human tlr8 is activated upon recognition of borrelia burgdorferi rna in the phagosome of human...

Human TLR8 is activated upon recognitionof Borrelia burgdorferi RNA in thephagosome of human monocytes

Jorge L. Cervantes,* Carson J. La Vake,* Bennett Weinerman,* Stephanie Luu,*Caitlin O’Connell,§ Paulo H. Verardi,§ and Juan C. Salazar*,†,‡,1

Departments of *Pediatrics and †Immunology, University of Connecticut Health Center, Farmington, Connecticut; ‡ConnecticutChildren’s Medical Center, Hartford, Connecticut; and §Department of Pathobiology and Veterinary Science, University of

Connecticut, Storrs, Connecticut

RECEIVED APRIL 15, 2013; REVISED JULY 5, 2013; ACCEPTED JULY 7, 2013. DOI: 10.1189/jlb.0413206

ABSTRACTPhagocytosed Borrelia burgdorferi (Bb), the Lyme dis-ease spirochete, induces a robust and complex innateimmune response in human monocytes, in which TLR8cooperates with TLR2 in the induction of NF-�B-medi-ated cytokine production, whereas TLR8 is solely re-sponsible for transcription of IFN-� through IRF7. Wenow establish the role of Bb RNA in TLR8-mediated in-duction of IFN-�. First, using TLR2-transfected HEK.293cells, which were unable to phagocytose intact Bb, weobserved TLR2 activation by lipoprotein-rich borreliallysates and TLR2 synthetic ligands but not in responseto live spirochetes. Purified Bb RNA, but not borrelialDNA, triggered TLR8 activation. Neither of these 2 li-gands induced activation of TLR7. Using purified hu-man monocytes we then show that phagocytosed liveBb, as well as equivalent amounts of borrelial RNA de-livered into the phagosome by polyethylenimine (PEI),induces transcription of IFN-� and secretion of TNF-�.The cytokine response to purified Bb RNA was mark-edly impaired in human monocytes naturally deficient inIRAK-4 and in cells with knockdown TLR8 expression bysmall interfering RNA. Using confocal microscopy weprovide evidence that TLR8 colocalizes with internal-ized Bb RNA in both early (EEA1) and late endosomes(LAMP1). Live bacterial RNA staining indicates that spi-rochetal RNA does not transfer from the phagosomeinto the cytosol. Using fluorescent dextran particles weshow that phagosomal integrity in Bb-infected mono-cytes is not affected. We demonstrate, for the firsttime, that Bb RNA is a TLR8 ligand in human monocytes

and that transcription of IFN-� in response to the spiro-chete is induced from within the phagosomal vacuolethrough the TLR8-MyD88 pathway. J. Leukoc. Biol. 94:000–000; 2013.

IntroductionLyme disease (LD), the most commonly reported vector-borneillness in the United States and Europe, is a multisystem, in-flammatory, infectious disorder caused by the extracellular spi-rochetal bacterium Borrelia burgdorferi (Bb) [1, 2]. LD has be-come a public health problem not only in the United States[2], but also globally [3]. The disease is often heralded in itsearly stage by erythema migrans, an expanding annular inflam-matory rash that develops after inoculation of spirochetes intothe skin at the site of tick feeding. If treated appropriately,the prognosis is excellent; however, if untreated, hematoge-nous dissemination of spirochetes may give rise to a widerange of clinical manifestations, including meningitis, en-cephalitis, arthritis, and even carditis [4]. Because Bb lacksorthologs of known exotoxins or the specialized secretorymachinery required for the delivery of noxious moleculesinto host cells [5], both local (skin) and systemic clinicalmanifestations of LD are the consequence of innate andcoevolving adaptive humoral and cellular immune re-sponses. Paradoxically, although these responses induce tis-sue damage, they are also deemed to be essential for theeradication of the bacterium.

For several years most efforts to understand how Bb initi-ates innate immune cell activation in tissues were focusedchiefly on the proinflammatory attributes of isolated spiro-chetal lipoproteins [6 –13], whereas less was done to definethe mechanisms underlying immune recognition of live spi-rochetes. The emphasis on borrelial lipoproteins as innateimmune agonists first emerged from the discovery that spi-

1. Correspondence: Juan C. Salazar, Connecticut Children’s Medical Center,Division of Infectious Diseases and Immunology, 282 Washington St.,Hartford, CT 06106. E-mail: [email protected]

Abbreviations: Bb�Borrelia burgdorferi; EEA1�early endosomal antigen 1;GFP�green fluorescent protein; HEK�human embryonic kidney;LAMP1�lysosomal-associated membrane protein 1; LD�Lyme disease;LPS� lipopolysaccharide; MOI�multiplicity of infection; PAMP�pathogen-associated molecular pattern; pDC�plasmacytoid dendritic cell;PEI�polyethylenimine; SEAP�secretory embryonic alkaline phosphatase;VV�vaccinia virus

The online version of this paper, found at www.jleukbio.org, includessupplemental information.

Article

0741-5400/13/0094-0001 © Society for Leukocyte Biology Volume 94, December 2013 Journal of Leukocyte Biology 1

Epub ahead of print August 1, 2013 - doi:10.1189/jlb.0413206

Copyright 2013 by The Society for Leukocyte Biology.

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rochetes express an abundance of these molecules [5, 14],many on their outer membrane, and that the borrelial cellenvelope lacks the potent gram-negative proinflammatoryglycolipid, lipopolysaccharide (LPS) [5]. Unlike LPS, whichsignals through TLR4 and CD14 [15], lipoproteins signalthrough TLR1/2 heterodimers [9, 11, 16 –18] in a CD14-dependent manner [10, 19, 20]. Collectively, these priorstudies led to the conclusion that innate immune cell activa-tion in LD occurred predominantly, if not exclusively,through the interaction of spirochetal lipoproteins withCD14 and TLR1/2 at the cell surfaces of monocytes, macro-phages, and dendritic cells [6 –13, 21, 22]. More recently,we have shown that phagocytosed live Bb is capable of in-ducing a more robust, diversified, and complex inflamma-tory response than can be simply attributed to TLR1/2 stim-ulation by individual borrelial lipoproteins [23–25]. To besure, we demonstrated that phagocytosed Bb also inducedTLR2-independent responses, most notably transcription ofIFN-� and several type I interferon-stimulated genes [25].In addition, we demonstrated that TLR8-IRF7-mediated sig-naling leads to the induction of IFN-� [26], whereas TLR2and TLR8 cooperate in the production of NF-�B-mediatedcytokines [26].

The type I IFNs are a family of specialized cytokines thatinstruct the innate immune responses to viruses [27] and bac-terial pathogens [28]. The receptors and signaling pathwaysthat link pathogen detection to induction of type I IFNs arelocalized to endosomal membranes (i.e., TLRs) or to the cellcytosol [27]. TLR7 and TLR8, the 2 principal endosomal TLRsexpressed in human monocytes [29–31], are capable of induc-ing production of type I IFNs. Although the role of TLR7 insensing bacterial RNA by immune cells is now well accepted[32, 33], a similar role for TLR8 has only began to be uncov-ered. Two relatively recent studies have shown that bacterialpathogen-associated molecular patterns (PAMPs) induce acti-vation of TLR8. In one study, TLR8 was up-regulated afterphagocytosis of Mycobacterium bovis in THP-1 cells [34]. In thesecond study, phagocytosis of Helicobacter pylori by THP-1 cellsinduced TLR8 activation [35]. Kariko et al. [36] provided evi-dence that Escherichia coli RNA could induce the production ofIL-8 in HEK.293 cells stably transfected with TLR7 and TLR8.To the best of our knowledge, no published studies have dem-onstrated whether bacterial RNA is capable of activating hu-man TLR8 in primary human cells.

Based on our collective findings, we have proposed a newmodel for Bb-induced monocyte activation, which highlightsthe importance of phagosomal signaling and the coopera-tive role of TLR2 and TLR8 sensing released spirochetalPAMPs [26, 37]. In the current study, using human embry-onic kidney (HEK) cells and freshly isolated human mono-cytes, we demonstrate that spirochetal RNA is the ligand forhuman TLR8 and that activation of this receptor occurssolely from within the phagosomal vacuole. The evidencealso suggests that in human monocytes the type I IFN re-sponses to live Bb do not require transfer of bacterial nu-cleic acids into the cell cytosol.

