The role of lncRNAs in innate immunity and inflammation

ABSTRACT

The innate immune system relies on a germ-line-encoded repertoire of pattern recognition receptors (PRRs), activated by deeply conserved pathogen signatures, such as bacterial cell wall components or foreign nucleic acids. To enable effective defence against invading pathogens and prevent from deleterious inflammation, PRR-driven immune responses are tightly controlled by a dense network of nuclear and cytoplasmic regulators. Long non-coding RNAs (lncRNAs) are increasingly recognized as important components of these regulatory circuitries, providing positive and negative control of PRR-induced innate immune responses. The present review provides an overview of the presently known roles of lncRNAs in human and murine innate antiviral and antibacterial immunity. The emerging roles in host defence and inflammation suggest that further mechanistic insights into the cellular functions of lncRNAs will decisively advance our molecular understanding of immune-associated diseases and open new avenues for therapeutic intervention.

KEYWORDS:

• Long non-coding RNA
• lncRNA
• innate immunity
• inflammation
• macrophage
• phagocyte
• infection
• pathogen
• bacteria
• virus

1. Introduction

By protecting from malignant cell transformation, participating in tissue repair upon injury and defeating infectious agents, the immune system plays a central role in the maintenance of organismal homoeostasis. While the adaptive immune system confers long-lasting, but delayed protection against newly emerging threats, the innate immune system provides immediate responsiveness to conserved pathogen- and danger-associated molecular patterns (PAMPs and DAMPs)[1,2]. The resulting inflammatory response, however, can cause irreversible tissue damage and even organ failure when not properly controlled [3]. Besides protein regulators, non-coding RNAs are increasingly recognized as important control-elements of the immune system enabling the adequate adjustment of the inflammatory response relative to the detected threat.

Regulatory non-coding RNAs are typically divided into two major groups. While microRNAs are approx. 20–24 nt long and typically act through miRISC-complex dependent recruitment of proteins decreasing target mRNA translation and stability, long non-coding RNAs (lncRNAs) are regulatory RNAs ≥ 200 nt and act through a plethora of different mechanisms and interactors [4–6]. A role of microRNAs in the immune system was already revealed in the first decade of the 2000s, at a time when long non-coding RNAs were still hardly explored [7]. Upon sensing of bacterial components by mammalian phagocytes, for instance, which play a central role in the innate immune system, microRNAs 146 and 155 were found to act as inducible feedback-regulators of the inflammatory response [8–11]. Other microRNAs adopt feed-forward regulatory functions during innate immune activation of phagocytes [12]. Similar regulatory implications in immune cells have recently been reported for lncRNAs. Between 2012 and 2017, the results of comprehensive transcriptome cataloguing approaches by the ENCODE and FANTOM consortia became publically available and revealed ~20.000 human lncRNA genes [13,14], most of which still remain functionally uncharacterized. Many basic questions, e.g. as to common modes of operation, subcellular localization patterns or the evolutionary conservation of lncRNAs, are still subject to intense debates. However, it is becoming increasingly clear that lncRNAs adopt important functions in a variety of cellular processes and in human diseases [4,15,16]. The already available mechanistic insights into the functions of immune-associated lncRNAs suggest important roles in the adjustment of pro-inflammatory signal-transduction events and in the differentiation and polarization of immune cells. The present review attempts to comprehensively depict the known contributions of lncRNAs to innate immunity in humans and mice and discusses emerging principles of lncRNA-based control in PAMP-triggered inflammation.

2. LncRNA classification and functional heterogeneity

Compared to mRNAs, lncRNAs are typically expressed at lower levels and are less conserved, but often undergo similar maturation steps, including splicing, 5ʹ-capping and 3ʹ-polyadenylation [13]. LncRNAs can be retained in in the nucleus or exported to the cytoplasm and consequently adopt a variety of different cellular functions, including transcriptional or translational regulation and control of protein and RNA stability [4,17]. In the literature, lncRNAs are often sub-classified according to their mode of operation or their site of transcription relative to coding genes. In this context, antisense lncRNAs, encoded on the reverse complement strand of an mRNA gene, intronic lncRNAs encoded within an intron of another gene, enhancer lncRNAs near or within an active enhancer element and intergenic long non-coding RNAs have been discriminated [5]. Intergenic lncRNAs (lincRNAs) form the largest of these sub-groups [13]. Mechanistically, lncRNAs may act in cis or in trans through interactions with nucleic acids or proteins. Their interactions with DNA, RNA or protein partners can fulfil decoying, guiding or scaffolding functions [4,5]. LncRNA PACER for instance acts as a decoy, binding to the p50 subunit of the NFκB transcription factor to prevent formation of repressive p50 homodimer complexes [18]. XIST and HOTAIR are classical examples of lncRNAs guiding chromatin-modifiers, such as the Polycomb Repressive Complex 2 (PRC2) to chromatin target sites, thereby regulating dosage compensation and developmental programmes, respectively [19,20]. In addition to its guiding function, HOTAIR also functions as a scaffold, bringing PRC2 and the LSD1-CoREST demethylase into close proximity during gene silencing [21]. Further lncRNAs bind several interaction partners at a time through different modular domains [5,21]. Besides the recruitment of chromatin-modifying enzymes, such interactions e.g. contribute to the assembly of multi-protein complexes during protein-ubiquitination or liquid-liquid phase-separation [5,17,22–24].

Despite the continuous improvement of mammalian gene catalogues, it remains debated how many of the annotated lncRNA genes are truly non-coding or functional. While the latter can only be determined individually through careful mutagenesis and biochemical studies, the former has recently been addressed by high-throughput analysis of ribosome-occupation. ~20-40% of lncRNAs turned out to associate and co-sediment with ribosomes in pull-down, ribosome-profiling and density gradient experiments [25–29]. Several of the lncRNAs in question were furthermore found to encode functional peptides, including in immune cells [25,26]. Coding potential analysis therefore remains a critical step during the experimental dissection of cellular lncRNA-mechanisms. Besides exclusion of protein-coding potential, the mechanistic investigation of lncRNAs typically involves the identification of lncRNA binding partners through affinity purification approaches, the determination of the subcellular localization by fractionation or in situ hybridization and loss-of-function studies using in vitro or in vivo models [17,30]. In the context of the innate immune system, the already reported molecular functions and interactors of lncRNAs illustrate a high degree of heterogeneity, ranging from control of signalling protein activity to mRNA stabilization and regulation of chromatin accessibility [27,31,32]. In the following sections, we first introduce the key principles of innate immune recognition, followed by the illustration of known functions of lncRNAs in professional phagocytes and in immune-activated tissue-anchored cell-types.

3. Key components and pathways of the innate immune system

Innate recognition of PAMPs and DAMPs is achieved through pattern recognition receptors (PRRs) spanning the plasma-membrane or locating to endosomes or the cytosol [1]. Different PRR classes can be discriminated based on their protein domain-structure, including the Toll-like receptors (TLRs), the Rig-I like receptors (RLRs), the NOD-like receptors (NLRs) and the C-type lectin receptors (CLRs) [1]. The pathogen structures sensed by the individual members of the different PRR classes range from bacterial cell wall components, such as lipopolysaccharides (TLR4), bacterial lipoproteins (TLR2) and peptidoglycans (NLRs), to bacterial and viral nucleic acids, such as dsRNA (e.g. TLR3, Rig-I, MDA5), CpG DNA (TLR9) or cytosolic DNA (cGAS) [33,34]. Different PRRs can activate distinct signalling cascades, culminating in the activation of master-transcription factors of the immune response, such as NFκB or the IRF protein family [1,33] (Fig. 1). While NFκB activates the majority of the acute pro-inflammatory genes, the IRF transcription factors selectively trigger the expression of interferons. The latter counteract intracellular infections by STAT transcription factor dependent activation of interferon-stimulated genes (ISGs), the products of which for instance interfere with viral transcription and translation pathways [1,33,35,36].

