Long non‐coding RNA expressed in macrophage co‐varies with the inflammatory phenotype during macrophage development and polarization

Abstract Advances in microarray, RNA‐seq and omics techniques, thousands of long non‐coding RNAs (lncRNAs) with unknown functions have been discovered. LncRNAs have presented a diverse perspective on gene regulation in diverse biological processes, especially in human immune response. Macrophages participate in the whole phase of immune inflammatory response. They are able to shape their phenotype and arouse extensive functional activation after receiving physiological and pathological stimuli. Emerging studies indicated that lncRNAs participated in the gene regulatory network during complex biological processes of macrophage, including macrophage‐induced inflammatory responses. Here, we reviewed the existing knowledges of lncRNAs in the processes of macrophage development and polarization, and their roles in several different inflammatory diseases. Specifically, we focused on how lncRNAs function in macrophage, which might help to discover some potential therapeutic targets and diagnostic biomarkers.

Next-generation RNA sequencing and omics techniques have displayed that most of the human genomes were transcribed into RNAs, and nearly 98% of them without coding for proteins. These 98% of RNAs are called non-coding RNAs (ncRNAs). 14 LncRNAs are ncRNAs with critical biological roles and a length of more than 200 nucleotides. [15][16][17] The mechanisms of lncRNAs remain unclear, 18 but increasing number of in vitro and in vivo studies have shown that ln-cRNAs can be expressed in macrophage during its development and control some gene expression. [19][20][21] However, a systematic review uncovered the interactions among lncRNAs expressed in macrophage is not available. Hence, we reviewed recent advances in elucidation of the variability of lncRNA function within macrophages, and mainly focused on the following four aspects: biology of lncRNAs, involvement of lncRNAs in human monocyte/macrophage differentiation, characteristics of lncRNAs in macrophage polarization and dysregulation of lncRNAs in macrophage involved inflammatory diseases. Overall, this review may help to discover some potential therapeutic targets and diagnostic biomarkers for multiple diseases. 22,23

| B I OG ENE S IS , S TRUC TURE S AND FUN C TI ON MANNER S OF LN CRNA S
Compared with other ncRNAs, the mechanisms of lncRNAs were still unclear because of its complexity and relatively poor conservation. 24 Increased researches have already extensively enhanced the knowledge of lncRNAs, and some articles have processed stemmatic reviews on its biology, structures and function manners. For details, see Beermann, 14 Fatica 25 or Quinn. 26 Briefly, biogenesis of lncRNAs are usually transcribed by RNA Pol II(RNPII) in the nucleus, and only a fraction are more likely to be transcribed by RNPIII. 24 LncRNAs can be transcribed from intergenic (between protein-coding genes, lincRNA), intronic, natural antisense transcripts (NATs) or promoters and enhancers. 27 Bio-functional activities of lncRNAs rely on base pairing upon the primary structure, or develop by higher-order configurations on the basis of the secondary structures. 28,29 Diversity of their structures enabled lncRNAs to perform a variety of functions. 30 LncRNAs share the same cap and polyadenylate tail structures with mRNAs, but express lower levels than mRNAs, and in generally exhibit more specific expression profiles and precise expression patterns. 31,32 The regulation of lncRNAs is also complex and diverse. Most transcribed lncRNAs tend to remain in the cell nucleus and be assembled into chromosomes during biological processes. 31 Biological functions of most lncRNAs in accordance with previous reports can be primarily classified into 3 types 14 and so on. 34 However, most lncRNAs work in a trans-regulatory way and play a repressor or activator role in some distant gene loci which are not even on the same chromosome. 35 LncRNAs can also originate from enhancer elements called eRNAs, acting on the regulation of enhancer activity. 36 (b) LncRNAs participate in posttranscriptional moderation in the cytoplasm. These lncRNAs can promote or weaken the translation of target mRNAs, even alter the stability of mRNAs and proteins, or change the protein translocation. 29,33,37,38 Moreover, they can function as competing endogenous RNAs (ceRNAs), also called microRNA (miRNA) sponges, to protect the target mRNAs expression by directly binding to miR-NAs or keeping miRNAs away from mRNAs. 39 (c) The last function manner of lncRNAs is secreted to extracellular. It has been displayed that lncRNAs could be packed in extracellular vesicles (EVs), such as exosomes, secreted out of cell alone or bound to proteins. 40 Circulating lncRNAs show advantages in biomarkers, regulating many pathophysiological processes 41 (shown in Figure 1).

