Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.

Recent advances in large-scale RNA sequencing (RNA-seq) have led to the identification of novel RNA species encoded in the genome, many of which are long noncoding RNAs (lncRNAs) (1, 2). lncRNAs are arbitrarily defined as having ≥200 nt, which discriminates them from small noncoding RNAs. These RNAs are further categorized based on their genomic location relative to neighboring protein-coding genes. Several comprehensive studies demonstrated that lncRNAs are important regulators of cell differentiation and developmental pathways (3, 4). A growing literature also supports their importance in immunity (5).

Natural antisense transcripts (NATs) are a class of lncRNAs defined as being complementary to one or more protein-coding genes (6). Approximately 50–70% of lncRNAs are classified as NATs, making them a substantial proportion of the noncoding genome (2, 6). Although the functions of some lncRNAs are well defined, relatively few NATs are functionally characterized. NATs can both activate and repress the expression of complementary coding genes (7, 8). Similar to the broader class of lncRNAs, NATs probably function in a cell type–specific manner. For example, in Th2 cells, lincR-Ccr2-5′AS regulates expression of Ccr2, Ccr3, and Ccr5 and trafficking of Th2 cells to the lung (9). However, the molecular functions of NATs in innate immunity are unknown.

Macrophages are the first line of defense against microbial pathogens. Following infection, host cells produce proinflammatory cytokines. In particular, IL-1α and IL-1β are rapidly induced and amplify inflammation via IL-1R complexes expressed on neighboring cells (10). Because these cytokines can cause significant tissue damage, their production must be tightly regulated. Caspase-1–dependent inflammasomes convert IL-1β into the mature biologically active cytokine. In contrast, IL-1α is active in its full-length form upon release from damaged or dying cells (11).

In this article, we describe a functional innate immune NAT, AS-IL1α, encoded within the IL-1α locus. Characterization of this NAT shows that AS-IL1α functions as an additional layer of regulation important in controlling IL-1α transcription.

C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) were maintained in specific pathogen–free conditions and used in accordance with the University of Massachusetts Medical School Institutional Animal Care and Use Committee. Listeria monocytogenes (clinical isolate 10403s) was from V. Boyartchuk (Norwegian University of Science and Technology). Mice were infected as described (12) for 24 h before harvesting the spleen. PBS was used as a control.

Single-cell suspensions from spleens were used to make total RNA, and 4 μg total RNA was used to generate libraries for RNA sequencing (RNA-seq) (Illumina unstranded RNA Kits). Samples were sized, quantified, and validated on a bioanalyzer. Libraries were sequenced on a HiSeq 2000 (Illumina, San Diego, CA) as paired-end 100 bp reads. Sequence reads were aligned to the mouse genome (NCBI m37/mm9) using TopHat. Gene-level read counts were calculated using HTSeq and the Ensembl 64 transcript annotation file. The DESeq package was used to normalize gene counts and calculate fold-change values and p values. The Circos program was used to visualize genome-wide gene-expression changes. As previously described (3), all protein-coding genes annotated with particular Gene Ontology IDs were classified as immune genes. All RNA-seq data are available from ArrayExpress under accession number E-MTAB-3315.

Primary bone marrow–derived macrophages (BMDMs) were generated and infected with Listeria or stimulated with LPS (100 ng/ml), Pam3CSK4 (100 nM), polyinosinic-polycytidylic acid [Poly(I:C)] (25 μg/ml), and IFN-stimulatory DNA (ISD; 3 μM) oligonucleotides (13), as described (12). After 2 h of incubation, total RNA was harvested from all conditions (RNeasy; QIAGEN).

Polysome profiling was performed as described (3, 4).

Lentiviral particles were generated using pLKO.1 TRC, as described (14). Knockdown efficiency was assessed by one-step quantitative RT-PCR (qRT-PCR; Bio-Rad). Sequences/primers are listed in Supplemental Table I.

Immunoblotting was performed using mouse IL-1α/IL-1F1 Ab (R&D Systems, cat. no. AB-400NA) and anti-goat IgG (Bio-Rad, cat. no. 172-1011).

nCounter CodeSets were constructed for detecting selected mouse-specific immune genes, and levels of RNA were measured using the nCounter Digital Analyzer, as described (3, 15).

