lnc-MRGPRF-6:1 Promotes M1 Polarization of Macrophage and Inflammatory Response through the TLR4-MyD88-MAPK Pathway

Background Macrophage-mediated inflammation plays an essential role in the development of atherosclerosis (AS). Long noncoding RNAs (lncRNAs), as crucial regulators, participate in this process. We identified that lnc-MRGPRF-6:1 was significantly upregulated in the plasma exosomes of coronary atherosclerotic disease (CAD) patients in a preliminary work. In the present study, we aim to assess the role of lnc-MRGPRF-6:1 in macrophage-mediated inflammatory process of AS. Methods The correlation between lnc-MRGPRF-6:1 and inflammatory factors was estimated firstly in plasma exosomes of CAD patients. Subsequently, we established lnc-MRGPRF-6:1 knockout macrophage model via the CRISPR/Cas9 system. We then investigated the regulatory effects of lnc-MRGPRF-6:1 on macrophage polarization and foam cell formation. Eventually, transcriptome analysis by RNA sequencing was carried out to explore the contribution of differential genes and signaling pathways in this process. Results lnc-MRGPRF-6:1 was highly expressed in the plasma exosomes of CAD patients and was positively correlated with the expression of inflammatory cytokines in plasma. lnc-MRGPRF-6:1 inhibition significantly reduced the formation of foam cells. The expression of lnc-MRGPRF-6:1 was upregulated in M1 macrophage, and lnc-MRGPRF-6:1 knockout decreased the polarization of M1 macrophage. lnc-MRGPRF-6:1 regulates macrophage polarization via the TLR4-MyD88-MAPK signaling pathway. Conclusions lnc-MRGPRF-6:1 knockdown can inhibit M1 polarization of macrophage and inflammatory response through the TLR4-MyD88-MAPK signaling pathway. lnc-MRGPRF-6:1 is a vital regulator in macrophage-mediated inflammatory process of AS.


Introduction
Coronary atherosclerotic disease (CAD) is a serious threat to human health, which is also one of the leading causes of human death worldwide [1]. Atherosclerosis (AS) is the pathological basis of CAD [2]. AS is a chronic inflammatory vascular disease, and macrophage plays a key role in the occurrence and development of AS. Apoptosis of macrophage is the process of forming necrotic core, and it is an inducer of long-term low-level inflammation of the intima, which can further promote the occurrence of AS [3]. Macrophage can secrete a variety of proinflammatory factors and chemokines to regulate the development of AS. After lipopolysaccharide (LPS) stimulation, macrophages initiate a series of inflammatory responses by activating a specific signaling cascade and releasing preinflammatory cytokines and mediators, including interleukin-(IL-) 6, tumor necrosis factor-(TNF-) α, reactive oxygen species (ROS), nitric oxide (NO), and prostaglandin E2 (PGE2) [4].
Long noncoding RNAs (lncRNAs) are a class of noncoding RNAs with a length of more than 200 nucleotides, which have important functions in many biological processes [5]. Previous studies show that lncRNAs are specifically expressed in various diseases, such as cancer, diabetes, cardiovascular disease, lung disease, and tissue fibrosis [6][7][8]. It is demonstrated that lncRNAs play important regulatory roles in inflammatory diseases by regulating macrophage function. It was reported that IL-7-AS promotes the expression of several inflammatory genes, including CCL2, CCL5, CCL7, and IL-6, by regulating NF-κB and MAPK signaling pathways in macrophages [9]. FOXP1-IT1 overexpression attenuates THP-1 cell differentiation and inhibits inflammatory development [10]. Therefore, lncRNA is an important regulator of macrophage activation. In our preliminary work, we identified that lnc-MRGPRF-6:1 was highly expressed in plasma exosomes of CAD patients. Interestingly, further research demonstrated lnc-MRGPRF-6:1 was positively correlated with TNF-α, TNF-β, and CXCL11 in CAD patients, which indicates that lnc-MRGPRF-6:1 may participate in the regulation of macrophage-mediated inflammation in AS.
Dysregulation of phenotypic transitions in macrophages prolongs inflammation and impedes tissue repair. There is evidence that the polarization imbalance of M1/M2 macrophage may contribute to inflammatory diseases, including AS [11]. The polarization of M1 and M2 macrophages largely determines the development direction of AS inflammation [12]. During the development of AS, M1 and M2 macrophages show different position preferences on the vascular wall. The plaque shoulder is dominated by atherogenic M1 cells, which are relatively fragile and prone to rupture but are rarely found in the fibrous cap area. The harmful effects of M1 macrophage were offset by the recovery of M2 macrophage and the protective effects of AS [13]. The plasticity of these subpopulations may have a considerable influence on the outcome of AS. Overactivation of M1 may preferentially promote persistent inflammation and plaque progression.
Coincidentally, several researches have indicated that lncRNAs are involved in the dysregulation of phenotypic transitions in macrophages. Cao et al. demonstrated that lncRNA-MM2P could affect macrophage M2 polarization by regulating the dephosphorylation of signal transducer and activator of transcription 6 (STAT6) [14]. Chi et al. suggested that lncRNA GAS5 could act as a ceRNA to facilitate SOCS3 expression by sponging miR-455-5p and result in macrophage M2 polarization in childhood pneumonia [15]. Li et al. proved that lnc-BAZ2B promoted M2 macrophage activation primarily by upregulating the transcription of interferon regulatory factor 4 (IRF4), a key transcription factor for M2 macrophage activation [16].
Taken together, the regulation of macrophage polarization largely determines macrophage-mediated inflammation development and AS progression, and maybe some lncRNAs are the keys to the lock of macrophage polarization. Therefore, in the present study, we aim to explore the role of lnc-MRGPRF-6:1 in macrophage polarization and macrophage-mediated inflammation.

