• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Mar 1, 2011; 108(9): 3683–3688.
Published online Feb 14, 2011. doi:  10.1073/pnas.1013776108
PMCID: PMC3048123
Immunology

Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-β

Abstract

Phagocytosed Borrelia burgdorferi (Bb) induces inflammatory signals that differ both quantitatively and qualitatively from those generated by spirochetal lipoproteins interacting with Toll-like receptor (TLR) 1/2 on the surface of human monocytes. Of particular significance, and in contrast to lipoproteins, internalized spirochetes induce transcription of IFN-β. Using inhibitory immunoregulatory DNA sequences (IRSs) specific to TLR7, TLR8, and TLR9, we show that the TLR8 inhibitor IRS957 significantly diminishes production of TNF-α, IL-6, and IL-10 and completely abrogates transcription of IFN-β in Bb-stimulated monocytes. We demonstrate that live Bb induces transcription of TLR2 and TLR8, whereas IRS957 interferes with their transcriptional regulation. Using confocal and epifluorescence microscopy, we show that baseline TLR expression in unstimulated monocytes is greater for TLR2 than for TLR8, whereas expression of both TLRs increases significantly upon stimulation with live spirochetes. By confocal microscopy, we show that TLR2 colocalization with Bb coincides with binding, uptake, and formation of the phagosomal vacuole, whereas recruitment of both TLR2 and TLR8 overlaps with degradation of the spirochete. We provide evidence that IFN regulatory factor (IRF) 7 is translocated into the nucleus of Bb-infected monocytes, suggesting its activation through phosphorylation. Taken together, these findings indicate that the phagosome is an efficient platform for the recognition of diverse ligands; in the case of Bb, phagosomal signaling involves a cooperative interaction between TLR2 and TLR8 in pro- and antiinflammatory cytokine responses, whereas TLR8 is solely responsible for IRF7-mediated induction of IFN-β.

Keywords: Lyme disease, endosomal receptors, type I interferons, phagocytosis

Lyme disease (LD), the most commonly reported vector-borne illness in the United States (1), is a multisystem infectious disorder caused by the spirochetal bacterium Borrelia burgdorferi (Bb). Monocytes and macrophages are considered to be critical cellular elements of the innate immune response to Bb (25). Recognition of the spirochete was previously thought to result primarily from the interactions of the bacterium's abundant outer membrane-associated lipoproteins with Toll-like receptor (TLR) 1/2 on the surface of innate immune cells (6). More recently, we and others have provided evidence that phagocytosed live Bb induces inflammatory signals that differ both quantitatively and qualitatively from those generated by lipoproteins (2, 4, 79). In addition to enhanced cytokine production, phagocytosed live Bb induced transcription of IFN-β and several IFN-stimulated genes (ISGs) in isolated human monocytes, whereas spirochetal lipoproteins were unable to do so (7).

Although production of type I IFNs was previously ascribed solely to antiviral immune responses (10), it now is well established that both intracellular (1114) and extracellular bacteria (11, 1517) also induce transcription of these cytokines. Bacteria can elicit type I IFNs either by activating TLRs (11, 12) or through TLR-independent recognition of bacterial pathogen-associated molecular patterns (PAMPs) within the host cell cytosol (11, 18). We and others have begun to demarcate the pathways by which live Bb induces type I IFNs in both mouse and human cells (7, 1922). Miller et al. (20) demonstrated that live Bb induces transcription of several ISGs in bone marrow-derived murine macrophages (BMDMs) independent of both MyD88 (20) and TRIF (21) yet requiring the transcription factor IFN regulatory factor (IRF) 3. Using Bb-infected human peripheral blood mononuclear cells (PBMCs), Petzke et al. (22) provided evidence that human plasmacytoid dendritic cells (pDCs) are a principal source of IFN-α in response to phagocytosed live spirochetes and demonstrated that transcription and secretion of this type I IFN was inhibited by blocking TLR7 and TLR9. These two TLRs, together with TLR8, make up a family of endosomal pattern recognition receptors that are capable of generating MyD88-dependent type I IFNs by sensing pathogen-derived nucleic acids (23, 24). In contrast to pDCs, human monocytes constitutively express TLR8, and although they also express TLR7, they do not express TLR9 (23, 2527). The role of TLR8 in modulating innate immune responses to Bb, including its involvement in the production of type I IFNs, has not been previously examined.

In this study, we used highly purified human monocytes to characterize more precisely the mechanisms whereby internalization of the LD spirochete induces production of pro- and antiinflammatory cytokines, including type I IFNs. We provide evidence that phagosomal signaling in Bb-infected monocytes involves a sequential and cooperative interaction between TLR2 and TLR8, which occurs exclusively by activation of TLR8 in the case of IFN-β. Our combined observations take us well beyond the simplistic notion that innate immune cell activation by Bb occurs only at the plasma membrane through TLR1/2 and CD14 signaling and, instead, reveal the importance of the phagosome as an efficient platform for the recognition of diverse bacterial PAMPs.

Results

Phagocytosis of Bb Is Required for Transcription of IFN-β in Bb-Infected Human Monocytes.