Cytosolic and nuclear fractions were prepared as described (16). The cell lysis buffer contained 0.15% Nonidet P-40; the sucrose cushion did not contain detergent. After fractionation, cytoplasmic and nuclear RNA were purified using QIAGEN RNeasy columns. Gapdh and 7SK were used for cytoplasmic and nuclear controls, respectively. Primers are listed in Supplemental Table I.

Cells were stimulated, as described, fixed in 1% formaldehyde, lysed, and sonicated. A total of 5 μg chromatin was immunoprecipitated with Dynabeads Protein G (Novex/Life Technologies, cat. no. 10009D) and Abs to RNA polymerase II (RNAPII; Active Motif, cat. no. 102660), H3K9-Ac (Abcam, cat. no. AB4441), or IgG isotype (Abcam, cat no. AB37415). Purified chromatin DNA was used for quantitative PCR amplification at the IL-1α, IP-10, or Gapdh promoter.

Statistical analysis was performed using a Student t test with GraphPad Prism software for bivariate studies and one-way ANOVA for multivariate studies. The p values ≤ 0.05 were considered statistically significant.

To assess lncRNA expression during an active infection, we infected C57BL/6 mice with L. monocytogenes and prepared libraries for RNA-seq from splenocytes isolated from infected and noninfected mice (24 h). Consistent with previous reports (17), Listeria induced expression of a wide range of protein-coding immune genes (Fig. 1A, inner track). We also detected many lncRNAs that were differentially expressed following Listeria infection (Fig. 1A, outer track).

FIGURE 1.

Identification of AS-IL1α. (A) Circos plot showing differentially expressed genes in splenocytes from L. monocytogenes–infected mice. Chromosomes are indicated along the outer track. The middle track shows log2 fold-change values for all lncRNAs and AS-IL1α (red). The inner track shows log2 fold-change values for protein-coding genes, with immune genes shown in red. (B) Schematic diagram of AS-IL1α in murine macrophages. AS-IL1α chromosomal localization overlaps with IL-1α protein-coding gene on chromosome 2 (indicated by vertical bar). (C) Macrophages infected with L. monocytogenes for 3 h at an multiplicity of infection (MOI) of 5 and 10. Data were normalized to Gapdh and fold change was calculated. Data are representative of two independent experiments. (D) Polysome profiling of Gapdh, IL-1α, and AS-IL1α transcripts. Immortalized macrophages were stimulated with LPS and treated with cycloheximide alone or harringtonine prior to cycloheximide treatment to determine the translational potential of AS-IL1α.

FIGURE 1.

Identification of AS-IL1α. (A) Circos plot showing differentially expressed genes in splenocytes from L. monocytogenes–infected mice. Chromosomes are indicated along the outer track. The middle track shows log2 fold-change values for all lncRNAs and AS-IL1α (red). The inner track shows log2 fold-change values for protein-coding genes, with immune genes shown in red. (B) Schematic diagram of AS-IL1α in murine macrophages. AS-IL1α chromosomal localization overlaps with IL-1α protein-coding gene on chromosome 2 (indicated by vertical bar). (C) Macrophages infected with L. monocytogenes for 3 h at an multiplicity of infection (MOI) of 5 and 10. Data were normalized to Gapdh and fold change was calculated. Data are representative of two independent experiments. (D) Polysome profiling of Gapdh, IL-1α, and AS-IL1α transcripts. Immortalized macrophages were stimulated with LPS and treated with cycloheximide alone or harringtonine prior to cycloheximide treatment to determine the translational potential of AS-IL1α.

Close modal

Strikingly, one of these lncRNAs (hereafter referred to as AS-IL1α) was induced 14-fold and was encoded by the opposite strand of the IL-1α locus on chromosome 2. We used PCR to confirm the orientation and gene structure of AS-IL1α (Fig. 1B, sequence accession KR095173 http://www.ncbi.nlm.nih.gov/genbank). Similar to the in vivo infection, AS-IL1α was induced in macrophages infected with Listeria (Fig. 1C). The Coding Potential Calculator (http://cpc.cbi.pku.edu.cn/) classified AS-IL1α as noncoding (score = −0.87), which was also supported by polysome profiling. Cells were treated with cycloheximide, which traps ribosomes along their RNA strands, and these RNA were detected in the heavier fractions of a sucrose density gradient. In parallel, cells were treated with harringtonine prior to cycloheximide, which inhibits translation and polysome formation by causing ribosomes to accumulate at their initiation sites, and is detected in the lighter fractions of a sucrose density gradient. As expected, harringtonine prevented polysome formation in both Gapdh and IL-1α mRNA (Fig. 1D), and both mRNAs shifted to lighter fractions in the sucrose gradient. In contrast, AS-IL1α was largely unaffected by harringtonine treatment, indicating that it is unlikely to associate with ribosomes and encode a protein product.