Figure 1. Roles of lncRNAs in PRR-triggered phagocyte immunity. LncRNAs have been implicated in PRR-MyD88-NFκB, PRR-TRIF-IRF3 and PRR-MAVS-IRF3 signalling (TLR4 and Rig-I pathway shown exemplarily), as well as in JAK/STAT-dependent ISG expression programmes triggered upon type I IFN release and auto- or paracrine IFNAR stimulation. Nuclear and cytoplasmic lncRNAs acting as positive and negative regulators of these pathways in murine (green) and human (blue) phagocytes are indicated

3.1. Pro-inflammatory gene regulation via NFκB

The lipopolysaccharide (LPS) sensor TLR4 is a prototypic plasma membrane spanning PRR and the only TLR capable of simultaneous activation of the intracellular MyD88 and TRIF signalling pathways [1,33] (Fig. 1). TLR4-signalling through the intracellular adaptor MyD88 triggers a complex ubiquitin-ligase (e.g. TRAF6) and kinase (e.g. IKK) dependent cascade culminating in the phosphorylation and degradation of the cytosolic NFκB-adaptor IκBα. Degradation of IκBα leads to NFκB nuclear translocation and pro-inflammatory gene expression [33]. A MAPK-dependent branch downstream of MyD88-TRAF6 furthermore activates the AP1 complex, a transcriptional co-activator of NFκB target genes [33]. Unlike the TLR4-MyD88 pathway, TLR4-signalling through the intracellular adapter TRIF is induced upon endocytosis of activated TLR4 [1]. TRIF-activation triggers a pathway which culminates in the activation of the IRF3 transcription factor (see below), but also of TRAF6, thereby fostering NFκB activation [33]. Different from TLR4, the remaining TLRs exclusively signal either through MyD88 or through TRIF. MyD88-dependent human TLRs and their agonists are TLR2 in conjunction with TLR1 or 6 (lipoproteins), TLR5 (flagellin), TLR7 and 8 (ssRNA) and TLR9 (CpG DNA). TLR3 signals through TRIF and senses dsRNA [33]. Other PRRs activate NFκB independent of MyD88 or TRIF. The NLR family members and cytosolic peptidoglycan receptors NOD1 and NOD2, for instance, signal through the adaptor protein RIP2 to trigger IκBα degradation and MAPK signalling dependent NFκB and AP1 activation [33]. Irrespective of the upstream pathway, nuclear translocation of NFκB leads to the transcription of a cell-type specific set of immune genes. This includes inflammation mediators, such as IL1β and TNFα, which in turn feedback-activate NFκB by autocrine cytokine receptor stimulation and signalling through the intracellular MyD88 and RIPK1 adapters, respectively [3,37,38]. NFκB induces the expression of numerous other soluble immune-mediators. Examples are IL8, which recruits immune cells from the periphery, COX2, which elicits diverse systemic adaptations, including fever and elevated pain sensitivity or IL6, which triggers acute phase protein production by hepatocytes and regulates B- and T-cell differentiation [38–40].

3.2. Interferon induction through IRF activation

In addition to the MyD88-NFκB dependent pathway, TLR4 can induce the expression of type I interferons (IFNs) through the TRIF-dependent pathway [1] (Fig. 1). TLR4 interaction with TRIF triggers TRAF3 dependent activation of the TBK1 kinase and phosphorylation of IRF3. This is followed by IRF3 dimerization, translocation to the nucleus and IRF3-dependent activation of type I IFN genes [1]. Unlike TLR4, dsRNA sensor TLR3 exclusively signals through TRIF and thus preferentially activates type I IFN transcription [1]. Different from TLR4 and TLR3, the RLR family members and dsRNA sensors Rig-I and MDA5 upon ligand binding activate type I IFN expression through a TRIF-independent pathway. In this case, ligand-induced receptor oligomerization is followed by binding to the adaptor protein MAVS at mitochondrial surfaces (Fig. 1), which triggers TRAF3-TBK1-IRF3 signal transduction and type I IFN expression [1]. Upon secretion, type I IFNs IFNβ and IFNα bind to the common receptor IFNAR on target cells, to induce pro-apoptotic programmes in infected cells or an antiviral and antimicrobial state within bystander cells through Janus kinase/signal transducers and activators of transcription (JAK/STAT) signalling [41].

3.3. Interferon-dependent JAK/STAT pathway activation

JAKs are a family of tyrosine kinases, which, except for Jak3, are ubiquitously expressed in mammalian cells and interact with the intracellular domains of diverse receptors, including the type I IFN-receptor IFNAR [35,42]. Upon IFNα or β binding to IFNAR, the dimeric receptor undergoes a conformational switch, which brings JAKs associated with each receptor monomer into special proximity, leading to JAK transphosphorylation and JAK-dependent STAT transcription-factor phosphorylation [43]. Activated STAT dimers translocate from the cytosol to the nucleus and promote expression of a variety of target genes [43] (Fig. 1). Similar to type I IFNs, the type II IFN IFNγ, which is produced primarily by T- and NK cells, activates a JAK-STAT dependent signalling cascade upon binding to its receptor IFNGR [44]. IFNAR and IFNGR activation typically triggers the phosphorylation and nuclear translocation of STAT1, which activates transcription of IFN stimulated genes (ISGs), such as IFIT1, OAS or ADAR1 [45]. JAK-STAT activation also amplifies expression of primarily NFκB-dependent inflammatory cytokines, such as IL6 and of class I and II HLAs, thus contributing to increased antigen presentation [41,44].

3.4. Division of labour in the innate immune system

Phagocytes, such as resident and recruited macrophages, dendritic cells or circulating monocytes play a central role in the orchestration of PAMP-triggered innate immune responses. They express a wide range of PRRs and are usually among the first cells to respond to an infection and transmit immune signals to the periphery. Phagocytes represent a major source of various soluble mediators such as IL6 and type I IFN [41,46]. Moreover, phagocytes are in constant exchange with surrounding cells, such as epithelial or endothelial cells, to produce and receive substances and signals that contribute to innate defence. Type I IFN, for example, stimulates an antiviral state in bystander cells surrounding infected phagocytes [41]. Via TNFα, phagocytes furthermore stimulate pro-inflammatory activation of epithelial and endothelial cells at the alveolar barrier [47]. Additionally, NK cells are recruited by phagocyte signals and can in turn boost the antimicrobial activity and antigen presentation capacity of phagocytes through IFNγ release [44]. Maintenance of the tissue phagocyte niche in turn involves recruitment- and differentiation-signals produced by endothelial cells and epithelial cells [48]. LncRNAs seem to vitally participate in the immune crosstalk of endothelial and epithelial cells, NK cells and phagocytes [49]. Thus, a comprehensive understanding of lncRNA functions in innate immunity and inflammation requires their mechanistic investigation not only in professional immune cells but also in cell types acting in the periphery of phagocytes during immune activation.

4. LncRNAs in phagocyte-driven innate immunity

Blood-derived (BDM) and bone-marrow-derived (BMDM) macrophages represent important phagocyte models, often used in basic immunology research. PRR-stimulation, e.g. with TLR4-agonist LPS, alters the expression of dozens of lncRNAs in these cells [27,50]. The functions of most of these lncRNAs presently remain unknown. Of note, attempts to systematically narrow-down phagocyte lncRNA functions suggest that in human and murine macrophages, ~23 and ~35% of the annotated lncRNAs associate with ribosomes, respectively [26,27]. Thus, investigation of the coding potential should represent a key step in the mechanistic dissection of the cellular functions of these lncRNAs. In human macrophages, lncRNAs absent from ribosomes are present both in the nucleus and cytoplasm and co-sediment with diverse macromolecular RNA-protein machineries on density gradients [27]. This fosters the concept that phagocyte lncRNAs, unlike other classes of non-coding RNA, exert their functions through a multitude of different regulatory RNA-protein interactions. In line with this notion, the already reported implications of lncRNAs in phagocyte immunity involve a variety of different nuclear and cytoplasmic mechanisms (Table 1), as outlined below. Of note, only few immune-associated phagocyte lncRNAs seem to be conserved at the primary sequence level between humans and mice [50]. We therefore separately depict lncRNAs involved in human and murine phagocyte immunity, respectively, in the following text.

