Negative feedback circuit for toll like receptor-8 activation in human embryonic Kidney 293 using outer membrane vesicle delivered bi-specific siRNA

BMC Immunology

Shishir Sharma⁴  Nawneet Mishra⁵  Rajendra Maan Shrestha¹  Hari Krishna Saiju⁴  Shravan Kumar Mishra³  Krishna Das Manandhar²  Birendra Prasad Gupta²  Anurag Adhikari⁴ 

[1] Padma Kanya Multiple Campus, Tribhuvan University, Kathmandu, Nepal;Central Department of Biotechnology, Tribhuvan University, Kathmandu, Nepal;National Public Health Laboratory, Teku, Kathmandu, Nepal;Asian Institute of Technology & Management, Purbanchal University, Knowledge village, Khumaltar, Satdobato, Lalitpur, Nepal;South Asian University, New Delhi, India

关键词: Negative feedback;    Inflammation;    p19;    Outer membrane vesicle;    INF type I;    TLR8;    Bi-siRNA;   

Others  :  1221112
DOI  :  10.1186/s12865-015-0109-9

 received in 2015-01-07, accepted in 2015-07-17,  发布年份 2015

【 摘 要 】

Background

TLR8 assists in antiviral approach by producing Type 1 INF via MyD88 dependent IRF7 pathway. However, over expression of INFα/β molecule poses threat by developing tolerance in chronic infection cases and enhancing inflammatory response. Here we report a bi-specific siRNA based complex which differentially activates and silences the TLR8 and MYD88 respectively in a negatively regulated fashion.

Results

Outer membrane vesicle from Escherichia coli used for siRNA delivery was observed more efficient when attached with invasive protein Ail along with OmpA (P < 0.001) in HEK293-TLR8 cell line. siRNA complexed with p19 protein was efficient in activating TLR8, confirmed by the increment of INFβ molecules (P < 0.001) in HEK293-TLR8 compared to its counterpart. Fusion of lipid bilayer of endosomal compartment was significant at pH 4.5 when fusogenic peptides (diINF-7) were incubated in membrane vesicle, thus facilitating the escape of siRNA complex to the host cytoplasm in order to silence MyD88 transcript (P < 0.001).

Conclusions

We investigated the activation of TLR8 by bi-specific si-RNA for the production of INFβ. In the same setting we showed that bi-specific si-RNA was able to silence MyD88 transcript in a delayed manner. For the cases of auto immune disease and inflammation where over activation of endosomal TLRs poses serious threat, bi specific siRNA could be used as negative feedback controlled system.

【 授权许可】

   
2015 Adhikari et al.

【 图 表 】

Fig. 1.