Materials and Methods
2.1. Cell Lines and Cell Culture. THP-1 cells (purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences) were incubated in RPMI Medium 1640 (Invitrogen, 11875-093) with 10% fetal bovine serum (Invitrogen, 21985). All cells were cultured in a moist environment at 37°C and 5% CO 2 .

Study
Population. This study enrolled 20 CAD patients in the Cardiology Department of the Affiliated Jiangning Hospital of Nanjing Medical University. Inclusion criteria were patients undergoing coronary angiography for chest pain or suspected CAD. Coronary angiography of the patient showed a lesion stenosis ≥ 50% in any coronary artery. And patients with congenital heart disease, cardiomyopathy, liver and kidney insufficiency, blood system diseases, malignant tumors, and other concomitant diseases were excluded. 20 healthy controls were selected from physical examination people during the same period. Plasma samples were collected and stored at liquid nitrogen immediately after collecting and centrifugation.

Isolation of Human Monocyte.
Human monocyte was isolated from venous blood by Ficoll (Solarbio, p8900). CD14 microbeads (Invitrogen, 11367D) and magnetic beads conjugated with antibodies against human CD14 were used for the monocyte separation.

Establishment of Macrophage Polarization and
Repolarization Models. THP-1 cells (2 × 10 5 /well) were inoculated in 24-well plates with 1 mL per well and incubated with PMA (Beyotime, S1819) with a final mass concentration of 100 ng/mL for 48 h. Human monocyte was incubated with macrophage colony-stimulating factor (M-CSF) (Beyotime, P5313) with a final mass concentration of 50 ng/mL for 7 days. M1 polarization of THP-1-derived macrophage and human monocyte-derived macrophage was performed using 20 ng/mL recombinant human IFN-γ (Beyotime, P5664) and 100 ng/mL LPS (Invitrogen, 00-4976). M2 polarization was performed using 20 ng/mL recombinant IL-4 (Beyotime, P5129). Nonpolarized PMA-activated cells were used as control. The polarization model of macrophage was established after 20 h of polarization. Based on the establishment of the macrophage polarization model, M1 macrophage was stimulated with 20 ng/ml IL-4 for 20 h and then induced to M2 polarization. M2 macrophage was induced to polarize toward M1 after 20 ng/mL IFN-γ and 100 ng/mL LPS stimulation for 20 h.  Mediators of Inflammation designed using CRISPOR (http://crispor.tefor.net) [17]. Two targets were designed for each location, and two locations were designed for lnc-MRGPRF  2.15. RNA Sequencing and Data Analysis. Total RNA was extracted from the cells using Trizol (Invitrogen, 15596018). RNA-seq was performed by BGISEQ-500 2.16. Statistical Analysis. GraphPad Prism 7 (GraphPad Software, USA) software and WPS Office 3 (Kingsoft Software, China) were used for statistical analysis. Quantitative data between the two groups were evaluated using Student's t -test and Mann-Whitney U tests. Spearman correlation analysis confirmed the correlation. P < 0:05 was considered statistically significant.