We previously showed that inhibition of phagocytosis diminished NF-κB–dependent cytokine production in Bb-stimulated PBMCs (2). More recently, we demonstrated that the monocyte is a major source of these cytokines and solely responsible for the production of IFN-β (7). We now provide evidence that internalization of the bacterium is essential for the induction of IFN-β and for optimal production of TNF-α in isolated human monocytes. When the actin filament disrupter Cytochalasin-D is used to block phagocytosis, transcription of ifnb is abrogated and secretion of TNF-α is significantly diminished (Fig. S1). Thus, if sensing of the spirochete is restricted to cell surface TLR2, induction of type I IFNs does not occur and production of NF-κB–mediated cytokines is significantly impaired.

Bb-Mediated Induction of IFN-β Cannot Be Explained by TLR2 Signaling.

TLR2 signals were previously believed to be incapable of inducing type I IFNs (28). Two recent studies have challenged this notion by demonstrating that activation of TLR2, by either viral (29) or bacterial (30) ligands, can induce transcription of IFN-β in inflammatory human monocytes and murine macrophages, respectively. In a previous study, we provided evidence that lipoprotein-rich borrelial lysates do not induce transcription of IFN-β by human monocytes (7). In the same study, we showed that production of IFN-β in Bb-infected murine BMDMs occurs independent of TLR2. Nonetheless, in light of the recent finding that a bacterial TLR2 ligand (Pam3CSK4) induces production of IFN-β in murine macrophages (30), we have reexamined herein the contribution of TLR2 activation in the production of this cytokine by human monocytes. We first established that transcription of IFN-β in Bb-stimulated monocytes does not begin in earnest until 2 h of incubation and increases noticeably after 4 h of stimulation (Fig. S2). In contrast to the robust type I IFN responses elicited by either LPS or live Bb, even high concentrations of Pam3CSSNA failed to induce transcription of IFN-β in monocytes (Fig. S3). These combined observations are indicative that TLR2 signaling does not play a significant role in the induction of type I IFNs in Bb-infected monocytes.

TLR8 Is Required for Transcription of IFN-β and Cooperates with TLR2 in Generating Pro- and Antiinflammatory Cytokine Production in Human Monocytes.

Having established that TLR2 signaling alone cannot explain the type I IFN responses to live Bb, we hypothesized that activation of an endosomal TLR could be responsible for the induction of IFN-β in the monocyte. To explore this possibility, we took advantage of the availability of specific inhibitory DNA–oligonucleotide sequences (IRSs) that have the ability to block ligation of TLR7, TLR8, or TLR9 (31, 32). These inhibitors were recently used by Petzke et al. (22) to demonstrate that induction of IFN-α in Bb-infected pDCs is dependent on TLR7 and TLR9. For these experiments, we first corroborated that healthy volunteers’ monocytes expressed TLR7 and TLR8 and very little TLR9 (27) (Table S1). As depicted in Fig. 1A, we then showed that inhibition of TLR8 by IRS957 eliminated transcription of IFN-β, whereas induction of this cytokine by the spirochete was unaffected by blocking TLR7 with IRS661 (P = 0.17 vs. no inhibitor) or TLR9 with IRS869 (P = 0.27 vs. no inhibitor). The specificity of IRS957 for TLR8 in human monocytes has been previously confirmed by exclusion of reactivity to TLR7 and TLR9 (32). Herein, we confirmed the specific inhibitory effect of IRS957 by demonstrating that production of IFN-β was markedly decreased in response to the TLR7/8 agonist R848 (Fig. S4) and the TLR8-specific ligand 3M-002 (Fig. S5), whereas TLR7 inhibition had no effect on 3M-002–mediated transcription of IFN-β. Moreover, the response to LPS, which induces IFN-β through TLR4 (28), was unaffected by the TLR8 inhibitor (Fig. 1A). In concert with the role of TLR8 in the production of NF-κB–dependent cytokines (3335), IRS957 also caused a significant decrease in secretion of TNF-α, IL-6, and IL-10 by Bb-infected monocytes (Fig. 1B). In parallel experiments, we verified using confocal microscopy that internalization of the spirochete was not significantly affected by blocking TLR8 (no inhibition, 19.2% ± 4.1 vs. TLR8 inhibited, 14.2% ± 1.2; P = 0.2). Taken as a whole, we interpreted these results as evidence that in Bb-infected monocytes, TLR8 cooperates with TLR2 in the production of NF-ĸB–mediated cytokines, whereas TLR8 alone is responsible for transcription of IFN-β.

Fig. 1.
Transcription of IFN-β is abrogated after TLR8 inhibition in human monocytes. (A) TLR8 inhibition by IRS-957 eliminated transcription of IFN-β in Bb-infected monocytes (10:1 multiplicity of infection), whereas a control ODN sequence did ...

