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. Author manuscript; available in PMC: 2010 Jan 15.
Published in final edited form as: J Infect Dis. 2009 Jan 15;199(2):236–242. doi: 10.1086/595833

Rickettsiae stimulate dendritic cells through TLR4, leading to enhanced NK cell activation in vivo

Jeffrey M Jordan 1, Michael E Woods 1, Lynn Soong 1,2, David H Walker 1,2,3
PMCID: PMC2613164  NIHMSID: NIHMS76374  PMID: 19072551

Abstract

Adoptive transfer of TLR4-stimulated dendritic cells induces protective immunity against an ordinarily lethal rickettsial challenge; however, the mechanism underlying this protection remains elusive. Therefore, we sought to determine the importance of TLR4 in early immunity to rickettsiae in vivo, particularly that conferred by TLR4-stimulated DC. Rickettsial growth proceeded logarithmically in mice lacking TLR4 function, whereas in TLR4-competent mice, rickettsial growth manifested a lag phase early, suggesting that TLR4 may initiate innate rickettsial immunity. TLR4-competent mice produced significant amounts of IFN-γ on day one of R. conorii infection, which was associated with significant expansion of activated NK cells. Moreover, NK cells from TLR4-competent mice produced significantly higher levels of IFN-γ and had greater cytotoxic activity than those from TLR4-deficient mice. Lastly, adoptive transfer of rickettsiae-exposed TLR4-stimulated DC activated NK cells in vivo. Together, these data reveal an important role for DC in recognizing rickettsiae through TLR4 and inducing early anti-rickettsial immunity.

Keywords: dendritic cells, natural killer cells, Toll-like receptor 4, bacterial infection

Background

Much attention on the induction of innate immunity to pathogens by dendritic cells (DC) has focused on the role of toll-like receptors (TLR). DC directly trigger NK cell function in vivo, and TLR-matured DC can activate NK cells in vivo via cell-cell contact and cytokine production (1). TLR ligation leads to DC production of IL-12, and IL-2 is produced by DC after TLR4-mediated LPS stimulation (2;3). Moreover, these DC-derived cytokines may enhance NK cell activity in vivo (4;5). Previously, we demonstrated that stimulation of DC with either LPS or rickettsiae leads to kinetically similar IL-2 production in vitro, suggesting that rickettsiae may ligate TLR4. The importance was further supported by the observation that TLR4-stimulated DC partially protect mice from lethal R. conorii challenge (6).

Owing to the obligately intracellular lifestyle of rickettsiae, cytotoxic T-lymphocytes and production of NO following IFN-γ and TNF-α activation of endothelium are critical in immunity (7;8). However, we do not fully understand the role of innate immunity in protection against rickettsiae. Early mobilization of NK cells may play a role in protection from rickettsial and other bacterial diseases. NK cell-derived IFN-γ production appears to play an important role in Shigella flexneri infection (9). Rickettsial antigens have been shown to increase human NK cell cytotoxicity in vitro, and depletion of NK cells leads to greater susceptibility to rickettsiae in mice (10;11). The spotted fever group rickettsia, Rickettsia africae, and the related organism, Wolbachia, can induce cellular activation through TLR4 and TLR2, for which the natural ligands are LPS and peptidoglycan, respectively (12;13). Nevertheless, the early triggers of NK cell-mediated innate immunity to rickettsiae, particularly the significance of TLR-ligation, have not been evaluated.

Stimulation of DC with LPS causes production of IL-12p40 in vitro and, upon transfer, NK cell mobilization in the draining lymph node. LPS-stimulated DC also induce NK cell proliferation in vivo (14). TLR9 is important in NK cell activation. Mice unable to signal through TLR9 have reduced NK cell-derived IFN-γ, and TLR9 expression in DC is important in inducing this activity (15;16). TLR9 ligation in DC directly induces NK cell activity in vivo (17), and IL-12 is required for the activation of NK cells in vivo. Therefore, while TLR ligation in DC may lead to NK cell activation in vivo, the importance of TLR4 ligation in DC-induced NK cell activation has not been conclusively demonstrated.

