SNHG15 is a negative regulator of inflammation by mediating TRAF2 ubiquitination in stroke-induced immunosuppression

Huiling Sun, Shuo Li, Zhaohan Xu, Chengfang Liu, Pengyu Gong, Qiwen Deng, Fuling Yan, Huiling Sun, Shuo Li, Zhaohan Xu, Chengfang Liu, Pengyu Gong, Qiwen Deng, Fuling Yan

Abstract

Background: Abnormal expression of long noncoding RNAs (lncRNAs) has been reported in the acute stage of acute ischemic stroke (AIS). This study aimed to explore differential lncRNA expression in the subpopulations of peripheral blood mononuclear cells (PBMCs) from AIS patients and further evaluate its underlying mechanisms in stroke-induced immunosuppression.

Methods: We reanalyzed lncRNA microarray data and investigated abnormally expressed lncRNAs in the subpopulations of PBMCs by magnetic cell sorting and real-time quantitative PCR. The potential mechanism of small nucleolar RNA host gene 15 (SNHG15) was explored through in vitro and in vivo approaches.

Results: The stroke-induced SNHG15 acted as a checkpoint to inhibit peripheral inflammatory responses. Functional studies showed that SNHG15 promoted M2 macrophage polarization. Mechanistically, SNHG15 expression was dysregulated through the Janus kinase (JAK)-signal transducer and activator of transcription 6 (STAT6) signaling pathway. SNHG15, localized in the cytoplasm, interfered with K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 2 and thereby repressed the activation of mitogen-activated protein kinase and nuclear factor kappa-B signaling pathways and prevented the production of proinflammatory cytokines. Administration of an adenovirus targeting SNHG15 improved stroke-induced immunosuppression in mice.

Conclusions: This study identified SNHG15 as a negative regulator of inflammation in stroke-induced immunosuppression, suggesting it as a novel biomarker and therapeutic target in stroke-associated infection. Trial registration ClinicalTrials.gov NCT04175691. Registered November 25, 2019, https://www.clinicaltrials.gov/ct2/show/NCT04175691 .

Keywords: Immunosuppression; Ischemic stroke; Long noncoding RNA; SNHG15; TRAF2.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2021. The Author(s).