We next used RT-PCR to examine the expression profile of TLR2 and TLR8 in Bb-infected monocytes and compared these responses with those elicited by synthetic TLR2 and TLR8 ligands. As shown in Table 1, live Bb differentially induced transcription of both TLR2 and TLR8, although it had no effect on TLR7 or TLR9. In parallel experiments, we demonstrated that up-regulation of both TLR2 and TLR8 was greatly diminished by blocking TLR8 (Table 1). The inhibitory effect of IRS957 on TLR8 protein levels was confirmed by Western blot analysis (Fig. S6). The regulatory effect of TLR8 stimulation on TLR2 is not at all unanticipated, given that IFN-β has been shown to up-regulate expression of TLR2 (36). In support of this idea, R848 induced transcription of both TLR8 and TLR2 (Table 1), whereas Pam3CSSNA only affected TLR2. The inhibitory effect of IRS957 on TLR8 transcription is indicative that TLR8 is self-regulated on engagement of its cognate spirochetal PAMP. These findings provide additional evidence that innate immune responses to Bb in human monocytes result from a cooperative interaction between TLR2 and TLR8.

Table 1.
TLR relative expression in stimulated human monocytes

TLR2 and TLR8 Colocalize with GFP-Bb to the Phagosomal Vacuole of the Monocyte.

Having established that phagocytosis is an essential element of the innate immune response to the spirochete and that TLR2 and TLR8 cooperate in generating phagosomal signals, we then used confocal microscopy to characterize the spatial-temporal distributions between phagocytosed Bb and TLR2 and TLR8 more precisely. We first showed that the baseline expression of TLR2 in unstimulated monocytes was greater than that of TLR8 (Fig. 2 and Fig. S7). TLR2 was visualized on the monocyte's surface as well as on the cytoplasm, whereas TLR8 was seen exclusively inside the cell. Within 30 min of coincubation with Bb, expression of both TLR2 and TLR8 increased in Bb-infected monocytes and to a lesser extent in bystander uninfected cells (Fig. 2 and Fig. S7). To capture spirochete-TLR localization at both early and later stages of phagocytosis, we analyzed multiple confocal images obtained at several times of incubation, beginning with the 30-min stimulation time point. TLR2 colocalized at the cell surface with spirochetes that were attached and/or partially embedded in the monocyte's membrane (Fig. 3A and Movie S1). TLR2 also colocalized to the phagosomal vacuole, with recently internalized spirochetes shown as tightly wound bacterial coils (Fig. 3B and Movie S2). TLR8 colocalized to phagosomal vacuoles containing fully degraded spirochetes in the shape of bright fluorescent blebs (Fig. 3C and Movie S3). Concurrent staining with TLR2 and TLR8 confirmed that both TLRs colocalize simultaneously to phagosomal vacuoles containing degraded spirochetes (Movie S4). Of particular significance, TLR2 colocalized with TLR8 in the cytoplasm of Bb-infected monocytes (Fig. 2, Inset and Movie S4), which suggests that TLR2 and TLR8 traffic to the phagosome from similar endosomal compartments. Taken together, these images provide visual evidence that Bb is initially sensed by TLR2 on the monocyte cell surface during binding and uptake, whereas partially and fully degraded spirochetes interact with both TLR2 and TLR8 in the phagosomal vacuole.

Fig. 2.
TLR2 and TLR8 cellular expression. Costaining for TLR2 (green) and TLR8 (red) is shown in unstimulated monocytes and in both infected and uninfected cells (bystander) within 30 min of incubation with Bb (10:1 multiplicity of infection). TLR2 fluorescent ...
Fig. 3.
Phagocytosed Bb colocalizes with TLR2 and TLR8 in human monocytes. A composite of representative confocal micrographs shows three consecutive Z-stack planes of GFP-Bb–stimulated human monocytes. Extracellular Bb interacts with TLR2 at the plasma ...

Live Bb Induces Transcription and Nuclear Translocation of IRF7.

We next sought to elucidate which transcription factor is associated with TLR8-mediated induction of IFN-β. We focused on IRF3, IRF5, and IRF7, all of which are associated with the induction of genes that encode type I IFNs (3741). We have previously demonstrated that IRF7 is the only IRF that is significantly up-regulated in PBMCs stimulated with live Bb (7). Herein, we show by RT-PCR that IRF7 (but not IRF3 or IRF5) is also induced in Bb-infected isolated human monocytes (Table 2). We also demonstrate that the transcriptional regulation of IRF7 is eliminated by blocking TLR8. Using epifluorescence microscopy, we then confirmed that IRF7 is translocated into the nucleus of Bb-infected monocytes and R848-stimulated cells (Fig. 4), a finding that is indicative of IRF7 activation through phosphorylation (42). A similar process did not occur in unstimulated monocytes or bystander uninfected cells. A two-step process involving IRF3 activation before activation of IRF7 does not seem plausible, because we did not observe any increase in IRF3 signal by microscopy or translocation of IRF3 into the nucleus (Fig. S8). Moreover, using Bb-infected monocytes in a time-chase experiment, we confirmed that relative increases in IRF7 sequentially preceded relative increases in IFN-β (Fig. S2). Blocking IRF7 phosphorylation using the kinase inhibitor staurosporine (43) completely abrogated IFN-β induction (Fig. S9A) and greatly impaired up-regulation of IRF7 (Fig. S9B). The amount of staurosporine used was enough to block nuclear translocation of IRF7 in the absence of apoptosis (Fig. S9C). Taken as a whole, these results provide evidence that IRF7 induces transcription of IFN-β in Bb-infected monocytes.

