Long non-coding RNA FENDRR regulates IFNγ-induced M1 phenotype in macrophages

Macrophages play an essential role in host defense and display remarkable plasticity in switching between classically (pro-inflammatory—M1) and alternatively activated (anti-inflammatory—M2) phenotypes. The molecular mechanisms of macrophage polarization are not fully understood. Long non-coding RNAs (lncRNAs) with a length of > 200 nucleotides have been shown to play diverse roles in biological processes. Aberrant expression of lncRNAs is associated with a variety of pathophysiological conditions such as cancer, diabetes, cardiovascular, pulmonary diseases, and tissue fibrosis. In this study, we investigated the role of lncRNA FENDRR in human and mouse macrophage polarization. Human THP-1 monocytes were activated with phorbol-12-myristate-13-acetate (PMA) and differentiated into M1 macrophages with IFNγ or M2 macrophages with IL4. Real-time PCR analysis revealed that FENDRR was expressed 80-fold higher in M1 macrophages than that in M2 macrophages. Overexpression of FENDRR in PMA-activated THP-1 cells increased the IFNγ-induced expression of M1 markers, including IL1β and TNFα at both mRNA and protein levels. Knockdown of FENDRR had an opposite effect. Similarly, FENDRR overexpression in primary mouse bone marrow-derived macrophages increased mRNA expression of M1 markers. FENDRR overexpression increased, while FENDRR knock-down decreased, the IFNγ-induced phosphorylation of STAT1 in PMA-activated THP-1 cells. Our studies suggest that FENDRR enhances IFNγ-induced M1 macrophage polarization via the STAT1 pathway.

Lentiviral FENDRR expression vector. FENDRR (transcript variant 3, GenBank# MK522493.1) was amplified by PCR using cDNA from human lung tissue and inserted into a lentiviral vector at the XhoI and EcoRI sites as described 23,24 . The control vector was constructed with a random genomic DNA insert that did not contain any known lncRNAs or mRNAs. All the inserts in the plasmid constructs were confirmed by DNA sequencing. Lentiviruses were produced using the Lenti-X™ HTX Packaging vectors (Clontech, Mountain View, CA) in HEK 293T cells. The virus titer was determined by infecting HEK 293T cells with a series of dilutions of the viral stock and counting the virus-infected green fluorescent protein (GFP)-positive cells.

FENDRR overexpression in non-activated suspension THP-1 cells (spinoculation of suspension cells).
Non-activated THP-1 cells (2 × 10 6 ) were resuspended into 2 mL of complete culture media containing 8 µg/mL polybrene (Sigma-Aldrich). FENDRR or control lentivirus was added at a multiplicity of infection (MOI) of 50 and incubated for 30 min at room temperature. After a brief mix by pipetting, cells were spun at 800×g for 2 h at 32 °C. Lentivirus-infected cells were seeded at 1 × 10 6 cells/well in a 6-well plate and incubated for 24 h at 37 °C. The media was replaced the next day with fresh complete culture media, and the cells were incubated for another 72 h. www.nature.com/scientificreports/ FENDRR overexpression in PMA-activated THP-1 macrophages. THP-1 cells (2 × 10 6 /well) were seeded in a 6-well plate and activated overnight with 320 nM PMA. Media was removed and 2 mL of fresh RPMI 1,640 media containing 8 µg/ mL polybrene. FENDRR or control lentivirus (MOI, 50) was added to the well. After a 24-h incubation at 37 °C, the media was replaced with fresh complete culture media, and the cells were incubated for another 72 h.