To determine the significance of TLR4 ligation in DC in the context of rickettsial infection, we utilized C3H/HeJ mice that are defective in TLR4 signaling and unresponsive to LPS (TLR4(Lps-d)) due to a single amino acid change in the cytoplasmic portion of TLR4 (18-20). We demonstrated that TLR4(Lps-d) mice were more susceptible to lethal R. conorii infection due to impaired adaptive immune responses when compared to genetically-related C3H/HeN mice, which possess functional TLR4 responses (21). Herein, we further show that rickettsial growth in TLR4(Lps-d) mice proceeds in a logarithmic fashion early in infection, whereas rickettsial growth experienced a lag phase in mice with TLR4 function, implying that TLR4(LPS-d) mice have impaired innate responses to rickettsiae. TLR4(LPS-d) mice have significantly lower levels of activated NK cells and serum IFN-γ. Lastly, we demonstrated that rickettsiae stimulate DC through TLR 4 and that transfer of TLR4-stimulated DC induced NK cell activity in vivo. Therefore, TLR4 ligation in DC is important in augmenting NK cell activity in vivo.

Methods

Mice and infections

Male C3H/HeN (H-2k) mice (Harlan Sprague Dawley, Indianapolis, Ind) and C3H/HeJ (H-2k) mice (Jackson Laboratories, Bar Harbor, Maine) between the ages of 6 and 12 weeks were housed under animal biosafety level-3, specific pathogen-free conditions according to a protocol approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch. Mice were infected intravenously with 8 × 103 PFU, which was approximately 1 LD50 in C3H/HeN mice (6;21).

Rickettsia

R. conorii, Malish 7 strain, (ATCC VR 613, Manassas, VA) was propagated in our laboratory in Vero cells from a 10% yolk sac stock (105 PFU per 150-cm2 flask) as described previously (6). Rickettsiae were then purified by discontinuous density gradient centrifugation in renografin (22). Viable rickettsiae were collected in sucrose-phosphate-glutamate buffer and stored at -80°C until used. Prior to use, rickettsiae were quantified by plaque assay.

Brain microvascular endothelial cell isolation

Isolation of mouse brain endothelial cells (MBEC) was adapted from previously reported protocols (23;24). Briefly, whole brains from male C3H/HeN or C3H/HeJ mice were removed aseptically and briefly soaked with 70% ethanol to render leptomeningeal vessels non-viable. The fresh brains were stored in ice-cold DMEM/F12 containing 2% FCS prior to homogenization. Brains were homogenized using a glass Dounce homogenizer and centrifuged. The resulting pellet was resuspended in 15% Dextran (M.W. 70 KDa) and centrifuged at 11,400 × g for 10 min at 4°C to remove myelin-containing cells. The pellet was washed once more in DMEM/F12 with 2% FBS and incubated at 37°C for 1.5 h with constant agitation in medium containing 1 mg/ml collagenase/dispase, 10 U/ml DNAse I, and 0.147 μg/ml Nα-toxyl-L-lysine chloromethyl ketone. Following digestion, the crude microvessels were washed in medium and plated on rat-tail collagen-coated plates in growth medium containing DMEM/F12, 10% FCS, 10% normal horse serum, 100 μg/ml endothelial cell growth supplement (Biomedical Technologies, Stoughton, MA), 100 μg/ml heparin, and 3 μg/ml puromycin. After three days of incubation, the puromycin was removed from the culture medium, and the cultures then consisted of pure brain endothelial cells. The cells were maintained at 37°C in 5% CO2.

NK cell cytotoxicity assay

Cytotoxicity was measured using the LIVE/DEAD® cell-mediated cytotoxicity kit for animal cells (Molecular Probes, Carlsbad, CA), and percent cytotoxicity was assessed by flow cytometry as described previously (25). Density-gradient-enriched lymphocytes were mixed with DiOC18-labeled target cells at effector-to-target (E:T) ratios of 50:1, 25:1, and 12.5:1 in duplicate. Propidium iodide (PI) was added to culture medium to allow for the determination of Yac-1 cell death after 3 h incubation at 37°C in 5% CO2. Percent cytotoxicity was assessed by flow cytometric analysis after collecting 3000 DiOC18+ events. Lysed (PI+, DiOC18+) and viable (DiOC18+ and PI-) Yac-1 cells were identified by their dual- or single-positive staining. Total cytotoxicity was determined as: [experimental % cytotoxicity] – [background % cytotoxicity in control Yac-1 cultures].

