The Journal of Immunology

Human Infected with Yersinia pestis Express Cell Surface TLR9 and Differentiate into Dendritic Cells1

Kamal U. Saikh,2 Teri L. Kissner, Afroz Sultana, Gordon Ruthel, and Robert G. Ulrich2

TLR9 recognizes DNA sequences containing hypomethylated CpG motifs and is a component of the highly conserved during eukaryotic evolution. Previous reports suggested that the expression of TLR9 is restricted to plasmacytoid dendritic cells and B . Our results indicate that low levels of TLR9 are present on the cell surface of freshly isolated human monocytes, and expression is greatly increased by infection with Yersinia pestis. Enhanced cell surface TLR9 coincided with elevated levels of cytoplasmic TLR9 and recruitment of MyD88. Infected monocytes differentiated into mature dendritic cells, expressed IFN-␣, and stimulated proliferative and cytotoxic responses specific to Y. pestis. Furthermore, uninfected B cells and monocytes both increased cell surface TLR9, CD86, and HLA-DR in response to treatment with CpG-containing oligonu- cleotides, whereas cell surface TLR9 was down-modulated on infected dendritic cells by the addition of agonist oligonucleotide. Our results suggest that increased expression of TLR9 on the surface of infected cells may serve a role as an activation signal to other cells of the immune system. The Journal of Immunology, 2004, 173: 7426–7434.

ost human infections caused by the Gram-negative ba- phenotype (10, 11). DC and other cells are activated when TLRs cillus Yersinia pestis are of the bubonic form, arising and other innate immune receptors respond to pathogen-associated M after the bite of a flea that previously fed on the blood molecular patterns (PAMPs) recognized on macromolecules of of an infected rodent (1). However, some cases of bubonic plague pathogens, for example lipids, , and nucleic acids (12). The progress to a pneumonic form, which has a high transmission and TLRs are defined by the presence of a cytosolic Toll/IL-1R do- fatality rate. Inhaling an infectious aerosol can also cause direct main, involved in , and an extracellular ligand- progression to pneumonic plague (2, 3). Monocytes and macro- binding domain comprised of leucine-rich repeats. Signals trans- phages are poised to interact with plague bacilli immediately upon duced through most TLRs trigger the induction of key injection of the bacteria by a flea bite (4, 5). At the earliest stage inflammatory and immune by activation of a common mo- of infection, Yersinia pestis bacilli are readily phagocytosed (6) lecular pathway that is anchored by the adaptor MyD88, and can grow in the phagolysozome of infected monocytes (5). and involves NF-␬B, MAPK p38, and JNK (13). In addition, cells Multiple virulence factors targeting host immunity are injected discriminate between different types of pathogens by combining into eukaryotic cells by Y. pestis using a cell contact-dependent TLR signals (14, 15). secretion apparatus (7) found also in Escherichia coli, Salmonella, TLR9 is an integral membrane protein (16) associated with en- Shigella spp., and several other pathogenic bacteria. Following a dosomal maturation (17) and is observed on the cell surface of B brief intracellular stage, Y. pestis proliferate extracellularly in lym- lymphocytes. In contrast, TLR9 expression by resting mouse mac- phatic tissues (8). The contribution of monocytes and - rophages is intracellular and restricted to endosomes (18). The derived dendritic cells (DCs)3 to controlling disease progression is presence of multiple leucine-rich repeats and a cysteine-rich do- unclear. main in all TLRs suggests a degree of conservation in protein Rapid DC recruitment is a hallmark of the acute inflammatory structure. However, there are no obvious sequence motifs indica- response to microbial infections (9). Activated DCs contribute sub- tive of sequestration in any one cellular organelle. Indeed, trans- stantially to inflammatory responses and subsequent resolution of fected cells expressing TLR9 on the cell surface respond to hy- infections, while infection may cause the DC to exhibit a mature pomethylated CpG-containing oligonucleotides (CpG NT) equivalently to naturally expressed receptors (16), confirming that the site of cellular localization does not affect biological activity. All TLRs, with the exception of TLR7 and TLR9, were reported to U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD 21702 be present in human myeloid DCs, whereas TLR7 and TLR9 were Received for publication June 23, 2004. Accepted for publication October 4, 2004. selectively expressed by plasmacytoid DCs (19–21), representing The costs of publication of this article were defrayed in part by the payment of page a possible dichotomy in function for these two DC subsets. Be- charges. This article must therefore be hereby marked advertisement in accordance cause activation of plasmacytoid DCs by TLR9 agonists results in with 18 U.S.C. Section 1734 solely to indicate this fact. release of type I IFNs (19), these cells are considered to be the 1 The views in this paper are those of the authors and do not purport to reflect official policy of the U.S. Government. primary in vivo targets for mediating the adjuvant effects of bac- 2 Address correspondence and reprint requests to Dr. Kamal U. Saikh or Dr. Robert terial DNA and synthetic CpG NT in humans. However, the mech- G. Ulrich, Laboratory of Molecular Immunology, U.S. Army Medical Research In- anisms controlling cell surface expression of TLR9 in B lympho- stitute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702. E-mail ad- cytes and the apparent intracellular retention of this in dress: [email protected] or [email protected] plasmacytoid DCs are currently unknown. Furthermore, insuffi- 3 Abbreviations used in this paper: DC, ; BSM, B lymphoblastoid cell line; DAPI, 4Ј,6-diamidino-2-phenylindole; CpG NT, hypomethylated CpG-contain- cient data are available concerning the cellular distribution and ing oligonucleotide; PAMP, pathogen-associated molecular pattern. function of TLR9 in actively infected cells. Although monocytes