MATERIALS AND METHODS

Cell linesHEK.293 cells stably expressing TLR2, TLR7, or TLR8 were purchasedfrom InvivoGen (San Diego, CA, USA). Cells were cultured in DMEM(Gibco; Invitrogen, CA, USA) containing 4.5 g/l l-glucose and 10% FBS.The activity of specific TLR agonists was assessed using the secretory embry-onic alkaline phosphatase (SEAP) reporter gene that is linked to NF-�Bactivation. Measurement of SEAP activity using the Quanti-Blue substrate(InvivoGen) after TLR agonist treatment was performed according to themanufacturer’s instructions in test medium (DMEM with 10% FBS) withoutantibiotics. NF-�B activation was expressed as a response ratio for eachstimulus relative to SEAP activity in unstimulated cells.

Human monocyte isolationAll procedures involving human subjects were approved by the institutionalreview board at the University of Connecticut Health Center and the Con-necticut Children’s Medical Center. After written informed consent wascontained, human monocytes were isolated using the human MonocyteIsolation Kit II (Miltenyi Biotec, Auburn, CA, USA), from LD-seronegativevolunteers as described previously [25]. In addition, we enrolled a 6-year-old child who has well characterized and genetically confirmed IRAK-4 de-ficiency (compound heterozygous mutation).

Ex vivo stimulation conditionsCells were incubated at 37°C in 5% CO2, with live, low-passage, tempera-ture-shifted green fluorescent protein (GFP)-Bb 297 at a multiplicity of in-fection (MOI) of 10:1 and correspondent stimuli. Freshly extracted BbRNA or DNA was delivered to the cells using polyethylenimine (PEI) Ex-Gen 500 in vitro transfection reagent (Fermentas, Hanover, MD, USA) andwere indicated N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl-sulfate (Avanti Polar Lipids, Inc., Alabaster, AL, USA) [38]. Incuba-tion times were 4 h in the case of human monocytes and 24 h in the caseof HEK.293 cells. To conduct the phagosomal-to-cytosol RNA transfer ex-periments, monocytes were pretreated with tetramethylrhodamine isothio-cyanate-dextran (average molecular weight 155,000; Sigma-Aldrich, St.Louis, MO, USA). Appropriate controls, including the TLR2 ligandPam3CSSNA (Bachem, Bubendorf, Switzerland), TLR8-ligand 3M-002 (Invivo-Gen), and TLR7/8 ligand R848 (Resiquimod; InvivoGen), were used asTLR-specific activation controls. All culture media and reagents were con-firmed to be essentially free of LPS contamination (�10 pg/ml) by Limulusamoebocyte lysate assay as described previously [25].

Small interfering (si) TLR8 nucleofectionTLR8 gene silencing was performed on isolated human monocytes vianucleofection of 20 pmol of psiRNA-hTLR8 plasmid (InvivoGen), followingthe Amaxa 4D-Nucleofector Protocol for Primary Human Monocytes(Lonza, Basel, Switzerland). Nucleofection was done in a Lonza 4D-Nucleo-fector (Lonza). Vector psiRNA-hTLR7 (InvivoGen) was used for TLR7 genesilencing, and a plasmid containing a scramble sequence psiRNA-h7SKgzScr (InvivoGen) was used as a control.

MyD88 blockingMyD88 inhibitory peptide (Pepinh-MYD) was acquired from InvivoGen.Human monocytes were pretreated with 40 �M MyD88 blocking peptidefor 1 h before stimulation, using a protocol described previously [39].

Bacteria and bacterial nucleic acid preparationBb RNA was extracted from liquid Bb cultures at 37oC, using the NucleoSpinII kit (Macherey-Nagel, Duren, Germany), following the manufacturer’s in-structions. Bb DNA was extracted using the DNeasy Mini Blood and Tissue Kit(Qiagen, Dusseldorf, Germany), following the manufacturer’s instructions.RNA extraction involved a DNase step. Samples used for DNA extraction were

2 Journal of Leukocyte Biology Volume 94, December 2013 www.jleukbio.org

treated with RNase A, as indicated by the manufacturer’s recommendations toeliminate copurification of RNA along with the DNA. Where indicated, BbDNA was digested using HindIII (Sigma-Aldrich) and BstXI (Promega, Madi-son, WI, USA). We evaluated the quality of Bb RNA preparations using theExperion Automated Electrophoresis System (Bio-Rad, Hercules, CA, USA).The average RNA integrity number for Bb RNA samples was 6.7 (range,5.9�7.6). The purity of Bb RNA was assessed using a NanoDrop 1000 spectro-photometer (NanoDrop Technologies, Montchanin, DE, USA), with an aver-age ratio of absorbance at 260/280 of 2.1 (range, 1.97�2.34). Bb RNA yieldscorrelated linearly with starting Bb counts, so that a live Bb MOI of 10:1 wasequivalent to 0.17 �g of Bb RNA (data not shown). DNA samples were run onan Agilent DNA 1000 chip (Agilent Technologies, Mannheim, Germany), forquality and also on a NanoDrop 1000 with an average ratio of absorbance at260/280 of 1.7 (range, 1.4�2.0).

Vaccinia virus (VV) DNA preparationVaccinia virus (strain Western Reserve) was purchased from American TypeCulture Collection (VR-1354; Manassas, VA, USA). Virus propagation, purifica-tion, and DNA isolation are described elsewhere [40]. In brief, VV was grownin HeLa S3 cells before titration on BS-C-1 cells. Titrated virus was then usedto infect HeLa S3 cells, followed by cell homogenization to release the virus,which was purified by sucrose cushion followed by sucrose gradient separation.Viral DNA was isolated using proteinase K and SDS, extracted with phenol andphenol/chloroform, and precipitated with ethanol.

Assessment of monocyte activation by quantitativeRT-PCR and cytokine secretionAt the conclusion of the respective incubation period, cells were harvested,and RNA was extracted using a total RNA isolation kit (Macherey-Nagel).cDNA was prepared from extracted RNA using a high-capacity cDNA RTkit (Applied Biosystems, Grand Island, NY, USA). RT-PCR conditions andreactions have been described previously [25]. Commercially availableprimers (Applied Biosystems) used for amplification included: ifn�

(Hs00277188_s1), tlr2 (Hs00610101_m1), tlr7 (Hs00152971_m1), tlr8(Hs00152972_m1), and gapdh (Hs99999905_m1). Expression levels of alltranscripts studied were normalized to the GAPDH level, and the relativechanges in gene expression generated were calculated using the 2��CT

method [41]. A cytokine bead array system (Human Inflammation Kit; BD,Franklin Lakes, NJ, USA) was used to assay TNF-�, IL-10, IL-6, and IL-1�

secreted cytokines in culture supernatants.

Confocal cellular localization of Bb nucleic acid andphagosomal receptorsTo track the cellular localization of nucleic acid delivered to the cell, a flu-orescent form of PEI, jetPEI-FluoF (Polyplus Transfection, Illkirch, France),was used. Whole live Bb RNA staining was generated by using 5-ethynyl uri-dine (Click-iT Nascent RNA capture kit; Molecular Probes, Grand Island,NY) using a non GFP-Bb. To visualize the cellular localization of phago-somal receptors in Bb-infected monocytes and HEK.293 cells, we used apreviously described immunofluorescence assay [26]. Primary antibodiesincluded rabbit anti-TLR8 polyclonal antibody (IMG-5653-1; Imgenex, SanDiego, CA, USA), rabbit anti-TLR2 polyclonal antibody (Rockland Immu-nochemicals, Gilbertsville, PA, USA), rabbit polyclonal anti-early endosomalantigen 1 (EEA1) and mouse monoclonal anti-lysosomal-associated mem-brane protein 1 (LAMP1; Abcam Inc., Cambridge, MA, USA). Secondaryantibodies were Texas Red-X-conjugated goat anti-rabbit antibody (T-6391;Invitrogen), and goat anti-mouse DyLight 594 (Thermo Scientific, Rock-ford, IL, USA) for LAMP1 staining. Lysosome staining was achieved usingLysoTracker Red (Invitrogen). Slides were examined by confocal micros-copy using a Zeiss LSM-510 microscope (Zeiss, Oberkochen, Germany)equipped with argon and HeNe lasers. Images were processed and ana-lyzed using ImageJ 1.44b (National Institutes of Health, Bethesda, MD,USA). Colocalization of Bb and TLRs was determined using ImageJ plug-ins: colocalization, colocalization finder, and JACoP. When necessary, thecell contour was drawn using the free-hand tool of ImageJ.