NK cell activation assay—determination of IFN-γ production

To determine NK cell-derived IFN-γ production, splenocytes (3 × 106) were co-cultured with 1 × 106 Yac-1 cells in 24-well plates in 2 ml complete RPMI-1640 medium for 18 h at 37°C. GolgiStop® (BD Pharmingen, San Diego, CA) was added followed by 6 h incubation. The percentages of IFN-γ-positive NK cells were determined by flow cytometric analysis.

Dendritic cell adoptive transfer

Bone marrow-derived dendritic cells (BMDC) were stimulated with heat-killed R. conorii (moi = 5), LPS (50 ng/ml), or SPG buffer diluted in complete IMDM (mock-infected), as described previously (6). Cells were harvested 24 h post-stimulation, washed three times in PBS, and resuspended at a concentration of 5 × 106 cells per ml. Mice were injected with DC suspension (5 × 105 cells per mouse) into the hind footpads (50 μl per foot), and challenged intravenously 24 h later with 3 LD50 (2.4 × 104 PFU) of R. conorii. Splenocytes were harvested at indicated time periods for determination of NK cell cytotoxicity.

Flow cytometry

Phenotypic analysis of cells was accomplished by staining cell suspensions in FACS buffer as described previously (6). The intensity of fluorescence was measured on a FACScalibur flow cytometer (BD Biosciences, Mountain View, CA) and analyzed using CellQuest (BD Biosciences) or FCS express (DeNovo Software, Thornhill, Ontario) software.

Determination of cytokine production by BioPlex assay

Serum cytokine levels were quantified using the Bio-Plex system, a bead-based array for simultaneous detection of up to 23 individual cytokines (Bio-Rad, Hercules, CA). Cytokine concentrations were determined in duplicate wells. Data were collected and evaluated on a Bio-Plex analyzer and associated software.

Real time-PCR quantitation of rickettsial loads

To determine bacterial loads, the brain, lung, and spleen were harvested on days 1, 3, and 5 post-infection, and 1-mm3 tissue pieces were stored at -20°C until processing. For in vitro rickettsial quantitation, BMEC were collected 24, 48, and 72 h post-infection and stored at −20°C until processing. Tissues were homogenized, and DNA was purified using the DNeasy Tissue Kit (Qiagen, Valencia, CA). Plasmids containing rickettsial gltA and murine β-actin PCR products were constructed using the TOPO 2.1 and TOPO 4 cloning kits, respectively. Rickettsial gltA was amplified using forward primer CS-5 (5′-GAGAGAAAATTATATCCAAATGTTGAT) and reverse primer CS-6 (5′-AGGGTCTTCGTGCATTTCTT). Murine β-actin was amplified using forward primer (5′-AGAGGGAAATCGTGCGTGAC) and reverse primer (5′-CAATAGTGATGACCTGGCCGT). Real-time PCR was performed using SYBR green Supermix, 1 μl DNA and primers (0.2 μM) on an iCycler real-time PCR apparatus (Bio-Rad). Standard curves were generated using plasmids containing cloned PCR products. All PCR reactions were performed using the following protocol: 95°C for 10 min followed by 40 cycles of 95°C for 30 sec, 50°C for 15 sec, and 60°C for 15 sec. Data are expressed as average copy number of rickettsial gltA per 10,000 copies of β-actin.

Statistical analyses

Data are expressed as mean ± standard error of mean or standard deviation, and the significant differences between two series of results were determined using Student’s unpaired t test. Values of p < 0.05 were considered significant.

