title: Human TLR8 is activated upon recognition of Borrelia burgdorferi RNA in the phagosome of human monocytes.
abstract: Phagocytosed Borrelia burgdorferi (Bb), the Lyme disease spirochete, induces a robust and complex innate immune response in human monocytes, in which TLR8 cooperates with TLR2 in the induction of NF-κB-mediated cytokine production, whereas TLR8 is solely responsible for transcription of IFN-β through IRF7. We now establish the role of Bb RNA in TLR8-mediated induction of IFN-β. First, using TLR2-transfected HEK.293 cells, which were unable to phagocytose intact Bb, we observed TLR2 activation by lipoprotein-rich borrelial lysates and TLR2 synthetic ligands but not in response to live spirochetes. Purified Bb RNA, but not borrelial DNA, triggered TLR8 activation. Neither of these 2 ligands induced activation of TLR7. Using purified human monocytes we then show that phagocytosed live Bb, as well as equivalent amounts of borrelial RNA delivered into the phagosome by polyethylenimine (PEI), induces transcription of IFN-β and secretion of TNF-α. The cytokine response to purified Bb RNA was markedly impaired in human monocytes naturally deficient in IRAK-4 and in cells with knockdown TLR8 expression by small interfering RNA. Using confocal microscopy we provide evidence that TLR8 colocalizes with internalized Bb RNA in both early (EEA1) and late endosomes (LAMP1). Live bacterial RNA staining indicates that spirochetal RNA does not transfer from the phagosome into the cytosol. Using fluorescent dextran particles we show that phagosomal integrity in Bb-infected monocytes is not affected. We demonstrate, for the first time, that Bb RNA is a TLR8 ligand in human monocytes and that transcription of IFN-β in response to the spirochete is induced from within the phagosomal vacuole through the TLR8-MyD88 pathway.
authors.affiliation: 1.Connecticut Children's Medical Center, Division of Infectious Diseases and Immunology, 282 Washington St., Hartford, CT 06106. jsalazar@nso1.uchc.edu.
authors.forename: Jorge L
authors.initials: JL
authors.lastname: Cervantes
authors.forename: Carson J
authors.initials: CJ
authors.lastname: La Vake
authors.forename: Bennett
authors.initials: B
authors.lastname: Weinerman
authors.forename: Stephanie
authors.initials: S
authors.lastname: Luu
authors.forename: Caitlin
authors.initials: C
authors.lastname: O'Connell
authors.forename: Paulo H
authors.initials: PH
authors.lastname: Verardi
authors.forename: Juan C
authors.initials: JC
authors.lastname: Salazar
mesh_terms: D025065:Borrelia burgdorferi / Q000276:immunology*; D005260:Female; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D016899:Interferon-beta / Q000235:genetics / Q000276:immunology; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics / Q000276:immunology; D008193:Lyme Disease / Q000235:genetics / Q000276:immunology* / Q000473:pathology; D008297:Male; D009000:Monocytes / Q000276:immunology* / Q000382:microbiology; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000276:immunology; D010588:Phagosomes / Q000276:immunology* / Q000382:microbiology; D012329:RNA, Bacterial / Q000276:immunology*; D051199:Toll-Like Receptor 7 / Q000235:genetics / Q000276:immunology; D051200:Toll-Like Receptor 8 / Q000235:genetics / Q000276:immunology*; D014158:Transcription, Genetic / Q000235:genetics / Q000276:immunology
chemical_list: C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D012329:RNA, Bacterial; C495348:TLR7 protein, human; C495349:TLR8 protein, human; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D016899:Interferon-beta; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases
keywords: Borrelia burgdorferi; TLR8; bacterial RNA; human monocytes
references.citation: Nature. 1997 Dec 11;390(6660):580-6
references.pmid: 9403685
references.citation: J Biol Chem. 1999 Nov 19;274(47):33419-25
references.pmid: 10559223
references.citation: Curr Top Microbiol Immunol. 2006;311:1-16
references.pmid: 17048703
references.citation: J Immunol Methods. 2008 Jul 20;336(1):45-55
references.pmid: 18456276
references.citation: J Innate Immun. 2010;2(3):280-7
references.pmid: 20375545
references.citation: Angew Chem Int Ed Engl. 2012 Mar 26;51(13):3143-6
references.pmid: 22323101
references.citation: Immunity. 2005 Aug;23(2):165-75
references.pmid: 16111635
references.citation: J Clin Invest. 2010 Oct;120(10):3651-62
references.pmid: 20811154
references.citation: Microsc Res Tech. 2012 May;75(5):691-7
references.pmid: 22095650
references.citation: J Immunol. 1999 Sep 1;163(5):2382-6
references.pmid: 10452971
references.citation: FEMS Microbiol Lett. 2009 Apr;293(2):278-84
references.pmid: 19260969
references.citation: Nat Immunol. 2008 Aug;9(8):847-56
references.pmid: 18604214
references.citation: Nat Med. 2002 Aug;8(8):878-84
references.pmid: 12091878
references.citation: J Immunol. 2005 Feb 1;174(3):1539-48
references.pmid: 15661914
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15779-84
references.pmid: 18840688
references.citation: Nat Immunol. 2009 Jun;10(6):587-94
references.pmid: 19430477
references.citation: Int Rev Immunol. 2011 Feb;30(1):16-34
references.pmid: 21235323
references.citation: Curr Opin Pharmacol. 2012 Aug;12(4):510-8
references.pmid: 22748800
references.citation: Expert Rev Vaccines. 2007 Oct;6(5):835-47
references.pmid: 17931162
references.citation: J Immunol. 1998 Jun 1;160(11):5485-92
references.pmid: 9605151
references.citation: Clin Cancer Res. 2012 Jan 15;18(2):499-509
references.pmid: 22128302
references.citation: PLoS One. 2011 Feb 02;6(2):e14647
references.pmid: 21311600
references.citation: J Immunol. 2001 Mar 15;166(6):4131-40
references.pmid: 11238663
references.citation: Nat Rev Microbiol. 2012 Jan 09;10(2):87-99
references.pmid: 22230951
references.citation: J Clin Microbiol. 2009 Nov;47(11):3427-34
references.pmid: 19726599
references.citation: Eur J Immunol. 2008 Mar;38(3):614-8
references.pmid: 18286571
references.citation: Nature. 2011 May 22;474(7351):385-9
references.pmid: 21602824
references.citation: PLoS Pathog. 2009 May;5(5):e1000444
references.pmid: 19461888
references.citation: Arch Med Sci. 2012 Dec 20;8(6):978-82
references.pmid: 23319969
references.citation: J Biol Chem. 2006 Dec 8;281(49):37427-34
references.pmid: 17040905
references.citation: J Immunol. 2008 Jun 1;180(11):7125-33
references.pmid: 18490711
references.citation: Vaccine. 2010 Aug 31;28(38):6273-81
references.pmid: 20637759
references.citation: J Immunol. 2006 Nov 15;177(10):6584-7
references.pmid: 17082568
references.citation: Int Immunopharmacol. 2007 Aug;7(8):1111-21
references.pmid: 17570328
references.citation: PLoS Pathog. 2007 Mar;3(3):e51
references.pmid: 17397264
references.citation: Eur J Immunol. 2009 Sep;39(9):2537-47
references.pmid: 19662634
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3683-8
references.pmid: 21321205
references.citation: PLoS Genet. 2008 Oct;4(10):e1000218
references.pmid: 18927625
references.citation: Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 Jun;109(6):844-50
references.pmid: 20399690
references.citation: Nat Immunol. 2010 May;11(5):395-402
references.pmid: 20351692
references.citation: J Immunol. 2002 May 1;168(9):4531-7
references.pmid: 11970999
references.citation: Nat Protoc. 2008;3(6):1101-8
references.pmid: 18546601