Table 2.
IRF Expression in Bb-stimulated human monocytes
Fig. 4.
Live Bb induces nuclear translocation of IRF7. IRF7 (red) is expressed in the cytoplasm of uninfected monocytes and translocates to the nucleus (DAPI, blue) in GFP-Bb–infected monocytes (indicated by black arrows) and in R848-stimulated controls. ...

Discussion

The experimental evidence presented herein indicates that the LD spirochete induces multifaceted innate inflammatory signals in human monocytes that can only be fully coordinated during internalization of the bacterium and formation of the phagosome. As maturation of the phagosome progresses, this unique vacuolar structure very quickly gains capabilities for the recognition of diverse spirochetal PAMPs and becomes an increasingly more efficient platform for signal generation, leading to enhanced production of NF-ĸB–mediated cytokines and MyD88-dependent induction of IFN-β. Our findings are unique in that they suggest that internalization and degradation of the spirochete by the monocyte facilitates sampling of liberated PAMPs through a sequential activation of cell surface TLR2, followed by signaling through TLR2 and TLR8 cooperatively at the phagosomal membrane.

Mammalian TLR8 is thought to signal exclusively through MyD88 after recognition of single-stranded RNA ligands (44). Human TLR8 can be activated by nucleic acids derived from a variety of RNA viruses (4548) as well as Escherichia coli RNA in genetically complemented HEK293 cells (49). To date, TLR8 has been linked to the recognition of two different intracellular bacteria, Mycobacterium bovis bacille Calmette–Guérin (bacillus Calmette–Guérin) (35) and Helicobacter pylori (50). In the case of bacillus Calmette–Guérin, TLR8 protein expression levels markedly increased in infected macrophages derived from a human acute monocytic leukemia cell line (THP-1 cells). Interestingly, a significant boost in TLR8 expression levels was also seen in the blood of patients with untreated Mycobacterium tuberculosis infection (50). Using activated THP-1 cells, Gantier et al. (35) demonstrated that expression of the two main human TLR8 variant mRNAs significantly increased following H. pylori phagocytosis. In this study, we reveal a previously unappreciated direct role for human TLR8 in transcription of IFN-β in Bb-infected monocytes and cooperativity with TLR2 in the production of several NF-κB–dependent cytokines. We hypothesize that endosomal TLR8 is activated in response to spirochetal mRNA released upon degradation of the bacterium into the phagosomal cargo. In contrast to bacillus Calmette–Guérin and H. pylori, Bb is an extracellular pathogen that is rapidly degraded once phagocytosed; thus, it is not surprising that engagement of TLR8 in the monocytes occurred soon after internalization of the bacterium. Given that induction of type I IFNs in Bb-infected pDCs is partially dependent on TLR9 (22), it is conceivable that borrelial DNA also may play a role in activating TLR8 in human monocytes. This premise is supported by the recent finding that murine TLR8, which is functional in mice in the context of a viral infection (51), can be activated by vaccinia virus (VV) DNA through recognition of poly(A)T-rich motifs (52). Like VV DNA, borrelial DNA also contains poly(A)T-rich motifs (53).

TLR2–spirochetal PAMP interactions were originally studied as cell surface phenomena (54). Several studies have now demonstrated that TLR2 is recruited to the phagosome, where it becomes available for signaling (14, 5557). Structurally, this model is not at all unanticipated, given that the phagosomal membrane is initially derived from the plasma membrane (58, 59). Consistent with this concept, Underhill et al. (55) found that in a mouse leukemic monocyte macrophage cell line (RAW-cells), cell surface TLR2 was recruited to phagosomes and colocalized with zymosan particles within minutes of their internalization, suggesting that TLR2-mediated signals occur within a circumscribed window of time during the initial phase of phagosome maturation. Studies conducted in Stephanie Vogel's laboratory (60), however, have revealed that prolonged TLR2-dependent signaling from the phagosomal vacuole takes place in murine macrophages infected with a mutant strain of Francisella tularensis (Ft), which is unable to escape the phagosome. The same group demonstrated that TLR2 and MyD88 colocalize to the phagosomal vacuole of RAW macrophages infected with a live vaccine Ft strain (14). TLR2 also can become available for signaling from within endosomal vacuoles in human monocytes stimulated with internalized staphylococcal lipoteichoic acid (56). O'Connell et al. (57) demonstrated that intracellular TLR2 was responsible for the initiation of signal transduction events by Chlamydia trachomatis. Herein, we validate that TLR2 senses live spirochetes at the plasma membrane during binding and uptake and provide previously undescribed evidence that TLR2 is also available for signaling at the phagosomal membrane of human monocytes. The finding that TLR2 colocalizes with TLR8 intracellularly in areas other than the phagosome (Fig. 4, region of interest) suggests that TLR2 traffics to the phagosomal vacuole from two different locations: most certainly from the plasma membrane but also from an as yet undefined cytosolic location/structure, similar to the intracellular trafficking of TLR8. Taken as a whole, we envision a model in which a superior TLR2-receptor density would become available for signaling in phagosomal vacuoles than can occur at the plasma membrane. The presence of specific “docking” molecules within the budding phagosomal vacuole would thus provide an effective scaffold for inserting spirochetal lipoproteins proximal to their cognate receptors. A higher receptor density, in combination with a large concentration of exposed and liberated lipoproteins, would thus greatly improve TLR2 signaling efficiency.