We next examined the expression of AS-IL1α in BMDMs stimulated with ligands for TLRs and other sensors. Fig. 2A depicts the location of our qRT-PCR primers, which do not overlap with IL-1α itself. AS-IL1α was induced by LPS (TLR4), Pam3CSK4 (TLR1/2), and Poly(I:C) (TLR3), but not ISD, which activates cGAS (Fig. 2B). We also measured expression of AS-IL1α in macrophages from IL-1α–deficient mice. In these cells, LPS induced expression of AS-IL1α but not IL-1α, indicating that transcription of AS-IL1α is regulated independently of IL-1α (Fig. 2C). We next compared LPS-inducible levels of AS-IL1α in wild-type and MyD88/TRIF double-knockout cells and cells treated with Bay11-7085, an inhibitor of NF-κB. In the absence of MyD88/TRIF or following inhibition of NF-κB, LPS failed to induce AS-IL1α (Fig. 2D, 2E).

FIGURE 2.

Inducibility of AS-IL1α by TLR ligands. (A) Schematic diagram of AS-IL1α and IL-1α and qRT-PCR primers for experiments in (B)–(E) (not to scale). (B) LPS, Pam3CSK4, Poly(I:C), or ISD were stimulated on immortalized BMDMs for 0, 1, 2, or 6 h, and AS-IL1α expression was measured by qRT-PCR. (C) Wild-type and IL-1α–knockout macrophages were stimulated with LPS for 6 h. AS-IL1α expression was induced, even in the absence of IL-1α. (D) Wild-type (WT) and MyD88/Trif double-knockout (dKO) macrophages were treated or not treated (NT) with LPS, Pam3CSK4, or Poly(I:C) [p(IC)] for 6 h. AS-IL1α expression was measured by qRT-PCR. ****p < 0.0001, **p < 0.01, one-way ANOVA. (E) An NF-κB inhibitor, Bay11-7082, was used to treat cells 1 h prior to LPS stimulation (6 h), and AS-IL1α expression was measured by qRT-PCR. Data were normalized to Gapdh, and fold change was calculated. Data are mean + SD and are representative of three replicates. ***p < 0.001, two-tailed t test.

FIGURE 2.

Inducibility of AS-IL1α by TLR ligands. (A) Schematic diagram of AS-IL1α and IL-1α and qRT-PCR primers for experiments in (B)–(E) (not to scale). (B) LPS, Pam3CSK4, Poly(I:C), or ISD were stimulated on immortalized BMDMs for 0, 1, 2, or 6 h, and AS-IL1α expression was measured by qRT-PCR. (C) Wild-type and IL-1α–knockout macrophages were stimulated with LPS for 6 h. AS-IL1α expression was induced, even in the absence of IL-1α. (D) Wild-type (WT) and MyD88/Trif double-knockout (dKO) macrophages were treated or not treated (NT) with LPS, Pam3CSK4, or Poly(I:C) [p(IC)] for 6 h. AS-IL1α expression was measured by qRT-PCR. ****p < 0.0001, **p < 0.01, one-way ANOVA. (E) An NF-κB inhibitor, Bay11-7082, was used to treat cells 1 h prior to LPS stimulation (6 h), and AS-IL1α expression was measured by qRT-PCR. Data were normalized to Gapdh, and fold change was calculated. Data are mean + SD and are representative of three replicates. ***p < 0.001, two-tailed t test.

Close modal

We next generated macrophage cell lines in which AS-IL1α expression was specifically silenced by short hairpin RNA (shRNA). We made two independent shRNAs that targeted AS-IL1α exons that did not overlap with those of IL-1α (Fig. 3A). We confirmed that AS-IL1α was significantly silenced in LPS-stimulated cell lines that expressed these shRNA (Fig. 3B). In shRNA-GFP lines, LPS strongly induced IL-1α mRNA levels, whereas LPS only induced low levels of IL-1α mRNA in cells expressing shRNA–AS-IL1α (Fig. 3C). LPS also failed to induce the high levels of IL-1α protein normally detected by immunoblotting (Fig. 3D). These results indicate that AS-IL1α is required for the inducible expression of IL-1α. We subsequently used RNA profiling (NanoString) to simultaneously analyze mRNA expression levels of a broader selection of immune genes following LPS stimulation (6 h) in control and AS-IL1α–silenced macrophages (Fig. 3E). IL-1α was the most significantly altered gene in the shRNA lines; IL-1β levels were decreased, albeit to a lesser extent.