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 7427 are important scavengers and hosts for intracellular infection with Cell isolation and culture Y. pestis, they are also an abundant source of precursors for mac- PBMC were obtained from consenting healthy donors. Blood components rophages and DCs. Our results indicate that human monocytes for in vitro research use were collected from healthy, consented volunteers express TLR9 and respond to infection by elevating cytoplasmic in accordance with the guidelines of an Institutional Review Board-ap- and cell surface levels of this innate immune receptor. In addition, proved research donor protocol. cell surface TLR9 expression may function as a proinflammatory Mononuclear cells were isolated by standard density gradient centrifu- gation with Ficoll-Hypaque (Pharmacia). Mononuclear cells were har- activation marker during the transition from monocyte to DCs. vested from the interface, washed, and suspended in RPMI 1640 medium. Monocytes (CD14ϩ) and B cells (CD19ϩ) were isolated, as previously Materials and Methods described (23). Briefly, unseparated mononuclear cells were suspended (107 cells/80 ␮l) in cold PBS supplemented with 2 mM EDTA and 0.5% Reagents BSA (fraction V; Sigma-Aldrich, St. Louis, MO). Paramagnetic beads Pooled human AB sera were obtained from Pel-Freez Biologicals (Brown Deer, WI). Mouse anti-human CD14, CD19, and CD3 mAbs conjugated with magnetic beads were purchased from Miltenyi Biotec (Auburn, CA). The FITC-conjugated mAbs Leu M3 (anti-CD14), Leu HLA-DR (anti- DR), anti-human CD19, anti-CCR7, and Ig isotype control Abs were pur- chased from BD Biosciences (San Jose, CA). Goat anti-mouse IgG, FITC labeled, was purchased from Zymed Laboratories (San Francisco, CA). PE-conjugated anti-CD83 and FITC-conjugated anti-CD86 were purchased from BD Pharmingen (San Diego, CA). Primary mAbs for TLR9, TLR2, and TLR4 were obtained from Active Motif (Carlsbad, CA) or from e- Biosciences (San Diego, CA). Anti-MyD88 Ab was obtained from Neo Markers (Freemont, CA). IL-15 was purchased from PeproTech (Rocky Hill, NJ). Ficoll-Hypaque was purchased from Pharmacia (Uppsala, Swe- den). CpG-NT 10103 is a previously reported (22) type B CpG oligonu- cleotide specifically optimized for vaccine applications and kindly pro- vided by C. Roy (U.S. Army Medical Research Institute of Infectious Diseases). Vectashield mounting medium containing 4Ј,6-diamidino-2- phenylindole (DAPI) was purchased from Vector Laboratories (Burlin- game, CA). Monoclonal (YPF19) mouse anti-F1 Ag Y. pestis was obtained from Research Diagnostics (Flanders, NJ).