Figures

Fig. 1
Fig. 1
Differentially expressed cytokines and lncRNAs in AIS patients and HCs. Blood samples were collected within 48 h after onset of stroke and were subsequently isolated within 2 h. The levels of IL-4 (A), IL-10 (B), IL-6 (C), and TNF-α (D) in plasma were measured by a Navios flow cytometer. Expression of IL-4 (E), IL-10 (F), IL-6 (G), and TNF-α (H) was detected by qRT-PCR in PBMCs. I The heat map shows 70 upregulated and 128 downregulated lncRNAs in PBMCs identified by lncRNA microarray analysis. J Disease enrichment analysis showed the association of these lncRNAs with stroke. K Pathway enrichment analysis suggested the correlations of these lncRNAs with inflammatory pathways (NF-κB and TNF signaling pathways). * P < 0.05, *** P < 0.001
Fig. 2
Fig. 2
Validation of SNHG15 expression and localization. The proportions of positive isolated monocytes (A) and negative isolated lymphocytes (B) after sorting by magnetic beads were determined by flow cytometry. Expression of IL-4 (C), IL-10 (D), IL-6 (E), and TNF-α (F) in monocytes/macrophages isolated from PBMCs was detected by qRT-PCR and calculated with ΔCT. G SNHG15 relative expression in subpopulations of PBMCs. The expression of SNHG15 in IL-4-induced (H) or LPS-induced (I) monocytes isolated from PBMCs. J The colocalization of SNHG15 and CD14 + monocytes was assessed by FISH. K SNHG15 expression in the cytoplasm and nucleus of monocytes/macrophages. * P < 0.05, ** P < 0.01, *** P < 0.001
Fig. 3
Fig. 3
SNHG15 expression in macrophages stimulated by IL-4 and LPS. Expression of SNHG15 in THP-1 and RAW264.7 cells was induced by IL-4 and LPS (A–D). E The localization of SNHG15 in THP-1 and RAW264.7 cells was assessed by FISH. SNHG15 expression in the cytoplasm and nucleus of THP-1 (F) and RAW264.7 (G) cells was detected by qRT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001
Fig. 4
Fig. 4
SNHG15 promotes M2 polarization of macrophages. (A–D) Effects of forced expression or silencing of SNHG15 on the expression of inflammatory factors induced by IL-4 or LPS in THP-1 cells. E–H Effects of forced expression or silencing of SNHG15 on the expression of inflammatory factors induced by IL-4 or LPS in RAW264.7 cells. * P < 0.05, ** P < 0.01, *** P < 0.001
Fig. 5
Fig. 5
SNHG15 inhibits TRAF2 auto-ubiquitination. A Sliver-stained SDS-PAGE gels showing separated proteins in macrophages that were pulled down using a biotin-labeled SNHG15 probe. The bands in the highlighted regions were assessed by mass spectrometry, verifying TRAF2 as a protein specifically interacting with SNHG15. B The interaction of SNHG15 and TRAF2 was analyzed with the catRAPID database. C Western blot analysis of biotin-labeled SNHG15 probe-bound proteins in THP-1 and RAW264.7 cells using an anti-TRAF2 antibody. D RNA immunoprecipitation was performed with an anti-TRAF2 antibody to assess SNHG15 expression in macrophages by qRT-PCR. IgG was used as a control. E The interaction of TRAF2 with the full-length sequence or fragments of SNHG15 was assessed by RNA pull-down assays. F The effect of SNHG15 on the transcription level of TRAF2 was assessed by qRT-PCR. G The localization of SNHG15 in THP-1 and RAW264.7 cells was assessed by FISH. H Immunoblotting was performed to assess the ubiquitination of TRAF2 in THP-1 and RAW264.7 cells. I Immunoblot analysis of the ubiquitination of TRAF2 in HEK293T cells transfected to express TRAF2 and HA-tagged K48- or K63-linked ubiquitin. Right panel: The influence of different concentrations of SNHG15 on the ubiquitination of TRAF2. J Effects of forced expression or silencing of SNHG15 on MAPK and NF-κB in THP-1 and RAW264.7 cells were determined by western blotting. *** P < 0.001
Fig. 6
Fig. 6
IL-4 induces SNHG15 expression in macrophages through JAK-STAT6 signaling. A The JASPAR CORE database was used to search for potential interacting regions in the SNHG15 promoter. B Effects of STAT6 overexpression on SNHG15 expression in macrophages. C Effects of STAT6 knockdown on SNHG15 expression in macrophages. D The interaction of STAT6 with the SNHG15 promoter was determined by luciferase reporter assays. E The STAT6 binding sites in the SNHG15 promoter were further determined by luciferase reporter assays. F STAT6 occupancy in the SNHG15 promoter region was analyzed by ChIP. ** P < 0.01, *** P < 0.001
Fig. 7
Fig. 7
SNHG15 is an anti-inflammatory molecule in mice. The levels of IL-4 (A), IL-10 (B), IL-6 (C), and TNF-α (D) in plasma were measured by ELISA. Expression of IL-4 (E), IL-10 (F), IL-6 (G), and TNF-α (H) was detected by qRT-PCR in PBMCs. I, J Effects of SNHG15 knockdown on cerebral injury 3 days post-stroke. K Neurological function was assessed 1, 3 and 7 days after tMCAO. The modified neurological severity score (mNSS) was used by investigators blinded to the tMCAO and control groups to assess neurological deficits; the assessment included the tail suspension test, locomotor test, beam walking test and reflex test. Neurological function was graded on a scale of 0 to 14 points, and higher scores indicated more severe neurological deficits. SNHG15 expression in PBMCs (L) and brain tissues (M). N Effects of SNHG15 knockdown on the STAT6, TRAF2, NF-κB, and MAPK pathways 3 days post-stroke. O–R Effects of silencing SNHG15 on the expression of inflammatory factors 3 days post-stroke. * P < 0.05, ** P < 0.01, *** P < 0.001
Fig. 8
Fig. 8
A schematic diagram of SNHG15-based signaling circuit in stroke-induced immunosuppression. Activation of IL-4–JAK-STAT6 signaling pathway contributes to the expression of SNHG15, and SNHG15 uniquely inhibits the K63-linked ubiquitination of TRAF2, thereby attenuating inflammatory responses and promoting M2 macrophage polarization after stroke