FIGURE 3.

AS-IL1α regulates IL-1α expression. (A) shRNA 1 and shRNA 2 target AS-IL1α in red regions (not to scale) that do not overlap with IL-1α. (B) AS-IL1α RNA levels (qRT-PCR) were reduced in both shRNA 1 and shRNA 2 cell lines. shRNA GFP is a control that targets the GFP gene. (C) IL-1α mRNA levels (qRT-PCR) were reduced in shRNA 1 and shRNA 2 cell lines. (D) IL-1α p33 protein expression (Western blot) was decreased in shRNA 1 and shRNA 2 cell lines stimulated with LPS. β-actin was used as a loading control. (E) NanoString analysis was performed on the cell lines to determine whether knocking down AS-IL1α affects the expression of other immune genes. Immune genes with a 15-fold change in expression following LPS stimulation are shown. Data were calculated as fold-change relative to Gapdh and are representative of three biological replicates. ****p < 0.0001, one-way ANOVA.

FIGURE 3.

AS-IL1α regulates IL-1α expression. (A) shRNA 1 and shRNA 2 target AS-IL1α in red regions (not to scale) that do not overlap with IL-1α. (B) AS-IL1α RNA levels (qRT-PCR) were reduced in both shRNA 1 and shRNA 2 cell lines. shRNA GFP is a control that targets the GFP gene. (C) IL-1α mRNA levels (qRT-PCR) were reduced in shRNA 1 and shRNA 2 cell lines. (D) IL-1α p33 protein expression (Western blot) was decreased in shRNA 1 and shRNA 2 cell lines stimulated with LPS. β-actin was used as a loading control. (E) NanoString analysis was performed on the cell lines to determine whether knocking down AS-IL1α affects the expression of other immune genes. Immune genes with a 15-fold change in expression following LPS stimulation are shown. Data were calculated as fold-change relative to Gapdh and are representative of three biological replicates. ****p < 0.0001, one-way ANOVA.

Close modal

To understand how AS-IL1α might regulate IL-1α, we first examined its localization by performing subcellular fractionation of nuclear and cytosolic compartments and analyzing RNA levels by qRT-PCR. We also measured levels of Gapdh, IL-1α, and the nuclear RNA 7SK. As expected, the mature IL-1α and Gapdh transcripts were localized to the cytosol, whereas 7SK RNA was confined to the nucleus. AS-IL1α was primarily nuclear (Fig. 4A).

FIGURE 4.

AS-IL1α regulates IL-1α transcription. (A) Localization of AS-IL1α in LPS-induced BMDMs. Cytosolic and nuclear fractions were separated via a sucrose gradient, and qRT-PCR was performed on fractionated RNA. Gapdh (cytosolic), IL-1α (cytosolic), and 7SK (nuclear) were also measured as controls. Data are the percentage of the cytosolic fraction or the nuclear fraction over cytosolic+nuclear (100%) total RNA. (B) qRT-PCR was performed on shRNA-GFP and two AS-IL1α–knockdown cell lines. Schematic diagram shows regions to be amplified by primers in mature IL-1α mRNA (exon–exon junction) and prespliced IL-1α mRNA. RNA levels are shown as a percentage of the expression in shRNA-GFP (representative of two experiments). (C) A comparison of poly-d(T) and random hexamer priming for reverse transcription to an AS-IL1α–specific primer to demonstrate specificity of the amplified region prior to qRT-PCR. (D) Chromatin immunoprecipitation was performed on the cell lines. Abs against RNAPII or IgG isotype control were used. Data were normalized with input chromatin before immunoprecipitation. Regions of the IL-1α gene, near the transcription start site (+22), were measured for RNAPII binding. RNAPII recruitment was significantly less in shRNA-AS-IL1a cell lines than shRNA-GFP cell lines (p = 0.0298, t test). RNAPII recruitment did not decrease in the respective shRNA cell lines for CXCL10/IP-10 (p = 0.127) or Gapdh promoter (p = 0.123) relative to shRNA-GFP.

FIGURE 4.

