International Immunology

International Immunology Advance Access originally published online on February 7, 2007
International Immunology 2007 19(3):311-320; doi:10.1093/intimm/dxl148

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Type 1 cytokine/chemokine production by mouse NK cells following activation of their TLR/MyD88-mediated pathways

Junko Sawaki^1,2, Hiroko Tsutsui^3,4, Nobuki Hayashi^2,4, Koubun Yasuda^2,4, Shizuo Akira⁵, Takakuni Tanizawa¹ and Kenji Nakanishi^2,4

¹ Department of Pediatrics
² Department of Immunology and Medical Zoology
³ Department of Microbiology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya 663-8501, Japan
⁴ Core Research of Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
⁵ Department of Host defenses, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan

Correspondence to: K. Nakanishi; E-mail: nakaken{at}hyo-med.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
It is well established that IL-18R- and toll-like receptor (TLR)-mediated signalings share a common signal pathway mediated by signal adaptor, MyD88, and that IL-18 synergizes with IL-12 for IFN- production by NK cells. Here, we investigated whether TLR agonists can replace IL-18 for production of IFN- by NK cells. Freshly isolated NK cells possessed functional LPS receptor composed of TLR4/MD2 complex and of CD14, and also expressed other various tlrs. Hepatic CD3^–DX5⁺ NK cells produced IFN- in response to TLR2 or TLR7 agonists only when co-stimulated with IL-12, indicating that TLR agonists synergize with IL-12 for IFN-. The tlr2^–/– or tlr7^–/– NK cells could not produce IFN- in response to IL-12 plus TLR2 or TLR7 ligands, respectively, indicating requirement of the corresponding TLRs. Furthermore, upon stimulation with these combinations, wild-type NK cells produced type 1 chemokines, such as CCL3, CCL4 and CCL5 as well. NK cells from bacterium (e.g. Propionibacterium acnes)-inoculated rag2^–/– mice, when compared with those from naive mice, exhibited significantly enhanced capacity to produce these CC chemokines and IFN-, suggesting that microbial infection enhances responsiveness of NK cells to TLR agonists. These results indicate that upon microbial infection, macrophages produce IL-12 that renders NK cells highly responsive to TLR agonists to produce IFN- and chemokines, which might in turn recruit and fully activate macrophages, leading to the development of inflammatory foci presumably necessary for efficient microbial eradication. Thus, NK cells, like T cells, induce orchestrated immune responses in collaboration with macrophages to show potent host defense effects during early infectious phase.

Keywords: CCL3, CCL4, IFN-, IL-12, NK lytic activity

	Introduction

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
NK cell is an important constituent of innate immunity that plays an essential role in host defense, particularly at early infectious phases (1, 2). Indeed, mice genetically lacking T cells and B cells, such as SCID or rag1^–/– mice, can eradicate pathological microbes in an IFN-- and NK cell-dependent manner (3, 4). It is well established that upon infection of mice with Listeria monocytogenes, a facultative gram-positive intracellular bacterium, dendritic cells (DCs) and macrophages produce IFN--inducing cytokines, such as IL-12 and IL-18, via activation of their toll-like receptor (TLR)/MyD88-mediated signal pathways, important innate immune signalings (5–7). IL-18 can activate NK cells to produce robust IFN- in combination with other IFN--inducing cytokine such as IL-12 (8–10). This IFN-, in turn, activates macrophages to accomplish their listeriocidal actions during the early infectious phase, eventually resulting in the early listerial clearance (1, 2). Indeed, the depletion of NK cells or IFN- gene renders mice highly vulnerable to L. monocytogenes. As previously reported, il18^–/– mice are susceptible to L. monocytogenes due to the impaired induction of IFN- (5). Intriguingly, il12^–/– mice are as vulnerable as ifng^–/–, but much more susceptible than il18^–/– mice, allowing us to propose the possibility that some factors other than IL-18 might cooperate with endogenous IL-12 for the NK cell production of IFN-.