Statistical analysisGeneral statistical analysis was conducted using GraphPad Prism 5.03(GraphPad Software, San Diego, CA, USA). NF-�B activation in HEK cellswas analyzed by comparing the response ratio between the various stimuli.Fold increase or decrease for each specific gene transcript assayed by quan-titative RT-PCR and cytokine concentrations were compared among thedifferent stimuli by using either a paired or unpaired Student’s t test or theequivalent nonparametric method (i.e., Mann-Whitney), whenever data didnot follow a normal distribution. To test whether values follow a Gaussiandistribution, the Kolmogorov-Smirnov test and, when applicable, theD’Agostino and Pearson normality test were used. For each experiment,both the sd and the se of the mean were calculated. A P value � 0.05 wasconsidered significant.

RESULTS

Single human TLR-expressing cell system reveals thatTLR8 is activated by Bb RNAWe reported previously that TLR2 and TLR8 are activated inresponse to phagocytosed Bb [26]. To decipher which borre-lial ligands are capable of activating individual TLRs, using apreviously established model [42, 43], we studied HEK.293cells stably transfected with each of the TLRs of interest(TLR2, TLR7, or TLR8). Using TLR2-transfected HEK.293cells, which lack the critical Bb-phagocytic receptor CR3 [44]and which were unable to phagocytose live Bb (data notshown), we show that live spirochetes do not activate TLR2(Supplemental Fig. 1). Conversely, stimulation of this samecell line with MOI equivalent concentrations of lipoprotein-rich borrelial lysates led to a robust activation of the TLR2-NF-�B pathway. Equivalence between live and lysed Bb wasconfirmed by SDS-PAGE and silver staining as previously re-ported by our group [25] (data not shown).

We then studied whether freshly extracted borrelial nucleicacids were capable of activating human TLR8. Although nakedRNA did not lead to NF-�B activation in HEK.293 TLR8-ex-pressing cells, delivery of the RNA into an intracellular com-partment using PEI-induced cellular activation (Fig. 1). As fur-ther evidence of the specificity of the TLR8 response to thisligand, activation was abrogated after pretreatment of Bb RNAwith RNase. Although IFN-� production by TLR8 seems to bemore prominent than NF-�B activation, it was sufficient to ob-serve a loss of activation when the Bb RNA extract was treatedwith RNase. In the same experiment, we show that TLR8 wasnot activated by lipoprotein-rich borrelial lysates, even whenthe ligand was delivered to an intracellular compartment bythe use of PEI. Because TLR7 is also expressed in humanmonocytes [29–33, 36], although not the main endosomalTLR in this cell line, we also wanted to explore whether borre-lial RNA activates human TLR7. As shown in SupplementalFig. 2A, stimulation of HEK.293 TLR7 cells by Bb RNA did notinduce their activation, even in the presence of very high con-centrations of the ligand.

Viral DNA and bacterial DNA are capable of inducing tran-scription of type I IFNs. Therefore, we also studied whether BbDNA could activate human TLR8. For these experiments wealso used VV DNA, which has been reported to activate TLR8in murine plasmacytoid dendritic cells (pDCs) [45] and similarto Bb DNA, VV DNA is A-T rich [5, 46]. As shown in Fig. 2, nei-

Cervantes et al. TLR8 recognition of Borrelia RNA

www.jleukbio.org Volume 94, December 2013 Journal of Leukocyte Biology 3

ther Bb DNA nor VV DNA induced activation of human TLR8in HEK.293 TLR8 cells. Because extracted DNA is a relativelylarger molecule than similarly extracted borrelial RNA, wequestioned whether predigestion of Bb DNA could facilitateligation and activation of TLR8. We first confirmed that theefficiency of internalization of PEI particles was comparable forRNA, DNA, and predigested DNA (data not shown). Asshown in Fig. 2, predigested borrelial DNA did not lead toTLR8 activation. In parallel experiments, we show that Bb

DNA did not induce activation of NF-�B through TLR7(Supplemental Fig. 2B).

Bb RNA-induced transcription of IFN-� in humanmonocytesHaving shown that Bb RNA activates human TLR8 in HEK.293cells, we then tested whether this ligand was also capable ofactivating TLR8 in freshly isolated human monocytes. Asshown in Fig. 3A, Bb RNA bound to a delivery vehicle (PEI)

Figure 1. Human TLR8 is activated by Bb RNA.HEK.293 cells stably expressing human TLR8(hTLR8) receptors were stimulated with 20 �MTLR8 ligand 3M-002. No activation of the NF-�Breporter on these cells was observed when 0.2�g/ml LPS, live Bb (MOI 10:1), Bb lysate ob-tained by sonication, with and without PEI, ornaked 0.1 �g of Bb RNA (an amount equivalentto an MOI 10:1 of live organism) was used asstimuli for 24 h. A significant activation was ob-served when the same amount of Bb RNAbound to PEI was used, and this stimulus disap-peared when the Bb RNA preparation wastreated with RNase A. Mann-Whitney test, *P �0.05. Average values of �3 independent experi-ments in triplicate. Error bars indicate se.

Figure 2. Human TLR8 is not activated by BbDNA or VV DNA. HEK.293 cells stably express-ing human TLR8 (hTLR8) receptors were stim-ulated with 20 �M TLR8 ligand 3M-002, BbDNA equivalent to an MOI 10:1, and 2 �g of BbDNA, with and without PEI. No activation of theNF-�B reporter on these cells was observed withthese stimuli, even when Bb DNA was digestedwith restriction enzymes. No activation was ob-served when another AT-rich DNA, VV DNA(1 �g), was used as a stimulus. Average valuesof �3 independent experiments in triplicate.Error bars indicate se.


induced robust transcription of IFN-� in a dose-dependentmanner, and the response was abrogated if Bb RNA was pre-treated with RNase. Furthermore, silencing of TLR8 expres-sion by siRNA caused a significant decrease in the transcrip-tion of IFN-� (Fig. 3B and C). Silencing of TLR7, on the otherhand, had no effect on transcription of IFN-� (SupplementalFig. 3). In parallel experiments we show that in contrast to liveBb and Bb RNA, neither Bb DNA nor VV DNA elicited tran-scription of IFN-� by the monocytes (Fig. 4). In line with ourprior finding that TLR8 activation by live spirochetes triggersproduction of a variety of NF-�B-associated cytokines, hereinwe show that Bb RNA also elicits secretion of TNF-� (Supple-mental Fig. 4).

Bb RNA colocalizes with TLR8 in the phagolysosomeWe previously demonstrated that transcription of IFN-� in re-sponse to Bb is phagocytosis dependent [25], while providingevidence that internalized and degraded spirochetes colocalizeat the phagosomal membrane with TLR8 [26]. To determinewhether internalized Bb RNA is delivered to the phagosomalvacuole, herein we used a fluorescent version of PEI as thetransport vehicle [47]. As shown in Fig. 5A, the fluorescentBb-RNA complex indeed colocalized inside the cell with TLR8.Providing further proof that TLR8 ligation occurs in an endo-somal structure, Bb-RNA colocalized first with the early endo-somal marker EEA1 and with the late endosomal markersLAMP1 and LysoTracker (Fig. 5B).