Table 1. lncRNAs in mammalian phagocyte immunity

lncRNA name	Cell type	Upstream pathway	Regula-tion	Function	Locali-zation	Orga-nism	PMID
INTERFERON-ASSOCIATED IMMUNITY:
MaIL1	Macrophage	TLR-MyD88	Up	Triggers OPTN dependent IRF3 phosphorylation & type I IFN expression	C	hsa	32,241,891
NRIR	Monocyte, macrophage	TLR-MyD88	Up	Promotes ISG expression	?	hsa	30,804,934 32,241,891
Lnczc3h7a	Macrophage	Virus sensors	Up	Triggers TRIM25-dependent Rig-I ubiquitination & type I IFN expression	C	mmu	31,036,902
Lnclrrc55-AS	Macrophage	JAK-STAT	Up	Promotes type I IFN expression through inactivation of the IRF3 inhibitor PP2A	C	mmu	31,213,650
Lnc-Lsm3b	Macrophage	JAK-STAT	Up	Inhibits Rig-I oligomerization & downstream type I IFN expression	C	mmu	29,706,547
Lfar1	Macrophage	JAK-STAT	Down	Suppression of NFκB dependent genes and NLRP3 inflammasome mediated pyroptosis	C/N	mmu	32,071,306
POSITIVE NFκB/AP1 CONTROL:
IL-1β-eRNA	Monocyte	TLR-MyD88	Up	Promotes expression of the pro-inflammatory cytokine IL1β	N	hsa	24,909,122
IL-1β-RBT46	Monocyte	TLR-MyD88	Up	Promotes expression of the pro-inflammatory cytokine IL1β	N	hsa	24,909,122
PACER	Macrophage	TLR-MyD88	Up	Decoys p50 & promotes NFκB p50/p65 heterodimer dependent immune responses	N	hsa	24,843,008
CARLR	Macrophage	TLR-MyD88	Up	Binds p65 to promote NFκB-dependent immune responses	C	hsa, mmu	28,626,066
ROCKI	Macrophage	TLR-MyD88	Up	Recruits HDAC1 to MARCKS promotor after interaction with APEX1	N	hsa	30,918,008
IL7-AS	Macrophage	TLR-MyD88	Up	Associates with p300 and SWI/SNF to promote immune gene expression	N	hsa	31,383,742
Lincrna-Tnfaip3	Macrophage	TLR-MyD88	Up	Forms a complex with HMGB1/NFκB to promote inflammatory gene expression	N	mmu	27,979,905
Lnc-epav	Macrophage	TLR-MyD88	Up	Promotes immune gene expression by sequestering SFPQ from the p65 promotor	N	mmu	31,363,026
AK170409	Macrophage	TLR-MyD88	Up	Fosters NFκB activation below the IκBα step	N	mmu	29,051,223
lincrna-Cox2	Macrophage	TLR-MyD88	Up	Positive or negative control of immune response through interaction with hnRNP A/B or A2/B1 or SWI/SNF	C/N	mmu	23,907,535 26,880,762 29,051,223
AS-IL1α	Macrophage	TLR-MyD88	Up	Promotes expression of IL1α by recruiting RNA polymerase II to its promotor	N	mmu	26,179,904
FIRRE	Macrophage	TLR-MyD88	Up	Promotes immune gene expression through hnRNPU dependent mRNA stabilization	C/N	mmu	28,993,514 31,723,143
Lncrna-CCL2	Macrophage	TLR	Up	Increases expression of immune genes. Counter-regulated by SIRT1	?	mmu	29,277,325
Lincrna-EPS	Macrophage	TLR-MyD88	Down	Together with hnRNPL fosters a repressive chromatin state at immune gene promoters	N	mmu	27,315,481
Lnc13	Macrophage	TLR-MyD88	Down	Suppresses inflammatory gene expression by interacting with hnRNPD and HDAC1	N	hsa, mmu	27,034,373
NEGATIVE NFκB/AP1 CONTROL:
THRIL	Macrophage	TLR/TNFR	?	Suggested to promote TNFα expression. Expression in immune cells remains debated.	N	hsa	24,371,310 28,900,427
Mirt2	Macrophage	TLR-MyD88	Up	Suppresses immune gene expression through inhibitory interaction with TRAF6	C	mmu	29,230,038
Anti-IL1β	Macrophage	TLR-MyD88	Up	Suppresses the expression of IL1β by modifying chromatin structure	N	mmu	23,677,478
SeT	Macrophage	TLR-MyD88	Up	Suppresses biallelic expression of TNFα	C	mmu	25,770,217 28,910,376
lncfao	Macrophage	TLR-MyD88	Up	Immune gene suppression through activation of HADHB-dependent fatty acid oxidation	C	mmu	32,513,690

C = cytoplasm; N = nucleus; mmu = mus musculus; hsa = homo sapiens

4.1. Control of interferon-dependent phagocyte immunity

The magnitude and kinetics of type I IFN expression is tightly regulated, to ensure adequate activation of ISG-dependent cellular defence against intracellular viruses and bacteria [41,45]. Recently, several lncRNAs were implicated in type I IFN production and ISG activation in professional cells of the innate immune system. In human macrophages, activation of the TLR4-MyD88-NFκB pathway by LPS triggers a rapid increase in MaIL1 lncRNA expression. MaIL1 constitutes an inducible component of the TLR4-TRIF signalling arm, which activates type I IFN production. Within the TLR4-TRIF pathway, MaIL1 forms a complex with the OPTN protein in the cytoplasm to promote ubiquitin-dependent aggregation of the OPTN signalling platform, which promotes TBK1 kinase-dependent IRF3 transcription factor phosphorylation [27]. Thus, up-regulation of lncRNA MaIL1 in human macrophages serves the MyD88-dependent positive control of TRIF-TBK1-IRF3-induced type I IFN expression. Suggesting an in vivo relevance in type I IFN immunity, MaIL1 levels linearly correlate with IFNβ levels in bronchoalveolar lavage from pneumoniae patients and loss of MaIL1 impedes type I IFN dependent cell-autonomous antibacterial defence in macrophages [27]. Like MaIL1, lncRNA NRIR is among the 10 most highly induced lncRNAs in LPS-activated human macrophages [27]. In monocytes, TLR4-dependent NRIR induction was reported to promote ISG (i.e. CXCL10) expression through a yet unknown mechanism. Expression of NRIR was found to be elevated in monocytes from systemic sclerosis patients and positively correlated with the patients ISG expression score [51]. Thus, both MaIL1 and NRIR function to leverage IFN-associated immune responses in TLR4-activated primary human phagocytes (Fig. 1).

In murine macrophages, lncRNA lnczc3h7a is induced in response to viral infection and acts as a molecular scaffold, bringing TRIM25 ubiquitin ligase and Rig-I into spacial proximity. This triggers TRIM25-dependent Rig-I ubiquitination and downstream IRF3-dependent type I IFN production [52]. Thus, reminiscent of the function of MaIL1 in human cells, lnczc3h7a regulates ubiquitin-dependent progression of PRR-signalling in the cytoplasm to promote type I IFN production by phagocytes. The response of murine macrophages to IFN stimulation is subsequently fine-tuned by additional lncRNAs, induced by the IFN-JAK-STAT signalling axis (Fig. 1). lncLrrc55-AS for instance, upon STAT-dependent activation, further promotes type I IFN production by supporting demethylation and inactivation of the IRF3 inhibitor PP2A by the PME1 protein [53]. Another murine lncRNA activated by type I IFN is lnc-Lsm3b, which binds to Rig-I monomers, this time however as a negative regulator, interfering with Rig-I signalling to limit further IFN production [54]. LncRNAs can also adopt broader regulatory functions that span both the IFN-dependent and the NFκB-dependent gene expression networks in murine phagocytes. LincRNA-EPS for instance is a suppressor of cytokine, chemokine and ISG expression. Downregulation of lincRNA-EPS upon TLR-MyD88-NFκB pathway activation thus lifts repression of immune gene activation. Mechanistically, lincRNA-EPS alters nucleosome positioning at target gene promoters through an interaction with the hnRNPL protein, to foster a repressive chromatin state [32]. LncRNA Lfar1 is also downregulated upon murine macrophage activation, however only upon IFNγ and not upon LPS stimulation, suggesting a JAK-STAT dependent regulation. Similar to lincRNA-EPS, Lfar1 functions as a negative regulator of the inflammatory response and its downregulation upon IFNγ stimulation relieves suppression of NFκB-dependent genes and NLRP3 inflammasome mediated pyroptosis [55].

Taken together, lncRNAs in human and murine phagocytes both positively and negatively regulate IFN-production and IFN-receptor triggered immune responses through a variety of mechanisms, ranging from control of protein ubiquitination, stability and activity in the cytoplasm to chromatin remodelling in the nucleus (Fig. 1).