FIGURE 2. Infection with Y. pestis transforms monocytes to DCs. A, Cell surface expression of DC markers on infected monocytes. Monocytes were harvested 48 h after infection and incubated with FITC-labeled anti HLA-DR, FITC-labeled anti-CD86, PE-labeled anti-CD83, or appropri- ately labeled isotype control mAbs. Alternatively, cells were labeled with an anti-CCR7 mAb, followed by secondary labeling with an FITC-conju- gated goat anti-mouse Ab. Ab binding was measured by flow cytometry. Thin lines represent isotype-matched control Abs, and thick lines represent FIGURE 1. Intracellular infection of human primary monocytes with cell surface Ag expression. B, Transcriptional activation of IFN-␣ by Y. ϩ Y. pestis. Monocytes (CD14ϩ) were infected with Y. pestis (50 bacteria/ pestis-infected monocytes. CD14 monocytes were infected with Y. pestis cell) for1hat37°C. Cells were then fixed, permeabilized, and stained (50 bacteria/cell) for1hat37°C, washed, and cultured for 24 h with RPMI with primary mouse anti-F1 Ab, followed by secondary goat anti-mouse 1640 containing 5% human AB serum. Total RNA was extracted from FITC-conjugated Ab to detect bacteria and Texas Red-conjugated pha- infected cells, and transcriptional activation of IFN-␣ was measured by loidin for cellular actin, and examined under confocal microscopy. RT-PCR. Results shown are representative of four experiments performed Bar ϭ 5 ␮M. with cells from four different donors. 7428 Y. pestis INFECTION OF MONOCYTES UP-REGULATES TLR9

anti-CD14 mAb, followed by separation in a magnetic column (as above). CD14ϩ monocytes were incubated 48 h with 100 ng/ml rIL-15 (Pepro- Tech) to prepare DCs, as previously described (23). Cell purities were Ͼ98% for isolated mononuclear cell subsets used in all reported experi- ments. Although representative results are presented, all experiments were performed at least three times.

Bacterial strain and growth conditions The attenuated Y. pestis strain CO92, PgmϪ, PlaϪ was previously de- scribed (24). Bacteria were grown from a single colony in heart infusion broth (Difco Laboratories, Detroit, MI) supplemented with 0.2% xylose

and 2.5 mM CaCl2. The cultures were grown for 12 h at 37°C, and bacteria were pelleted by centrifugation and washed with RPMI 1640 medium be- fore use in experiments.

