially.

Triggering receptor expressed on myeloid cell-1 (TREM-1) is a kind of immunoglobulin mainly expressed on the cell membrane of neutrophils, monocytes and macrophages. TREM-1 amplifies toll-like receptor-initiated responses to potentiate the role of pro-inflammatory chemokines and cytokines [2]. Like many membrane bound receptors, TREM-1 also exists in the soluble form (sTREM-1) in human body fluids, which can be tested and measured. Studies reported that sTREM-1 levels can be potentially used for early diagnosis and as a prognosis marker of some infectious diseases. For example, in patients with bacterial pneumonia, sTREM-1 levels in bronchoalveolar lavage fluid were significantly increased [3]. In lipopolysaccharide (LPS)-induced acute lung injury mice, TREM-1 depletion resulted in decreased systematic cytokine production and improved survival [4]. Elevated serum sTREM-1 levels reflected higher severity and poor prognosis in COPD patients [5]. Obviously, TREM-1 is involved in inflammation and interfering with it might ameliorate the inflammation.

Statins are inhibitors of 3-Hydroxymethyl-3-glutaryl Coenzyme A reductase, used clinically as lipid control regime for hyperlipemia especially hypercholesterolemia. In addition to their lipid-lowering property, statins reportedly possess some anti-inflammatory and immunomodulatory effects. In a vitro study, statin reduced the inflammatory cytokine production (IL-6, -8) in air interface Calu-3 cells after LPS stimulation [6]. Clinical studies demonstrated that in patients with inflammatory lung diseases such as asthma, lung injury, and COPD, statins were promising treatment [[7], [8], [9]]. For example, in COPD patients, statins could reduce the pulmonary-related mortality [10]. Meanwhile, other study presented the different conclusions [11]. Indeed, the role and underlying cellular mechanism of statins on inflammatory cells have been poorly explored to date. In this study, we observed whether pravastatin sodium could attenuate TREM-1-mediated inflammation in human PBMCs and if the attenuation works via NF-κB signaling pathway.

Section snippets

Isolation and culture of PBMCs

The protocol of this study was approved by the Ethic Committee of Anhui Provincial Hospital, and an agreement was received by each of the patient. PBMCs were obtained by density gradient centrifugation using the lymphocyte separation solution according to the manufacturer's instruction. Blood samples from stable stage COPD patients were collected in EDTA tubes. Blood was diluted 1:1 with sterile 1XPBS and layered over sterile Lymphoprep™. Samples were centrifuged (550 g, 20 min, 25 °C). The

Cytotoxicity

To determine a non-cytotoxic concentration of the pravastatin sodium, gradient concentrations of pravastatin sodium were tested and cell viability was observed. The results showed that cells survived greater than 90% when treated with pravastatin sodium at a concentration less than 100 μM. Higher doses of pravastatin led to lower viability. Therefore, we chose the concentration of 10, 50, and 100 μM for the subsequent experiments.

Effect of pravastatin sodium on LPS-upregulated TREM-1 expression in PBMCs

Referring to the reported article [12], we detected TREM-1 mRNA

Discussion

COPD is a multi-component disease involving long-term inflammation in the lung. The chronic inflammatory responses were persistent, leading to tissue damage and organ dysfunction. PBMCs could be attracted and recruited by the local inflammatory mediators in the lung tissue and play a critical role in the inflammatory process. In this study, we observed the changes of stimulated PBMCs in vitro, concentrating on the effects of pravastatin sodium on TREM-1-mediated inflammatory responses and the

Conflicts of interest

No conflicts exist for the specified authors.

Acknowledgements

This study was supported by the Natural Science Foundation of Anhui Province, China (1308085MH115) and Science and Technology Foundation of Anhui Province, China (1704f0804007).

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  • Cited by (5)

    1

    The authors contributed equally to this work.

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