We now know that the TLR-mediated pathways share a signal cascade with IL-18 signaling (6, 7, 11, 12). Both IL-18 and TLR ligands require MyD88, a signal adaptor, to activate NF-B. Therefore, it is plausible that TLR agonists can replace IL-18 and synergize with IL-12 for IFN- production by NK cells. Furthermore, in analog with IL-18 induction of chemokines (13, 14) by macrophages, TLR agonists seem to induce chemokine production by NK cells (6). In this study, we addressed these questions and found that murine NK cells constitutively express functional TLRs and produce IFN- in response to TLR ligands, like to IL-18 in the presence of IL-12 in a corresponding TLR- and MyD88-dependent manner. Furthermore, the NK cells simultaneously produced robust CC chemokines, CCL3, CCL4 and CCL5, which can recruit various subtypes of leukocytes including macrophages (15). Finally, we assessed physiological roles of the TLR-mediated NK cell activation. Naive NK cells produced little or a small amount of those chemokines upon stimulation with TLR agonists without IL-12. By contrast, bacterium-elicited NK cells were well armed and capable of producing much larger amounts of the chemokines, indicating that microbial infection commits NK cells competent to directly respond to TLR ligands. These results provide new insights into NK cell responses early in infection.

	Methods

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Mice
C57BL/6 mice were purchased from Crea Japan (Osaka, Japan). rag2^–/– mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). tlr2^–/– mice (16), tlr7^–/– mice (17) and myd88^–/– mice (11) were backcrossed with C57BL/6 mice, and F8 to F10 generations were used. The il12^–/– il18^–/– mice were shown elsewhere (5). All mice were kept under SPF conditions and received humane care as outlined in the Guide for the Care and Use of Experimental Animals (Hyogo Medical College Animal Care Committee).

Reagents
LPS derived from Escherichia coli O11:B5 was purchased from Sigma (St Louis, MO, USA). TLR2 agonist, peptidoglycan (PGN), and TLR3 agonist, poly (I):poly (C), were from Fluka (Bachs, Sweden) and Amersham Pharmacia Biotech (Freiburg, Germany), respectively. TLR7 agonist, Loxoribine (18), was purchased from InvivoGen (San Diego, CA, USA). Anti-DX5-bound microbeads and anti-FITC-bound microbeads were purchased from Miltenyi Biotec Inc. (Auburn, CA, USA). Recombinant mice IL-12 and IL-18 were obtained from R&D (Minneapolis, MN, USA) and MBL (Nagoya, Japan), respectively. Recombinant human IL-15 was from Peprotech (London, UK). Recombinant human IL-2 was kindly provided by Shionogi Pharmaceuticals (Osaka, Japan). FITC-conjugated anti-CD3 and PE-conjugated DX5 were purchased from PharMingen (San Diego, CA, USA). FITC-conjugated anti-TLR4-MD2 complex was kindly provided by Dr. K. Miyake at the University of Tokyo (Tokyo, Japan). Propionibacterium acnes were killed at 60°C for 4 h as previously shown (19). RAW264.7, mouse macrophage cell line, was used (20). The culture medium generally used in this study is RPMI 1640 containing 10% FCS, 100 U ml^–1 penicillin, 100 µg ml^–1 streptomycin, 50 µM of 2-ME and 2 mM L-glutamine.

Preparation of NK cells
CD3^–DX5⁺ cells were prepared by using autoMACS (Miltenyi Biotec Inc.) as described before (21, 22) or were sorted by FACSaria (Becton Dickinson, Mountain View, CA, USA). Hepatic CD3^–DX5⁺ cells prepared from P. acnes-primed mice (19) were incubated with 300 ng ml^–1 of rIL-15 for 10 days and used as IL-15-expanded NK cells. The purity of CD3^–DX5⁺ cells expanded was >85%. Splenic DX5⁺ cells were also prepared from rag2^–/– treated with or without P. acnes using autoMACS. The cells were incubated with PGN (100 and 10 µg ml^–1), poly I:C (100 and 10 µg ml^–1) or loxoribine (1000 and 100 nM) in the presence of various recombinant cytokines for 48 h, and supernatants were collected.

Assay for cytokines and chemokines
Concentrations of IFN-, IL-13, tumor necrosis factor (TNF)-, CCL3, CCL4 and CCL5 in each supernatant were measured by a commercially available kit (R&D).

FACS
Cells were incubated with PE-conjugated anti-DX5, Allophycocyanin-conjugated anti-CD3 and FITC-conjugated anti-TLR4-MD2 complex or anti-CD14 after FcR blocking (22). Stained cells were analyzed using FACScalibur (Becton Dickinson) (23).