AS-IL1α regulates IL-1α transcription. (A) Localization of AS-IL1α in LPS-induced BMDMs. Cytosolic and nuclear fractions were separated via a sucrose gradient, and qRT-PCR was performed on fractionated RNA. Gapdh (cytosolic), IL-1α (cytosolic), and 7SK (nuclear) were also measured as controls. Data are the percentage of the cytosolic fraction or the nuclear fraction over cytosolic+nuclear (100%) total RNA. (B) qRT-PCR was performed on shRNA-GFP and two AS-IL1α–knockdown cell lines. Schematic diagram shows regions to be amplified by primers in mature IL-1α mRNA (exon–exon junction) and prespliced IL-1α mRNA. RNA levels are shown as a percentage of the expression in shRNA-GFP (representative of two experiments). (C) A comparison of poly-d(T) and random hexamer priming for reverse transcription to an AS-IL1α–specific primer to demonstrate specificity of the amplified region prior to qRT-PCR. (D) Chromatin immunoprecipitation was performed on the cell lines. Abs against RNAPII or IgG isotype control were used. Data were normalized with input chromatin before immunoprecipitation. Regions of the IL-1α gene, near the transcription start site (+22), were measured for RNAPII binding. RNAPII recruitment was significantly less in shRNA-AS-IL1a cell lines than shRNA-GFP cell lines (p = 0.0298, t test). RNAPII recruitment did not decrease in the respective shRNA cell lines for CXCL10/IP-10 (p = 0.127) or Gapdh promoter (p = 0.123) relative to shRNA-GFP.

Close modal

Because AS-IL1α was localized to the nucleus, we hypothesized that it regulated the transcription of IL-1α. We used qRT-PCR primers that targeted intron-containing sequences of the IL-1α pre-mRNA, as well as primers targeting spliced IL-1α transcripts. Consistent with an effect on transcription, both spliced and unspliced pre-mRNA IL-1α levels were decreased in the three AS-IL1α shRNA lines (Fig. 4B). We also made reverse-transcription primers that targeted AS-IL1α only, and compared the results with our normal method that uses poly-d(T) and random hexamer primers to confirm specific amplification of our qRT-PCR amplicon from AS-IL1α cDNA. The trends were comparable, indicating that the abrogated AS-IL1α expression levels were specific to AS-IL1α and not the result of promiscuous priming (Fig. 4C).

We also performed chromatin immunoprecipitation–PCR assays to measure RNAPII occupancy at the region surrounding the transcription start site of the IL-1α locus (+22 nt). As expected, LPS treatment in the shRNA-GFP control cells resulted in robust RNAPII binding at this region. This was greatly reduced in macrophages expressing AS-IL1α shRNA (Fig. 4D). We observed similar results when we measured the epigenetic mark H3K9-acetylation, an indicator of active transcription at this same locus (Supplemental Fig. 1). The effect of AS-IL1α shRNA was specific to the IL-1α locus, because recruitment of RNAPII to Cxcl10 or Gapdh was unaffected in the knockdown line. These results indicate that AS-IL1α is required to facilitate RNAPII recruitment to the IL-1α locus during LPS-induced transcription.

lncRNAs are emerging as important regulators of gene expression in diverse biological contexts, including immunity. LPS was shown to induce widespread changes in lncRNA expression in immune cells (18). lncRNAs also were shown to promote or repress inflammatory gene expression in immune cells (19). The identification of AS-IL1α as a natural antisense lncRNA that enhances IL-1α gene transcription adds to our understanding of cytokine-mediated inflammation. Indeed, disruption of AS-IL1α function could limit IL-1α transcription and potentially alleviate the damaging effects of excessive IL-1α levels during infection and inflammatory disease.

This work was supported by grants from the National Institutes of Health (T32 Training Grant A1095213 to J.C. and Grant AI067497 to K.A.F.), the American Heart Association (to M.A.), the German Research Foundation (to R.E.), and the Arthritis National Research Foundation (to S.C.).

The sequences presented in this article have been submitted to the ArrayExpress database under accession number E-MTAB-3315 and to the Genbank database under accession number KR095173.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMDM

bone marrow–derived macrophage

ISD

IFN-stimulatory DNA

lncRNA

long noncoding RNA

NAT

natural antisense transcript

Poly(I:C)

polyinosinic-polycytidylic acid

qRT-PCR

quantitative RT-PCR

RNAPII

RNA polymerase II

RNA-seq

RNA sequencing

shRNA

short hairpin RNA.

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The authors have no financial conflicts of interest.

Supplementary data