4.2. Positive regulation of TLR-NFκB driven phagocyte immunity

Large parts of the pro-inflammatory innate defence programmes rely on TLR-triggered NFκB and AP1 transcription-factor activation [1]. Similar to the IFN-associated immune circuitries, lncRNAs have been implicated in both positive and negative control of TLR-NFκB signalling events in human phagocytes, to confer productive immune-activation levels. In human monocytes for instance, the lncRNAs IL-1β-eRNA and IL-1β-RBT46, originating from the IL1β gene locus, are induced following TLR4-MyD88-NFκB activation by bacterial LPS and promote expression of the pro-inflammatory cytokine and MyD88-signalling amplifier IL1β [56]. Another TLR4-inducible nuclear lncRNA is PACER, which decoys NFκB p50 to suppress p50 homodimer formation and promote NFκB p50/p65 heterodimer formation and thus NFκB-dependent immune gene expression in human macrophages [18]. Similarly, lncRNA CARLR is induced in human macrophages upon LPS-treatment and promotes pro-inflammatory gene expression by binding to NFκB, this time, however, to the p65 subunit in the cytoplasm [57]. Other lncRNAs involved in positive control of TLR-MyD88 dependent gene expression in human macrophages are ROCKi and IL7-AS [58,59]. IL7-AS is one of the few lncRNAs implicated in both human and murine phagocyte immunity [50]. Upon TLR-MAPK/NFκB activation, it associates with p300 and SWI/SNF and contributes to histone acetylation and promoter remodelling to foster the expression of soluble immune mediators such as CCL2 and IL6 [59].

Like in human cells, several lncRNAs were found to confer positive control of TLR-NFκB responses in murine macrophages. Besides IL7-AS and CARLR, which are involved both in human and murine phagocyte immunity, lincRNA-Tnfaip3 is induced in a TLR4-NFκB dependent manner and promotes expression of various soluble mediators, such as IL6. Mechanistically, lincRNA-Tnfaip3 associates with chromatin regulator HMGB1 to form a lincRNA/HMGB1/NFκB complex and promote NFκB binding at target gene loci [60]. Similar to CARLR and lincRNA-Tnfaip3, another TLR-inducible lncRNA, lnc-EPAV, directly controls NFκB activity in murine macrophages. Lnc-EPAV is encoded within an endogenous retrovirus locus and induced upon stimulation with viral dsRNA mimic poly(I:C). Lnc-EPAV supports the production of immune mediators, such as IL6, TNFα or type I IFN by decoying the inhibitory SFPQ protein from the p65 promoter, thereby leveraging NFκB p65 dependent gene expression [61]. Using an NFκB-GFP reporter system, further lncRNAs were found to regulate NFκB-dependent gene expression in murine macrophages [62]. While nuclear TLR-induced lncRNA AK170409 was found to foster NFκB activity below the IκBα step, the partially cytosolic lncRNA lincRNA-Cox2 was found to promote IκBα degradation, which causes NFκB-nuclear translocation and pro-inflammatory gene expression [62]. LincRNA-Cox2 had previously been shown to be highly induced upon TLR-MyD88 pathway activation, to negatively regulate TLR-induced ISG CCL5 expression, independent of type I IFN. Mechanistically, linc-Cox2 binds to hnRNPA/B and A2/B1 in the nucleus and inhibits RNA polymerase II recruitment to the CCL5 promoter. In the same study, lincRNA-Cox2 was also shown to promote TLR-dependent expression of a subset of pro-inflammatory genes [63]. In another follow-up study, positive control of NFκB-dependent gene expression by lincRNA-Cox2 was reported to involve SWI/SNF dependent chromatin remodelling at target gene promoters [64]. Together, these reports suggest that lincRNA-Cox2 both positively and negatively regulates NFκB-dependent gene expression in murine macrophages through different, cytoplasmic and nuclear mechanisms. Another lncRNA acting at the chromatin level is AS-IL1α, which is induced upon TLR-activation with LPS, Pam3CSK4 or poly(I:C) and recruits RNA polymerase II to the IL1α promoter [65]. Expression of lncRNA FIRRE is also increased upon macrophage activation with LPS and promotes hnRNPU dependent stabilization of immune-associated mRNAs, including IL1β, IL12b and VCAM1 [66]. In vivo, mice overexpressing FIRRE displayed increased pro-inflammatory cytokine levels (TNFα, IL12-p40, MIP-2) when compared to WT mice [67]. Surprisingly, in FIRRE knockout mice cytokine levels remained unaffected, questioning the physiological relevance of FIRRE in this context [67]. Haematopoietic phenotypes of ΔFIRRE mice, however, argue for an in vivo role of this lncRNA in leukocyte driven immunity [67]. Another murine lncRNA reported to promote activation of the macrophage inflammatory response is lncRNA-CCL2 [68]. The mechanism, through which LPS-inducible lncRNA-CCL2 supports expression of inflammation markers such as IL1β, TNFα or IL6, however, remains to be determined. Expression of this lncRNA is counter regulated by SIRT1, a protein downregulated upon LPS stimulation and under septic shock conditions [68].

Besides PRR-inducible lncRNAs, several lncRNAs are downregulated upon TLR-activation of murine macrophages. As described above (4.1), downregulation of lincRNA-EPS upon TLR-MyD88-NFκB activation lifts repression of inflammatory gene activation [32]. In line with this observation, macrophages from lincRNA-EPS deficient mice, infected with L. monocytogenes display increased expression of pro-inflammatory genes and nitric oxid production and are less susceptible to Listeria monocytogenes infection [69]. Another lncRNA downregulated in a TLR-MyD88-NFκB dependent manner is lnc13. Downregulation of lnc13 relieves suppression of a subset of NFκB-dependent genes. Thereby, lnc13 contributes to murine macrophage immune activation very similar to lincRNA-EPS. Mechanistically, lnc13 regulates its target genes by fostering hnRNPD and HDAC1 induced repressive chromatin states [70]. Of note, decreased lnc13 levels were observed in intestinal biopsy samples from coeliac disease patients [70], suggesting a role of this lncRNA in human inflammatory diseases as well.

In summary, the reports available so far suggests that lncRNAs vitally contribute to human and murine phagocyte activation, as induced positive-regulators and supressed negative regulators of PRR-triggered pro-inflammatory gene expression programmes (Fig. 1).

4.3. Negative regulation of TLR-NFκB driven phagocyte immunity

Besides supporting the activation of TLR-NFκB dependent gene expression, lncRNAs are also involved in processes restraining the activity of this signalling axis. In a human macrophage cell line model, lncRNA THRIL, for instance, was reported to be downregulated upon Pam3CSK4 or TNFα stimulation. It was suggested that a THRIL/hnRNPL ribonucleoprotein complex positively regulates TNFα expression at the promoter level [71]. In this case, THRIL downregulation would counter-regulate TNFα production. Independent expression profiling studies, however, failed to confirm the presence of this lncRNA in primary human phagocytes [27,50].

In murine LPS-treated macrophages, lncRNA Mirt2 was found to be induced both via the TLR-MyD88 and the TLR-TRIF signalling pathway. Mirt2 was shown to negatively regulate TLR-triggered pro-inflammatory gene expression by interacting with the TLR-MyD88 pathway component TRAF6 and negatively regulating TRAF6 auto-ubiquitination and oligomerization in the cytoplasm [72]. This observation puts Mirt2 on par with other cytoplasmic lncRNAs such as MaIL1 or lnczc3h7a, which regulate the immune response via ubiquitination of PRR-signalling components. Another TLR4-induced negative regulator, besides Mirt2, is Anti-IL1β, which inhibits IL1β production, probably through deposition of promoter H3K4 tri-methylation marks [73]. LncRNA SeT is also induced in murine macrophages upon LPS stimulation and supresses biallelic TNFα expression [74]. In SeT deficient mice, increased steady-state TNFα levels and biallelic expression seem to result in increased mortality upon LPS-injection [75], suggesting a protective role of SeT in endotoxemia. LncRNA lncFAO is another negative regulator of murine macrophage immune responses, which governs mitochondrial energy balance – a process that is tightly linked to macrophage inflammatory activity [76]. LncFAO is induced late upon LPS-stimulation to counter-regulate expression of pro-inflammatory mediators such as IL1β and contribute to inflammation resolution. Mechanistically, lncFAO associates with the fatty acid oxidation promoting enzyme HADHB at mitochondria and stabilizes HADHB protein levels. LncFAO binding promotes mitochondrial HADHB activity and fatty acid oxidation, which in turn decreases macrophage pro-inflammatory activity [77].

Thus, lncRNAs confer both positive and negative control of TLR-NFκB driven immunity in human and murine phagocytes through control of mitochondrial metabolism, protein and mRNA stability, and chromatin modification and remodelling (Fig. 1).