Assay for NK lysis
IL-15-expanded hepatic CD3^–DX5⁺ cells were incubated with PGN (100 µg ml^–1), poly I:C (100 µg ml^–1) or loxoribine (1 µM) in the presence of IL-12 (200 pg ml^–1) for 24 h. NK lytic activity against YAC-1 cells was determined by the method described previously (24).

EMSA
Double-stranded, NF-B-specific oligonucleotide, the consensus sequence (5'-TCG-AGG-GCT-GGG-GAT-TCC-CCA-TCT-C-3'), was labeled with [³²P] dCTP and used as probe. Cells were incubated with 1 µg of LPS or 10 ng of IL-18 for the indicated hours, and nuclear localization of NF-B was determined by using the cell extract as previously described (25). Briefly, nuclear extracts (20 µg) were incubated with the probe and electrophoresed, and gel was subsequently dried and visualized by autoradiography.

Reverse transcription–PCR
Total RNA was extracted, and mRNA for TLR1, 2, 3, 4, 5, 6, 7, 9 and ß-actin was determined by reverse transcription–PCR (25). The individual primers and amplifying cycle were shown in Table 1.

View this table: [in this window] [in a new window]   Table 1. Reverse transcription–PCR for murine TLRs

 
Statistics
All data are shown as the mean ± SD of triplicate samples. Significance between the experimental and control groups was examined by the unpaired Student's t-test.

	Results

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
NF-B activation in response to LPS
First, we investigated NK cell expression of TLRs. Hepatic CD3^–DX5⁺ cells freshly isolated from naive wild-type (WT) mice expressed tlr1, tlr2, tlr3, tlr4, tlr6, tlr7, tlr8 and tlr9 (Fig. 1A). This was also the case for splenic NK cells (data not shown). These results indicate that mouse NK cells express various TLRs under normal conditions. As both TLR4/MD2 complex and CD14 are required for the TLR4-mediated signal pathway (26, 27), we analyzed their expressions of these molecules. FACS-sorted hepatic CD3^–DX5⁺ NK cells moderately expressed these receptors on their surface (Fig. 1B). The cells incubated with control mAbs showed the same intensity for those molecules as the backgrounds (data not shown). NK cells expressed less amounts of TLR4/MD2 complex than RAW cells, mouse macrophage cell line (Fig. 1B).

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1. Constitutive expressions of tlrs in NK cells. (A) Total RNA was extracted from freshly isolated, FACS-sorted hepatic CD3–DX5+ cells (fresh) or the cells that were expanded from the same cell fraction with IL-15 for 10 days (IL-15-expanded), and reverse transcription–PCR was performed for various TLRs by using corresponding primers. (B) Purity of CD3–DX5+ cells in ‘fresh’ and ‘IL-15-expanded’ was determined. Insets indicate cell percentage of each quadrant. fresh, IL-15-expanded cells and RAW cells were incubated with anti-TLR4/MD2 complex or anti-CD14 mAmAb (solid lines). Dotted lines indicate background staining. (C) IL-15-expanded hepatic CD3–DX5+ NK cells were incubated with LPS (100 ng ml–1) or IL-18 (10 ng ml–1) for the indicated periods. Nuclear proteins were extracted, and EMSA for NF-B was performed. Representative photographs were shown (A). The data are representative of three independent experiments with the similar results.

 
As NF-B activation is a hallmark of the TLR-mediated signalings (6, 9, 10, 28), we examined whether NK cells show the DNA-binding activity of NF-B promptly after stimulation with LPS, TLR4 agonist. Because freshly isolated NK cells showed too low yield to examine further, we expanded them with IL-15 (29). The IL-15-expanded cells still had CD3^–DX5⁺ cells at >85% (Fig. 1B). The IL-15-expanded hepatic NK cells expressed various tlrs except for tlr5 and tlr8 (Fig. 1A) and TLR4/MD2 and CD14 on their surface as well (Fig. 1B). Thus, we used IL-15-expanded cells thereafter to examine NK cell potential via activating their TLRs. Like IL-18, LPS could induce nuclear NF-B accumulation in NK cells (Fig. 1C). These results indicated that the IL-15-expanded hepatic NK cells are able to respond to LPS at least in terms of NF-B activation without help from other cell types. This was also true for splenic NK cells (data not shown). These results suggested that NK cells as well as macrophages and DCs might be able to sense TLR ligands.