Bb RNA does not transfer from the phagosome ofhuman monocytesTo explore whether Bb RNA transfers from the phagosomeinto the cell cytosol to engage receptors capable of generatingtype I IFNs [48, 49], we elected to use 2 complementary ap-proaches. First, we labeled live spirochetal RNA by ethynyl uri-dine incorporation (Click-iT methodology) [50, 51] andtracked its location by microscopy. As shown in Fig. 6, inter-nalized fluorescent Bb RNA colocalized with LysoTracker toan endosomal location and could not be visualized in the cellcytosol. We then used fluorescent dextran particles to assessphagosomal integrity in Bb-infected monocytes. Dextran parti-cles colocalized to the phagosomal vacuole with degradedGFP-Bb and with live Bb RNA stained with Click-iT

Figure 3. Bb RNA induced transcription of IFN-� in humanmonocytes. (A) Isolated human monocytes were stimulated for 4 hwith the TLR-8 ligand 3M-002, live Bb (MOI 10:1), and increasingdoses of Bb RNA bound to PEI. A dose response was observed for thetranscription of IFN-�, which was markedly impaired when the BbRNA preparation was treated with RNase A. (B) siTLR8 on humanmonocytes interferes with transcription of IFN-�. (C) Shows a non-statistically significant reduction in TLR8 transcription after siTLR8 onhuman monocytes. Mann-Whitney test, *P � 0.0005. Average values of�3 independent experiments in triplicate. Error bars indicate se.

Figure 4. Bb DNA is not a major inducer of IFN-� transcription in humanmonocytes. Isolated human monocytes were stimulated for 4 h with theTLR8 ligand 3M-002, live Bb (MOI 10:1), 1 �g of Bb RNA, 1 �g of Bb DNA,and 1 �g of VV DNA, all delivered with PEI. A great induction of IFN-� tran-scription was observed when 3M-002, whole live Bb, and Bb RNA were usedas stimuli, but not with either of the AT-rich DNA preparations, Bb DNA andVV DNA. Mann-Whitney test, *P � 0.05, and ** P � 0.005. Average values of�3 independent experiments in triplicate. Error bars indicate se. N/S, notsignificant.



(Fig. 7A and B). The diffuse cytosolic dextran pattern, evidentlyseen in silica microsphere-treated monocytes (Fig. 7C) [52, 53],was not present in GFP-Bb infected monocytes. Absence of cyto-solic receptor activation is also supported by the lack of up-regu-lation of IRF3 or IRF5 in Bb-infected monocytes (Fig. 8), whichare activated by cytosolic nucleic acid receptors [54–56]. On theother hand, Bb RNA stimulation up-regulated IRF7, similarly towhat we reported with whole Bb [26].

Bb RNA signals through MyD88 and IRAK-4 inhuman monocytesIt is well accepted that TLR8-mediated production of type I IFNsin humans is highly dependent on the availability of MyD88.With this in mind, we first analyzed whether Bb RNA-mediatedtranscription of IFN-� is affected by blocking MyD88. To do so,we treated human monocytes with a commercially availableMyD88 inhibitory peptide as described previously [39]. We thenstimulated pretreated cells and controls with Bb RNA and aTLR8-specific ligand (3M-002). As shown in Fig. 9, transcriptionof IFN-� was markedly decreased in pretreated cells in response

to Bb RNA and 3M-002. A similar effect was not observed in pre-treated monocytes stimulated with LPS, which induces transcrip-tion of IFN-� through the MyD88-independent TRIF pathway[57]. We then studied monocytes obtained from a 6-year-old pa-tient with a known compound heterozygous mutation in thegene that codes for IRAK-4 (data not shown), a kinase that playsan essential role in downstream MyD88-dependent TLR-mediatedsignaling [58, 59]. Compared with healthy control monocytesisolated in parallel, transcription of IFN-� in response to Bb-RNAwas noticeably decreased in IRAK-4-deficient cells (Fig. 10). Ofinterest, transcription of IFN-� in LPS-stimulated IRAK-4-deficientmonocytes was lower than that of the same day healthy controlcells. Although not fully understood, a similar decrease inLPS has been observed in neutrophils of IRAK-4-deficientpatients [60].

DISCUSSION

Live Bb induces a complex innate immune response in humanmonocytes, which we have shown to be highly dependent on

Figure 5. Bb RNA colocalizes with TLR8 in the phagolysosome of human monocytes. (A) Staining for TLR8 (red) and fluorescent PEI (green) bound to BbRNA is shown in stimulated monocytes after 3 h of incubation. TLR8 colocalizes with fluorescent PEI when both channels are merged (shown in yellow) and aswhite pixels after using a colocalization plug-in from ImageJ. A differential interference contrast microscopy (DIC) image showing the localization of the inter-nalized Bb RNA-PEI-Fluo complex is shown. (B) Bb RNA bound to PEI-Fluo (green) is shown colocalizing with EEA1, LAMP1, and LysoTracker red. Differen-tial interference contrast microscopy images show localization of the internalized Bb RNA-PEI-Fluo complex.

Figure 6. Fluorescently labeled BbRNA (Click-iT) is internalized inthe phagolysosome. Whole live BbRNA was stained by means of ethy-nyl uridine incorporation (Click-iT). Fluorescent Bb RNA (green) isshown colocalizing with Lyso-Tracker red in human monocytes.


phagocytosis of the bacterium [23–25]. The immunologicevents that follow internalization and degradation of the spiro-chete, which we have called “phagosomal signaling,” can beexplained by 3 different mechanisms: (1) recruitment and acti-vation of TLR1/2 receptors to the phagosome for optimal rec-ognition of released spirochetal lipoproteins; (2) recruitmentand activation of endosomal TLR8 by released spirochetal nu-cleic acids; and (3) cooperative TLR2 and TLR8 signalingfrom within the phagosomal vacuole. We now provide evi-dence that Bb RNA, a viability-associated PAMP [61], activateshuman TLR8, leading to production of type I IFNs. This studyprovides further confirmation that innate immune recognitionof released spirochetal PAMPs occurs principally at thephagolysosomal compartment and does not involve transferinto the cell cytosol to induce production of IFN-�.

The role of TLR7 in sensing bacterial RNA by immune cellsis now well accepted [32, 33]; however, a similar role for TLR8has only begun to be uncovered. TLR7 recognizes RNA fromgroup B streptococcus in conventional dendritic cells [32] andfrom E. coli in murine pDCs [33]. In peripheral blood mono-nuclear cells, bacterial ribosomal RNA induces the activationof TLR7 [33]. Upon stimulation with purified E. coli RNA,HEK cells stably transfected with either TLR7 or TLR8 se-creted IL-8 [36]. TLR8 was previously shown to be up-regu-lated after phagocytosis of M. bovis by THP-1 cells [34]. In thatsame study, the investigators provided evidence that there wasan association of TLR8 gene variants with susceptibility to pul-monary tuberculosis. Likewise, phagocytosis of H. pylori byTHP-1 cells was shown to induce activation of TLR8 [35]. Wewere the first to show that phagocytosis of live B. burgdorferi

Figure 7. Bb RNA does not transfer from the phagosomeinto the cytosol. Human monocytes were treated with redfluorescent dextran before stimulation with (A) Click-iTstained Bb or (B) Bb RNA bound to PEI-Fluo (green).The diffuse cytoplasmic localization pattern observedwhen fluorescent dextran transfers from the phagosomeinto the cytosol after the use of (C) silica particles (red)was not observed in either Bb Click-iT stained or Bb RNAwith PEI-Fluo, which retained the punctuate endosomaldistribution that colocalizes with both of the stimuli. (D)Shows punctuate pattern of fluorescent dextran in un-stimulated cells.



induced transcription of IFN-� in human monocytes [25] andthat transcription of this cytokine was entirely dependent onthe availability of TLR8 [26]. In the current study we confirmthat borrelial RNA is indeed the principal bacterial ligand forhuman TLR8.

Over the years TLR8 has received less attention than otherendosomal TLRs [37, 62]. This is not surprising, given the ini-tial observation that TLR8 was inactive in mice [63–65]. Morerecent studies have demonstrated that TLR8 is actually respon-sive to the TLR8 ligand 3M-002 in combination with polyT oli-gonucleotides in murine peripheral blood mononuclear cells[66] and to VV DNA in murine pDCs [45]. Although highlyresponsive to Bb RNA, herein, we show that human TLR8 wasnot activated by either VV DNA or Bb DNA. In concert withour findings, it was recently reported that IFN-� production inresponse to VV is TLR independent [67]. Because of this dis-

crepancy, we hypothesized that the overall size of the DNAconstruct could play a factor in the efficiency of the nucleicacid delivery by PEI; however, even digested Bb DNA was un-able to activate TLR8. In addition, neither the PEI nucleicacid particle size, nor its uptake, differed when the deliveryvehicle was bound to any of the nucleic acid ligands (data notshown). Given that real-time PEI trafficking studies confirmthat PEI/DNA nanoparticles are actively transported to earlyand late endosomes [68] and that over time the transfectantsaccumulate in late endosomes/lysosomes [68, 69], it is un-likely that the unresponsiveness to DNA ligands by humanTLR8 was due to an inability to deliver the ligand to an endo-somal location.