Results

Rickettsial growth increases at a logarithmic rate in mice deficient in TLR4 function

To examine the effect of TLR4 ligation and innate immunity on rickettsial infection, we determined the kinetics of rickettsial infection in vivo. After i.v. inoculation, increases in rickettsial titers occurred logarithmically in the brain and lung of TLR4(LPS-d) mice, whereas mice that possessed functional TLR4 responses controlled the initial rickettsial proliferation during the first three days, resulting in a lag phase of rickettsial growth early in infection (Figure 1a, b). These data suggest that defective TLR4 signaling results in significant delay in initiation of immunity to rickettsiae.

Figure 1. Early rikettsial proliferation in vivo is blunted through a TLR4- dependent mechanism.

Figure 1

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To determine the influence that TLR4 has on the kinetics of rickettsial growth in vivo, mice were infected and rickettsial titer was determined by quantitative real-time PCR on brain (A) and lung (B). Data represent a mean of 3 mice per time point ± SD. Primary brain microvascular endothelial cells obtained from TLR4(LPS-d) and TLR4-competent mice were infected in vitro with renografin-purified R. conorii, and rickettsial titers were quantituted by real-time PCR (C). Following R conorii chemokine and cytokins production (D) was determined by Bio-Plex assay. ND, none detected.

Enhanced resistance to rickettsial disease is independent of rickettsial growth or NO production in endothelial cells

The kinetic differences of rickettsial proliferation in vivo would be attributed to TLR4-mediated activation of innate immune responses, or to TLR4 ligation in the vascular endothelium (main rickettsial target cells) that leads directly to an endothelial anti-rickettsial effect. To determine if TLR4 ligation on vascular endothelium played a role in rickettsial proliferation, we cultured primary brain microvascular endothelial cells in vitro to determine the rate of proliferation of rickettsiae in the absence of immune pressure. As shown in Figure 1c, bacteria replicated logarithmically with comparable growth rates in endothelium obtained from TLR4(LPS-d) and TLR4-competent mice, although at 24 h post-infection TLR4-competent mice had significantly greater rickettsial loads than TLR4(LPS-d) mice (p < 0.05). These data argue that growth differences in vivo were due to early initiation of innate immunity that required cells other than endothelium.

NO production is an important, innate effector response against rickettsiae in endothelium. Although TLR4 ligation is important in inducing NO production in macrophages, the role of TLR4 ligation in endothelium after rickettsial infection is unknown. To address this issue, we used R. conorii-infected primary endothelial cells obtained from TLR4(LPS-d) and TLR4-competent mice, and did not observe significant differences in NO production, as judged by nitrite concentrations in supernatants after 48 h of infection in vitro (data not shown). Although R. conorii did not stimulate significant NO production in endothelium through a TLR4-dependent mechanism, we did observe that several cytokines and chemokines were produced in endothelium by a TLR4-dependent mechanism after rickettsial infection. Supernatants obtained from primary TLR4-competent but not TLR4(LPS-d) brain microvascular endothelial cells 48 h after infection with R. conorii contained markedly elevated levels of IL-1α, MCP-1, MIP-1a, RANTES, and TNF-α (Figure 1d). Although TLR4 ligation does not play a direct role in limiting rickettsial growth, TLR4-dependent production of cytokines and chemokines in endothelium may cooperate with other immune effectors in inducing anti-rickettsial immunity in vivo.

Elevated levels of IFN-γ in the sera of R. conorii-infected TLR4-competent mice correlate with NK cell activation

The comparable growth rates of rickettsiae in TLR4-deficient and -competent endothelial cells in vitro led us to examine TLR4-mediated activation of innate immune responses and the production of IFN-γ and TNF-α in vivo. On day one post-infection with R. conorii, we detected significantly higher concentrations of IFN-γ in sera of TLR4-competent mice (Figure 2a). However, the levels of IL-12p40 and IL-12p70 were not significantly elevated, although levels of IL-17 were marginally increased (p = 0.056) (Figure 2a).

Figure 2. Rickettsias induce significant activation of NK cells in vivo through a TLR4-dependent mechanism.