Infection of monocytes, intracellular staining, and confocal microscopy Freshly isolated CD14ϩ monocytes were allowed to adhere to sterile glass coverslips in a 6-well cell culture plate (Corning Glass, Corning, NY) and FIGURE 3. Proinflammatory production by Y. pestis-infected were then incubated (1 h, 37°C) with 0.1 ml of a Y. pestis suspension in ϩ monocytes. Monocytes (CD14 ) were infected with Y. pestis (50 bacteria/ RPMI 1640 medium. Cells were gently washed with PBS (pH 7.4, 37°C) cell) for 1 h (37°C), washed, and cultured (24 h in RPMI 1640, 5% human to remove nonadherent cells and bacteria, and were fixed with 1% para- AB serum). Cells were harvested, and culture supernatants were tested for formaldehyde (Tousimis Research, Rockville, MD), 0.1% glutaraldehyde . Results shown in the figure were from one representative ex- (Sigma-Aldrich) in PBS (5 min, 20°C). The fixed cells were washed with periment of three, using separate donors. PBS containing 0.5% BSA and incubated (30 min, 20°C) in a permeabi- lization solution (BD Biosciences). Cells were then washed and incubated (15 min, 20°C) with PBS containing 5% BSA to block nonspecific Ab binding. Cells were incubated with labeled primary anti-F1 Ab for 1 h, washed (PBS, 0.5% BSA), followed by FITC-conjugated goat anti-mouse coated with anti-CD3 or anti-CD19 mAbs (Miltenyi Biotec) were mixed secondary Ab, and counterstained with DAPI to detect bacteria within the with the mononuclear cells (107 cells/20 ␮l). The Ab-labeled cells were monocytes. Cells were mounted by inverting the coverslips on a slide, and incubated for 15 min (4°C), washed, and passed through a type LS or MS labeled cells were imaged using a Bio-Rad (Hemel Hempstead, U.K.) Ra- iron-fiber column placed within a strong magnetic field (Miltenyi Biotec). diance 2000 MP laser scanning confocal system connected to a Nikon CD19ϩ B cells bound to the column were eluted with buffer. Monocytes (Melville, NY) TE 300 inverted microscope. FITC fluorescence was visu- were isolated from cells that were not bound to the anti-CD3 column, using alized using 488-nm wavelength excitation from a krypton/argon laser, and

FIGURE 4. Monocytes and DCs infected with Y. pestis activate Ag-specific T cells. Monocytes (CD14ϩ) or mature DCs (HLA-A2) were infected with Y. pestis. Gentamicin (10 ␮g/ml) was added to cultures after2hofinfection. Monocytes or DCs were cultured for 26 h and washed extensively to remove extracellular bacteria before adding autologous T cells to all cultures. Primary T cell cultures were then collected after 6 days of incubation. Proliferation to purified recombinant Yersinia F1 Ag presented by fresh autologous DCs was measured after an additional 6 days of culture by incorporation of [3H]thymidine. Alternatively for CTL assays, specific BSM target cells (HLA-A2) were sensitized by incubating (1 h, 37°C) with the F1 Ag of Y. pestis. Target cells (5 ϫ 103/well) were cultured (4 h; 100 ␮l volume) with activated T cells. Final Ag concentration ϭ 10 ␮g/ml. Target cell lysis was assessed by measuring ATP remaining in metabolically active cells after CTL killing, followed by subtraction of experimental from maximum lysis of target cells. The data represent stimulation index or percent cytotoxicity Ϯ SE of means of triplicate wells. Statistical analyses of proliferation of T cells (cpm) without or with F1 Ag (1, 10, and 100 ␮g/ml), the two-tailed p value from Student’s t test equals 0.0103, 0.0001, and 0.08, respectively. Statistical analyses of percent of cytotoxicity, i.e., cytolysis of target cells pulsed without or with F1 Ag at target:effector 1:1, 1:5, and 1:10 were all statistically significant, the two-tailed p value from Student’s t test Ͻ0.05. Results presented in the figure were representative of three experiments and three separate donors. The Journal of Immunology 7429

FIGURE 5. Increased expression of TLR9 and MyD88 in human monocytes infected with Y. pestis. A, Differential cell surface expression of CD14 and TLRs on infected or control monocytes treated with CpG NT. Monocytes (CD14ϩ) were infected with Y. pestis (50 bacteria/cell) for1hat37°C, washed, and cultured for 48 h in RPMI 1640 containing 5% human AB serum. The monocytes were harvested and labeled with PE-conjugated anti-CD14, or alternatively, cells were labeled with anti-TLR2, anti-TLR4, or anti-TLR9 mAbs, followed by secondary labeling with an FITC-conjugated goat anti-mouse Ab, and cell surface expression was measured by flow cytometry. The thin line represents histogram for isotype-matched control Ab. The data shown are from a representative experiment (total of four experiments with different donors). B, Intracellular expression of TLR9 and MyD88 within infected monocytes. Monocytes (CD14ϩ) were infected with Y. pestis (10 bacteria per cell) for 1 h, noninternalized bacteria were removed by washing, and infected cells were cultured for 24 h. Cells were fixed, permeabilized, and labeled with primary anti-TLR9 Ab and primary anti-MyD88 Ab, followed by appropriate FITC- or PE-conjugated Ab. Cells were covered with mounting fluid containing DAPI for DNA staining, and examined using confocal microscopy. Bar ϭ 10 ␮M. The data shown are from a representative experiment (total of three experiments, separate donors). 7430 Y. pestis INFECTION OF MONOCYTES UP-REGULATES TLR9