4.4. LncRNAs in phagocyte differentiation and polarization

Many lncRNAs display highly tissue- and cell-type specific expression patterns, which suggests their implication in cell lineage specification and differentiation [13,78]. In the immune system, besides their well-established roles in PRR-triggered immunity, lncRNAs indeed contribute to haematopoiesis and peripheral blood cell differentiation. Myeloid-specific human transcript HOTAIRM1, for instance, is up-regulated in a PU.1 transcription factor dependent manner during granulocytic differentiation and promotes expression of the myeloid differentiation markers CD11b and CD18 [79,80]. Both proteins together form the myeloid-specific integrin α_Mβ₂, which regulates myeloid cell adhesion and induction of which is also PU.1-dependent [81]. Another lncRNA involved in myeloid cell differentiation is Morrbid. This lncRNA is up-regulated in murine macrophages upon LPS-treatment and inhibits macrophage apoptosis. Mechanistically, Morrbid negatively regulates expression of the pro-apoptotic factor Bim in cis, by binding to PRC2 component EZH2 and promoting PRC2 binding to the Bim promoter and deposition of repressive H3K27me3 chromatin marks [82]. In Morrbid-deficient mice, eosinophil, neutrophil, and monocyte counts were reduced, suggesting a general role of this lncRNA in the control of myeloid cell life-span. Morrbid expression was found to be elevated in eosinophils of hyper-eosinophilic syndrome patients, possible prolonging eosinophil life-span [82]. Another lncRNA involved in the granulocytic lineages is EGO. This lncRNA is generated from an intronic region within the ITPR1 gene and is highly expressed in mature eosinophils. RNA silencing revealed EGO to promote the expression of eosinophil markers such as MBP and EDN [83]. A function in the differentiation of dendritic cells from human monocytes was reported for lnc-DC. This lncRNA binds to STAT3, a transcription factor required for dendritic cell differentiation, and promotes STAT3 phosphorylation by preventing its interaction with and dephosphorylation by SHP1 [84]. Lnc-DC additionally increases IL12 production by dendritic cells upon activation with LPS [84]. Furthermore, NEAT1 was found to be required for proper LPS-dependent maturation of dendritic cells [85].

Besides their roles in differentiation and longevity, lncRNAs were also reported to affect the polarization state of phagocytes. Unlike classically activated macrophages, M2-like macrophages are generated in response to Th2 cell cytokines (see section 7) and adopt rather anti-inflammatory and tissue repair functions [86]. Upon human macrophage differentiation with M-CSF and Th2 cytokines IL4 and IL13, expression levels of lncRNA lnc-M2 were found to be increased compared to non-activated, M-CSF-differentiated M0 macrophages. Lnc-M2 is induced by Th2 cytokines in a STAT3-dependent manner and forms a complex with PKA to promote PKA phosphorylation and PKA/CREB pathway-dependent M2 polarization [87]. In mice, the lncRNA AK085865 was reported to support M2 polarization. AK085865 deficient animals display fewer M2 macrophages, reduced eosinophil counts and attenuated allergic airway inflammation in an asthmatic mouse model [88].

Taken together, besides their emerging roles in PRR-driven phagocyte immunity, lncRNAs adopt important functions in human and murine myeloid cell differentiation and polarization, thereby contributing to pathogen eradication and tissue repair, but also to inflammation-associated disorders when falsely regulated.

5. LncRNAs in innate immune responses beyond the phagocyte system

Professional immune cells, such as monocytes or macrophages are key to the adequate activation and subsequent resolution of innate inflammatory responses to pathogen- and danger-signals. These cells however do not act autonomously but rather receive a multitude of signals from the periphery, including their surrounding tissue environment [47,48]. To capture the complex intercellular communication events within human tissue under infection conditions, we recently developed an engineered model of the human intestinal barrier, comprising an epithelial and an endothelial compartment equipped with peripheral blood leukocytes. RNA-seq analysis of epithelial cells, endothelial cells, monocytes and natural killer cells isolated from epithelially infected (Salmonella enterica Typhimurium) or control-treated tissue models, revealed dozens of up- and downregulated lncRNAs in all cell types during innate immune crosstalk at the intestinal barrier [49]. While the majority of these lncRNAs currently remains uncharacterized, several studies have already provided insights into the roles of individual lncRNAs in tissue-anchored cell types, such as endothelial cells, epithelial cells or fibroblasts, during immune activation and inflammation (Fig. 2, Table 2).

Table 2. lncRNAs in mammalian immune responses beyond the phagocyte system

lncRNA name	Cell type	Upstream pathway	Regula-tion	Function	Locali-zation	Orga-nism	PMID
POSITIVE CONTROL OF INTERFERON IMMUNITY:
NEAT1	HeLa, HUVEC	Rig-I	Up	Induces type I IFN expression by displacing SFPQ from the Rig-I promoter or by promoting IRF3 activation in complex with Hexim1	N	hsa	28 712 728 28 202 761
IVRPIE	Lung epithelial	TLR3	Up	Promotes type I IFN expression by hnRNPU dependent chromatin changes	N	hsa	32 153 544
NRAV	Lung epithelial	?	Down	Down-regulation withdraws a repressive interaction with ZONAB and promotes antiviral MxA expression	N	hsa	25 525 793
Lnc-ITPRIP	Hepatocytes	JAK/STAT	Up	Promotes oligomerization of MDA5, resulting in type I IFN expression	C/N	hsa	29 899 107
Lnc13	Pancreatic ß-cells	TLR3	Up	Stabilizes STAT1 mRNA by promoting its interaction with PCBP2	C	hsa	32 284 404
NEGATIVE CONTROL OF INTERFERON IMMUNITY:
TSPOAP1‐AS1	Lung epithelial	TLR3	Up	Suppresses the expression of antiviral ISGs	N	hsa	30 968 963
Lnc-MxA	Lung epithelial	JAK/STAT	Up	Supresses IFNβ expression by blocking promoter binding of p65 and IRF3	N	hsa	31 434735
LncATV	Hepatocytes	JAK/STAT	Up	Reduces type I IFN expression through an inhibitory interaction with Rig-I	C	hsa	31 379 885
EGOT	Hepatocytes	Rig-I	Up	Negatively regulates the interferon-response	N	hsa	27 283 940
NRIR	Hepatocytes	JAK/STAT	Up	Negatively regulates the interferon-response	N	hsa	25 122 750
lncRNA # 32	Hepatocytes	TLR3	Down	Promotes ISG expression via an interaction with hnRNPU and ATF2	?	hsa	27 582 466
POSITIVE NFκB/AP1 CONTROL:
PACER	Mammary epithelial	?	?	Promotes expression of pro-inflammatory mediator COX2	N	hsa	24 843 008
CARLR	Intestinal epithelial	TLR4	Up	Pro-inflammatory role in macrophages (see Table 1)	C	hsa	28 626 066
FIRRE	Intestinal epithelial	TLR-MyD88	Up	Stabilizes immune-associated mRNAs through interaction with hnRNPU	C/N	hsa	28 993 514
IL7-AS	Intestinal epithelial	TLR-MyD88	Up	Promotes p300 and SWI/SNF dependent cytokine expression	N	hsa	31 383 742
IL7-AS	Lung epithelial	IL1R-MyD88	Up	Promotes IL6 expression	N	hsa	28 900 427
NEAT1	HeLa epithelial cells	TLR3	Up	Promotes IL8 expression by displacement of SFPQ from the promoter	N	hsa	24 507 715
NEAT1	HeLa epithelial cells	Salmo-nella	Up	NEAT1 stabilization fosters paraspeckle formation and immune gene expression	N	hsa	29 880 601
NEGATIVE NFκB/AP1 CONTROL:
NKILA	Mammary epithelial	TLR4	Up	Suppresses immune gene expression by preventing IκB phosphorylation	C	hsa	25 759 022
TMC3-AS1	Intestinal epithelial	TLR4	Up	Suppresses expression of IL10 by sequestering p65 from the promotor	N	hsa	32 774 335
Linc-Cox2	Intestinal epithelial	TNFR	Up	Supresses IL12b induction by Mi2/NuRD repressor complex recruitment	N	mmu	26 578 685
Lnc-IL7R	HUVEC	TLR4	Up	Suppresses immune gene expression through promoter H3K7 tri-methylation	N	hsa	24 723 426
Lethe	MEF	TNFR	Up	Supresses inflammatory gene expression by sequestering p65 away from the promotor	N	mmu	23 898 399