FENDRR overexpression in mouse bone marrow-derived macrophages.
Bone marrow-derived macrophages (BMDM) were isolated from 8 to 10 weeks old C57Bl/6 mice according to Inés Pineda-Torra et al. 25  Western blotting analysis of phosphorylated STAT1. Macrophages were lysed in lysis buffer (T-PER, Thermo Fisher Scientific) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 30 min on ice. Cell debris was removed by centrifugation at 15,000×g for 10 min at 4 °C. Protein concentration in the cell lysate was determined using a BioRad protein assay kit (BioRad, Hercules, California, USA). The proteins in each sample (10 µg) were separated by 10% SDS-PAGE, and subsequently transferred onto a nitrocellulose membrane using the BioRad Turbo Trans system. After blocking with 5% skim milk for 1 h in TTBS (20 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5), membranes were incubated with primary antibodies, anti-phospho STAT1-Y701 (1:1,000 dilution, Cell Signaling, Beverly, MA, USA), or mouse anti-human β-actin (1:3,000 dilution, Thermo Fisher Scientific) overnight and then for 1 h with the respective secondary antibodies (1:3,000 dilution, goat anti-rabbit and goat anti-mouse HRP conjugated, Jackson Immuno Research, USA). Blots were developed using Super Signal West Pico (Thermo Fisher Scientific), and signals were detected with Amersham Imager 600. Intensity of the bands was quantified by ImageJ densitometry with β-actin as a loading control. , scale bar-50 µm. M1 phenotype is associated with an elongated cell shape, while M2 phenotype has a flattened and rounded shape. (G) Shows that lncRNA FENDRR was expressed 80 times higher in M1 (IFNγ) than that in M2 (IL4) polarized macrophages. The results were normalized to GAPDH and expressed as a fold change to 4 h. Data are presented as the mean ± SD. n = 3. ***P < 0.001, # P < 0.05, ### P < 0.001, &&& P < 0.001, $$ P < 0.01, $$$ P < 0.001, @@@ P < 0.001. Two-way ANOVA, followed by Tukey's post hoc test. CTRL: control cells.
Scientific RepoRtS | (2020) 10:13672 | https://doi.org/10.1038/s41598-020-70633-7 www.nature.com/scientificreports/ Statistical analysis. All experiments were repeated three times. Data were shown as the mean ± standard deviation (SD). One-way and two-way ANOVA, followed by a Tukey's post hoc test were performed for multiple group comparisons using GraphPad Prism software. P < 0.05 was considered statistically significant.  (Fig. 1D,E). We also observed that the THP-1 macrophages that polarized toward different phenotypes exhibited dramatic changes in cell shape: IL4-induced M2 cells had a rounded shape with elongated filopodia, while IFNγ-induced M1 cells adopted an elongated, spindle-shaped cell morphology (Fig. 1F). These results confirmed the M1 and M2 polarization models. We next examined the expression of FENDRR in IFNγ-and IL4-polarized THP-1 macrophages. Our data showed that IFNγ treatment of THP-1 macrophages significantly increased FENDRR expression at 24 and 48 h (Fig. 1G). However, IL-4 had no effects on FENDRR expression. These results suggest that FENDRR may play a role in IFNγ-induced M1 macrophage polarization.

Effect of FENDRR overexpression on M1 macrophage polarization. Because FENDRR expression
was significantly increased by IFNγ treatment, we wanted to know if FENDRR overexpression can induce M1 macrophage phenotype. We first determined whether FENDRR can directly convert THP1 monocytes to M1 macrophages. THP1 cells were infected with a FENDRR lentivirus by spinoculation. GPF images showed a high infection efficiency ( Fig. 2A). FENDRR overexpression was confirmed in the lentivirus-treated THP1 cells compared to virus control-infected or blank cells (Fig. 2B). There were no significant differences in the expression of M1 macrophage markers, TNFα, IL1β, and IL6, and M2 macrophage marker, IL10 between the FENDRR overexpressing and control groups (Fig. 2C-F), suggesting that FENDRR does not induce M1 and M2 phenotypes in non-activated monocytes.
We then determined whether overexpression of FENDRR in the PMA-activated THP-1 macrophages can induce the M1 macrophage phenotype. The high infection efficiency and overexpression of FENDRR in the PMA-activated THP1 macrophages are shown in Fig. 3A,B. FENDRR overexpression increased the mRNA expression of M1 markers, IL1β, TNFα and IL6 and IL1β, TNFα protein levels released into the culture media, but had no significant effects on the mRNA expression of M2 markers, IL10 and CCL22 compared to the virus control ( Fig. 3C-I), suggesting that FENDRR induces M1 but not M2 polarization. FENDRR appears to increase the IL10 mRNA level compared to blank control. This is likely due to the effects of the lentiviral system that we used to overexpress FENDRR since the control virus also increased IL10 expression and there was no significant difference in IL10 levels between the virus control and FENDRR group. www.nature.com/scientificreports/ Since THP-1 is derived from acute monocytic leukemia 26,27 , we examined whether FENDRR also induced M1 polarization in primary mouse bone marrow-derived macrophages (BMDM). We confirmed the high infection efficiency and overexpression of FENDRR in BMDM using the lentiviral expression system (Fig. 4A,B). Similar to PMA-activated THP-1 macrophages, we observed that FENDRR overexpression in BMDM increased the mRNA expression of mouse M1 markers, IL1β, TNFα and CXCL10 (Fig. 4C-E) and did not significantly affect the mRNA expression of mouse M2 markers, arginase 1 (ARG1) and found in inflammatory zone 1 (FIZZ1) (Fig. 4F,G).
Finally, we determined whether FENDRR can enhance IFNγ-induced M1 polarization. The PMA-activated THP1 cells were infected with a FENDRR lentivirus for 48 h and then treated with IFNγ or IL4 for another 48 h. Once again, infection efficiency and overexpression of FENDRR was confirmed (Fig. 5A,B). FENDRR overexpression further increased IFNγ-induced mRNA expression of IL1β, TNFα and IL6 but had little effect on IL10 expression compared to control virus (Fig. 5C-F). Using ELISA, we also observed that FENDRR increased IFNγ-induced IL1β and TNFα proteins released into the media (Fig. 5G,H). Our data suggest that IFNγ and FENDRR had a synergic effect on M1 polarization.