| LN CRNA S INVOLVE IN H UMAN MONOC Y TE/MACROPHAG E D IFFERENTIATI ON
Monocytes/macrophages originated from haematopoietic stem cells (HSCs) play a major role in non-specific immunity and inflammatory response. 42 Monocyte is a population of mononuclear leucocyte in circulating blood cells. It can transfer to diverse tissues and then differentiate into macrophage. This complex differentiation process requires a coordinated expression of transcription factors, cytokines and ncRNAs. 43 Although many of well-defined miRNAs have been recognized as key regulators involved in haematopoietic differentiation, there were few reports about lncR-NAs regulating monocyte/macrophage differentiation. 44 Chen et al have revealed that phorbol-12-myristate-13-acetate (PMA) could raise the long non-coding monocytic RNA (lnc-MC) expression in human THP-1 cell line, HL-60 cell line and CD34+ haematopoietic stem-progenitor cells (HSPCs). Mechanically, lnc-MC acted as a ceRNA to soak up miR-199a-5p and release activin A receptor type 1B (ACVR1B), 45 then enhanced the effect of haematopoiesis-specific transcription factor PU.1 on suppressing miR-199a-5p and eventually facilitated the differentiation process through activating the transforming growth factor β (TGF-β). 46 Once monocyte recruiting from bone marrow to peripheral blood, it can differentiate into macrophage with phenotypic variability. 47 LncRNA NTT was found to be expressed in resting human primary monocyte, monocyte-derived macrophage and the THP-1 cell line.  Table 1).