Although cross-talk between TLR2 and TLR8 occurs following stimulation of PBMCs by their individual TLR ligands (61), the transcriptional and translational responses shown here distinctively demonstrate that coordination between TLR2 and TLR8 does occur in response to a live bacterium. How these integrated responses facilitate control of the LD spirochete and whether or not they are beneficial or detrimental to individuals with LD have only begun to be uncovered. Evidence from recently published studies suggests that endosomal TLR activation, including TLR8, can have an impact on the ability of the host cell to respond to stimulation of a different TLR (62, 63), including TLR2 (62). Given that type I IFNs can shape a variety of downstream inflammatory responses through positive and/or negative regulation of the expression of hundreds of additional genes involved in secondary host defenses, TLR8 activation is likely to play a critical role in clearance of the spirochete and, most importantly, disease control. Type I IFNs also can promote inflammatory cell death of host cells (50, 64), a phenomenon that we previously described in Bb-infected monocytes (4), and thus may constitute an additional mechanism for controlling spirochetal replication. Whether or not the presence of tlr8 SNPs in patients with LD affects spirochetal clearance and/or dissemination, and whether these SNPs are linked to disease progression and/or the severity of the initial clinical presentation, needs to be studied.

Materials and Methods

Human Monocyte Isolation and Stimulation Conditions.

All procedures involving human subjects were approved by the Institutional Review Board at the University of Connecticut Health Center. After obtaining written informed consent, monocytes were isolated from LD-seronegative volunteers as previously described (7). Except where noted, cells were incubated for 4 h at 37 °C/5% (vol/vol) CO2 with live, low-passage, temperature-shifted GFP-Bb 1479 at various multiplicities of infection. Appropriate controls, including a synthetic TLR2 ligand (Pam3CSSNA), were used in the ex vivo stimulation model as previously described (7). All culture media and reagents were confirmed to be essentially free of LPS contamination (<10 pg/mL) by Limulus amoebocyte lysate assay (Cambrex). In selected experiments, Cytochalasin-D was used as previously described (2).

Endosomal TLR Blocking.

Specific immunoregulatory DNA sequences (IRSs; Integrated DNA Technologies) (31) were used to block TLR7 signaling (IRS661: 5′-TGCTTGCAAGCTTGCAAGCA-3′), TLR7 and TLR8 (IRS957: 5′-TGCTTG-ACATCCTGGAGGGGTTGT-3′), and TLR9 (IRS869: 5′-TCCTGGAGGGGTTGT-3′) as described by Petzke et al. (22). A non–TLR-specific oligodeoxyribonucleotide sequence was used as a control, in the same concentration as the TLR inhibitor. R848 (Resiquimod; InvivoGen), a known synthetic ligand for human TLR7 and TLR8 (32) and a potent inducer of IFN-β (65), was used to study the response in monocytes. The specificity of the TLR7 and TLR8 inhibitors was assessed with the TLR8-specific ligand 3M-002 (InvivoGen) (66).

Assessment of Monocyte Activation by Quantitative RT-PCR and Cytokine Secretion.

At the conclusion of the 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 RT kit (Applied Biosystems). RT-PCR conditions and reactions have been described previously (7). Commercially available primers (Applied Biosystems) used for amplification included the following: ifnb (Hs00277188_s1), tlr2 (Hs00610101_m1), tlr7 (Hs00152971_m1), tlr8 (Hs00152972_m1), tlr9 (Hs00152973_m1), irf3 (Hs01547283_m1), irf5 (Hs00158114_m1), irf7 (Hs00185375_m1), and gapdh (Hs99999905_m1). Expression levels of all transcripts studied were normalized to the GAPDH level, and the relative changes in gene expression generated were calculated using the 2−ΔΔCT method. A cytokine bead array system (Human inflammation kit; Becton Dickinson) was used to assay cytokines secreted by TNF-α, IL-10, IL-6, and IL-1β in culture supernatants. Secretion of IFN-β was determined using a commercially available ELISA method (VeriKine-HS; PBL Biomedical Laboratories).

Western Blot Analysis of TLR8 Protein Expression.

TLR8 expression was measured by Western blot analysis. Briefly, 40 μg of protein was loaded in each correspondent lane, and protein was transferred to a PVDF membrane. After blocking, the membrane was probed with TLR8 antibody (catalog no. H00051311-MO1; Abnova). Membranes were washed, and secondary antibodies were added. After 1 h, membranes were washed with PBS-Tween and developed. The same membrane was stripped, reblocked, reprobed, and developed for human GAPDH (Santa Cruz Biotechnology, Inc.) Densitometric analysis of obtained bands was done using a Flurchem 8900 Chemiluminescence and Gel Imager (Alpha Innotech).

Cellular Localization of IRF7/IRF3, TLR8/TLR2, and GFP-Bb.