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【 参考文献 】

• [1]Sarvestani ST, Williams BR, Gantier MP. Human Toll-like receptor 8 can be cool too: implications for foreign RNA sensing. J Interferon Cytokine Res. 2012; 32(8):350-361.
• [2]Gantier MP, Williams BR. Making sense of viral RNA sensing. Mol Ther. 2011; 19(9):1578-1581.
• [3]Walsh KB, Teijaro JR, Zuniga EI, Welch MJ, Fremgen DM, Blackburn SD, von Tiehl KF, Wherry EJ, Flavell RA, Oldstone MB. Toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection. Cell Host Microbe. 2012; 11(6):643-653.
• [4]Heil F, Ahmad-Nejad P, Hemmi H, Hochrein H, Ampenberger F, Gellert T, Dietrich H, Lipford G, Takeda K, Akira S et al.. The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol. 2003; 33(11):2987-2997.
• [5]Hornung V, Guenthner-Biller M, Bourquin C, Ablasser A, Schlee M, Uematsu S, Noronha A, Manoharan M, Akira S, de Fougerolles A et al.. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 2005; 11(3):263-270.
• [6]Vasilakos JP, Tomai MA. The use of Toll-like receptor 7/8 agonists as vaccine adjuvants. Expert Rev Vaccines. 2013; 12(7):809-819.
• [7]Smits EL, Ponsaerts P, Berneman ZN, Van Tendeloo VF. The use of TLR7 and TLR8 ligands for the enhancement of cancer immunotherapy. Oncologist. 2008; 13(8):859-875.
• [8]Meyer K, Kwon YC, Liu S, Hagedorn CH, Ray RB, Ray R. Interferon-alpha inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection. Sci Rep. 2015; 5:9012.
• [9]Nakao K, Nakata K, Yamashita M, Tamada Y, Hamasaki K, Ishikawa H, Kato Y, Eguchi K, Ishii N. p48 (ISGF-3gamma) is involved in interferon-alpha-induced suppression of hepatitis B virus enhancer-1 activity. J Biol Chem. 1999; 274(40):28075-28078.
• [10]Su H, Yee JK. Regulation of hepatitis B virus gene expression by its two enhancers. Proc Natl Acad Sci U S A. 1992; 89(7):2708-2712.
• [11]Tur-Kaspa R, Teicher L, Laub O, Itin A, Dagan D, Bloom BR, Shafritz DA. Alpha interferon suppresses hepatitis B virus enhancer activity and reduces viral gene transcription. J Virol. 1990; 64(4):1821-1824.
• [12]Peng G, Lei KJ, Jin W, Greenwell-Wild T, Wahl SM. Induction of APOBEC3 family proteins, a defensive maneuver underlying interferon-induced anti-HIV-1 activity. J Exp Med. 2006; 203(1):41-46.
• [13]Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, Stephens EB, Guatelli J. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe. 2008; 3(4):245-252.
• [14]Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature. 2004; 427(6977):848-853.
• [15]Sandler NG, Bosinger SE, Estes JD, Zhu RT, Tharp GK, Boritz E, Levin D, Wijeyesinghe S, Makamdop KN, del Prete GQ et al.. Type I interferon responses in rhesus macaques prevent SIV infection and slow disease progression. Nature. 2014; 511(7511):601-605.
• [16]Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol. 2014; 5:461.
• [17]Saitoh T, Satoh T, Yamamoto N, Uematsu S, Takeuchi O, Kawai T, Akira S. Antiviral protein Viperin promotes Toll-like receptor 7- and Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells. Immunity. 2011; 34(3):352-363.
• [18]Huang L, Lieberman J. Production of highly potent recombinant siRNAs in Escherichia coli. Nat Protoc. 2013; 8(12):2325-2336.
• [19]Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996; 173(1 Spec No):33-38.
• [20]Kadurugamuwa JL, Beveridge TJ. Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion. J Bacteriol. 1995; 177(14):3998-4008.
• [21]Kesty NC, Kuehn MJ. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles. J Biol Chem. 2004; 279(3):2069-2076.
• [22]Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol. 2005; 17(1):1-14.
• [23]O'Neill LA, Golenbock D, Bowie AG. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol. 2013; 13(6):453-460.
• [24]Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010; 11(5):373-384.
• [25]Van Heel DA, McGovern DP, Jewell DP. Crohn’s disease: genetic susceptibility, bacteria, and innate immunity. Lancet. 2001; 357(9272):1902-1904.
• [26]Kobayashi K, Hernandez LD, Galan JE, Janeway CA, Medzhitov R, Flavell RA. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell. 2002; 110(2):191-202.
• [27]Kinjyo I, Hanada T, Inagaki-Ohara K, Mori H, Aki D, Ohishi M, Yoshida H, Kubo M, Yoshimura A. SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. Immunity. 2002; 17(5):583-591.
• [28]Zhang G, Ghosh S. Negative regulation of toll-like receptor-mediated signaling by Tollip. J Biol Chem. 2002; 277(9):7059-7065.
• [29]Wald D, Qin J, Zhao Z, Qian Y, Naramura M, Tian L, Towne J, Sims JE, Stark GR, Li X. SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol. 2003; 4(9):920-927.
• [30]Gringhuis SI, van der Vlist M, van den Berg LM, den Dunnen J, Litjens M, Geijtenbeek TB. HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells. Nat Immunol. 2010; 11(5):419-426.
• [31]Rakoff-Nahoum S, Paglino J, Eslami-Varzaneh F, Edberg S, Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell. 2004; 118(2):229-241.
• [32]Metcalf D, Di Rago L, Mifsud S, Hartley L, Alexander WS. The development of fatal myocarditis and polymyositis in mice heterozygous for IFN-gamma and lacking the SOCS-1 gene. Proc Natl Acad Sci U S A. 2000; 97(16):9174-9179.
• [33]Marine JC, Topham DJ, McKay C, Wang D, Parganas E, Stravopodis D, Yoshimura A, Ihle JN. SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality. Cell. 1999; 98(5):609-616.
• [34]Hirata Y, Ohmae T, Shibata W, Maeda S, Ogura K, Yoshida H, Kawabe T, Omata M. MyD88 and TNF receptor-associated factor 6 are critical signal transducers in Helicobacter pylori-infected human epithelial cells. J Immunol. 2006; 176(6):3796-3803.
• [35]Rad R, Ballhorn W, Voland P, Eisenacher K, Mages J, Rad L, Ferstl R, Lang R, Wagner H, Schmid RM et al.. Extracellular and intracellular pattern recognition receptors cooperate in the recognition of Helicobacter pylori. Gastroenterology. 2009; 136(7):2247-2257.
• [36]Obonyo M, Sabet M, Cole SP, Ebmeyer J, Uematsu S, Akira S, Guiney DG. Deficiencies of myeloid differentiation factor 88, Toll-like receptor 2 (TLR2), or TLR4 produce specific defects in macrophage cytokine secretion induced by Helicobacter pylori. Infect Immun. 2007; 75(5):2408-2414.
• [37]Xiao B, Liu Z, Li BS, Tang B, Li W, Guo G, Shi Y, Wang F, Wu Y, Tong WD et al.. Induction of microRNA-155 during Helicobacter pylori infection and its negative regulatory role in the inflammatory response. J Infect Dis. 2009; 200(6):916-925.
• [38]Tang B, Xiao B, Liu Z, Li N, Zhu ED, Li BS, Xie QH, Zhuang Y, Zou QM, Mao XH. Identification of MyD88 as a novel target of miR-155, involved in negative regulation of Helicobacter pylori-induced inflammation. FEBS Lett. 2010; 584(8):1481-1486.
• [39]Triantafilou K, Orthopoulos G, Vakakis E, Ahmed MA, Golenbock DT, Lepper PM, Triantafilou M. Human cardiac inflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR) 8-dependent. Cell Microbiol. 2005; 7(8):1117-1126.
• [40]Steenholdt C, Andresen L, Pedersen G, Hansen A, Brynskov J. Expression and function of toll-like receptor 8 and Tollip in colonic epithelial cells from patients with inflammatory bowel disease. Scand J Gastroenterol. 2009; 44(2):195-204.