It has become increasingly evident that human TLR8 has afundamental role in a variety of innate and adaptive immuneresponses to viral and bacterial pathogens. TLR-mediated rec-

Figure 8. Bb RNA induces transcription of IRF7. Rela-tive IRF transcription in human monocytes after stimu-lation with the TLR8 ligand 3M-002, live Bb (MOI 10:1), and 0.1 �g Bb RNA bound with PEI. All 3 stimuliincreased transcription of IRF7, but not of IRF3 orIRF5. Relative expression refers to relative changes ingenes irf3, irf5, and irf7 transcription compared withunstimulated after normalizing to gapdh. Mann-Whit-ney test, *P � 0.05 Average values of 3 independentexperiments. Error bars indicate se.

Figure 9. Bb RNA signals throughMyD88 in human monocytes. Iso-lated human monocytes were pre-treated with a MyD88 blockingpeptide (40 �M) for 1 h beforestimulation for 4 h with 3M-002,LPS (0.2 �g/ml), and 0.1 �g of BbRNA. MyD88 blockade affectedtranscription of IFN-� in responseto 3M-002 and Bb-RNA but notLPS. Average values of �3 inde-pendent experiments. Error barsindicate se.


ognition of bacterial ligands in the phagosome promotes opti-mal MHC class II bacterial antigen presentation and triggersinduction of stimulatory molecules and cytokines necessary foractivation and differentiation of T lymphocytes [70]. TLR8 isexpressed in most immune cells; thus, it is not unexpectedthat TLR8 agonists have been shown to be important vaccineadjuvants [71–75]. In addition, TLR8 has the ability to cross-talk with other TLRs [76, 77], and, thus, activation of this re-ceptor will modulate the responsiveness of other TLRs to theircognate PAMPs. In line with this premise, we showed that Bb-mediated activation of TLR8 enhanced expression of TLR2[26], which we hypothesized occurred in response to the auto-crine stimulation of the interferon receptor by secreted IFN-�[78]. To be sure, preexposure of THP-1 cells with the TLR8ligand 3M-002 resulted in enhanced TLR2 activation by a syn-thetic TLR2 ligand [79]. TLR8 variants also have a regulatoryeffect of TLR8 function in CD16�CD14� differentiated mono-cytes [35], a subset with enhanced proinflammatory properties[80]. Lastly, genetic variations in TLR8 could alter the host’ssusceptibility to a given human pathogen, including Mycobacte-rium tuberculosis [34].

Cytosolic receptors such as the retinoic acid-inducible gene,the retinoic acid-inducible gene I-like receptors, melanomadifferentiation-associated protein 5, and LGP2, are all capableof recognizing RNA to induce type I IFNs [48, 49]. Transfer ofbacterial RNA has the ability to activate cytosolic receptors[81] and thus could potentially contribute to the induction ofIFN-�. Using Bb RNA stained by Click-it and fluorescent dex-tran, we demonstrate that in our ex vivo monocyte stimulationsystem Bb RNA does not appeared to be transferred from thephagosomal compartment into the cell cytosol. Moreover, inBb-infected monocytes, we did not observe up-regulation ofIRF3 or IRF5, two interferon regulators that are linked to cyto-solic receptor activation [54–56]. On the contrary, up-regula-

tion of IRF7 was observed after Bb RNA stimulation, which isconcordant with our previous findings on TLR8 signalingupon phagocytosis of Bb [26]. In addition, the response toBb-RNA was markedly diminished in the presence of a syn-thetic MyD88 blocker, by interfering with the expression ofTLR8 and in IRAK-4-deficient human monocytes. Our com-bined results signify that in human monocytes, the type I IFNresponse to Bb RNA is MyD88 dependent and does not re-quire activation of a cytosolic receptor.

The overall role of type I IFNs in LD remains poorly under-stood. In experimentally infected mice, the development ofLyme arthritis is associated with an IFN-induced gene expres-sion signature while blocking type I IFN signaling had a pro-tective effect in the development of arthritis [82]. Whetherproduction of type I IFNs via TLR8 is beneficial or protectivein humans is not yet known. It is known that activation ofTLR8 leads to inhibited expression of TLR7 and TLR9 [83]and, conversely, TLR8 deficiency leads to overexpression ofTLR7 and TLR9, with subsequent development of autoim-munity and autoantibodies [84, 85]. It is possible that TLR8variants could be deleterious in the context of LD and per-haps could have a role in the pathogenesis of the more se-vere inflammatory manifestations of LD (i.e., arthritis) orpersistent chronic inflammatory symptoms in some Lymedisease patients. It is conceivable that future therapeuticmeasures for LD could be aimed at both enhancing or sup-pressing bacterial recognition and the ensuing TLR8-medi-ated inflammatory response.

The validation of the phagosomal signaling model and thesubstantiation for the role of Bb RNA as a TLR8 ligand, asshown herein, significantly advance our understanding for howthe LD spirochete triggers the inflammatory processes that,under actual disease conditions, cause tissue damage and/orpromote bacterial clearance. Our findings provide a new para-digm for innate immune recognition of Bb and lay thegroundwork for understanding how innate immune recogni-tion of the bacterium affects the clinical outcome of LD inhumans.

ACKNOWLEDGMENTS

Special thanks are given to Dr. Justin Radolf [University ofConnecticut Health Center (UCHC)] and Kate Fitzgerald(University of Massachusetts) for their scientific guidance, toRenee Gilberti (UCHC) and Sivapriya Kailasan-Vanaja (TuftsUniversity) for their technical assistance, and to Drs. UzelGulbu and Steve Holland (National Institutes of Health), fortheir help characterizing our IRAK-4 deficient patient. Thiswork was supported by NIAID grant AI090166 (to J.C.S.).

AUTHORSHIP

J.L.C. designed and carried out experiments, interpreted re-sults, and wrote the paper. C.J.L.V., B.W., and S.L. helpedcarry out experiments. C.O. and P.H.V. provided the VVDNA. J.C.S. planed experiments, interpreted results and wrotethe paper.

Figure 10. Bb RNA signals through IRAK-4 in human monocytes. Iso-lated human monocytes from an IRAK-4-deficient (IRAK-4 def) patientand from a healthy control (extracted and run in parallel) were stimu-lated for 4 h with LPS (0.2 �g/ml) and 0.1 �g of Bb RNA. Absence ofIRAK-4 affected transcription of IFN-� responses to Bb RNA (N�1).



DISCLOSURES

The authors declare no conflict of interest.

REFERENCES

1. Radolf, J. D., Caimano, M. J., Stevenson, B., Hu, L. T. (2012) Of ticks,mice and men: understanding the dual-host lifestyle of Lyme diseasespirochaetes. Nat. Rev. Microbiol. 10, 87–99.

2. Levi, T., Kilpatrick, A. M., Mangel, M., Wilmers, C. C. (2012) Deer,predators, and the emergence of Lyme disease. Proc. Natl. Acad. Sci.U. S. A. 109, 10942–10947.

3. Smith, R., Takkinen, J. (2006) Lyme borreliosis: Europe-wide coordi-nated surveillance and action needed? Euro Surveill. 11, E060622.

4. Biesiada, G., Czepiel, J., Lesniak, M. R., Garlicki, A., Mach, T. (2012)Lyme disease: review. Arch. Med. Sci. 8, 978–982.