references.citation: Proc Natl Acad Sci U S A. 2005 Oct 18;102(42):15190-4
references.pmid: 16219698
references.citation: Hum Gene Ther. 1997 Sep 1;8(13):1585-94
references.pmid: 9322091
references.citation: Proc Natl Acad Sci U S A. 2012 Jan 24;109(4):1228-32
references.pmid: 22232682
references.citation: AIDS. 2010 Jul 31;24(12):1841-8
references.pmid: 20588172
references.citation: J Clin Invest. 2009 Aug;119(8):2231-44
references.pmid: 19620771
references.citation: PLoS Pathog. 2009 Jul;5(7):e1000500
references.pmid: 19578435
references.citation: J Immunol. 2002 Jan 1;168(1):348-55
references.pmid: 11751980
references.citation: J Control Release. 2002 Aug 21;82(2-3):441-54
references.pmid: 12175756
references.citation: Nat Immunol. 2002 Jun;3(6):499
references.pmid: 12032557
references.citation: EMBO J. 2012 Nov 5;31(21):4153-64
references.pmid: 23064150
references.citation: Immunity. 2011 May 27;34(5):637-50
references.pmid: 21616434
references.citation: Curr Protoc Mol Biol. 2001 May;Chapter 16:Unit16.17
references.pmid: 18265124
references.citation: Infect Immun. 1995 Jun;63(6):2154-63
references.pmid: 7768594
references.citation: J Exp Med. 2007 May 14;204(5):1025-36
references.pmid: 17470642
references.citation: Infect Immun. 1996 Sep;64(9):3845-52
references.pmid: 8751937
references.citation: Hum Mutat. 2010 Sep;31(9):1069-79
references.pmid: 20652908
references.citation: Infect Immun. 2008 Jan;76(1):56-70
references.pmid: 17938216
references.citation: Vector Borne Zoonotic Dis. 2002 Winter;2(4):275-8
references.pmid: 12804169
references.citation: Cell Mol Immunol. 2012 Nov;9(6):434-8
references.pmid: 23085951
references.citation: J Leukoc Biol. 2009 Apr;85(4):664-72
references.pmid: 19164128
references.citation: Blood. 2007 Jan 15;109(2):619-25
references.pmid: 16973959
references.citation: Immunity. 2006 Sep;25(3):373-81
references.pmid: 16979569
references.citation: J Immunol. 2005 Jul 1;175(1):494-500
references.pmid: 15972684
references.citation: Infect Immun. 1999 Jan;67(1):140-7
references.pmid: 9864208
references.citation: Semin Immunol. 2004 Feb;16(1):3-9
references.pmid: 14751757
references.citation: Exp Mol Pathol. 2008 Aug;85(1):20-7
references.pmid: 18462718
references.citation: Infect Immun. 1995 Apr;63(4):1507-15
references.pmid: 7890417
references.citation: Infect Immun. 2005 May;73(5):2951-7
references.pmid: 15845501
references.citation: Science. 1999 Jul 30;285(5428):736-9
references.pmid: 10426996
references.citation: Science. 2013 Mar 22;339(6126):1426-9
references.pmid: 23520111
references.citation: Nat Med. 2007 May;13(5):552-9
references.pmid: 17479101
references.citation: J Immunol. 2006 Dec 1;177(11):8164-70
references.pmid: 17114492
references.citation: Infect Immun. 2007 Apr;75(4):2046-62
references.pmid: 17220323
references.citation: J Leukoc Biol. 2007 Mar;81(3):584-92
references.pmid: 17135573
references.citation: J Immunol. 2008 Mar 15;180(6):3729-38
references.pmid: 18322178
references.citation: J Immunol. 2005 Dec 1;175(11):7407-18
references.pmid: 16301648
references.citation: Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):10942-7
references.pmid: 22711825
references.citation: Euro Surveill. 2006 Jun 22;11(6):E060622.1
references.pmid: 16819127
references.citation: Cell Microbiol. 2010 Jul;12(7):881-90
references.pmid: 20482555
references.citation: J Immunol. 1999 Aug 15;163(4):2049-56
references.pmid: 10438943
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6442-7
references.pmid: 20308556
references.citation: J Immunol. 2008 Feb 15;180(4):2117-24
references.pmid: 18250417
references.citation: Science. 1999 Jul 30;285(5428):732-6
references.pmid: 10426995