Figure 2

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Serum eytoline concentrations were determined in TLR4-competent and TLR4(LPS-d) mice on day one post-infection with R. conorii (A). Splenocytes were obtained from rickettsiae-infected mice and percent activated NK (CD69+ DX5+ CD3-) cells (B) and percent total NK cells (C) were determined by flow cytometric analysis. (D) Splenocytes from TLR4(LPS-d) and TLR4-competent mice were obtained on day 3 post-infection. Splenocytes, from uninfected and R. conorii-infected mice were stimulated in vitro with Yac-1 cells, and IFN-γ production was assessed by flow cytometry. Bar graphs are representative of 3 mice per timepoint ± SD. *,P <0.05.

Our laboratory has previously shown that early IFN-γ production is largely due to activated NK cells, and that IFN-γ and TNF-α are important in limiting rickettsial growth (8;11;26). To test whether increased IFN-γ on day one post-infection in TLR4-competent mice was due to increased NK cell proliferation and activation, we determined the percentage, absolute number, and activation status of splenic NK cells during the course of infection by flow cytometry. Although there was no significant increase in splenic NK cells on day one post-infection (data not shown), NK cells obtained from TLR4-competent mice were activated to a greater extent than those obtained from TLR4(LPS-d) mice, based on CD69+ phenotype (Figure 2b). On day three post-infection, TLR4-competent mice showed significant increases in total numbers and percentages of splenic NK cells as compared with TLR4 defective mice (Figure 2c). These results were in agreement with serum IFN-γ levels, suggesting the production of IFN-γ by activated NK cells at early stages of infection in TLR4-competent mice,

To confirm the role of NK cells, we determined the ability of NK cells to produce IFN-γ after stimulation with Yac-1 cells in vitro. NK cells from both TLR4-competent and TLR4(LPS-d) infected mice produced markedly more IFN-γ than uninfected mice; however, NK cells obtained from TLR4-competent mice on day 3 post-infection produced significantly more IFN-γ than those obtained from TLR4(LPS-d) mice (Figure 2d). Together, these data suggest that activated NK cells contribute to the increased serum IFN-γ in infected TLR4-competent mice.

Mice defective in TLR4 signaling have impaired NK cell cytotoxicity activity

On days 1 and 3 post-infection, NK cells obtained from R. conorii-infected TLR4-competent mice had significantly greater Yac-1 cell cytotoxicity than those obtained from TLR4-deficient mice (Figure 3). These data imply a direct correlation between resistance to rickettsial infection and NK cell functions (IFN-γ production and cytotoxic activity). However, they did not rule out TLR4-independent mechanisms in the induction of NK cell cytotoxic activity, because R. conorii-infected mice had significantly greater NK cytotoxicity than uninfected controls, regardless of ability to signal through TLR4.

Figure 3. TLR4-competent mice have significantly greater NK cell cytotoxicity activity.

Figure 3

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Splenocytes were obtained from R. conorii-infected TLR4(LPS-d) or TLR4-compentent mice or mock-infected controls. Splenocytes were co-cultured with DiOC-labeled Yac-1 cells, and percent killing was determined by flow cytometry. *,p < 0.05.

Dendritic cells activate NK cells through a TLR4-dependent mechanism in vivo

Given that TLR4-competent mice have significantly greater NK cell activities after R. conorii infection and that adoptive transfer of TLR4-stimulated DC could protect mice from an ordinarily lethal challenge (6), we speculated that TLR4-mediated signaling in DC could lead to NK cell activation in vivo. To test this hypothesis, we stimulated BMDC from TLR4(LPS-d) and TLR4-competent mice with R. conorii in vitro. At 24 h post-stimulation, DC were injected into the footpads of TLR4-competent mice, and splenocytes were harvested 24 h after cell transfer for determination of splenic NK cell activity via Yac-1 cell cytotoxicity. As shown in Figure 4, DC capable of signaling through TLR4 induced significantly greater levels of NK cell cytotoxicity after R. conorii infection than the counterpart DC derived from TLR4(LPS-d) mice. These data suggest TLR4-dependent, DC-mediated NK cell activation during R. conorii infection in vivo.

Figure 4. Ligation of TLR4 in DC induces NK cell activation in vivo.

Figure 4

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Bone marrow-derived DC were obtained from TLR4(LSP-d) and TLR4-competent mice and stimulated in vitro with R. conorii (moi = 5), LPS(50 ng/ml), or mock-infection for 24 h. DC were then injected into mice (5 × 105 cells per mouse), and splenocytes were harvested 24 h later to determine Yac-1 cell cytotoxicity. *,p <0.05; **,p < 0.005.