5. Fraser, C. M., Casjens, S., Huang, W. M., Sutton, G. G., Clayton, R.,Lathigra, R., White, O., Ketchum, K. A., Dodson, R., Hickey, E. K.,Gwinn, M., Dougherty, B., Tomb, J. F., Fleischmann, R. D., Richardson,D., Peterson, J., Kerlavage, A. R., Quackenbush, J., Salzberg, S., Hanson,M., van Vugt, R., Palmer, N., Adams, M. D., Gocayne, J., Weidman, J.,Utterback, T., Watthey, L., McDonald, L., Artiach, P., Bowman, C., Gar-land, S., Fuji, C., Cotton, M. D., Horst, K., Roberts, K., Hatch, B., Smith,H. O., Venter, J. C. (1997) Genomic sequence of a Lyme disease spiro-chaete, Borrelia burgdorferi. Nature 390, 580–586.

6. Lien, E., Sellati, T. J., Yoshimura, A., Flo, T. H., Rawadi, G., Finberg,R. W., Carroll, J. D., Espevik, T., Ingalls, R. R., Radolf, J. D., Golenbock,D. T. (1999) Toll-like receptor 2 functions as a pattern recognition re-ceptor for diverse bacterial products. J. Biol. Chem. 274, 33419–33425.

7. Norgard, M. V., Riley, B. S., Richardson, J. A., Radolf, J. D. (1995) Der-mal inflammation elicited by synthetic analogs of Treponema pallidumand Borrelia burgdorferi lipoproteins. Infect. Immun. 63, 1507–1515.

8. Norgard, M. V., Arndt, L. L., Akins, D. R., Curetty, L. L., Harrich, D. A.,Radolf, J. D. (1996) Activation of human monocytic cells by Treponemapallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptidesproceeds via a pathway distinct from that of lipopolysaccharide but in-volves the transcriptional activator NF-�B. Infect. Immun. 64, 3845–3852.

9. Aliprantis, A. O., Yang, R. B., Mark, M. R., Suggett, S., Devaux, B.,Radolf, J. D., Klimpel, G. R., Godowski, P., Zychlinsky, A. (1999) Cellactivation and apoptosis by bacterial lipoproteins through Toll-like re-ceptor-2. Science 285, 736–739.

10. Sellati, T. J., Bouis, D. A., Caimano, M. J., Feulner, J. A., Ayers, C., Lien,E., Radolf, J. D. (1999) Activation of human monocytic cells by Borreliaburgdorferi and Treponema pallidum is facilitated by CD14 and correlateswith surface exposure of spirochetal lipoproteins. J. Immunol. 163, 2049–2056.

11. Brightbill, H. D., Libraty, D. H., Krutzik, S. R., Yang, R. B., Belisle, J. T.,Bleharski, J. R., Maitland, M., Norgard, M. V., Plevy, S. E., Smale, S. T.,Brennan, P. J., Bloom, B. R., Godowski, P. J., Modlin, R. L. (1999) Hostdefense mechanisms triggered by microbial lipoproteins through Toll-like receptors. Science 285, 732–736.

12. Wooten, R. M., Ma, Y., Yoder, R. A., Brown, J. P., Weis, J. H., Zachary,J. F., Kirschning, C. J., Weis, J. J. (2002) Toll-like receptor 2 is requiredfor innate, but not acquired, host defense to Borrelia burgdorferi. J. Immu-nol. 168, 348–355.

13. Salazar, C. A., Rothemich, M., Drouin, E. E., Glickstein, L., Steere, A. C.(2005) Human Lyme arthritis and the immunoglobulin G antibody re-sponse to the 37-kilodalton arthritis-related protein of Borrelia burgdorferi.Infect. Immun. 73, 2951–2957.

14. Radolf, J. D., Goldberg, M. S., Bourell, K., Baker, S. I., Jones, J. D., Nor-gard, M. V. (1995) Characterization of outer membranes isolated fromBorrelia burgdorferi, the Lyme disease spirochete. Infect. Immun. 63, 2154–2163.

15. Akira, S. (2006) TLR signaling. Curr. Top. Microbiol. Immunol. 311, 1–16.16. Wooten, R. M., Ma, Y., Yoder, R. A., Brown, J. P., Weis, J. H., Zachary,

J. F., Kirschning, C. J., Weis, J. J. (2002) Toll-like receptor 2 plays a piv-otal role in host defense and inflammatory response to Borrelia burgdor-feri. Vector Borne Zoonotic Dis. 2, 275–278.

17. Alexopoulou, L., Thomas, V., Schnare, M., Lobet, Y., Anguita, J.,Schoen, R. T., Medzhitov, R., Fikrig, E., Flavell, R. A. (2002) Hypore-sponsiveness to vaccination with Borrelia burgdorferi OspA in humans andin TLR1- and TLR2-deficient mice. Nat. Med. 8, 878–884.

18. Hirschfeld, M., Kirschning, C. J., Schwandner, R., Wesche, H., Weis,J. H., Wooten, R. M., Weis, J. J. (1999) Cutting edge: inflammatory sig-naling by Borrelia burgdorferi lipoproteins is mediated by Toll-like recep-tor 2. J. Immunol. 163, 2382–2386.

19. Wooten, R. M., Morrison, T. B., Weis, J. H., Wright, S. D., Thieringer,R., Weis, J. J. (1998) The role of CD14 in signaling mediated by outermembrane lipoproteins of Borrelia burgdorferi. J. Immunol. 160, 5485–5492.

20. Benhnia, M. R., Wroblewski, D., Akhtar, M. N., Patel, R. A., Lavezzi, W.,Gangloff, S. C., Goyert, S. M., Caimano, M. J., Radolf, J. D., Sellati, T. J.(2005) Signaling through CD14 attenuates the inflammatory responseto Borrelia burgdorferi, the agent of Lyme disease. J. Immunol. 174, 1539–1548.

21. Sellati, T. J., Waldrop, S. L., Salazar, J. C., Bergstresser, P. R., Picker,L. J., Radolf, J. D. (2001) The cutaneous response in humans to Trepo-nema pallidum lipoprotein analogues involves cellular elements of bothinnate and adaptive immunity. J. Immunol. 166, 4131–4140.

22. Giambartolomei, G. H., Dennis, V. A., Lasater, B. L., Philipp, M. T.(1999) Induction of pro- and anti-inflammatory cytokines by Borreliaburgdorferi lipoproteins in monocytes is mediated by CD14. Infect. Immun.67, 140–147.

23. Moore, M. W., Cruz, A. R., LaVake, C. J., Marzo, A. L., Eggers, C. H.,Salazar, J. C., Radolf, J. D. (2007) Phagocytosis of Borrelia burgdorferi andTreponema pallidum potentiates innate immune activation and induces �interferon production. Infect. Immun. 75, 2046–2062.

24. Cruz, A. R., Moore, M. W., La Vake, C. J., Eggers, C. H., Salazar, J. C.,Radolf, J. D. (2008) Phagocytosis of Borrelia burgdorferi, the Lyme diseasespirochete, potentiates innate immune activation and induces apoptosisin human monocytes. Infect. Immun. 76, 56–70.

25. Salazar, J. C., Duhnam-Ems, S., La Vake, C., Cruz, A. R., Moore, M. W.,Caimano, M. J., Velez-Climent, L., Shupe, J., Krueger, W., Radolf, J. D.(2009) Activation of human monocytes by live Borrelia burgdorferi gener-ates TLR2-dependent and -independent responses which include induc-tion of IFN-�. PLoS Pathog. 5, e1000444.

26. Cervantes, J. L., Dunham-Ems, S. M., La Vake, C. J., Petzke, M. M., Sa-hay, B., Sellati, T. J., Radolf, J. D., Salazar, J. C. (2011) Phagosomal sig-naling by Borrelia burgdorferi in human monocytes involves Toll-like re-ceptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated inductionof IFN-�. Proc. Natl. Acad. Sci. U. S. A. 108, 3683–3688.

27. Stetson, D. B., Medzhitov, R. (2006) Type I interferons in host defense.Immunity 25, 373–381.

28. Monroe, K. M., McWhirter, S. M., Vance, R. E. (2010) Induction of typeI interferons by bacteria. Cell. Microbiol. 12, 881–890.

29. Armstrong, M. E., Gantier, M., Li, L., Chung, W. Y., McCann, A., Baugh,J. A., Donnelly, S. C. (2008) Small interfering RNAs induce macrophagemigration inhibitory factor production and proliferation in breast can-cer cells via a double-stranded RNA-dependent protein kinase-depen-dent mechanism. J. Immunol. 180, 7125–7133.

30. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B.,Giese, T., Endres, S., Hartmann, G. (2002) Quantitative expression ofToll-like receptor 1�10 mRNA in cellular subsets of human peripheralblood mononuclear cells and sensitivity to CpG oligodeoxynucleotides.J. Immunol. 168, 4531–4537.

31. Gantier, M. P., Tong, S., Behlke, M. A., Xu, D., Phipps, S., Foster, P. S.,Williams, B. R. (2008) TLR7 is involved in sequence-specific sensing ofsingle-stranded RNAs in human macrophages. J. Immunol. 180, 2117–2124.

32. Mancuso, G., Gambuzza, M., Midiri, A., Biondo, C., Papasergi, S., Akira,S., Teti, G., Beninati, C. (2009) Bacterial recognition by TLR7 in thelysosomes of conventional dendritic cells. Nat. Immunol. 10, 587–594.

33. Eberle, F., Sirin, M., Binder, M., Dalpke, A. H. (2009) Bacterial RNA isrecognized by different sets of immunoreceptors. Eur. J. Immunol. 39,2537–2547.

34. Davila, S., Hibberd, M. L., Hari Dass, R., Wong, H. E., Sahiratmadja, E.,Bonnard, C., Alisjahbana, B., Szeszko, J. S., Balabanova, Y., Drobniewski,F., van Crevel, R., van de Vosse, E., Nejentsev, S., Ottenhoff, T. H.,Seielstad, M. (2008) Genetic association and expression studies indicatea role of Toll-like receptor 8 in pulmonary tuberculosis. PLoS Genet. 4,e1000218.

35. Gantier, M. P., Irving, A. T., Kaparakis-Liaskos, M., Xu, D., Evans, V. A.,Cameron, P. U., Bourne, J. A., Ferrero, R. L., John, M., Behlke, M. A.,Williams, B. R. (2010) Genetic modulation of TLR8 response followingbacterial phagocytosis. Hum. Mutat. 31, 1069–1079.

36. Kariko, K., Buckstein, M., Ni, H., Weissman, D. (2005) Suppression ofRNA recognition by Toll-like receptors: the impact of nucleoside modifi-cation and the evolutionary origin of RNA. Immunity 23, 165–175.

37. Cervantes, J. L., Weinerman, B., Basole, C., Salazar, J. C. (2012) TLR8:the forgotten relative revindicated. Cell. Mol. Immunol. 9, 434–438.

38. Song, Y. K., Liu, F., Chu, S., Liu, D. (1997) Characterization of cationicliposome-mediated gene transfer in vivo by intravenous administration.Hum. Gene. Ther. 8, 1585–1594.

39. Toshchakov, V. U., Basu, S., Fenton, M. J., Vogel, S. N. (2005) Differen-tial involvement of BB loops of Toll-IL-1 resistance (TIR) domain-con-taining adapter proteins in TLR4- versus TLR2-mediated signal transduc-tion. J. Immunol. 175, 494–500.

40. Earl, P. L., Moss, B., Wyatt, L. S., Carroll, M. W. (2001) Generation ofrecombinant vaccinia viruses. Curr. Protoc. Mol. Biol. 43, 16.16.1–16.17.19.

41. Schmittgen, T. D., and Livak, K. J. (2008) Analyzing real-time PCR databy the comparative CT method. Nat. Protoc. 3, 1101–1108.

42. Kikkert, R., de Groot, E. R., Aarden, L. A. (2008) Cytokine induction bypyrogens: comparison of whole blood, mononuclear cells, and TLR-transfectants. J. Immunol. Methods 336, 45–55.

43. Huang, L. Y., Dumontelle, J. L., Zolodz, M., Deora, A., Mozier, N. M.,Golding, B. (2009) Use of Toll-like receptor assays to detect and identifymicrobial contaminants in biological products. J. Clin. Microbiol. 47,3427–3434.

44. Hawley, K. L., Olson, C. M., Jr., Iglesias-Pedraz, J. M., Navasa, N., Cer-vantes, J. L., Caimano, M. J., Izadi, H., Ingalls, R. R., Pal, U., Salazar,


J. C., Radolf, J. D., Anguita, J. (2012) CD14 cooperates with comple-ment receptor 3 to mediate MyD88-independent phagocytosis of Borreliaburgdorferi. Proc. Natl. Acad. Sci. U. S. A. 109, 1228–1232.

45. Martinez, J., Huang, X., Yang, Y. (2010) Toll-like receptor 8-mediatedactivation of murine plasmacytoid dendritic cells by vaccinia viral DNA.Proc. Natl. Acad. Sci. U. S. A. 107, 6442–6447.

46. Gautam, A., Hathaway, M., Ramamoorthy, R. (2009) The Borrelia burg-dorferi flaB promoter has an extended �10 element and includes a T-rich �35/�10 spacer sequence that is essential for optimal activity.FEMS Microbiol. Lett. 293, 278–284.

47. Cubillos-Ruiz, J. R., Engle, X., Scarlett, U. K., Martinez, D., Barber, A.,Elgueta, R., Wang, L., Nesbeth, Y., Durant, Y., Gewirtz, A. T., Sentman,C. L., Kedl, R., Conejo-Garcia, J. R. (2009) Polyethylenimine-basedsiRNA nanocomplexes reprogram tumor-associated dendritic cells viaTLR5 to elicit therapeutic antitumor immunity. J. Clin. Invest. 119,2231–2244.

48. Kawai, T., Akira, S. (2011) Toll-like receptors and their crosstalk withother innate receptors in infection and immunity. Immunity 34, 637–650.

49. Kumar, H., Kawai, T., Akira, S. (2011) Pathogen recognition by the in-nate immune system. Int. Rev. Immunol. 30, 16–34.

50. Jao, C. Y., Salic, A. (2008) Exploring RNA transcription and turnover invivo by using click chemistry. Proc. Natl. Acad. Sci. U. S. A. 105, 15779–15784.

51. Dumont, A., Malleron, A., Awwad, M., Dukan, S., Vauzeilles, B. (2012)Click-mediated labeling of bacterial membranes through metabolicmodification of the lipopolysaccharide inner core. Angew. Chem. Int. Ed.Engl. 51, 3143–3146.

52. Hornung, V., Bauernfeind, F., Halle, A., Samstad, E. O., Kono, H.,Rock, K. L., Fitzgerald, K. A., Latz, E. (2008) Silica crystals and alumi-num salts activate the NALP3 inflammasome through phagosomal desta-bilization. Nat. Immunol. 9, 847–856.

53. Costantini, L. M., Gilberti, R. M., Knecht, D. A. (2011) The phagocytosisand toxicity of amorphous silica. PLoS One 6, e14647.

54. Rathinam, V. A., Jiang, Z., Waggoner, S. N., Sharma, S., Cole, L. E.,Waggoner, L., Vanaja, S. K., Monks, B. G., Ganesan, S., Latz, E., Hor-nung, V., Vogel, S. N., Szomolanyi-Tsuda, E., Fitzgerald, K. A. (2010)The AIM2 inflammasome is essential for host defense against cytosolicbacteria and DNA viruses. Nat. Immunol. 11, 395–402.

55. Herskovits, A. A., Auerbuch, V., Portnoy, D. A. (2007) Bacterial ligandsgenerated in a phagosome are targets of the cytosolic innate immunesystem. PLoS Pathog. 3, e51.

56. Pandey, A. K., Yang, Y., Jiang, Z., Fortune, S. M., Coulombe, F., Behr,M. A., Fitzgerald, K. A., Sassetti, C. M., Kelliher, M. A. (2009) NOD2,RIP2 and IRF5 play a critical role in the type I interferon response toMycobacterium tuberculosis. PLoS Pathog. 5, e1000500.

57. Takeda, K., Akira, S. (2004) TLR signaling pathways. Semin. Immunol. 16,3–9.

58. Kim, T. W., Staschke, K., Bulek, K., Yao, J., Peters, K., Oh, K. H., Van-denburg, Y., Xiao, H., Qian, W., Hamilton, T., Min, B., Sen, G.,Gilmour, R., Li, X. (2007) A critical role for IRAK4 kinase activity inToll-like receptor-mediated innate immunity. J. Exp. Med. 204, 1025–1036.