Knockdown of FENDRR inhibits IFNγ-induced M1 macrophage polarization.
To further confirm the effects of FENDRR on M1 macrophage polarization, we knocked down FENDRR by infecting the PMA-activated THP-1 cells with a lentivirus containing shRNA targeting FENDRR, followed by IFNγ-induced M1 polarization. FENDRR expression was effectively reduced by the shRNA (Fig. 6A). The reduction of FEN-DRR blocked IFNγ-induced IL1β, TNFα and IL6 mRNA expression but had no effects on IL-10 expression (Fig. 6B-E), further supporting that FENDRR promotes M1 macrophage polarization. www.nature.com/scientificreports/ FENDRR acts via STAT1 signaling. STAT1 is the primary mediator for IFNγ signaling 28 . To gain insight into the underlying mechanism of FENDRR-mediated M1 polarization, we examined whether FENDRR influences the STAT1 phosphorylation. The results showed that IFNγ increased the phosphorylation of STAT1, and FENDRR overexpression further increased STAT1 phosphorylation, as demonstrated by western blot using anti-phosphoSTAT1 (Tyr701) (Fig. 7A,B). On the other hand, silencing FENDRR reduced STAT1 phosphorylation (Fig. 7C,D). These results suggest that FENDRR-induced M1 polarization functions via STAT1.