Synergistic action of TLR agonists with IL-12 for IFN- production
Next, we wanted to know biological products of TLR agonist-activated NK cells. As IL-18 is a potent IFN--inducing cytokine, we focused on the action of TLR agonists on IFN- production by NK cells. We prepared hepatic NK cells expanded with IL-15 for 10 days and incubated them with various TLR agonists. PGN (TLR2), poly I:C (TLR3) or loxoribine (TLR7) stimulation solely could not induce IFN- (Fig. 2). This was also the case for NK cells expanded with IL-15 for 6 days (Supplementary Figure 1, available at International Immunology Online). However, the TLR agonists synergized with IL-12 for induction of production of IFN- (Fig. 2), which is similar to the synergism between IL-18 and IL-12 (9, 30, 31). Although IL-18 shows somewhat synergy with IL-2 or IL-15 (9), TLR agonists did not synergize with these cytokines for IFN- production by NK cells (Fig. 2). Since NK cells can produce TNF- or IL-13 (9), we examined whether TLR agonists, in combination with IL-12 or solely, induce these cytokines in NK cells. The cells produced little IL-13 and TNF- (Fig. 3). Intriguingly, they could not produce TNF- (Fig. 3) or IL-6 (data not shown), strongly suggesting that the NK cell preparation seems not to be contaminated with the cells that produce TNF- and/or IL-6 in response to the TLR agonist, such as macrophages and DCs. We next tested whether IL-12 dose dependently induce IFN- when in conjunction with TLR ligands. Expectedly, we found the dose dependency (Fig. 4A). This does not exclude the possibility that TLR-expressing macrophages are contaminated in the NK cell preparation and produce IL-18 in response to TLR agonists, which eventually induce IFN- in collaboration with exogenous IL-12. To neglect this possibility, we stimulated NK cells prepared from il12^–/– il18^–/– mice with the TLR agonists plus IL-12 and found their production of IFN- (Fig. 4B). Those characteristics were also found in IL-15-expanded splenic NK cells (data not shown). These results indicated that NK cells have potential to respond to TLR agonists in the presence of IL-12 by production of IFN-.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2. IL-12, but not IL-2 or IL-18, synergizes with TLR agonists for IFN- by NK cells. IL-15-expanded NK cells were incubated with PGN (100 µg ml–1), poly I:C (100 µg ml–1) or loxoribine (Lox, 1000 µM) in the presence or absence of IL-12 (2000 pg ml–1), IL-2 (500 U ml–1) or IL-18 (10 ng ml–1) for 48 h. IFN- in each supernatant was measured by ELISA. The data are expressed as mean ± SD of triplicates. The data are representative of three independent experiments with the similar results.

 

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3. NK cells do not produce IL-13 or TNF- in response to IL-12 plus TLR agonists. IL-15-expanded hepatic NK cells were incubated with the TLR agonists shown in the legend to Fig. 2 in the presence of IL-12 (2000 pg ml–1) for 48 h. IL-13 and TNF- concentrations in each supernatant was measured. ND indicates that concentrations of IL-13 and TNF- are <0.01 ng ml–1. The data are expressed as mean ± SD and representative of three independent experiments with the similar results.

 

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4. IL-12 dose dependently induces IFN- in collaboration with various TLRs. IL-15-expanded hepatic NK cells were incubated with the TLR ligands shown in the legend to Fig. 2 in the presence of various doses of IL-12. IFN- concentration in each supernatant was measured. The data are expressed as mean ± SD and representative of three independent experiments with the similar results.

 
Requirement of corresponding TLRs and MyD88 for the IFN- production
We next investigated whether TLR agonists/IL-12-induced IFN- requires the corresponding TLRs. We manipulated hepatic CD3^–DX5⁺ NK cells prepared from WT, tlr2^–/– and tlr7^–/– mice. The tlr2^–/– NK cells produced IFN- in response to poly I:C and loxoribine, but not PGN (Fig. 5), indicating requirement of TLR2 for PGN induction of IFN-. The tlr7^–/– cells produced IFN- in response to poly I:C and PGN, but not loxoribine (Fig. 5), indicating requirement of TLR7 for loxoribine induction of IFN-. Therefore, corresponding TLRs are essential.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5. TLR- and MyD88-dependent IFN- production. IL-15-expanded hepatic NK cells were prepared from WT, tlr2–/–, tlr7–/– or myd88–/– mice and incubated with the various doses of TLR agonists in the presence of IL-12 for 48 h. IFN- concentration in each supernatant was measured. The data are expressed as mean ± SD and representative of three independent experiments with the similar results.

