LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b

Ti et al. J Transl Med (2015) 13:308 DOI 10.1186/s12967-015-0642-6 Open Access RESEARCH LPS‑preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome‑shuttled let‑7b Dongdong Ti1†, Haojie Hao1†, Chuan Tong1, Jiejie Liu1, Liang Dong1, Jingxi Zheng1, Yali Zhao2, Huiling Liu1, Xiaobing Fu1* and Weidong Han1* Abstract Background: Within the last few years, it has become evident that LPS-preconditioned mesenchymal stromal cells (LPS pre-MSCs) show enhanced paracrine effects, including increased trophic support and improved regenerative and repair properties. MSCs may release large amounts of exosomes for cell-to-cell communication and maintain a dynamic and homeostatic microenvironment for tissue repair. The present study assesses the therapeutic efficacy and mechanisms of LPS-preconditioned MSC-derived exosomes (LPS pre-Exo) for chronic inflammation and wound healing. Methods: We extracted exosomes from the supernatant of LPS pre-MSCs using a gradient centrifugation method. In vitro, THP-1 cells were cultured with high glucose (HG, 30 mM) as an inflammatory model and treated with LPS preExo for 48 h. The expression of inflammation-related cytokines was detected by real-time RT-PCR, and the distribution of macrophage subtype was measured by immunofluorescence. Next, the miRNA expression profiles of LPS pre-Exo were evaluated using miRNA microarray analysis. The molecular signaling pathway responsible for the regenerative potential was identified by western blotting. In vivo, we established a cutaneous wound model in streptozotocininduced diabetic rats, and LPS pre-Exo were injected dispersively into the wound edge. The curative effects of LPS pre-Exo on inflammation and wound healing were observed and evaluated. Results: LPS pre-Exo have a better ability than untreated MSC-derived exosomes (un-Exo) to modulate the balance of macrophages due to their upregulation of the expression of anti-inflammatory cytokines and promotion of M2 macrophage activation. Microarray analysis of LPS pre-Exo identified the unique expression of let-7b compared with un-Exo, and the let-7b/TLR4 pathway served as potential contributor to macrophage polarization and inflammatory ablation. Further investigation of the mechanisms that control let-7b expression demonstrated that a TLR4/NF-κB/ STAT3/AKT regulatory signaling pathway plays a critical role in the regulation of macrophage plasticity. Knockdown of AKT in THP-1 cells similarly abolished the immunomodulatory effect of LPS pre-Exo. In vivo, LPS pre-Exo greatly alleviated inflammation and enhanced diabetic cutaneous wound healing. Conclusion: LPS pre-Exo may have improved regulatory abilities for macrophage polarization and resolution of chronic inflammation by shuttling let-7b, and these exosomes carry much immunotherapeutic potential for wound healing. Keywords: Mesenchymal stromal cells, Macrophage polarization, Exosome, LPS preconditioning *Correspondence: ; † Dongdong Ti and Haojie Hao contributed equally to this work 1 Institute of Basic Medicine Science, College of Life Science, Chinese PLA General Hospital, No. 28 Fuxing Road, Beijing 100853, China Full list of author information is available at the end of the article © 2015 Ti et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ti et al. J Transl Med (2015) 13:308 Background Impairment of cutaneous wound healing represents a serious complication of diabetes and is associated with significant morbidity and a massive socioeconomic burden [1]. Evidence suggests that chronic cutaneous wounds are characterized by an abnormal inflammatory state, with prolonged accumulation of macrophages and an increased release of pro-inflammatory cytokines [2, 3]. Therefore, orchestrating the inflammatory response might be a promising strategy to promote proper wound healing. As the primary effectors of inflammation in tissue injury, macrophages can be polarized into the “classically activated” M1 phenotype or the “alternatively activated” M2 phenotype according to signals in the microenvironment [4, 5]. The proinflammatory responses of M1 macrophages depend on Toll-Like Receptors (TLRs) and the activation of NFκB, leading to pathogen phagocytosis, oxidative burst and intracellular killing. Conversely, activation of M2 macrophages leads to the recruitment of STAT3 or other transcription factors, resulting in dampened inflammation and promoting tissue remodeling. Previous studies have shown that in diabetic mouse models, high glucose can enhance unrestrained M1 cells but impair M2 cell polarization, produce a large number of pro-inflammatory mediators, and drive chronic inflammation [6]. Furthermore, dysregulated inflammation in diabetic mellitus allows for the hyper-induction of M1 macrophages and the aberrant production of inducible nitric oxide synthase (iNOS), causing de novo tissue destruction and recapitulating chronic wounds [7]. Therefore, the appropriate balancing of macrophage polarization plays a crucial role in regulating inflammation and subsequently accelerates tissue repair and homeostasis. Regenerative medicine strategies using mesenchymal stromal cells (MSCs) are expected to be a hopeful alternative approach to the treatment of a wide variety of pathological conditions, such as chronic inflammation. Pelizzo and colleagues found that MSCs may relieve the inflammatory response by inducing the wound’s capacity to progress in regeneration of skin architecture and not regress to a chronic wound state, and this biological property of MSCs to promote the transition from inflammatory to the proliferative phase is very crucial for treating wounds where high levels of inflammation inhibit healing [8]. Furthermore, MSCs switched from infiltration of pro-inflammatory to anti-inflammatory macrophages for alleviating inflammation and augmenting cardiac regeneration [9]. In a sepsis model, MSCs can secrete certain growth factors to increase the percentage of reparative M2 macrophages and improve Page 2 of 14 organ function [10]. Studies have found that the exposure of MSCs to the pharmacological agent lipopolysaccharide (LPS) can increase their trophic effects and functional properties to defend against the harsh inflammatory environment [11–13]. Liu et al. have confirmed that LPS-primed MSCs have a superior therapeutic ability to preserve skin flap survival in a diabetic rat model compared to unprimed MSCs [1 (...truncated)