To determine the cellular localization of TLR2 and TLR8 in Bb-infected monocytes, we used a modified version of the method described by Ma et al. (67). Briefly, monocytes were plated in poly-d-lysine–treated culture slides (BioCoat; BD Biosciences). Following different time points of stimulation (0 min, 30 min, 60 min, 90 min, 3 h, and 4 h), slides were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature (RT); permeabilized with 0.2% saponin for 10 min; blocked with Connaught Medical Research Laboratories (CMRL) 10% FCS for 2 h at RT; incubated overnight at 4 °C with a rabbit anti-human IRF7 polyclonal or mouse anti-human IRF3 monoclonal antibody (Santa Cruz Biotechnology, Inc.), a rabbit anti-TLR8 polyclonal antibody (IMGENEX), or a rabbit anti-TLR2 polyclonal antibody (Rockland Immunochemicals) (1:100 dilution for all); and subsequently incubated with Texas RedR-X–conjugated goat anti-rabbit antibody (1:200 dilution; Invitrogen) for 1 h at RT or goat anti-mouse DyLight 594 (Thermo Scientific). For TLR2 and TLR8 immunofluorescent costaining, we used the following antibodies: a primary monoclonal mouse antibody to human TLR8 (IMGENEX) followed by goat anti-mouse AlexaFluor 546 (Invitrogen) and a primary polyclonal rabbit anti-TLR2 antibody (Rockland Immunochemicals) followed by goat anti-rabbit AlexaFluor 635 (Invitrogen). Slides were washed and mounted in Vectashield antifade reagent (Invitrogen) containing DAPI. Images were acquired on an LSM-510 confocal microscope using a 63× (1.4 N.A.) oil immersion objective at a pixel resolution of 512 × 512. Image analysis was performed using ImageJ (National Institutes of Health); pixel colocalization was displayed using the colocalization plug-in. Mander's overlap colocalization coefficients (M1 and M2) were determined using the JACoP plug-in (68). Movies were generated using ImageJ and Quicktime Pro software (Apple, Inc.). To measure TLR2 and TLR8 cellular expression, ~100 images of human monocytes, stained for either TLR2 or TLR8, were acquired at a magnification of 100× in an Olympus BX-41 epifluorescence microscope equipped with a digital camera (Q Imaging), using equal exposure time and gain parameters. Regions of interest corresponding to individual cellular contours were drawn using the Freehand selection tool of the ImageJ software, and mean fluorescence intensity and cellular area were calculated.

Statistical Methods.

General statistical analysis was performed using GraphPad Prism 4.0. The fold increase or decrease for each specific gene transcript assayed by quantitative RT-PCR and cytokine concentrations was compared among the different stimuli using either a paired or unpaired Student's t test or the equivalent nonparametric method (i.e., Mann–Whitney U test). To analyze changes over time, we used a nonparametric statistical test for trend analysis (Kruskal–Wallis test), because a Gaussian distribution could not be assumed. A P value <0.05 was considered significant.

Supplementary Material

Supporting Information:

Acknowledgments

We thank the staff, Tom Kiely in particular, at the General Clinical Research Center at the University of Connecticut Health Center for their help in recruiting and enrolling healthy volunteers into our study. We thank Drs. J. Anguita and P. von Landenberg for their technical assistance. This work was supported, in part, by Public Health Service Grants AI-62439-03 (to J.C.S.), AI-090166-01 (to J.C.S.), AI-29735 (to J.D.R.), M01RR006192 (to the University of Connecticut Health Center), AI-054546 (to T.J.S.), U54 AI057159 (to S.M.D.-E.); and by the Connecticut Children's Medical Center's Arrison and Burr Curtis Research Funds (to J.C.S.).

Footnotes

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1013776108/-/DCSupplemental.

