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Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes.

IIott NE, Heward JA, Roux B, Tsitsiou E, Fenwick PS, Lenzi L, Goodhead I, Hertz-Fowler C, Heger A, Hall N, Donnelly LE, Sims D, Lindsay MA - Nat Commun (2014)

Bottom Line: Early reports indicate that long non-coding RNAs (lncRNAs) are novel regulators of biological responses.Crucially, we demonstrate that knockdown of nuclear-localized, NF-κB-regulated, eRNAs (IL1β-eRNA) and RBT (IL1β-RBT46) surrounding the IL1β locus, attenuates LPS-induced messenger RNA transcription and release of the proinflammatory mediators, IL1β and CXCL8.We predict that lncRNAs can be important regulators of the human innate immune response.

View Article: PubMed Central - PubMed

Affiliation: 1] CGAT Programme, MRC Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, UK [2].

ABSTRACT
Early reports indicate that long non-coding RNAs (lncRNAs) are novel regulators of biological responses. However, their role in the human innate immune response, which provides the initial defence against infection, is largely unexplored. To address this issue, here we characterize the long non-coding RNA transcriptome in primary human monocytes using RNA sequencing. We identify 76 enhancer RNAs (eRNAs), 40 canonical lncRNAs, 65 antisense lncRNAs and 35 regions of bidirectional transcription (RBT) that are differentially expressed in response to bacterial lipopolysaccharide (LPS). Crucially, we demonstrate that knockdown of nuclear-localized, NF-κB-regulated, eRNAs (IL1β-eRNA) and RBT (IL1β-RBT46) surrounding the IL1β locus, attenuates LPS-induced messenger RNA transcription and release of the proinflammatory mediators, IL1β and CXCL8. We predict that lncRNAs can be important regulators of the human innate immune response.

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IL1β-eRNA and IL1β-RBT46(+) regulate LPS-induced IL1β and CXCL8 expression and release.Human monocytic THP-1 cells (Anti-RBT46+) were transfected with two negative control LNAs or an LNA antisense against either IL1β-eRNA (Anti-eRNA) or IL1(β)-RBT46(+) (Anti-RBT46+) at a final concentration of 30 nM. Cells were then treated with either buffer (non-stimulated) or LPS prior to quantification of (a) IL1β-eRNA1 and IL1β-RBT46(+) expression at 2 h (b) IL1β mRNA, CXCL8 mRNA and IL6 mRNA at 24 h, (c) IL1β, CXCL8 and IL6 protein release at 24 h and (d) IL1α mRNA and IL1RN mRNA at 24 h. Data are the mean±s.e.m. of nine independent experiments. Statistical significance was determined using a one-way analysis of variance with a Dunnett’s post test, where *P<0.05, **P<0.01 and ***P<0.001.
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f7: IL1β-eRNA and IL1β-RBT46(+) regulate LPS-induced IL1β and CXCL8 expression and release.Human monocytic THP-1 cells (Anti-RBT46+) were transfected with two negative control LNAs or an LNA antisense against either IL1β-eRNA (Anti-eRNA) or IL1(β)-RBT46(+) (Anti-RBT46+) at a final concentration of 30 nM. Cells were then treated with either buffer (non-stimulated) or LPS prior to quantification of (a) IL1β-eRNA1 and IL1β-RBT46(+) expression at 2 h (b) IL1β mRNA, CXCL8 mRNA and IL6 mRNA at 24 h, (c) IL1β, CXCL8 and IL6 protein release at 24 h and (d) IL1α mRNA and IL1RN mRNA at 24 h. Data are the mean±s.e.m. of nine independent experiments. Statistical significance was determined using a one-way analysis of variance with a Dunnett’s post test, where *P<0.05, **P<0.01 and ***P<0.001.

Mentions: Given their genomic position (Fig. 4d) and nuclear localization (Fig. 6c), we speculated that IL1β-eRNA and IL1β-RBT46 might regulate the transcription of IL1β. To examine this hypothesis, we designed a panel of five locked nucleic acid (LNA)-based antisense inhibitors against IL1β-eRNA and IL1β-RBT46(+) and transfected them into the monocytic THP-1 cells. Following LPS stimulation, we found that in the case of both IL1β-eRNA and IL1β-RBT46(+), only one (of the five) attenuated lncRNA production (Fig. 7a). However, these LNA antisense inhibitors, but not two negative controls, reduced LPS-induced IL1β-eRNA and IL1β-RBT46(+) generation by 85±9% and 53±9%, respectively (Fig. 7a). Of relevance, we also failed to attenuate LPS-induced IL1β-eRNA production using a panel of four siRNAs (Supplementary Fig. 4a) despite showing a 64±4% reduction in LPS-induced IL6 mRNA production using a positive control siRNA (Supplementary Fig. S4b). In contrast, we were able to show knockdown using all five LNA antisense inhibitors targeted against the constitutively expressed lncRNA, OIP5-mf-lncRNA (Supplementary Fig. S4c). These studies suggested that unlike previous reports that have successfully employed both LNA antisense and siRNA for the knockout of constitutively expressed lncRNAs and eRNAs313233, this approach is more problematic when applied to lncRNAs that are located within the nucleus and rapidly induced upon exposure to LPS.


Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes.