References

    1. Gu HQ, Yang X, Wang CJ, Zhao XQ, Wang YL, Liu LP, et al. Clinical Characteristics, Management, and In-Hospital Outcomes in Patients With Stroke or Transient Ischemic Attack in China. JAMA network Open. 2021;4(8):e2120745. doi: 10.1001/jamanetworkopen.2021.20745.
    1. Suda S, Aoki J, Shimoyama T, Suzuki K, Sakamoto Y, Katano T, et al. Stroke-associated infection independently predicts 3-month poor functional outcome and mortality. J Neurol. 2018;265(2):370–375. doi: 10.1007/s00415-017-8714-6.
    1. Ji R, Shen H, Pan Y, Wang P, Liu G, Wang Y, et al. Novel risk score to predict pneumonia after acute ischemic stroke. Stroke. 2013;44(5):1303–1309. doi: 10.1161/STROKEAHA.111.000598.
    1. Silvestri L, van Saene HK, Milanese M, Ros S, Zandstra DF. Parenteral antibiotics are not enough to prevent pneumonia in stroke. Lancet. 2015;386(9994):653. doi: 10.1016/S0140-6736(15)61501-X.
    1. Brogan E, Langdon C, Brookes K, Budgeon C, Blacker D. Respiratory infections in acute stroke: nasogastric tubes and immobility are stronger predictors than dysphagia. Dysphagia. 2014;29(3):340–345. doi: 10.1007/s00455-013-9514-5.
    1. Hoffmann S, Harms H, Ulm L, Nabavi DG, Mackert BM, Schmehl I, et al. Stroke-induced immunodepression and dysphagia independently predict stroke-associated pneumonia - The PREDICT study. J Cerebr Blood Flow Metab. 2017;37(12):3671–3682. doi: 10.1177/0271678X16671964.
    1. Huang YY, Li X, Li X, Sheng YY, Zhuang PW, Zhang YJ. Neuroimmune crosstalk in central nervous system injury-induced infection and pharmacological intervention. Brain Res Bull. 2019;153:232–238. doi: 10.1016/j.brainresbull.2019.09.003.
    1. McCulloch L, Smith CJ, McColl BW. Adrenergic-mediated loss of splenic marginal zone B cells contributes to infection susceptibility after stroke. Nat Commun. 2017;8:15051. doi: 10.1038/ncomms15051.
    1. Deng QW, Yang H, Yan FL, Wang H, Xing FL, Zuo L, et al. Blocking sympathetic nervous system reverses partially stroke-induced immunosuppression but does not aggravate functional outcome after experimental stroke in rats. Neurochem Res. 2016;41(8):1877–1886. doi: 10.1007/s11064-016-1899-8.
    1. Chamorro A, Meisel A, Planas AM, Urra X, van de Beek D, Veltkamp R. The immunology of acute stroke. Nat Rev Neurol. 2012;8(7):401–410. doi: 10.1038/nrneurol.2012.98.
    1. Faura J, Bustamante A, Miro-Mur F, Montaner J. Stroke-induced immunosuppression: implications for the prevention and prediction of post-stroke infections. J Neuroinflammation. 2021;18(1):127. doi: 10.1186/s12974-021-02177-0.
    1. Palazzo AF, Koonin EV. Functional long non-coding RNAs evolve from junk transcripts. Cell. 2020;183(5):1151–1161. doi: 10.1016/j.cell.2020.09.047.
    1. Kopp F, Mendell JT. Functional classification and experimental dissection of long noncoding RNAs. Cell. 2018;172(3):393–407. doi: 10.1016/j.cell.2018.01.011.
    1. Zhang S, Zhang Y, Wang N, Wang Y, Nie H, Zhang Y, et al. Long non-coding RNA MIAT impairs neurological function in ischemic stroke via up-regulating microRNA-874-3p-targeted IL1B. Brain Res Bull. 2021;175:81–89. doi: 10.1016/j.brainresbull.2021.07.005.
    1. Chen J, Jin J, Zhang X, Yu H, Zhu X, Yu L, et al. Microglial lnc-U90926 facilitates neutrophil infiltration in ischemic stroke via MDH2/CXCL2 axis. Mol Ther. 2021;29(9):2873–2885. doi: 10.1016/j.ymthe.2021.04.025.
    1. Song L, Li H, Suo M, Sun Y, Su M, Song Y, et al. A functional variant of the long noncoding RNA AL110200 is associated with the risk of ischaemic stroke recurrence. Eur J Neurol. 2021;28(8):2708–2715. doi: 10.1111/ene.14895.