References

1. Bacon RM, Kugeler KJ, Mead PS. Centers for Disease Control and Prevention (CDC) Surveillance for Lyme disease—United States, 1992-2006. MMWR Surveill Summ. 2008;57:1–9. [PubMed]
2. Moore MW, et al. Phagocytosis of Borrelia burgdorferi and Treponema pallidum potentiates innate immune activation and induces gamma interferon production. Infect Immun. 2007;75:2046–2062. [PMC free article] [PubMed]
3. Salazar JC, et al. (2003) Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans. J Immunol. 171:2660–2670. [PubMed]
4. Cruz AR, et al. Phagocytosis of Borrelia burgdorferi, the Lyme disease spirochete, potentiates innate immune activation and induces apoptosis in human monocytes. Infect Immun. 2008;76:56–70. [PMC free article] [PubMed]
5. Montgomery RR, Nathanson MH, Malawista SE. The fate of Borrelia burgdorferi, the agent for Lyme disease, in mouse macrophages. Destruction, survival, recovery. J Immunol. 1993;150:909–915. [PubMed]
6. Norris SJ, Coburn J, Leong JM, Hu LT, Hook M. Pathobiology of Lyme Disease. In: Samuels DS, Radolf JD, editors. Borrelia: Molecular Biology, Host Interaction and Pathogenesis. Norfolk, United Kingdom: Caister Academic; 2010. pp. 299–331.
7. Salazar JC, et al. Activation of human monocytes by live Borrelia burgdorferi generates TLR2-dependent and -independent responses which include induction of IFN-beta. PLoS Pathog. 2009;5:e1000444. [PMC free article] [PubMed]
8. Shin OS, et al. Distinct roles for MyD88, TLR2, TLR5, and TLR9 in phagocytosis of Borrelia burgdorferi and inflammatory signaling. Infect Immun. 2008;76:2341–2351. [PMC free article] [PubMed]
9. Dennis VA, et al. Live Borrelia burgdorferi spirochetes elicit inflammatory mediators from human monocytes via the Toll-like receptor signaling pathway. Infect Immun. 2009;77:1238–1245. [PMC free article] [PubMed]
10. Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors. Nat Immunol. 2010;11:373–384. [PubMed]
11. Monroe KM, McWhirter SM, Vance RE. Induction of type I interferons by bacteria. Cell Microbiol. 2010;12:881–890. [PMC free article] [PubMed]
12. Stetson DB, Medzhitov R. Type I interferons in host defense. Immunity. 2006;25:373–381. [PubMed]
13. Reimer T, Schweizer M, Jungi TW. Type I IFN induction in response to Listeria monocytogenes in human macrophages: Evidence for a differential activation of IFN regulatory factor 3 (IRF3) J Immunol. 2007;179:1166–1177. [PubMed]
14. Cole LE, et al. Toll-like receptor 2-mediated signaling requirements for Francisella tularensis live vaccine strain infection of murine macrophages. Infect Immun. 2007;75:4127–4137. [PMC free article] [PubMed]
15. Mancuso G, et al. Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells. Nat Immunol. 2009;10:587–594. [PubMed]
16. Charrel-Dennis M, et al. TLR-independent type I interferon induction in response to an extracellular bacterial pathogen via intracellular recognition of its DNA. Cell Host Microbe. 2008;4:543–554. [PMC free article] [PubMed]
17. Gratz N, et al. Group A streptococcus activates type I interferon production and MyD88-dependent signaling without involvement of TLR2, TLR4, and TLR9. J Biol Chem. 2008;283:19879–19887. [PMC free article] [PubMed]
18. Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clin Microbiol Rev. 2009;22:240–273. [PMC free article] [PubMed]
19. Miller JC, Ma Y, Crandall H, Wang X, Weis JJ. Gene expression profiling provides insights into the pathways involved in inflammatory arthritis development: Murine model of Lyme disease. Exp Mol Pathol. 2008;85:20–27. [PMC free article] [PubMed]
20. Miller JC, et al. A critical role for type I IFN in arthritis development following Borrelia burgdorferi infection of mice. J Immunol. 2008;181:8492–8503. [PMC free article] [PubMed]
21. Miller JC, Maylor-Hagen H, Ma Y, Weis JH, Weis JJ. The Lyme disease spirochete Borrelia burgdorferi utilizes multiple ligands, including RNA, for IRF3-dependent induction of type I IFN-responsive genes. Infect Immun. 2010;78:3144–3153. [PMC free article] [PubMed]
22. Petzke MM, Brooks A, Krupna MA, Mordue D, Schwartz I. Recognition of Borrelia burgdorferi, the Lyme disease spirochete, by TLR7 and TLR9 induces a type I IFN response by human immune cells. J Immunol. 2009;183:5279–5292. [PubMed]
23. Kawai T, Akira S. TLR signaling. Cell Death Differ. 2006;13:816–825. [PubMed]
24. Kawai T, Akira S. TLR signaling. Semin Immunol. 2007;19:24–32. [PubMed]
25. Chuang TH, Ulevitch RJ. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw. 2000;11:372–378. [PubMed]
26. Latz E, Visintin A, Espevik T, Golenbock DT. Mechanisms of TLR9 activation. J Endotoxin Res. 2004;10:406–412. [PubMed]
27. Hornung V, et al. Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J Immunol. 2002;168:4531–4537. [PubMed]
28. Toshchakov V, et al. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol. 2002;3:392–398. [PubMed]
29. Barbalat R, Lau L, Locksley RM, Barton GM. Toll-like receptor 2 on inflammatory monocytes induces type I interferon in response to viral but not bacterial ligands. Nat Immunol. 2009;10:1200–1207. [PMC free article] [PubMed]
30. Dietrich N, Lienenklaus S, Weiss S, Gekara NO. Murine toll-like receptor 2 activation induces type I interferon responses from endolysosomal compartments. PLoS ONE. 2010;5:e10250. [PMC free article] [PubMed]
31. Barrat FJ, et al. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med. 2005;202:1131–1139. [PMC free article] [PubMed]