Conclusions

These data provide evidence that DC recognize rickettsiae through TLR4 and promote NK cell activation in vivo (Figure 5). Additionally, we have demonstrated, for the first time, that TLR4 stimulation is important both in initiation of anti-rickettsial innate immunity, namely the expansion of NK cells, and in expansion of Th1 cells (21). TLR4-ligated DC induce recruitment of NK cells to draining lymph nodes (6;14); furthermore, this recruitment and the production of NK cell-derived IFN-γ in draining lymph nodes are important in augmenting the Th1 immune response (27-29). Our results also argue strongly for an important role of TLR4-induced innate immunity. After R. conorii infection in TLR4(LPS-d) mice, rickettsial growth proceeded immediately into logarithmic phase, as compared with the lag phase observed in mice possessing functional TLR4. However, we found that rickettsial proliferation rates and NO production were comparable in primary brain microvascular endothelial cells derived from TLR4(LPS-d) and TLR4-competent mice. These data therefore suggest that the synergistic action of IFN-γ and TNF-α in inducing rickettsicidal NO in endothelium may be more important than TLR4-mediated NO production in rickettsiae-infected endothelial cells.

Figure 5. Proposed mechanism of DC-mediated activation of NK cells in vivo.

Figure 5

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TLR4-activated DC induce activation and proliferation of NK cells and subsequent production of IFN-γ.Additionally, TNF-α is produced in endothelium and macrophages. IFN-γ and TNF-α then cooperatively stimulate NOS2 and the subsequent production of rickettsicidal nitric oxide, limiting rickettsial infection.

Importantly, endothelial cells do produce significant levels of TLR4-dependent proinflammatory cytokines. Previous research had shown that rickettsia-infected endothelial cells did not produce significant amounts of TNF-α. However, those studies were performed with HUVEC cells, which do not contain significant quantities of TLR4; therefore, our results suggest that endothelium may play a greater role in the immune response than previously thought. Of note, we have observed significant production of NO in cultured endothelium in response to R. rickettsii via a TLR4-dependent mechanism (Woods and Jordan, data not shown). Thus, different rickettsial organisms may differentially ligate TLR, leading to differences in immune responses and pathogenesis. These findings are biologically important, because R. rickettsii is more pathogenic than R. conorii, with a significantly higher case fatality rate (33;34).

It has been documented recently that TLR4 stimulation is also important in inducing NK cell activation in vivo (35). Consistent with this report, we demonstrated that the quantity and percentage of NK cells in spleen were consistently lower in TLR4(LPS-d) mice than TLR4-competent mice after R. conorii infection. Additionally, splenocytes from TLR4(LPS-d) mice had less NK cell cytotoxic activity than TLR4-competent mice in vitro. Moreover, TLR4-competent mice also had significantly greater levels of IFN-γ in the sera during early infection; and subsequent investigation demonstrated that NK cells in TLR4-competent mice produced significantly greater amounts of IFN-γ after stimulation in vitro. Given these data, we suggest that this IFN-γ production is important in inducing early NO production in infected endothelial cells (Figure 5). This NO production is likely an important factor leading to the lag phase of rickettsial growth observed in TLR4-competent mice. In sum, this study indicates that TLR4 ligation is an important step in limiting early rickettsial proliferation by activating NK cell cytotoxicity and IFN-γ production, and that TLR4 signaling plays a significant role in both innate and adaptive protective immunity against R. conorii infection.

Acknowledgments

Financial Support: National Institute of Allergy and Infectious Diseases (grant AI21242 to D.H.W.); James W. McLaughlin Predoctoral Fellowship (to J.M.J); National Institute of Allergy and Infectious Diseases (T32 AI007526 Emerging and Tropical Infectious Diseases Pre-doctoral Training Grant to J.M.J).

Footnotes

Conflict of Interest Statement: The authors do not have any conflict of interest.

Presented in Part: 2007 American Association of Immunologists, Miami, FL., May 18-22, 2007.

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