C = cytoplasm; N = nucleus; mmu = mus musculus; hsa = homo sapiens

Figure 2. LncRNAs in NFκB- and type I IFN-activating PRR responses outside the phagocyte system. Murine (green) and human (blue) lncRNAs, reported to positively or negatively control activation of NFκB- or type I IFN-driven innate immune responses in intestinal, vascular, alveolar, pancreatic, hepatic, mammary or fibroblast cells are shown

5.1. Positive control of interferon-immunity in epithelial, endothelial and β-cells

While phagocytes represent a predominant source of type I IFN during acute immune responses, cells of the endo- and epithelia may produce IFNs as well. In addition, the endo- and epithelia represent important targets of type I IFN induced metabolic adaptations and desensitization against viral and intracellular bacterial infections [35,41,89]. IFN production by endothelial cells during viral infection is under positive control by the nuclear lncRNA NEAT1. Upon infection, a ribonucleoprotein particle, containing NEAT1 and the Hexim1 protein as core components, undergoes extensive remodelling, leading to release of paraspeckle proteins, such as SFPQ, and recruitment of STING [90]. The latter functions as a cyclic dinucleotide sensor and protein component of antiviral IFN-induction pathways [1,34,91]. Consequently, NEAT1-Hexim1 complex remodelling upon infection results in STING-dependent IRF3 phosphorylation and type I IFN expression [90]. During Hantavirus infection of endothelial cells NEAT1 was found to be upregulated via the PRR Rig-I to promote type I IFN expression by displacing SFPQ from the Rig-I promoter [92]. Thus, dynamic nuclear NEAT1-SFPQ complexes are involved in different IFN-induction pathways in cells of the blood vessel linings during viral infection. In lung epithelial cells, human lncRNA IVRPIE is upregulated upon Influenza A virus infection and poly(I:C) stimulation to promote IFNβ and ISG expression. Mechanistically, IVRPIE fosters the deposition of activatory H3K4 tri-methylation marks at target gene promoters, potentially through an interaction with hnRNPU [93]. Also in lung epithelial cells, lncRNA NRAV is down-regulated upon IAV infection through a yet to be determined pathway. NRAV down-regulation promotes expression of several ISGs, including the antiviral protein MxA. Mechanistically, NRAV interacts with the protein ZONAB, which functions as a positive regulator of MxA expression. Thus, NRAV down-regulation likely serves to withdraw a repressive interaction of the lncRNA with ZONAB and promote antiviral defence [94]. Of note, viruses such as IAV may also utilize lncRNAs has host-factors, promoting replication in lung epithelial cells, as exemplified by lncRNA-ACOD1 [95,96]. A closer investigation of the endogenous roles of such lncRNAs, e.g. in resolution of antiviral responses, could provide further insights into the implications of lncRNAs in lung epithelial immunity. In human hepatocytes, lncRNA ITPRIP was reported to be upregulated upon type I IFN stimulation and HCV infection to promote oligomerization of the PRR MDA5, thereby enhancing type I IFN production [97]. Thus, several lncRNAs function as positive regulators of type I IFN production in the cytoplasm and nucleus of human endothelial and epithelial cells during viral infections.

Upon their release, type I IFNs bind to the ubiquitous surface receptor IFNAR to trigger JAK-STAT signalling and ISG expression [35,45]. In human pancreatic β cells, lnc13 is transferred from the nucleus to the cytoplasm upon stimulation with a viral dsRNA mimic. Lnc13 subsequently stabilizes STAT1 mRNA in the cytoplasm by promoting its interaction with the PCBP2 RNA-binding protein [31]. Thus, different from its reported function in phagocytes (4.2), in β cells, lnc13 does not act in the nucleus as a repressor but rather as a cytoplasmic positive-regulator of the STAT-pathway, suggesting cell-type specific roles of this lncRNA.

5.2. Negative control of interferon-immunity in epithelial cells

Uncontrolled IFN immunity can have detrimental effects, by, for example, contributing to the development of autoimmune diseases and cellular exhaustion [35,41]. Besides the contribution of lncRNAs to the activation of type I IFN immunity, several lncRNAs therefore act as negative regulators of type I IFN production and signalling within and beyond the leukocyte system. In human lung epithelial cells, lncRNA TSPOAP1‐AS1 for instance is induced following poly(I:C) stimulation in an NFκB-dependent manner. TSPOAP1‐AS1 is largely retained in the nucleus and counteracts Influenza A virus-induced IFNβ expression [98]. Similarly, human lnc-MxA is upregulated upon Influenza A infection and poly(I:C) or IFNβ stimulation of lung epithelial cells to restrain IFNβ expression. This involves lnc-MxA association with the IFNβ promoter and interference with IRF3 and p65 transcription factor binding [99]. Like in lung epithelial cells, lncRNAs contribute to negative control of type I IFN production and signalling in human hepatocytes. LncATV negatively regulates type I IFN and ISG expression by associating with Rig-I and preventing downstream signal transduction [100]. Rig-I induced nuclear lncRNA EGOT restrains ISG expression through an as yet unknown mechanism. Knockdown of EGOT decreases HCV replication and expression of EGOT was found to be increased in liver biopsies from HCV patients [101]. NRIR is induced upon IFNα stimulation of hepatocytes and negatively regulates ISG expression, again through an unknown mechanism [102]. This regulation mode differs from its reported role in phagocytes, thus similar to lnc13, NRIR might adopt cell-type specific functions. Different from LncATV, EGOT and NRIR, lncRNA # 32 is downregulated upon poly(I:C) or type I IFN stimulation in human hepatocytes. LncRNA # 32 promotes ISG expression through interaction with the ATF2 transcription factor. Thus, the downregulation of lncRNA # 32 counteracts type I IFN induced ISG expression [103]

Taken together, type I IFN production and downstream signalling is promoted and limited by a complex network of lncRNAs during immune-activation of cells of the blood vessels, lung, liver and pancreas (Fig. 2). Further lncRNAs acting as regulators of type I IFN immunity in other organ systems are to be expected.

5.3. Positive control of TLR-NFκB driven immunity in epithelial and endothelial cells

Besides IFN immunity, classic NFκB-driven pro-inflammatory gene expression needs to be tightly controlled, not only in cells of the immune system, to prevent from ineffective immune responses but also from vascular damage or disruption of the epithelial barriers [104,105]. Several lncRNAs seem to assist in the activation of NFκB-induced gene expression in epithelial cells. PACER for instance is not only active in phagocytes (4.2) but also in human mammary epithelial cells and promotes expression of pro-inflammatory mediator COX2 [18]. In human intestinal epithelial cells, CARLR is induced upon LPS-treatment. In human intestinal biopsy cells from coeliac disease compared to control patients CARLR is furthermore transferred from the nucleus to the cytoplasm. Its NFκB p65 binding activity and role as a positive regulator of inflammatory gene expression in macrophages predicts that CARLR adopts a similar function in the intestinal epithelium [57]. Again in human intestinal epithelial cells, lncRNA FIRRE, which is also active in macrophages (4.2), is induced upon TLR4-NFκB activation and stabilizes immune-associated mRNAs (IL1b, IL12b, VCAM1) through interaction with hnRNPU [66]. Furthermore, expression of IL7-AS is elevated upon TLR-MAPK/NFκB pathway activation in intestinal epithelial cells, to promote expression of a set of cytokines (but not IFNs) through the association with p300 and SWI/SNF dependent histone acetylation and promoter remodelling complexes [59]. In lung epithelial cells, IL7-AS is induced upon stimulation with the pro-inflammatory, NFκB activating cytokine IL1β and promotes IL6 expression, similar to its role described in intestinal epithelial cells [50]. In HeLa cells, NEAT1 was found to be induced upon TLR3-p38 pathway activation to dislocate SFPQ from the IL8 promoter and tether it into paraspeckles, thereby promoting IL8 expression [106]. This function is reminiscent of the displacement of SFPQ from the Rig-I promoter by NEAT1 in endothelial cells [92], (5.1). Also in HeLa cells, NEAT1, is stabilized upon Salmonella infection through diminished nuclear RNA decay and promotes paraspeckle formation and expression of immune-associated genes such as TNFSF9 or CCL2, thereby contributing to antibacterial defence [107]. Thus, positive control of the TLR-NFκB axis in human mammary, intestinal and cervical epithelial cell models is provided by several lncRNAs directly acting on NFκB, promoting permissive chromatin states or stabilizing mRNAs (Fig. 2).