| lncRNAs expression profile in M1-like macrophage
Granulocyte macrophage colony stimulating factor (GM-CSF), LPS and Toll-like receptor (TLR) ligands always contribute to M1 polarization in macrophage. That macrophage might be polarized through the JNK pathway after binding to CCR2 and nuclear factor-κB (NF-κB), or through the activation of PI3K with increased RelA/NF-κB activity. 8 Functionally, M1-type macrophage promotes Th1 response and produces copious amounts of pro-inflammatory cytokines or reactive oxygen species (ROS) to kill pathogens. 44 TLR-triggered NF-κB is one of the best studied pathways participated in the conversion of macrophage to M1-like phenotype, 49 which is shown in Figure 3. [50][51][52][53] All TLRs, excluding TLR3, mainly activate the NF-κB-dependent or IRF7-dependent type I IFN (TLR7-9) pro-inflammatory signallings via the adapter protein myeloid differentiation marker 88 (MyD88). 54,55 Many transcriptome sequencing analyses of macrophages stimulated with TLR ligands have shown that numerous genes and lncRNAs involved in macrophage polarization ( Figure 3 and Table 1), and these macrophages tended to be induced into M1 type, along with high levels of pro-inflammatory mediators. 56 FIGURE 1 Biogenesis, structures and functions of lncRNAs. EBF: enhancer-binding factors. The biogenesis of lncRNAs is mainly transcribed by RNPII in the nucleus. Bio-functional activities of lncRNAs rely on base pairing upon the primary structure, or develop by higher-order configurations on the basis of the secondary structures including helices, hairpin loops, bulges and pseudoknots. Biological functions of lncRNAs can be primarily classified into 3 types: acting as regulator of genomic transcription in the nucleus via cis-or trans-acting lncRNAs and eRNAs; participating in post-transcriptional moderation in the cytoplasm and involving in peripheral circulation. [28][29][30][31][32][33][34][35][36][37][38][39][40][41] LncRNAs promoted biological processes showed in green arrow, and negative regulated displayed in red line In response to TLR4 (LPS) activation, lncRNA-Nfkb2 and ln-cRNA-Rel, located near some classical pro-inflammatory transcription factors, were up-regulated and co-regulated with inflammatory response in mouse bone marrow-derived macrophages (BMDMs). 57 AS-IL1α was a NAT located in the nucleus of macrophage and expressed at a low level in resting cells. It was highly up-regulated after stimulating by TLRs via NF-ĸB signal, and then recruited RNPII to the IL-1α promoter. In the presence of abundant AS-IL1α, elevated acetylation of histone H3 at lysine 9 (H3K9) promoted the combination of RNPII and the promoter of IL-1α, but it could not control the gene's expression. 58 LncRNA cox-2 (also has been called PACER) was located at upstream of the cyclooxygenase-2 (cox-2) gene in both human and murine macrophages. PACER could directly interact with the p50/p50 homodimeric, an inhibitor of NF-κB family and subsequently resulted in activation of the cox-2 promoter. 49,59 Moreover, lnc IL1β-eRNA and IL1β-RBT46 were found to be transcribed from enhancer regions via NF-ĸB in THP-1 cells, and predominantly localized to the nucleus, functioning as eRNAs to promote the expression of IL1-β and CXCL8. 60 After LPS stimulation, lincRNA-Tnfaip3, which is located at the tumour necrosis factor a-induced protein 3 (Tnfaip3) gene, mediated early response through NF-κB signalling in murine macrophages. LincRNA-Tnfaip3 could non-covalently unite to the high-mobility group box 1 protein (HMGB1), DNA and RNA molecules, then assembled into a complex to facilitate Hmgb1-associated histone modification and the combination of DNA and p65/ p50, eventually promoted the transcription of many inflammatory and defence genes in macrophages, 61 as well as other mice tissues induced by LPS. 62 Sirtuin-1 (SIRT1) is a conserved histone deacetylase, which can inhibit macrophage response by deacetylating H3K9 in lncRNA-CCL2 loci and transcriptionally suppressing the lncRNA-CCL2 level. 63 LncRNA-CCL2 was significantly upregulated in sepsis mice or LPS-stimulated macrophages, but the SIRT1 expression was down-regulated. 64  HSPCs. It could act as a ceRNA to soak up miR-199a-5p, and eventually facilitate the differentiation process through activating the transforming growth factor β (TGF-β) signalling pathway. 45,46 In response to various stimuli, macrophages mainly polarized into two types: classical M1 and alternative M2 type, and dynamically switched its function to adapt to the specific environmental changes. LPS induced up-regulation of lncRNA Mirt2 in the cytoplasm, but the increase in Mirt2 was restricted by sustained and excessive activation of inflammatory responses at the late stage. Restoring Mirt2 expression in later stage promoted the IL-4 induced M2 polarization with a remarkable increased level of M2 markers. 53 HOTTIP expression was associated with TLR tolerance, and has been speculated to skew macrophage polarization to a "M2-like" phenotype. 82 KCNQ1OT1 could function as a miR-21a-5p decoy to up-regulate IL-10, induced polarization of macrophages into M2 type. 83 F I G U R E 3 Involvement and regulation of lncRNAs in M1-like macrophage polarization. Many toll-like receptor (TLR)-triggered polarization of M1-like macrophage mainly depends on nuclear factor-κB (NF-κB) signalling pathway. At the plasma membrane, the binding of myeloid differentiation marker 88 (MyD88) to TLRs results in the recruitment and phosphorylation of IL-1 receptor-associated kinases (IRAKs), which facilitates oligomerization (via K63) and auto-ubiquitination (via E2 ubiquitin ligase Ubc13) of TNF receptor-associated factor 6 (TRAF6). Ubiquitinated TRAF6 subsequently activates other signalling proteins, such as transforming growth factor β-activated kinase (TAK1). Then TAK1 activates the inhibitor of κB α (IκBα) kinases (IKKs), leading to degradation of IκBα and activation of NF-κB, immune and inflammatory responses. LncRNAs promote the development of M1 macrophage via NF-κB or other pathways showed in red font, whereas negative regulated displayed in blue font [50][51][52][53] However, it did not seem to involve in regulating the expression of overlapped IL7R gene and viral infection. 73 Similarly, lincRNA THRIL as a negative regulation of NF-κB could interact with hnRNPL at the Tnfα gene and ensure the expression of TNF-α in dosage control, but it down-regulated in human macrophages after TLR2 activation. 74,75 Encoded by the Tnfα loci, lncRNA SeT increased in LPS-treated murine macrophage, 76 with unstable Tnfα mRNA. 20 The above lncRNAs are important to reduce TNFα expression in macrophages. NKILA was highly expressed to restrain the NF-ĸB-driven inflammation in response to TNF-α irritant. NKILA could decorate the phosphorylation of IĸBα to inhibit its degradation and prevent NF-ĸB from being transferred to the nucleus. 77