    1. Deng QW, Li S, Wang H, Sun HL, Zuo L, Gu ZT, et al. Differential long noncoding RNA expressions in peripheral blood mononuclear cells for detection of acute ischemic stroke. Clin Sci. 2018;132(14):1597–1614. doi: 10.1042/CS20180411.
    1. Li S, Lu G, Wang D, He JL, Zuo L, Wang H, et al. MicroRNA-4443 regulates monocyte activation by targeting tumor necrosis factor receptor associated factor 4 in stroke-induced immunosuppression. Eur J Neurol. 2020;27(8):1625–1637. doi: 10.1111/ene.14282.
    1. Du M, Yuan L, Tan X, Huang D, Wang X, Zheng Z, et al. The LPS-inducible lncRNA Mirt2 is a negative regulator of inflammation. Nat Commun. 2017;8(1):2049. doi: 10.1038/s41467-017-02229-1.
    1. Varfolomeev E, Goncharov T, Maecker H, Zobel K, Komuves LG, Deshayes K, et al. Cellular inhibitors of apoptosis are global regulators of NF-kappaB and MAPK activation by members of the TNF family of receptors. Sci Signal. 2012;5(216):22. doi: 10.1126/scisignal.2001878.
    1. Mathelier A, Fornes O, Arenillas DJ, Chen CY, Denay G, Lee J, et al. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2016;44(D1):D110–D115. doi: 10.1093/nar/gkv1176.
    1. Daidone M, Cataldi M, Pinto A, Tuttolomondo A. Non-coding RNAs and other determinants of neuroinflammation and endothelial dysfunction: regulation of gene expression in the acute phase of ischemic stroke and possible therapeutic applications. Neural Regen Res. 2021;16(11):2154–2158. doi: 10.4103/1673-5374.310607.
    1. Pan Y, Jiao Q, Wei W, Zheng T, Yang X, Xin W. Emerging role of LncRNAs in ischemic stroke-novel insights into the regulation of inflammation. J Inflamm Res. 2021;14:4467–4483. doi: 10.2147/JIR.S327291.
    1. Zuo L, Zhang L, Zu J, Wang Z, Han B, Chen B, et al. Circulating Circular RNAs as biomarkers for the diagnosis and prediction of outcomes in acute ischemic stroke. Stroke. 2020;51(1):319–323. doi: 10.1161/STROKEAHA.119.027348.
    1. Chen S, Shen X. Long noncoding RNAs: functions and mechanisms in colon cancer. Mol Cancer. 2020;19(1):167. doi: 10.1186/s12943-020-01287-2.
    1. Shuai Y, Ma Z, Lu J, Feng J. LncRNA SNHG15: A new budding star in human cancers. Cell Proliferation. 2020;53(1):e12716. doi: 10.1111/cpr.12716.
    1. Zhu QQ, Lai MC, Chen TC, Wang X, Tian L, Li DL, et al. LncRNA SNHG15 relieves hyperglycemia-induced endothelial dysfunction via increased ubiquitination of thioredoxin-interacting protein. Lab Invest. 2021;101(9):1142–52. doi: 10.1038/s41374-021-00614-5.
    1. Li J, Tian J, Lu J, Wang Z, Ling J, Wu X, et al. LncRNA GAS5 inhibits Th17 differentiation and alleviates immune thrombocytopenia via promoting the ubiquitination of STAT3. Int Immunopharmacol. 2020;80:106127. doi: 10.1016/j.intimp.2019.106127.
    1. Chassin C, Goujon JM, Darche S, du Merle L, Bens M, Cluzeaud F, et al. Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways. J Immunol. 2006;177(7):4773–4784. doi: 10.4049/jimmunol.177.7.4773.
    1. Su X, Min S, Cao S, Yan H, Zhao Y, Li H, et al. LRRC19 expressed in the kidney induces TRAF2/6-mediated signals to prevent infection by uropathogenic bacteria. Nat Commun. 2014;5:4434. doi: 10.1038/ncomms5434.
    1. Jeon SH, Zhu GQ, Kwon EB, Lee KW, Cho HJ, Ha US, et al. Extracorporeal shock wave therapy decreases COX-2 by inhibiting TLR4-NFkappaB pathway in a prostatitis rat model. Prostate. 2019;79(13):1498–1504.
    1. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity. 2014;41(1):14–20. doi: 10.1016/j.immuni.2014.06.008.
    1. Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003;3(1):23–35. doi: 10.1038/nri978.

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