5.4. Negative control of TLR-NFκB driven immunity in epithelial cells, endothelial cells and fibroblasts

Failure to restrain NFκB activity may not only provoke inflammation-associated tissue damage [104,105] but also promote oncogenesis [108]. In a breast cancer cell line model, human cytoplasmic lncRNA NKILA is induced by LPS and acts as a negative feedback regulator, interacting with NFκB and blocking IκB phosphorylation. NKILA levels are decreased in breast cancer patients, which predicts a role of NKILA in NFκB activation in this disease [109]. Similarly, in human intestinal epithelial cells, lncRNA TMC3-AS is induced by LPS and interacts with NFκB p65 to prevent from p65 binding to the IL10 promoter [110]. In human endothelial cells, lnc-IL7R is upregulated upon LPS-treatment and negatively regulates inflammatory gene expression (e.g. IL6, VCAM-1 or E-selectin) by promoting H3K27 tri-methylation at gene promoters [111]. Thus, several human lncRNAs prevent from exaggerated NFκB activation in human epithelial and endothelial cells.

Among the few lncRNAs implicated in immune-modulation in murine cells outside the phagocyte system, Lethe was described as a negative regulator induced upon TNFα stimulation of mouse embryonic fibroblasts. Lethe functions as a negative feedback regulator, restraining NFκB activity by interacting with NFκB p65 and inhibiting p65 binding to DNA [112]. Also upon TNFα stimulation, linc-Cox2 is upregulated in murine intestinal epithelial cells to suppress IL12b induction through Mi2/NuRD repressor complex recruitment to the IL12b promoter [113].

Taken together, similar to their functions in professional immune cells, lncRNAs are engaged in positive and negative control circuitries of epithelial cells, endothelial cells and fibroblasts (Fig. 2), promoting and restraining inflammatory gene expression through a variety of nuclear and cytoplasmic regulatory mechanisms.

6. Emerging patterns of lncRNA-mediated control in the innate immune system

The above-depicted functions of lncRNAs in the immune system suggest recurring higher-level regulatory principles through which the inflammatory process is modulated and adapted to external signals. Irrespective of their subcellular localization and molecular interactors, regulatory RNAs can be sub-grouped into feedback and feed-forward regulators. Feedback-regulators help to stabilize a given signalling pathway at an optimal level through either negative or positive feedback [114]. Feed-forward regulators on the other hand may cause a bi-phasic or ultrasensitive response to an immune stimulus [114]. LncRNA Mirt2, for instance, acts as a negative feedback regulator, which inhibits PRR-signal transduction upon its TLR-dependent induction, by interfering with TRAF6 activation in the cytoplasm. PACER on the other hand provides positive feedback, upon NFκB-dependent activation, by promoting NFκB p50/p65 heterodimer formation. Several other lncRNAs introduced above, similarly act as negative or positive feedback regulators, helping to stabilize PRR, cytokine and IFN signalling networks (Fig. 3). Besides lncRNAs providing direct feedback to their upstream pathways, feed-forward control seems to be a major principle of lncRNA-mediated regulation of inflammatory responses. The majority of innate-immune associated lncRNAs described today seems to be engaged in coherent feed-forward control, where a positive regulator is induced or a repressor is withdrawn, to amplify the response to a given stimulus, not necessarily involving a direct feedback function (Fig. 3). Examples are MaIL1 and linc-Cox2, but also lnc13 and lincRNA-EPS (Fig. 3). Finally, incoherent feed-forward control, e.g. involving the induction of a repressor of a given pathway, is another prominent regulation mode, through which for instance linc-Cox2, IL7-AS or SeT impact the innate immune response (Fig. 3).

Figure 3. LncRNA-based feedback and feed-forward mechanisms in PRR-triggered immunity. LncRNAs can provide positive or negative feedback to their upstream PRR-, cytokine- or interferon-signalling pathways (left). Feed-forward control promotes, sensitizes or re-shapes receptor triggered immune-signalling programmes. In this context, lncRNA have been implicated in coherent (elevated positive-regulator or down-regulated repressor) and incoherent (induced repressor) feed-forward control (right). Murine lncRNAs shown in green, human lncRNAs in blue

The majority of immune-associated lncRNAs characterized to date seems to be involved in positive control (feedback or feed-forward) of PRR, cytokine- and IFN-receptor signalling pathways (Fig. 3), suggesting an important contribution of lncRNAs to the initiation and amplification of the immune response. This is reminiscent of the previously known feed-forward control of the macrophage inflammatory response by microRNAs of the let-7 family [12]. Negative regulators, including several lncRNAs, such as lncFAO, but also microRNAs, such as miR-146 [10,11], subsequently level the inflammatory response or contribute to inflammation resolution. Thus, it is becoming increasingly clear, that mammalian innate immune responses to pathogen- and danger-signals are tightly controlled and modulated by a dense network of regulatory RNAs, preventing both from overshooting and attenuated inflammation through feed-back and feed-forward control mechanisms (Fig. 3, Tables 1 and 2).

7. LncRNAs at the interface between innate and adaptive immunity

Besides the above-portrayed functions of lncRNAs in the innate immunity, lncRNAs vitally participate in the adaptive immune system by affecting differentiation and activation of T- and B-lymphocytes [115]. Phagocytes actively communicate with T- and B-cells to regulate their activity. Among others, this involves the secretion of cytokines affecting T-cell differentiation, or the presentation of antigens recognized by T-cell receptors (TCRs) [116]. T-cell activation upon TCR ligation to antigens presented e.g. by dendritic cells or on the surface of various other cell-types through MHC-complexes involves a calcium-signalling dependent pathway leading to the activation of the transcription factor NFAT [116,117]. NFAT nuclear translocation is counter-regulated by lncRNA NRON [118]. This lncRNA serves as a scaffold redirecting NFAT to a cytoplasmic complex consisting of the IQGAP protein and several kinases, which prevent NFAT from being dephosphorylated and imported into the nucleus [119]. Resolution of T cell responses involves the process of activation-induced cell death (AICD), which is regulated by lncRNA NKILA. TCR activation triggers STAT-dependent expression of NKILA, which sensitizes cytotoxic T-lymphocytes to AICD [120]. In epithelial cells, NKILA was reported counteract LPS-induced NFκB activation (see 5.4). Thus, NKILA is an example of an lncRNA engaged both in innate and adaptive immunity.

T-cell polarization is actively shaped by macrophages, dendritic cells and other cell types producing cytokines such as IL6 or IL12 [116]. Via IL12 release, activated macrophages and dendritic cells stimulate NK- and T-cells for production of interferon-γ (IFNγ) [116], which in turn triggers ISG expression in phagocytes and promotes increased antigen presentation and antimicrobial responses [121]. Production of IFNγ by CD4+ Th1-polarized cells and CD8 + T-cells is under positive control by the lncRNA IFNG-AS1 (also known as NeST, or Tmevpg1) [122–125]. Mechanistically, murine IFNG-AS1 interacts with the histone-methyltransferase WDR5 to promote deposition of activating histone methylation marks at the IFNγ locus [126]. Supporting a physiological role in inflammation, the IFNG-AS gene overlaps with an Inflammatory Bowel Disease susceptibility SNP and its expression is increased in ulcerative colitis patients [123,127]. Moreover, in mice, IFNG-AS1 was shown to abate Salmonella enterica induced pathologies and reduce bacterial burden in an IFNγ dependent manner [126]. Besides its role in CD4+ Th1 cells and CD8 + T-cells, IFNG-AS1 also promotes IFNγ production by NK cells, which participate both in innate and adaptive immunity [128] (see below). Another lncRNA regulating IFNγ production is MORRBID. Originally described to regulate phagocyte differentiation and longevity (see 4.4), in CD8 + T-cells MORRBID is induced upon TCR activation and type I IFN stimulation during viral infection [129]. Unlike IFNG-AS1, MORRBID functions to restrain IFNγ production [129].

While CD8 + T-cells and CD4+ Th1 cells regulate phagocyte activity via IFNγ, CD4+ Th2 cells, upon their activation produce IL4, which promotes macrophage polarization towards an anti-inflammatory and tissue-repair associated phenotype [86,116]. Similar to IFNγ-production by Th1 cells, IL4 generation by Th2 cells depends on several lncRNAs, including lincR-Crr2-5ʹAS, GATA3-AS1 and TH2-LCR [115]. In macrophages, IL4 dependent polarization in turn is regulated by lncRNA lnc-M2 (see 4.4). Thus, the communication between T-cells and phagocytes is controlled by lncRNAs on both sides.