32. Hurst J, et al. TLR7 and TLR8 ligands and antiphospholipid antibodies show synergistic effects on the induction of IL-1beta and caspase-1 in monocytes and dendritic cells. Immunobiology. 2009;214:683–691. [PubMed]
33. Bekeredjian-Ding I, et al. T cell-independent, TLR-induced IL-12p70 production in primary human monocytes. J Immunol. 2006;176:7438–7446. [PubMed]
34. Heil F, 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;33:2987–2997. [PubMed]
35. Gantier MP, et al. Genetic modulation of TLR8 response following bacterial phagocytosis. Hum Mutat. 2010;31:1069–1079. [PubMed]
36. Guo Z, et al. A distal regulatory region is required for constitutive and IFN-beta-induced expression of murine TLR9 gene. J Immunol. 2005;175:7407–7418. [PubMed]
37. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science. 2004;303:1529–1531. [PubMed]
38. Schoenemeyer A, et al. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J Biol Chem. 2005;280:17005–17012. [PubMed]
39. Pandey AK, et al. NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis. PLoS Pathog. 2009;5:e1000500. [PMC free article] [PubMed]
40. Yasuda K, et al. Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production. J Immunol. 2007;178:6876–6885. [PubMed]
41. Stetson DB, Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity. 2006;24:93–103. [PubMed]
42. Honda K, Taniguchi T. IRFs: Master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol. 2006;6:644–658. [PubMed]
43. Smith EJ, Marié I, Prakash A, García-Sastre A, Levy DE. IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked by Vaccinia virus E3L protein. J Biol Chem. 2001;276:8951–8957. [PubMed]
44. Diebold SS. Recognition of viral single-stranded RNA by Toll-like receptors. Adv Drug Deliv Rev. 2008;60:813–823. [PubMed]
45. Gringhuis SI, et al. HIV-1 exploits innate signaling by TLR8 and DC-SIGN for productive infection of dendritic cells. Nat Immunol. 2010;11:419–426. [PubMed]
46. Forsbach A, et al. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J Immunol. 2008;180:3729–3738. [PubMed]
47. Triantafilou K, et al. TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1. Eur J Immunol. 2005;35:2416–2423. [PubMed]
48. Wang JP, et al. Toll-like receptor-mediated activation of neutrophils by influenza A virus. Blood. 2008;112:2028–2034. [PMC free article] [PubMed]
49. Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005;23:165–175. [PubMed]
50. Davila S, et al. Genetic association and expression studies indicate a role of toll-like receptor 8 in pulmonary tuberculosis. PLoS Genet. 2008;4:e1000218. [PMC free article] [PubMed]
51. Gorden KK, Qiu XX, Binsfeld CC, Vasilakos JP, Alkan SS. Cutting edge: Activation of murine TLR8 by a combination of imidazoquinoline immune response modifiers and polyT oligodeoxynucleotides. J Immunol. 2006;177:6584–6587. [PubMed]
52. Martinez J, Huang X, Yang Y. Toll-like receptor 8-mediated activation of murine plasmacytoid dendritic cells by vaccinia viral DNA. Proc Natl Acad Sci USA. 2010;107:6442–6447. [PMC free article] [PubMed]
53. Gautam A, Hathaway M, Ramamoorthy R. The Borrelia burgdorferi 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. 2009;293:278–284. [PMC free article] [PubMed]
54. Lien E, et al. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem. 1999;274:33419–33425. [PubMed]
55. Underhill DM, et al. The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature. 1999;401:811–815. [PubMed]
56. Nilsen NJ, et al. Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling: Role of CD14 and CD36. J Leukoc Biol. 2008;84:280–291. [PMC free article] [PubMed]
57. O'Connell CM, Ionova IA, Quayle AJ, Visintin A, Ingalls RR. Localization of TLR2 and MyD88 to Chlamydia trachomatis inclusions. Evidence for signaling by intracellular TLR2 during infection with an obligate intracellular pathogen. J Biol Chem. 2006;281:1652–1659. [PubMed]
58. Desjardins M. ER-mediated phagocytosis: A new membrane for new functions. Nat Rev Immunol. 2003;3:280–291. [PubMed]
59. Stuart LM, Ezekowitz RA. Phagocytosis: Elegant complexity. Immunity. 2005;22:539–550. [PubMed]
60. Cole LE, et al. Macrophage proinflammatory response to Francisella tularensis live vaccine strain requires coordination of multiple signaling pathways. J Immunol. 2008;180:6885–6891. [PMC free article] [PubMed]
61. Ghosh TK, et al. TLR-TLR cross talk in human PBMC resulting in synergistic and antagonistic regulation of type-1 and 2 interferons, IL-12 and TNF-alpha. Int Immunopharmacol. 2007;7:1111–1121. [PubMed]
62. Mureith MW, Chang JJ, Lifson JD, Ndung'u T, Altfeld M. Exposure to HIV-1-encoded Toll-like receptor 8 ligands enhances monocyte response to microbial encoded Toll-like receptor 2/4 ligands. AIDS. 2010;24:1841–1848. [PMC free article] [PubMed]
63. Wang J, et al. The functional effects of physical interactions among Toll-like receptors 7, 8, and 9. J Biol Chem. 2006;281:37427–37434. [PubMed]
64. Lehner M, Felzmann T, Clodi K, Holter W. Type I interferons in combination with bacterial stimuli induce apoptosis of monocyte-derived dendritic cells. Blood. 2001;98:736–742. [PubMed]
65. Yang K, et al. Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda is IRAK-4 dependent and redundant for protective immunity to viruses. Immunity. 2005;23:465–478. [PubMed]
66. Gorden KB, et al. Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8. J Immunol. 2005;174:1259–1268. [PubMed]
67. Ma Y, et al. Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. J Cell Biol. 2006;175:209–215. [PMC free article] [PubMed]
68. Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006;224:213–232. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...