 
Next, we examined whether MyD88 (6) is required for these IFN- production. As expected, myd88^–/– cells exhibited little increase in production of IFN- in response to the TLR2/7 agonists in the presence of IL12 (Fig. 5), demonstrating that the TLR2- and TLR7-mediated IFN- production by NK cells requires MyD88. Consistent with the previous reports (6, 7), MyD88 is dispensable for TLR3-mediated IFN- production (Fig. 5). Those characteristics were also true for IL-15-expanded splenic NK cells (data not shown). Collectively, NK cells have potential to produce IFN- via activation of their corresponding TLRs and MyD88.

Lack of up-regulating action of TLR agonists onto NK lytic activity
Since IL-18 up-regulates NK lytic activity, we next examined effects of the TLR agonists. NK cells did not show increased lytic activity against YAC-1 cells, authentic murine NK target cells, after stimulation with the agonists either in the presence or in the absence of IL-12 (Fig. 6). Thus, NK killing activity is not profoundly affected by the treatment with TLR agonists.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6. Lack of enhancing effects of TLR agonists on NK cytolytic activity. IL-15-expanded hepatic NK cells were incubated with various TLR ligands in the presence or absence of IL-12. Their cytotoxicity against YAC-1 cells was evaluated. The data are expressed as mean and representative of three independent experiments with the similar results.

 
Type 1 chemokine productions
It has been demonstrated that NK cells become to cytoplasmically possess CCL3 (MIP-1), CCL4 (MIP-1ß) and CCL5 (RANTES) in vivo after infection of mice (32–34) but the mechanism underlying is still to be elucidated. Thus, we assumed that microbial TLR agonists directly activate NK cells under influence of IL-12. We measured those chemokine levels in the supernatants of NK cells stimulated with TLR agonists in vitro. CD3^–DX5⁺ NK cells produced CCL3, CCL4 and CCL5 in response to the TLR2, TLR3 and TLR7 agonists plus IL-12 (Fig. 7). Those characteristics were also found by using IL-15-expanded splenic NK cells (data not shown). Separately, we observed that IL-15-expanded NK cells could produce IFN- and CCL3 in response to TLR4 and TLR9 ligands in the presence of IL-12 (Supplementary Figure 2, available at International Immunology Online), indicating TLR4- and TLR9-mediated activation of NK cells for production of type 1 cytokine/chemokine. These results indicated again that TLR agonists synergize with IL-12 for induction of production of the chemokines by NK cells.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7. NK cell production of CCL3, CCL4 and CCL5 in response to IL-12 plus TLR agonists. IL-15-expanded hepatic NK cells were incubated with the various TLR agonists shown in Fig. 2 in the presence of IL-12 for 48 h. CCL3, CCL4, CCL5 and CCL11 in each supernatant were measured. The data are expressed as mean ± SD and representative of three independent experiments with the similar results.

 
Enhancement of TLR-mediated NK cell responses by in vivo administration of heat-killed bacterium
All the results indicate that NK cells have potential to respond to various TLR agonists by producing type 1 cytokine/chemokines. These results led us to know physiological roles of the TLR-mediated activation of NK cells. To address this, we investigated whether microbial infection enhances this NK cell response particularly in the innate immune phase. To exclude the possible interference with harmful exotoxins and/or enzymes produced by live bacterium and evaluate responses only to the bacterial components, we systemically administered heat-killed P. acnes, a gram-positive bacterium, into rag2^–/– mice and compared responses of their NK cells with PGN, TLR agonists of gram-positive bacteria, with those in naive mice. NK cell purity of the MACS-sorted DX5⁺ cells prepared from naive and P. acnes-primed rag2^–/– mice were 96 and 94%, respectively (Fig. 8A). As compared with naive cells, P. acnes-elicited NK cells produced five to 10 times more amounts of CCL3 and CCL4 in response to PGN (Fig. 8B), indicating that in vivo treatment with P. acnes renders NK cells more sensitive to PGN. However, either P. acnes-elicited or naive NK cells did not produce IFN- in response to PGN in the absence of IL-12 (Fig. 8B). Intriguingly, in the presence of IL-12, the former cells turned to produce IFN- at larger amounts (Fig. 8C), indicating that P. acnes-elicited NK cells still require IL-12 for production of IFN- and again augmenting effects of P. acnes treatment on the TLR-mediated IFN- production from NK cells. These results indicated that infected macrophages augment their anti-microbial functions by arming NK cells with enhanced production of IFN- and chemokines upon TLR agonist challenge, that in combination induces strong innate immune responses.