DAPI images were collected using 2-photon excitation from a Ti/sapphire Cytokine analysis laser tuned to 800 nm. Images were collected using Bio-Rad LaserSharp software. Images taken for the purpose of comparing uninfected with Y. Cytokines in culture supernatants were measured using BD Cytometric pestis-infected cells were collected using identical settings, and any sub- Bead Array (BD Pharmingen) using capture beads coated with Abs sequent processing of the images was likewise performed identically for specific for cytokines and flow cytometry analysis, according to the man- the two conditions. ufacturer’s method. Briefly, the cytokine standards were reconstituted in the assay dilution buffer for 15 min, and then were diluted by serial dilution Flow cytometry from 5 to 5000 pg/ml. The cytokine capture beads were mixed together (10 ␮l/test) and then transferred, 50 ␮l of mixed beads to each assay tube. The To examine cell surface expression of proteins on primary cells or after PE detection reagent was added next to each assay tube (50 ␮l/test). The infection, cells were incubated (20 min, 4°C) with FcR-blocking reagent standard dilutions and test samples were added to the appropriate sample (Miltenyi Biotec), washed twice with HBSS containing 1% BSA, and then tubes (50 ␮l/test). The capture beads, PE-conjugated detection Abs, which incubated (30 min, 4°C) with FITC-labeled or PE-labeled control or iso- form a sandwich complex, and recombinant standards or test samples were type-matched mAbs. For unlabeled mAbs (detecting TLR9, CCR7, F1), incubated together for3hatroom temperature and protected from the . cells were first incubated (30 min, 4°C) with primary Ab, washed briefly by After the 3-h incubation, the samples were washed with 1 ml of wash buffer centrifugation, and then incubated with goat anti-mouse IgG (FITC or PE and centrifuged for 10 min at ϳ200 ϫ g. The supernatant was decanted, conjugated). Unbound Ab was removed by washing the cells with HBSS and 300 ␮l of wash buffer was added to each assay tube for analysis. After (4°C) and centrifugation. After two additional washes, the labeled cells sample data acquisition by the BD FACSCalibur flow cytometer, the sam- were fixed with 1% paraformaldehyde in PBS, and the cell-associated im- ple results were plotted in graphical and tabular format by the BD Cyto- munofluorescence was measured by flow cytometry (FACScan; BD metric Bead Array analysis software. Biosciences). T cell proliferation assay Results ϩ For detecting T cell proliferative responses to Yersinia Ags, CD14ϩ mono- CD14 monocytes differentiate into DCs in response to Y. cytes from an HLA-A2 donor were infected with Y. pestis at the start of pestis infection ␮ culture. Gentamicin (10 g/ml) was added to cultures after2hofinfection. Resting monocytes were selected for our study from peripheral Monocytes were washed extensively to remove extracellular bacteria be- fore adding autologous T cells to the culture at 24 h. Primary T cell cultures blood of volunteers, based on expression of the LPS-binding re- were then collected after 6 days of incubation. After 6 days of culture with ceptor CD14. Fresh cultures of Y. pestis, grown at 37°C under purified recombinant Yersinia F1 Ag presented by fresh DCs, cultures were optimal conditions for production of bacterial capsules and most 3 pulsed with 1 ␮Ci of [ H]thymidine/well for 10 h, and proliferation was virulence factors, were washed in cell culture medium and mixed measured by harvesting the cells onto microplate unifilters and measuring