Results
3.1. lnc-MRGPRF-6:1 Is Highly Expressed in the Plasma Exosomes of CAD Patients and Is Positively Correlated with the Expression of Inflammatory Cytokines in Plasma. We successfully isolated exosomes and characterized the vesicles by TEM. TEM shows the enriched fraction of exosome-like vesicles (Figure 1(e)). A total of 40 subjects (20 CAD patients and 20 healthy controls) were selected for the study. It was shown that the lnc-MRGPRF-6:1 levels in the plasma exosomes of CAD patients were significantly higher than those of the control group (Figure 1(a)). Compared with the control group, as shown in Table 2, mRNA levels of TNF-α, TNF-β, and CXCL11 in the plasma of CAD patients were significantly increased with U6 as an internal reference. Further research demonstrated that lnc-MRGPRF-6:1 was positively correlated with TNF-α (Spearman r = 0:544, P = 0:001), TNF-β (Spearman r = 0:469, P = 0:005), and CXCL11 (Spearman r = 0:376, P = 0:018) in plasma (Figures 1(b)-1(d)).
These facts suggest that the expression of lnc-MRGPRF-6:1 was upregulated in M1 macrophage, and the expression will change with the phenotype transformation of the macrophage.  (Figures 4(a)-4(c)). Furthermore, the increases in KO were inferior to control. In the meantime, compared with control, TNF-α and TNF-β secretions were increased apparently in both KO and control, and the increasing levels in KO were also less than those in control (Figures 4(d) and 4(e)).
Meanwhile, induced by IL-4, the CCL17 and ARG1 levels in both KO and control were dramatically increased, and the increases in KO were great than those in the control group (Figures 4(g) and 4(h)). Similarly, the IL-10 secretions in both KO and control were also markedly increased (Figure 4(f)). Interestingly, the difference of IL-10 secretion level between KO and control was not statistically significant.

Mediators of Inflammation
These results suggest that lnc-MRGPRF-6:1 knockout effectively inhibits the polarization of M1 macrophage.
As shown in Figure 5(b), functions and processes associated with immune were enriched after lnc-MRGPRF-6:1 knockout, including immune system processes, innate immune response, and inflammatory response. In innate immunity, macrophages produce proinflammatory mediators by activating several receptors that recognize pathogens, including the toll-like receptor family (TLRs) [18]. Coincidentally, the toll-like receptor signaling pathway was enriched by KEGG pathway analysis ( Figure 5(c)). After LPS stimulation, activated TLR4 can trigger the downstream MAPK signaling pathway through the MyD88-dependent pathway [19]. Fascinatingly, according to RNA sequencing data, expressions of TLR4, MyD88, and P38 were   Mediators of Inflammation significantly changed after lnc-MRGPRF-6:1 knockout ( Figure 5(d)).
Considering MAPK including JNK, ERK, and P38 signal transduction pathway, we still verified JNK and ERK pathways, although RNA-seq showed no significant changes in JNK and ERK expression in MAPK after lnc-MRGPRF-6:1 knockout.

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Mediators of Inflammation      14 Mediators of Inflammation in the protein level, which suggested that lnc-MRGPRF-6:1 might regulate macrophage polarization through the TLR4-MyD88-MAPK signaling pathway.

Discussion
AS, as a chronic inflammatory vascular disease, is the pathological basis of CAD, which is a serious threat to human health. It is demonstrated that lncRNAs play important regulatory roles in inflammatory diseases, including AS [20]. In a preliminary work, we identified that lnc-MRGPRF-6:1 was significantly upregulated in the plasma of CAD patients. We want to explore whether there is a relationship between lnc-MRGPRF-6:1 and AS.
Foam cells derived from macrophages are associated with the initial stage of AS [21]. Therefore, we investigated the effect of lnc-MRGPRF-6:1 expression on macrophage