| Characteristics of lncRNAs in M2-like macrophage, M1/M2 switch or TMAs
Macrophage activated through an opposite manner is known as M2 type. M2 macrophage plays an essential role in adaptive response and contributes to Th2-type response involved in acute tissue damage and repair. 44,47 However, studies on the expression of lncRNAs in M2 macrophage are still rare, and the exact mechanism of action is still unclear. Du et al found that lncRNA Mirt2 was up-regulated in the cytoplasm of macrophage after LPS stimulating, then inhibited the oligomerization and ubiquitination of TNF receptor-associated factor 6 (TRAF6) and relieved inflammatory responses at the late stage.
They speculated that Mirt2 might hide the ubiquitination sites for the E2 ubiquitin ligase Ubc13, and induce a decreased ubiquitination level of TRAF6 ( Figure 2 and Table 1). At the early stage, IL-4 could reduce Mirt2 via the Jak-Stat6 pathway. After restoring the expression of Mirt2, IL-4 treated macrophage was inclined to M2 polarization with a high level of M2 molecule markers. However, the elaborate mechanisms of macrophage polarization induced by Mirt2 are still unknown. 53 Murphy et al conducted real-time PCR in LPS-tolerized macrophage and found increased expression of lncRNA HAR1A and HAR1B but decreased levels of PCGEM1 and HOTTIP lncRNAs. 50 Regulating the expression of HOTTIP is associated with TLR tolerance, and has been speculated to promote macrophage into M2 phenotype. 82 In polymethyl methacrylate (PMMA)-induced BMDMs, the expression of TNF-α, iNOS and miR-21a-5p were increased, but the expression of IL-10, Arg1 and lncRNA KCNQ1OT1 were suppressed. KCNQ1OT1 might act as a miR-21a-5p decoy to up-regulate IL-10 expression, 83 and then prompt macrophages into M2 type. 84  ( Figure 2 and Table 1).
Tumour-associated macrophages (TAMs) have a particular polarization state which is deemed as M2-like macrophage. 47  In addition to M1, M2 type and TAMs, some macrophage subsets with unique characteristics and functions have also been reported, such as CD169+ or TCR+ macrophage. 47 However, it is still not clear about these newly discovered macrophage types, and more research is needed to prove its origin, biomarkers and differentiation processes.
Hence, we only reviewed classical M1, M2 macrophage and TAMs.

| Atherosclerosis and cholesterol transport
Macrophages take up oxidized low-density lipoproteins (oxLDL), causing apoptosis to form foam cells, which is the basis of atherosclerosis. 95   Lethe was decreased in macrophages, and released more free p65-NF-κB to the nucleus, which promoted ROS and NOX2 production, eventually impaired wound healing. 115

| Coeliac disease
Coeliac disease (CeD) is a immune-mediated primary intestinal malabsorption syndrome caused by gluten intolerance, and the pathological feature is small intestinal mucosal lesions. 116

| Other relevant diseases
Silicosis is one of the most severe occupational diseases, and shows common histological changes in persistent inflammation and pulmonary fibrosis. Studies have indicated that silica particles could promote the liberation of copious oxidants and inflammatory factors in macrophages and epithelial cells, which caused a deposition in extracellular matrix, and finally led to pulmonary fibrosis. 122 Silica-induced pulmonary fibrosis, macrophages exposed to silica or fibroblasts exposed to TGF-β were decreased the miR-489 level. 123  However, the effects of circRNAs in macrophage differentiation and polarization have not to be widely explored. Whether circRNA involved in the activation of macrophages and whether it affect the development of macrophages by regulating lncRNAs require more further experiments to detect. 126 Unfortunately, there is a defect in our paper that many studies on the mechanisms of lncRNAs expressed in macrophages have not been studied intensively. It is expected that more exploration about lncRNAs in regulating innate immune response and macrophage-associated biological processes will be carried out. In conclusion, lncRNAs harmonizes in the development and function of macrophages, which can prevent the human body from being damaged by internal and external stimuli without generating an excessive immune response. Increased or depressed lncR-NAs in macrophages might be regarded as potential therapeutic targets and diagnostic biomarkers, and provided a new sight into the treatment of inflammatory diseases and cancers.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.