Of note, naïve CD4 + T-cell activation critically depends on dendritic cell presented antigens [116]. Dendritic cell differentiation and activation by innate immune agonists such as LPS is controlled by lncRNAs lnc-DC and NEAT1 (see 4.4). It remains to be determined, whether lncRNAs participating in the PRR-responses of other phagocytes, including macrophages (see section 4), are also active in dendritic cells and contribute to their bridging functions in innate and adaptive immunity. Likewise, the roles of lncRNAs in NK cells, a type of innate lymphoid cells, that do not rearrange immunoglobulin and TCR genes but can kill target cells through immunoglobulin-binding [130,131], are still poorly understood. Besides the role of IFNG-AS1 in IFNγ production by NK cells (see above), Lnc-CD56 was found to promote expression of the important NK-cell marker and adhesion molecule CD56 and to contribute to NK-cell differentiation [132]. Further lncRNAs are expressed and dynamically regulated under infection conditions in NK cells [49,132] but presently remain uncharacterized. Functional characterization of the still poorly studied lncRNA repertoires of dendritic cells and NK cells should further improve our understanding of the regulatory implications of lncRNAs at the interface between innate and adaptive immunity.

8. Conclusions

LncRNAs are critically involved in many cellular processes [4]; thus, it is not surprising that these molecules also play a central role in innate immune responses of professional phagocytes and other cell types in their periphery. The literature available so far suggests a plethora of different mechanisms through which lncRNAs can interfere with or promote cellular inflammatory responses. These mechanisms range from nuclear processes, such as transcription factor positioning and chromatin remodelling to cytoplasmic processes, including protein ubiquitination and mRNA stabilization. However, despite the recent progress in our understanding of the roles of lncRNAs in innate immunity, the field is still at its infancy and the majority of immune-associated lncRNAs remains mechanistically uncharacterized. Moreover, only few of the known immune-relevant lncRNAs seem to be conserved between mice and humans at the primary sequence level [50]. This could hint at the redundancy of many immunoregulatory lncRNAs, as regulatory RNAs whose function is redundant with other regulators are expected to display little evolutionary sequence conservation [133]. LncRNA redundancy, however, does not necessarily indicate lack of importance at an evolutionary scale. Rather, the volatility of lncRNA repertoires was speculated to contribute to robustness and evolvability of species [134]. This might be particularly true for immune-modulatory lncRNAs, since infectious agents constitute an important driver of evolution [135]. Besides the possibility of redundancy as an explanation for little lncRNA conservation, lncRNA genes might also accumulate neutral mutations that dissipate the primary sequence over the course of evolution without affecting RNA secondary structure and function [136]. Such structure-neutral mutations are well documented for ribosomal RNAs, which display higher conservation at the structural than at the primary sequence level [137]. Besides sequence- and structure-conservation, the simple act of transcription from syntenic loci, independent of the lncRNA sequence, might explain the function of some lncRNAs. Combinations of sequence- and structure-conservation and syntenic transcription could contribute to an lncRNAs overall functional conservation [138]. An important task in the coming years will be to determine structure-conserved orthologues, functional equivalents and syntenic loci corresponding to immune-regulatory lncRNAs such as linc-Cox2 and MaIL1, the study of which is presently limited to rodents and primates, respectively.

Independent of their evolutionary conservation, the already obtained mechanistic insights into the roles of lncRNAs in human and murine cells suggest a vital participation in the TLR-TIRF-, RLR-MAVS- and STING-dependent type I IFN induction pathways and in JAK-STAT- dependent responses to type I IFNs. Furthermore, lncRNAs participate in TLR-MyD88, TLR-TRIF and cytokine-receptor dependent activation of the inflammation master regulator NFκB. Of note, lncRNA-mediated feedback and feed-forward control of cellular immune responses is not limited to professional innate immune cells, but also contributes to the adequate immune activation of epithelial cells, endothelial cells and fibroblasts in different organ systems. Many of the immune-regulatory lncRNAs identified to date, seem to interact with chromatin-regulatory (e.g. PRC2 or SWI/SNF) and paraspeckle proteins (e.g. SFPQ). Further lncRNAs impact the immune response through interaction with RNA binding proteins controlling mRNA stability (e.g. hnRNPU or PCBP2) or ubiquitination dependent signalling (e.g. TRIM25 or OPTN). It would be of great interest to the community to learn about the global RNA substrates of these RNA binding proteins (RBPs), e.g. through unbiased CLIP-seq methods which could reveal larger RBP-lncRNA networks and lncRNA binding hubs involved in cellular innate immunity. Co-sedimentation analysis using density gradients indeed suggest that lncRNAs in immune-activated phagocytes form discriminable subgroups, potentially interacting with shared protein components [27]. Besides their functions in response to PRR agonists, relatively little is known about the role of lncRNAs in the differentiation and polarization of immune cells. Many lncRNAs exhibit highly tissue- and cell-type-specific expression patterns [13,78] and these lncRNAs might play important roles in leukocyte differentiation and in shaping the cellular responsiveness to immune stimuli. Further efforts are required to map lineage specific lncRNAs involved in leukocyte differentiation and function and potentially contributing to haematological disorders.

Besides the important progress already made, many open questions as to the functions of lncRNAs in the innate immune system and the strategies to study lncRNA candidates remain. The lower copy numbers of lncRNAs per cell compared to mRNAs, for example, raise questions about possible lncRNA mechanisms. Whereas individual lncRNAs, such as MALAT1 are highly expressed in many cell types [138], the median expression levels reached by lncRNAs are ~10-fold below those reached by mRNAs [139]. This should, for instance affect the decoying capacity of lncRNAs such as PACER, which occludes NFκB p50 from the COX-2 promoter. Besides serving as protein decoys, several lncRNAs were described to function as competing endogenous RNAs, which capture microRNAs and reduce their ability to bind to other target RNAs [4,140]. The copy number of an lncRNA relative to mRNAs targeted by the same microRNA would be highly relevant to the determination of its ability to serve as a ceRNA. So far, however, such stoecheometries are poorly documented for immune-associated lncRNAs. Apart from the decoy function, low-level lncRNA expression does not necessarily indicate little regulatory potential, as exemplified by lncRNA VELUCT, which profoundly affects cell survival despite very low copy numbers [141]. Mechanistically, lowly expressed lncRNAs could for instance serve as starting points for the assembly of larger signalling complexes, such as the OPTN-TBK1 platform [27] or Myddosomes [142]. Once triggered, such complexes might continue to assemble independent of additional RNA copies. Besides their lower expression compared to mRNAs, the association of a substantial number of lncRNAs with ribosomes [26,27] raises questions about their potential cellular functions. Both in human and murine cells, annotated lncRNAs were found to encode previously unknown peptides, including in phagocytes [25,26]. Of note, ribosome-association alone does not predict coding potential. Long read-sequencing and ribosome-profiling data are therefore required to fine-map lncRNA transcript architectures and ribosome-occupied regions. Thereby, falsely annotated lncRNAs encoding peptides could be discriminated from true lncRNAs associating with ribosomes to control immune-relevant ribosomal functions. In addition to the protein-coding potential of lncRNAs, it remains unclear, to which degree the already published lncRNA functions hint towards recurring regulatory principles in the innate immune system, especially since most immune-regulated lncRNAs in phagocytes and other cell types have not yet been functionally characterized [27,49]. One reason for this could be the challenging de novo identification of RNA interaction partners, which renders mechanistic lncRNA research tedious. However, emerging technologies, such as gradient-sequencing and mass spectrometry (Grad-seq), RNA antisense affinity purification and mass-spectrometry (RAP-MS) or proximity labelling methods promise to accelerate the fine-mapping of lncRNA-protein interactions [27,143–145]. Together with the recent improvements of the ENCODE eCLIP, ChIP-seq and chromatin 3D structure catalogues [146], these methodologies may substantially facilitate the dissection of lncRNA-mechanisms involved in the control of immune mediator production and inflammation. The already available mechanistic insights predict that further research on inflammation-associated lncRNAs will decisively advance our understanding of the control-mechanisms of the innate immune system and thus of the pathomechanisms underlying prevalent human diseases.

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Deutsche Forschungsgemeinschaft [SFB TR-84].