59. Li, X. (2008) IRAK4 in TLR/IL-1R signaling: possible clinical applica-tions. Eur. J. Immunol. 38, 614–618.

60. van Bruggen, R., Drewniak, A., Tool, A. T., Jansen, M., van Houdt, M.,Geissler, J., van den Berg, T. K., Chapel, H., Kuijpers, T. W. (2010) Toll-like receptor responses in IRAK-4-deficient neutrophils. J. Innate Immun.2, 280–287.

61. Sander, L. E., Davis, M. J., Boekschoten, M. V., Amsen, D., Dascher,C. C., Ryffel, B., Swanson, J. A., Muller, M., Blander, J. M. (2011) Detec-tion of prokaryotic mRNA signifies microbial viability and promotes im-munity. Nature 474, 385–389.

62. Tanji, H., Ohto, U., Shibata, T., Miyake, K., Shimizu, T. (2013) Struc-tural reorganization of the Toll-like receptor 8 dimer induced by agonis-tic ligands. Science 339, 1426–1429.

63. Jurk, M., Heil, F., Vollmer, J., Schetter, C., Krieg, A. M., Wagner, H.,Lipford, G., Bauer, S. (2002) Human TLR7 or TLR8 independently con-fer responsiveness to the antiviral compound R-848. Nat. Immunol. 3,499.

64. Gorden, K. K., Qiu, X., Battiste, J. J., Wightman, P. P., Vasilakos, J. P.,Alkan, S. S. (2006) Oligodeoxynucleotides differentially modulate activa-tion of TLR7 and TLR8 by imidazoquinolines. J. Immunol. 177, 8164–8170.

65. Forsbach, A., Nemorin, J. G., Montino, C., Muller, C., Samulowitz, U.,Vicari, A. P., Jurk, M., Mutwiri, G. K., Krieg, A. M., Lipford, G. B.,Vollmer, J. (2008) Identification of RNA sequence motifs stimulating

sequence-specific TLR8-dependent immune responses. J. Immunol. 180,3729–3738.

66. Gorden, K. K., Qiu, X. X., Binsfeld, C. C., Vasilakos, J. P., Alkan, S. S.(2006) Cutting edge: activation of murine TLR8 by a combination ofimidazoquinoline immune response modifiers and polyT oligodeoxy-nucleotides. J. Immunol. 177, 6584–6587.

67. Zhu, J., Martinez, J., Huang, X., Yang, Y. (2007) Innate immunityagainst vaccinia virus is mediated by TLR2 and requires TLR-indepen-dent production of IFN-beta. Blood 109, 619–625.

68. Suh, J., An, Y., Tang, B. C., Dempsey, C., Huang, F., Hanes, J. (2012)Real-time gene delivery vector tracking in the endo-lysosomal pathway oflive cells. Microsc. Res. Tech. 75, 691–697.

69. Bieber, T., Meissner, W., Kostin, S., Niemann, A., Elsasser, H. P. (2002)Intracellular route and transcriptional competence of polyethylenimine-DNA complexes. J. Control Release 82, 441–454.

70. Connolly, D. J., and O’Neill, L. A. (2012) New developments in Toll-likereceptor targeted therapeutics. Curr. Opin. Pharmacol. 12, 510–518.

71. Tomai, M. A., Miller, R. L., Lipson, K. E., Kieper, W. C., Zarraga, I. E.,Vasilakos, J. P. (2007) Resiquimod and other immune response modifi-ers as vaccine adjuvants. Expert Rev. Vaccines 6, 835–847.

72. Du, J., Wu, Z., Ren, S., Wei, Y., Gao, M., Randolph, G. J., Qu, C. (2010)TLR8 agonists stimulate newly recruited monocyte-derived cells into po-tent APCs that enhance HBsAg immunogenicity. Vaccine 28, 6273–6281.

73. Lu, H., Dietsch, G. N., Matthews, M. A., Yang, Y., Ghanekar, S., Ino-kuma, M., Suni, M., Maino, V. C., Henderson, K. E., Howbert, J. J., Di-sis, M. L., Hershberg, R. M. (2012) VTX-2337 is a novel TLR8 agonistthat activates NK cells and augments ADCC. Clin. Cancer Res. 18, 499–509.

74. Kanzler, H., Barrat, F. J., Hessel, E. M., Coffman, R. L. (2007) Thera-peutic targeting of innate immunity with Toll-like receptor agonists andantagonists. Nat. Med. 13, 552–559.

75. Wille-Reece, U., Flynn, B. J., Lore, K., Koup, R. A., Kedl, R. M., Matta-pallil, J. J., Weiss, W. R., Roederer, M., Seder, R. A. (2005) HIV Gag pro-tein conjugated to a Toll-like receptor 7/8 agonist improves the magni-tude and quality of Th1 and CD8� T cell responses in nonhuman pri-mates. Proc. Natl. Acad. Sci. U. S. A. 102, 15190–15194.

76. Ghosh, T. K., Mickelson, D. J., Solberg, J. C., Lipson, K. E., Inglefield,J. R., Alkan, S. S. (2007) TLR-TLR cross talk in human PBMC resultingin synergistic and antagonistic regulation of type-1 and 2 interferons,IL-12 and. TNF-�. Int. Immunopharmacol. 7, 1111–1121.

77. Makela, S. M., Strengell, M., Pietila, T. E., Osterlund, P., Julkunen, I.(2009) Multiple signaling pathways contribute to synergistic TLR ligand-dependent cytokine gene expression in human monocyte-derived mac-rophages and dendritic cells. J. Leukoc. Biol. 85, 664–672.

78. Guo, Z., Garg, S., Hill, K. M., Jayashankar, L., Mooney, M. R., Hoel-scher, M., Katz, J. M., Boss, J. M., Sambhara, S. (2005) A distal regula-tory region is required for constitutive and IFN-�-induced expression ofmurine TLR9 gene. J. Immunol. 175, 7407–7418.

79. Mureith, M. W., Chang, J. J., Lifson, J. D., Ndung’u, T., Altfeld, M.(2010) Exposure to HIV-1-encoded Toll-like receptor 8 ligands en-hances monocyte response to microbial encoded Toll-like receptor 2/4ligands. AIDS 24, 1841–1848.

80. Ziegler-Heitbrock, L. (2007) The CD14� CD16� blood monocytes: theirrole in infection and inflammation. J. Leukoc. Biol. 81, 584–592.

81. Abdullah, Z., Schlee, M., Roth, S., Mraheil, M. A., Barchet, W., Bottcher,J., Hain, T., Geiger, S., Hayakawa, Y., Fritz, J. H., Civril, F., Hopfner,K. P., Kurts, C., Ruland, J., Hartmann, G., Chakraborty, T., Knolle, P. A.(2012) RIG-I detects infection with live Listeria by sensing secreted bac-terial nucleic acids. EMBO J. 31, 4153–4164.

82. Miller, J. C., Ma, Y., Crandall, H., Wang, X., Weis, J. J. (2008) Gene ex-pression profiling provides insights into the pathways involved in inflam-matory arthritis development: murine model of Lyme disease. Exp. Mol.Pathol. 85, 20–27.

83. Wang, J., Shao, Y., Bennett, T. A., Shankar, R. A., Wightman, P. D.,Reddy, L. G. (2006) The functional effects of physical interactionsamong Toll-like receptors 7, 8, and 9. J. Biol. Chem. 281, 37427–37434.

84. Demaria, O., Pagni, P. P., Traub, S., de Gassart, A., Branzk, N., Murphy,A. J., Valenzuela, D. M., Yancopoulos, G. D., Flavell, R. A., Alexopoulou,L. (2010) TLR8 deficiency leads to autoimmunity in mice. J. Clin. Invest.120, 3651–3662.

85. Zheng, L., Zhang, Z., Yu, C., Yang, C. (2010) Expression of Toll-like re-ceptors 7, 8, and 9 in primary Sjögren’s syndrome. Oral Surg. Oral Med.Oral Pathol. Oral Radiol. Endod. 109, 844–850.

KEY WORDS:TLR8 � bacterial RNA � Borrelia burgdorferi � human monocytes



human tlr8 is activated upon recognition of borrelia burgdorferi rna in the phagosome of human...

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