IIott NE, Heward JA, Roux B, Tsitsiou E, Fenwick PS, Lenzi L, Goodhead I, Hertz-Fowler C, Heger A, Hall N, Donnelly LE, Sims D, Lindsay MA - Nat Commun (2014)

IL1β-eRNA and IL1β-RBT46(+) regulate LPS-induced IL1β and CXCL8 expression and release.Human monocytic THP-1 cells (Anti-RBT46+) were transfected with two negative control LNAs or an LNA antisense against either IL1β-eRNA (Anti-eRNA) or IL1(β)-RBT46(+) (Anti-RBT46+) at a final concentration of 30 nM. Cells were then treated with either buffer (non-stimulated) or LPS prior to quantification of (a) IL1β-eRNA1 and IL1β-RBT46(+) expression at 2 h (b) IL1β mRNA, CXCL8 mRNA and IL6 mRNA at 24 h, (c) IL1β, CXCL8 and IL6 protein release at 24 h and (d) IL1α mRNA and IL1RN mRNA at 24 h. Data are the mean±s.e.m. of nine independent experiments. Statistical significance was determined using a one-way analysis of variance with a Dunnett’s post test, where *P<0.05, **P<0.01 and ***P<0.001.
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Related In: Results  -  Collection

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f7: IL1β-eRNA and IL1β-RBT46(+) regulate LPS-induced IL1β and CXCL8 expression and release.Human monocytic THP-1 cells (Anti-RBT46+) were transfected with two negative control LNAs or an LNA antisense against either IL1β-eRNA (Anti-eRNA) or IL1(β)-RBT46(+) (Anti-RBT46+) at a final concentration of 30 nM. Cells were then treated with either buffer (non-stimulated) or LPS prior to quantification of (a) IL1β-eRNA1 and IL1β-RBT46(+) expression at 2 h (b) IL1β mRNA, CXCL8 mRNA and IL6 mRNA at 24 h, (c) IL1β, CXCL8 and IL6 protein release at 24 h and (d) IL1α mRNA and IL1RN mRNA at 24 h. Data are the mean±s.e.m. of nine independent experiments. Statistical significance was determined using a one-way analysis of variance with a Dunnett’s post test, where *P<0.05, **P<0.01 and ***P<0.001.
Mentions: Given their genomic position (Fig. 4d) and nuclear localization (Fig. 6c), we speculated that IL1β-eRNA and IL1β-RBT46 might regulate the transcription of IL1β. To examine this hypothesis, we designed a panel of five locked nucleic acid (LNA)-based antisense inhibitors against IL1β-eRNA and IL1β-RBT46(+) and transfected them into the monocytic THP-1 cells. Following LPS stimulation, we found that in the case of both IL1β-eRNA and IL1β-RBT46(+), only one (of the five) attenuated lncRNA production (Fig. 7a). However, these LNA antisense inhibitors, but not two negative controls, reduced LPS-induced IL1β-eRNA and IL1β-RBT46(+) generation by 85±9% and 53±9%, respectively (Fig. 7a). Of relevance, we also failed to attenuate LPS-induced IL1β-eRNA production using a panel of four siRNAs (Supplementary Fig. 4a) despite showing a 64±4% reduction in LPS-induced IL6 mRNA production using a positive control siRNA (Supplementary Fig. S4b). In contrast, we were able to show knockdown using all five LNA antisense inhibitors targeted against the constitutively expressed lncRNA, OIP5-mf-lncRNA (Supplementary Fig. S4c). These studies suggested that unlike previous reports that have successfully employed both LNA antisense and siRNA for the knockout of constitutively expressed lncRNAs and eRNAs313233, this approach is more problematic when applied to lncRNAs that are located within the nucleus and rapidly induced upon exposure to LPS.

Bottom Line: Early reports indicate that long non-coding RNAs (lncRNAs) are novel regulators of biological responses.Crucially, we demonstrate that knockdown of nuclear-localized, NF-κB-regulated, eRNAs (IL1β-eRNA) and RBT (IL1β-RBT46) surrounding the IL1β locus, attenuates LPS-induced messenger RNA transcription and release of the proinflammatory mediators, IL1β and CXCL8.We predict that lncRNAs can be important regulators of the human innate immune response.

View Article: PubMed Central - PubMed

Affiliation: 1] CGAT Programme, MRC Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford OX1 3QX, UK [2].

ABSTRACT
Early reports indicate that long non-coding RNAs (lncRNAs) are novel regulators of biological responses. However, their role in the human innate immune response, which provides the initial defence against infection, is largely unexplored. To address this issue, here we characterize the long non-coding RNA transcriptome in primary human monocytes using RNA sequencing. We identify 76 enhancer RNAs (eRNAs), 40 canonical lncRNAs, 65 antisense lncRNAs and 35 regions of bidirectional transcription (RBT) that are differentially expressed in response to bacterial lipopolysaccharide (LPS). Crucially, we demonstrate that knockdown of nuclear-localized, NF-κB-regulated, eRNAs (IL1β-eRNA) and RBT (IL1β-RBT46) surrounding the IL1β locus, attenuates LPS-induced messenger RNA transcription and release of the proinflammatory mediators, IL1β and CXCL8. We predict that lncRNAs can be important regulators of the human innate immune response.

Show MeSH
Related in: MedlinePlus