radioactivity in a liquid scintillation counter (Packard Instrument, with monocytes. Bacteria labeled with an Ab reacting with the Meriden, CT). capsular protein F1 were clearly visible within the cytoplasm of infected monocytes by1hofculture (Fig. 1), and we observed a Cytotoxicity assay consistently high level of monocytes infected with bacteria (Ͼ87% Cytolytic activity was measured by a luciferase-based assay of ATP average). The infected monocytes remained viable after 48 h of present in metabolically active target cells (Lumitech, Nottingham, U.K.). infection, and acquired a DC-like morphology, exhibited a loss Briefly, monocytes were obtained from an HLA-A2 donor. To transform of CD14, and increased cell surface expression of HLA-DR, monocytes to DCs, monocytes were cultured with IL-15 for 48 h, as pre- viously described (23). Cells were washed extensively to remove the IL-15 CD86, and the DC maturation markers CD83 and CCR7 (Figs. and any nonadherent cells. Monocytes were also cultured in the absence of 2A and 5A). These phenotypic changes indicated that infection IL-15 and remained as monocytes throughout the culture. Autologous hu- resulted in differentiation of the monocytes into DCs. The Y. man T cells and Ags (10 ␮g/ml) were added to the monocyte or DC culture pestis-infected monocytes also expressed IFN-␣ (Fig. 2B). Sig- and incubated for 6 days. T cells were collected and used as effector cells nificant levels of TNF-␣, IL-1␤, and IL-6 were produced by in cytotoxicity assays. HLA-A2-matched B lymphoblastoid cell line (BSM) were pulsed with soluble recombinant Ag (10 ␮g/ml, final Ag con- monocytes infected for 24 h (Fig. 3), suggesting a possible con- centration), or no Ag for1hat37°C and used as target cells. Target cells tribution of proinflammatory cytokines to maturation of the in- (BSM cells, 5 ϫ 103/well) were incubated (4 h; 100 ␮l volume) with fected monocytes to DCs. specific effector T cells (1:1, 1:5, and 1:10 of BSM:T cell). Cells were then lysed with 100 ␮l of a nucleotide-releasing agent (Lumitech); then 20 ␮lof an ATP-monitoring agent was added. Light emission was measured in a T cell response to protective Ags from Y. pestis presented by luminometer (Wallac 1420 Victor; PerkinElmer, Shelton, CT). Cytotoxic- infected monocytes and DCs ity was measured after CTL killing, after subtraction of experimental from maximum lysis of target cells. The capsular F1 protein of Y. pestis is a major protective Ag, and immunity to F1 Ag confers protection from plague infection by RT-PCR analysis either the s.c. or respiratory route of exposure (26). We cocultured Total RNA was extracted from untreated and Y. pestis-infected mono- Y. pestis-infected monocytes or DCs with autologous T cells to cytes using Tri-Reagent (Molecular Research Center, Cincinnati, OH) evaluate Yersinia-specific T cell responses. T cells primed in and reverse transcribed into cDNA with Maloney murine leukemia vi- vitro were isolated and tested with F1 Ag in proliferation and rus reverse transcriptase (Promega, Madison, WI), according to the cytotoxicity assays. Our results showed that Y. pestis-infected manufacturer’s instructions. Amplification of IFN-␣-specific and ␤-ac- tin control sequences was performed, as previously described (25). PCR DCs induced F1-specific T cell proliferation (Fig. 4). In addi- products were run on a 1.5% agarose gel, stained with ethidium bro- tion to Th responses, monocytes and DCs infected with Y. pestis mide, and photographed. stimulated F1 Ag-specific CTL responses (Fig. 4). These results