View larger version (14K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Propionibacterium acnes treatment induces responsiveness of NK cells to TLRs agonists. NK cells freshly isolated from naive or P. acnes-primed mice were stained with anti-DX5 and anti-CD3 for determination of NK cell purity (A). The cells were incubated with PGN in the presence (C) or absence (B) of IL-12 for 48 h. IFN-, CCL3 and CCL4 concentrations in each supernatant were measured. The data are expressed as mean ± SD and representative of three independent experiments with the similar results.

 

	Discussion

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
In this study, we demonstrated that NK cells are activated through their TLRs for production of IFN-, CCL3, CCL4 and CCL5, which are essential for efficient microbial clearance. Various TLR agonists could activate NK cells to produce those type 1 cytokine/chemokines in synergy with IL-12, but not with IL-18 or IL-2, and these responses depended on the corresponding TLRs and MyD88 (Figs 4, 5 and 7). Importantly, these actions were extensively enhanced by the treatment of mice with heat-killed P. acnes (Fig. 8). Indeed, P. acnes-elicited NK cells produced much larger amounts of those CC chemokines upon stimulation with P. acnes-derived TLR agonist, PGN, than naive cells (Fig. 8). These results suggested that microbial infection of mice induces NK cells to become competent to directly respond to the microbial TLR ligands. Based on these observations, one can imagine the following scenario for the early microbial eradication. Following microbial infection, tissue macrophages and DCs produce IL-12 in response to the microbial TLR ligands, and IL-12-stimulated NK cells become highly susceptible to these ligands. Therefore, there is positive feedback between TLR-mediated IL-12 and TLR ligand responsiveness of IL-12-stimulated NK cells. After recognizing the microbial TLR ligands, the NK cells in the infectious sites produce robust CCL3, CCL4 and CCL5, which eventually induces migration of TLR-expressing macrophages around the NK cells in the infectious sites. These migrated macrophages recognize the microbial TLR ligands of the infectious sites and increase production of IL-12 and other IFN--inducing cytokines, which eventually and fully activate the NK cells to further produce IFN-. Furthermore, IL-12-stimulated NK cells increase production of IFN- and chemokines in response to the microbial TLR agonists at the infected sites. Thus, IFN- production from NK cells is positively regulated by TLR agonists and IL-12. These events might converge into the histological formation of inflammatory foci around the microbe and the generation of a powerful positive circuit between NK cells and macrophages, leading to the efficient early host defense. It is noted that IL-12 is solely capable of activating naive and P. acnes-elicited NK cells to produce IFN- (Fig. 8), but poorly the chemokines (data not shown), indicating that IL-12 by itself cannot activate the positive circuit. Convincingly, microbial infection renders NK cells competent to the microbial TLR agonists via induction of DC or macrophage IL-12 production.

The mechanism underlying the increased responsiveness of NK cells to TLR agonists during P. acnes treatment is still to be elucidated. To investigate possible involvement of IFN- in this response, we administered neutralizing anti-IFN- into mice on and after P. acnes treatment. However, IFN- blockade did not reduce their responsiveness to PGN (data not shown), indicating minor role of endogenous IFN- in the facilitated responsiveness of NK cells to TLR agonists. We need further study to conclude that endogenous IL-12 contributes to this differentiation of NK cells.