Discussion
The highly dynamic phenotype and function of macrophages can be shaped by different environmental signals 29 . Progress has been made in defining the mechanisms underlying macrophage polarization 30 . However, the role of lncRNAs in macrophage polarization is less known. In this study, we identified lncRNA FENDRR as a positive regulator of M1 macrophage polarization. Among the multiple factors involved in the regulation of macrophage polarization, noncoding RNAs have been recognized as important regulatory molecules. MicroRNAs (miRNAs) have emerged as positive or negative regulators of M1 polarization 31 . For example, miR-21, miR-29a and let-7b were found to induce TNFα and IL6 in microglia and macrophages by binding TLR7 (TLR8 in humans) 32,33 , suggesting that they may be involved in M1 macrophage polarization. These miRNAs function as a TLR7 ligand as they have a similar GU content and length as the known TLR7 ligand, ssRNA40. Several miRNAs have been shown to regulate macrophage polarization by modulating transcription factors and signaling pathways involved in M1 and M2 polarization 34 . miR-125b increases macrophage responsiveness to IFNγ by targeting the transcription factor, IRF4 that inhibits NF-kB activity, thereby promoting M1 phenotype macrophages 35 . miR-27 and miR-130 promote pro-inflammatory macrophage polarization by interacting with PPARγ, while miR-155 and miR-21 enhanced pro-inflammatory responses by activating STAT1 and STAT3 pathways [36][37][38][39] . Most recently, miR-216a was found to enhance M1 and suppress M2 macrophage polarization by regulating telomerase activity through SMAD3/NF-kB pathway 40 . www.nature.com/scientificreports/ Many lncRNAs are expressed in a cell type-and state-specific manner, and their expression is tightly regulated by various cellular signals 11,41 . Our current study shows that IFNγ, but not IL4, induces FENDRR expression in human macrophages. Hundreds of intergenic lncRNAs are modulated by JAK-STAT signaling in T helper cells 12 . LncRNA BANCR expression is induced in human retinal pigment epithelial cells by IFN-γ, but not TNFα or IL1β and a JAK inhibitor blocks this effect 42 . Using the PROMO online software, we identified two STAT3 binding sites in the 5 kb FENDRR promoter, suggesting that IFNγ may regulate FENDRR expression in macrophage through STAT1/3 heterodimers. FENDRR also enhances IFNγ-mediated STAT1 phosphorylation, indicating a forward feedback regualtion of FENDRR expression by IFNγ (Fig. 8).
Compared to miRNAs, much less are known regarding the roles of lncRNAs in macrophage polarization. LncRNA THRIL mediates the pro-inflammatory response of PMA-activated THP1 macrophages by interacting with heterogeneous nuclear ribonucleoproteins 43 . LncRNA GAS5 promotes M1 polarization through sponging miR-455-5p 44 . LncRNA Malat1 enhances M1 macrophage polarization, but inhibits M2 phenotype. Myeloid specific knockout of Malat1 in mice has a reduced LPS-induced lung inflammation, but an increased lung fibrosis caused by bleomycin 45 . lncRNA-MMP2 is upregulated in M2 polarized macrophages and is required for M2 polarization through STAT6 activation. However, the mechanism of lncRNA-MM2P-mediated STAT6 phosphorylation remains unknown 46 .
Our current studies uncovered lncRNA FENDRR as a new positive regulator of M1 macrophage polarization. It has been long appreciated that IFNγ-induced signaling typically augments M1 macrophage polarization through a STAT1-dependent mechanism. IFNγ, the sole member of the type II interferon family, acts via binding the IFNγ receptor and signaling through the JAK/STAT pathway, leading to STAT1 phosphorylation, nuclear translocation and induction of transcription of STAT1-regulated genes 47 . It has been reported that lncRNA MacORIS inhibits JAK2 and STAT1 phosphorylation 48 . Our data shows that overexpression of FENDRR increased and knock-down of FENDRR reduced IFNγ-mediated phosphorylation of STAT1, suggesting that enhancement of M1 polarization by FENDRR occur via the STAT1-dependent pathway. www.nature.com/scientificreports/ How FENDRR regulates STAT1 signaling and M1 macrophage polarization remains to be determined. Based on literature, we speculated several possibilities. The first possibility may involve epigenetic regualtion. Fendrr has been shown to interact with both PRC2 and TrxG/Mll complexes via dsDNA/RNA triplex formation at target regulatory elements and to increase PRC2 occupancy at these sites, in addition to enhanced trimethylation of histone 3 lysine 4 (H3K4me3) and histone 3 lysine 27 (H3K27me3) at the promoter site of the target genes 49 . This mechanism is consistent with two previous findings: (a) histone methyltransferase MLL is upregulated in M1 macrophages, which increases H3K4me3 at the promoter of pro-inflammatory cytokine CXCL10 50 . (b) PRC2mediated EZH2-dependent H3K27 methylation suppresses several anti-inflammatory genes such as MERTK, PPARG and RANK in IFNγ-polarized macrophages 51 .  The results are presented as the mean ± SD. n = 3. *P < 0.05, ***P < 0.001, # P < 0.05, ### P < 0.001. One-way ANOVA, followed by www.nature.com/scientificreports/ The second possibility is that FENDRR may function as a miRNA sponge. FENDRR has been recently shown to act as a molecular sponge for miRNA-18a-5p and miR-126 in prostate, gallbladder cancer and human brain microvascular endothelial cells [52][53][54] .
The third possibility is that FENDRR may promote M1 macrophage polarization through iron-mediated repression of STAT1 signaling. Iron metabolism genes are differentially expressed in M1 and M2 macrophages. Compared to M2 macrophages, M1 macrophages have a higher expression of ferritin (iron storage) and a lower expression of ferroportin (iron export), transferrin receptor (iron import) and iron regulatory protein 1 and 2 55,56 . Iron suppresses M1 polarization in Raw 264.7 macrophages, mouse bone marrow-derived macrophages and THP-1 monocyte-derived macrophages [57][58][59] and promotes M2 polarization in THP-1 monocyte-derived macrophages 59,60 . However, one study reported opposite results showing that iron increases M1 macrophage markers, but inhibits IL-4-induced M2 macrophage markers in mouse bone marrow-derived macrophages 61 . Iron decreases STAT1 phosphorylation in IFNγ-treated RAW 246.7 macrophages 57 , which is consistent with the iron-mediated inhibition of M1 macrophage polarization. We have recently shown that FENDRR reduces iron levels in lung fibroblasts by interacting with iron regulatory protein-1 to inhibit fibroblast activation 62 . Thus, it is possible that FENDRR also reduces iron levels in macrophages and the decrease in iron levels in turn activates STAT1 signaling and thus promotes M1 macrophage polarization.
In summary, our results suggest that FENDRR promotes M1 macrophage polarization by modulating STAT1 activation pathway. Targeting FENDRR may provide a potential therapeutic benefit for the treatment of disorders associated with macrophage polarization.