FIGURE 6. Similar regulation of cell surface molecules on human monocytes and B cells responding to TLR9 agonist. A, Monocytes, CD14 and TLR9; B cells, CD19 and TLR9. Live cells were incubated with fluorochrome-conjugated Abs, fixed, and then analyzed by flow cytometry. B, Activation of B cells by TLR9 agonists. Cell surface expression of critical markers on B cells (CD19ϩ) or monocytes (CD14ϩ) treated with CpG NT. Cell surface receptors were labeled with FITC-conjugated mAbs specific to each Ag. Thick lines represent cell surface expression of Ags, and thin lines represent histogram for isotype-matched controls. Results are from a representative experiment (total of three experiments). The Journal of Immunology 7431 7432 Y. pestis INFECTION OF MONOCYTES UP-REGULATES TLR9 indicated that DCs derived from infected monocytes were func- surface expression was not dependent on phagocytosis. Collec- tionally equivalent to DCs infected after maturation from cul- tively, our results suggest that agonist-induced changes in ex- tured monocytes. pression of costimulatory molecules and TLR9 are similar in monocytes and B lymphocytes. Increased cell surface expression of TLR9 on monocytes infected with Y. pestis Discussion Infection of monocytes with Y. pestis resulted in reduced cell sur- A thorough understanding of the initial interactions between host face CD14, whereas levels of CD14 on cells treated only with CpG and pathogen is essential for devising methods of controlling the NT remained similar to untreated control cultures (Fig. 5A). Be- high mortality associated with Y. pestis infections. Our results cause molecular signatures of pathogens are recognized by TLRs, demonstrate that human monocytes infected with Y. pestis differ- which in turn activate DCs, we next examined changes in expres- entiate into DCs, suggesting that adaptive immunity may still func- sion of TLRs on monocytes from multiple donors. Primary mono- tion during the intracellular stage of plague. Primary helper and cytes were infected for 48 h with Y. pestis and examined by flow CTLs were activated by monocytes matured to DCs by infection cytometry. Infection with Y. pestis increased cell surface TLR9, and by myeloid-derived DCs that were directly infected with Y. whereas no change was observed for expression of TLR2 or TLR4 pestis. The capsular F1 protein, which is a major protective Ag and compared with levels in untreated cells (Fig. 5A). Similarly, treat- vaccine component (27), was recognized by lymphocytes respond- ment with CpG NT, containing motifs optimal for human biolog- ing to infected monocytes. Activation of cells expressing TLR9 by ical activity, increased cell surface expression of TLR9. Adding hypomethylated CpG NT motifs present in bacterial DNA stimu- CpG NT agonists of TLR9 to infected monocytes decreased cell lates a rapid inflammatory response (28). Our results also indicate surface TLR9 (Fig. 5A). A significantly increased colocalization of that Y. pestis infection activates the TLR9 signal transduction path- MyD88 with TLR9 was observed in the cytoplasm of cells har- way, resulting in a significant increase in expression of TLR9, boring intracellular bacteria (Fig. 5B), whereas most uninfected both intracellularly and at the cell surface. Expression of intra- monocytes retained a low, but detectable, level of TLR9 with little cellular MyD88 was also activated by infection, suggesting that colocalization of MyD88. We also noted that levels of intracellular increased amounts of this adaptor may be necessary to accom- MyD88 were quite high compared with the noninfected controls modate the elevated levels of TLR9 resulting from the host (Fig. 5B), suggesting that neoexpression of the adaptor protein immune response to infection. Phagocytic internalization of may be essential for the TLR9-mediated response to infection. bacteria is also strongly promoted by TLR9 (29). In addition, These results indicated that TLR9 signaling was activated by in- we conclude that both monocytes and myeloid DCs express fection and coincided with DC differentiation. functional TLR9, a property previously thought to be restricted to B cells and plasmacytoid DCs.