It has been reported that various chemokines and their receptors contribute to host defense. In particular, CCR5 and its ligands, CCL3, CCL4 and CCL5, are important. The ccl3^–/– mice have defects in protection against viral infection with concomitant numerical reduction of infiltrated NK cells and abolition of elevated IFN- in the infection sites (32, 35), indicating the importance of CCL3 for the recruitment of IFN--producing NK cells and presumably of IL-12-producing DCs and macrophages. Indeed, DCs and macrophages as well as NK cells express CCR5 (15, 36, 37). Expectedly, mice deficient in CCR5, like ccl3^–/– mice, are highly susceptible to infection with intracellular pathogenic fungi and various viruses (37, 38). It was previously reported that following viral infection, NK cells produce CCL3 and CCL4 via activation of their activating receptors and/or in response to endogenous type I IFN (34, 39, 40). Our present results clearly demonstrate a third chemokine-inducing tool equipped by NK cells. NK cells produce those CCR5 ligands in response to TLR agonists (Fig. 8). Therefore, it is convincing that the TLR/MyD88-mediated NK cell production of these chemokines might also contribute to the eradication of various types of microbes.

Synergy of IL-18 and IL-12 for IFN- is partly attributed to the up-regulated expression of IL-18R by IL-12R and vice versa (9, 29). In analog with this, we investigated whether TLR expressions on NK cells are also increased after stimulation with IL-12. However, IL-12 did not induce increase of TLR4/MD2 or CD14 expressions in NK cells (data not shown). It was shown that the synergy occurs at the signaling levels via activation of both NF-B and stat4, which are induced by IL-18 and IL-12, respectively (9, 41). TLR ligands, like IL-18, can activate NF-B as well (Fig. 1C). Therefore, synergistic action of TLR agonists and IL-12 for their IFN- might be regulated at their signaling levels similar to that of IL-18 and IL-12 (9, 41).

TLR ligands, unlike IL-18, did not augment murine NK cytotoxicity even in the presence of IL-12 (Fig. 6). As previously reported, murine NK cells show increased perforin- or Fas ligand-dependent NK lytic activities after stimulation with IL-18 (24, 42). IL-12, like IL-18, up-regulates perforin-dependent NK activity both in vitro (24). However, unlike for IFN- production, these two cytokines do not synergize for increase of NK cytolitic activity (24). Furthermore, IL-18 can augment NK lytic activity in the absence of IL-12 (24), strongly indicating that IL-18 signaling for up-regulating NK cell killing activity might be independent of that for IFN- production (9, 41). Accordingly, TLR ligands can replace IL-18 for IFN- production presumably via activation of NF-B, but not activate the signal pathway for NK cytolysis induced by IL-18. Further study is required for the identification of this signal pathway of IL-18 up-regulation of NK lysis, that is not shared by the TLR-mediated signal pathway. Importantly, in contrast to our results of mouse NK cells, human NK cells show increased NK lytic activity upon stimulation with TLR agonists (43–46). We need further study to clarify the molecular mechanisms differentially underlying those two species.

There is a possibility that IL-15-expanded CD3^–DX5⁺ population is contaminated with non-NK cells that might produce the upstream cytokines that might induce type 1 cytokine/chemokine production from NK cells. To exclude this possibility, we used mouse-cloned NK cells, namely, LNK5E6 cells and analyzed their potentials (42). The LNK cells similarly expressed various TLRs as in freshly isolated and IL-15-expanded NK cells (Supplementary Figure 3, available at International Immunology Online). Furthermore, the cells synergistically produced IFN- in response to TLR7 ligands and IL-12. These results support our conclusion that NK cells are directly activated through their TLRs for production of type 1 cytokine/chemokine, which may eventually lead to the early microbial clearance.

In summary, our present results clearly showed that NK cells produce robust IFN- and CCR5 ligands in response to TLRs plus IL-12 and suggested that the NK cells activated by this combination might be essential for the efficient early eradication of pathogenic microbes. Although we need further studies to evaluate in vivo precise roles of these NK cell responses, our present observations still provide new insights into NK cell activation in the host defense, particularly at the early infectious phase.

	Supplementary data

Top Abstract Introduction Methods Results Discussion Supplementary data References

 
Supplementary figures 1, 2 and 3 are available at International Immunology Online.

	Acknowledgements

 
This study was partly supported by Grants and a Hitec Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Ms Shizue Yumikura-Futatsugi and Ms Noriko Nakano for excellent technical assistance.

	Abbreviations

 

DC, dendritic cell

PGN, peptidoglycan

TLR, toll-like receptor

TNF, tumor necrosis factor

WT, wild type

	Notes

 
Transmitting editor: T. Hamaoka

Received 23 October 2006, accepted 22 December 2006.

	References

Top Abstract Introduction Methods Results Discussion Supplementary data References

 

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