The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation

Nature Communications, Dec 2017

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) with a crucial function in innate immune responses. Activation of TLR4 signaling at the plasma membrane by lipopolysaccharide (LPS) stimulates proinflammatory signaling pathways dependent on the E3 ubiquitin ligase TRAF6. Here we show the LPS-induced long non-coding RNA (lncRNA) Mirt2 functions as a checkpoint to prevent aberrant activation of inflammation, and is a potential regulator of macrophage polarization. Mirt2 associates with, and attenuates Lys63 (K63)-linked ubiquitination of, TRAF6, thus inhibiting activation of NF-κB and MAPK pathways and limiting production of proinflammatory cytokines. Adenovirus mediated gene transfer of Mirt2 protects mice from endotoxemia induced fatality and multi-organ dysfunction. These findings identify lncRNA Mirt2 as a negative feedback regulator of excessive inflammation.

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The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation

Abstract Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) with a crucial function in innate immune responses. Activation of TLR4 signaling at the plasma membrane by lipopolysaccharide (LPS) stimulates proinflammatory signaling pathways dependent on the E3 ubiquitin ligase TRAF6. Here we show the LPS-induced long non-coding RNA (lncRNA) Mirt2 functions as a checkpoint to prevent aberrant activation of inflammation, and is a potential regulator of macrophage polarization. Mirt2 associates with, and attenuates Lys63 (K63)-linked ubiquitination of, TRAF6, thus inhibiting activation of NF-κB and MAPK pathways and limiting production of proinflammatory cytokines. Adenovirus mediated gene transfer of Mirt2 protects mice from endotoxemia induced fatality and multi-organ dysfunction. These findings identify lncRNA Mirt2 as a negative feedback regulator of excessive inflammation. Introduction Innate immune responses have the capacity to both combat infectious microbes and drive pathological inflammation, which contributes to diseases such as sepsis, atherosclerosis, obesity, autoimmunity and cancer1,2,3. Toll-like receptors (TLR) are pattern recognition receptors (PRR) in the innate immune system, and each TLR recognizes specific pathogen-associated molecular patterns (PAMP)4. Lipopolysaccharide (LPS) is a natural adjuvant synthesized by Gram-negative bacteria that stimulates cells through TLR4, and has profound effects on immune responses5. TLR4-triggered signaling depends on the adaptor proteins myeloid differentiation marker 88 (MyD88) and Toll–interleukin-1 (IL-1) receptor (TIR) domain–containing adaptor-inducing IFNβ (TRIF), which mediate distinct responses that are classified as MyD88-dependent and TRIF-dependent signaling pathways6. At the plasma membrane, the binding of MyD88 to TLR4 results in the recruitment and phosphorylation of IL-1 receptor-associated kinase 1 (IRAK1) and IRAK4, which facilitate oligomerization and auto-ubiquitination of TNF receptor–associated factor 6 (TRAF6)7, 8. Ubiquitinated TRAF6 subsequently engages other signaling proteins, such as transforming growth factor β–activated kinase (TAK1), to activate the inhibitor of κB (IκB) kinase (IKK) and mitogen-activated protein kinase (MAPK) kinase (MKK), leading ultimately to activation of transcription factors such as nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1) to induce immune and inflammatory responses9, 10. Long non-coding RNAs (lncRNA) are a large class of non-protein-coding transcripts that are greater than 200 bases in length11. They are involved in many physiological and pathological processes that include genomic imprinting, embryonic development, cell differentiation, tumor metastasis and regulation of the cell cycle12,13,14. Although a number of lncRNAs have been reported to have crucial functions in diverse processes and diseases, only a few lncRNAs have been show to regulate the immune system15,16,17. In this study, we investigate global lncRNA expression profiles using microarray analysis of macrophages treated with LPS, and propose a model whereby TLR signaling induces the up-regulation of lncRNA-Mirt2, which serves as a repressor of inflammatory responses through interaction with TRAF6, and inhibition of its oligomerization and auto-ubiquitination. Results Differentially expressed lncRNAs in LPS-activated macrophages To identify the lncRNAs that are involved in the innate immune response, we performed a microarray analysis in primary cultured peritoneal macrophages obtained from C57BL/6 mice. LPS, which is a TLR4 ligand, induced numerous differentially expressed lncRNAs. In the volcano plot, 64221 lncRNAs were represented, of which, 2070 were significantly upregulated (red plots) and 1750 were downregulated (blue plots) when filtered with a threshold of a fold change ≥2 and q < 0.05 (Fig. 1a). Differentially expressed lncRNAs, between the control and LPS treatment group, were explored by using more stringent criteria (Student’s t test, P < 0.01, fold change > 20) and filtered according to transcript abundance. In this way we identified the 145 lncRNAs that were most highly induced by LPS stimulation, of which 98 were upregulated and 47 were down regulated (Fig. 1b). LncRNA-Mirt2 was among the most highly induced upregulated lncRNAs and was abundantly expressed in macrophages. Fig. 1 Differentially expressed lncRNAs in macrophages treated with LPS. a Primary cultured peritoneal macrophages were treated with LPS (1 μg/mL) for 24 h and then evaluated to determine their lncRNA profiles using an lncRNA expression microarray. A volcano plot showing the relationship between the q values and the magnitude of the differences in the expression values of the samples in the different groups. b The cluster heatmap shows lncRNAs with expression change fold > 20 from microarray data (P < 0.01). c The expression of Mirt2 in cultured peritoneal macrophages treated with LPS of c (...truncated)


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Meng Du, Lin Yuan, Xin Tan, Dandan Huang, Xiaojing Wang, Zhe Zheng, Xiaoxiang Mao, Xiangrao Li, Liu Yang, Kun Huang, Fengxiao Zhang, Yan Wang, Xi Luo, Dan Huang, Kai Huang. The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation, Nature Communications, 2017, Issue: 8, DOI: 10.1038/s41467-017-02229-1