ϩ The recruitment of MyD88 to endosomes containing TLR9 con- Freshly isolated CD14 monocytes express TLR9 firmed that these receptors of innate immunity were functional in Because our results showed a significant increase in cell surface Y. pestis-infected monocytes. All TLR9-mediated responses are expression of TLR9 with Y. pestis infection, this prompted us to dependent on MyD88 (30), and colocalization of TLR9, MyD88, examine expression on freshly isolated human blood cells. By flow and CpG NT in late endosomes is necessary for signaling (17, 28). cytometry, cell surface TLR9 was observed on 5–7% of the The colocalization of agonist, adaptor, and receptor was reported CD14ϩ monocytes and B cells (Fig. 6A), varying from donor to to be inhibited by wortmannin, suggesting a critical involvement of donor (n ϭ 16). Similar results were obtained with two indepen- PI3K (31). Agonists of TLR9 bind to the plasma membrane (32), dent mAbs against human TLR9 (data not shown). These results while other reports suggest that CpG NT-mediated signaling oc- confirmed that freshly isolated monocytes are poised to respond to curs in acidified endocytic vesicles (33, 34). The precise cellular TLR9 ligands. mechanism delivering TLR9 to the cell surface is presently not known. Furthermore, it is possible that TLR9 turnover from cyto- plasm to the cell surface is not critical to function and that signal B cells and monocytes exhibit similar regulation of TLR9 transduction only occurs in endosomes. phagosomes surface expression are formed by direct association and fusion of endoplasmic retic- As expression of TLR9 is a prerequisite for CpG NT responsive- ulum to the plasma membrane during early phagocytosis (35). ness in B cells, we next examined TLR9 in primary cultures of B Both endoplasmic reticulum and endosomes contribute ele- (CD19ϩ) lymphocytes, depleted of monocytes. A low level of ments to the phagosome membrane (35), bringing proteins from TLR9 expression was observed by flow cytometry (Fig. 6), which these intracellular compartments to the cell surface during appeared to be greater than that displayed by monocytes (Fig. 5A). phagocytosis. Therefore, it is possible that phagocytosis may We observed a dramatic increase in CD86 and HLA-DR, and a cause TLR9 to cycle between endosomal and plasma mem- more modest increase in CD83 expression, in B cells responding to branes. However, phagocytosis of inert beads by monocytes did TLR9 agonist treatment (Fig. 6). Like monocytes, the change in not result in activation of TLR9 expression or turnover, and a surface markers in response to CpG NT was dose dependent (data previous study of 293 cells transfected with a hemagglutinin- not shown). Also similar to the results obtained with monocytes tagged TLR9 demonstrated receptor in the plasma membrane of (Fig. 3), B cells treated with CpG NT (specific for human re- these nonphagocytic cells (28). Taken together, these observa- ceptor) exhibited a significant increase in cell surface TLR9 tions suggest a potential role for TLR9 in innate immune rec- (Fig. 6B). Because the CpG NT-induced increase in CD86 ex- ognition of both intracellular and extracellular PAMPs, perhaps pression by B cells was so dramatic, we re-examined expression serving an additional regulatory function in vivo. Interestingly, of this costimulatory molecule in monocytes (Fig. 6B). An in- TLR2 can bind Ags at the cell surface (36) and thus contribute crease in CD86 was observed in CD14ϩ cells treated with CpG to CD4ϩ T cell recognition of cognate Ag (36). In a similar NT, although the level was somewhat less than that obtained manner, the high expression level of TLR9 on infected mono- with B cells. 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Toll-like was reported to be induced by in vitro infection of human myeloid receptors and innate cytokine expression patterns of human CD56ϩ T cells are similar to natural killer cells in response to infection with Venezuelan equine DCs with the intracellular organism Listeria monocytogenes (44). encephalitis virus replicons. J. Infect. Dis. 188:1562. DC maturation during infection may be mediated by proinflam- 26. Andrews, G. P., D. G. Heath, G. W. Anderson, Jr., S. L. Welkos, and matory cytokines, for example TNF-␣, IL-1␤, and IL-6 measured A. M. Friedlander. 1996. Fraction 1 capsular antigen (F1) purification from Yer- in our study and in previous reports (45), or IL-15, as previously sinia pestis CO92 and from an Escherichia coli recombinant strain and efficacy against lethal plague challenge. Infect. Immun. 64:2180. reported (23). In contrast, virulent Mycobacterium tuberculosis 27. Brandler, P., K. U. Saikh, D. Heath, A. Friedlander, and R. G. Ulrich. 1998. 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