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Direct TLR2 signaling is critical for NK cell activation and function in response to vaccinia viral infection.

Martinez J, Huang X, Yang Y - PLoS Pathog. (2010)

Bottom Line: However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood.In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets.We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections.

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The NKG2D pathway is required for efficient NK activation and function in response to VV infection.(A) WT or TLR2−/− DX5+CD3− NK cells were co-cultured with WT CD11c+ DCs and stimulated with VV alone (+VV), VV in the presence of anti-NKG2D (VV+anti-NKG2D) or NKp46-Fc chimera (VV+NKp46-Fc), or left uninfected (Uninfected). 24 hr later, NK cells were assayed for intracellular IFN-γ and Granzyme B. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is shown. (B–C) WT or TLR2−/− mice were infected with VV (+VV) or left uninfected (Naïve). Some mice were pre-treated with anti-NKG2D antibodies 24 and 6 h prior to infection with VV (VV+anti-NKG2D). 48 h after infection, splenic NK cells were analyzed for IFN-γ and Granzyme B production. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is indicated (n  =  4 per group) (B). The ovaries of female mice were harvested for measurement of viral load. Data represents the mean viral titer ± SD as pfu per ovary (n  =  4 per group) (C). (D) 48 h after infection, splenocytes from WT mice were assayed for NK cell lytic activity on VV-infected L929 cells in the presence of anti-NKG2D antibodies (+anti-NKG2D) or NKp46-Fc chimera (+NKp46-Fc), for 4 hr at different effector∶target ratios. The mean percentage ± SD of specific lysis is indicated (n  =  4 per group). Data shown is representative of two independent experiments.
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ppat-1000811-g006: The NKG2D pathway is required for efficient NK activation and function in response to VV infection.(A) WT or TLR2−/− DX5+CD3− NK cells were co-cultured with WT CD11c+ DCs and stimulated with VV alone (+VV), VV in the presence of anti-NKG2D (VV+anti-NKG2D) or NKp46-Fc chimera (VV+NKp46-Fc), or left uninfected (Uninfected). 24 hr later, NK cells were assayed for intracellular IFN-γ and Granzyme B. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is shown. (B–C) WT or TLR2−/− mice were infected with VV (+VV) or left uninfected (Naïve). Some mice were pre-treated with anti-NKG2D antibodies 24 and 6 h prior to infection with VV (VV+anti-NKG2D). 48 h after infection, splenic NK cells were analyzed for IFN-γ and Granzyme B production. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is indicated (n  =  4 per group) (B). The ovaries of female mice were harvested for measurement of viral load. Data represents the mean viral titer ± SD as pfu per ovary (n  =  4 per group) (C). (D) 48 h after infection, splenocytes from WT mice were assayed for NK cell lytic activity on VV-infected L929 cells in the presence of anti-NKG2D antibodies (+anti-NKG2D) or NKp46-Fc chimera (+NKp46-Fc), for 4 hr at different effector∶target ratios. The mean percentage ± SD of specific lysis is indicated (n  =  4 per group). Data shown is representative of two independent experiments.

Mentions: Previous studies have shown that NKG2D is partially involved in NK cell-mediated control of mousepox virus in vivo [27], and that recognition of VV-infected cells by human NK cells is, in part, mediated by natural cytotoxicity receptors, NKp30, NKp44 and NKp46 [26]. Among natural cytotoxicity receptors, only NKp46 is expressed in mice. Thus, we investigated whether NKG2D or NKp46 contributed to TLR2-independent NK cell activation in response to VV infection. We first tested this in vitro with the NK-DC co-culture system. Purified NK cells from WT or TLR2−/− mice were co-cultured in vitro with WT CD11c+ DCs in the presence of a blocking anti-NKG2D antibody or a blocking NKp46-Fc fusion protein, followed by infection with VV, the activation of NK cells was analyzed 24 h later. The production of IFN-γ and granzyme B by WT NK cells was significantly (p <0.01) decreased in the presence of anti-NKG2D compared to the control without anti-NKG2D (Figure 6A). In addition, the production of IFN-γ and granzyme B by TLR2−/− NK cells was completely abolished with the NKG2D blocking (Figure 6A). However, blocking with NKp46-Fc had no effect on activation of WT or TLR2−/− NK cells (Figure 6A). These results indicate that NKG2D, but not NKp46, is also involved in NK cell activation upon VV infection.


Direct TLR2 signaling is critical for NK cell activation and function in response to vaccinia viral infection.

Martinez J, Huang X, Yang Y - PLoS Pathog. (2010)

The NKG2D pathway is required for efficient NK activation and function in response to VV infection.(A) WT or TLR2−/− DX5+CD3− NK cells were co-cultured with WT CD11c+ DCs and stimulated with VV alone (+VV), VV in the presence of anti-NKG2D (VV+anti-NKG2D) or NKp46-Fc chimera (VV+NKp46-Fc), or left uninfected (Uninfected). 24 hr later, NK cells were assayed for intracellular IFN-γ and Granzyme B. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is shown. (B–C) WT or TLR2−/− mice were infected with VV (+VV) or left uninfected (Naïve). Some mice were pre-treated with anti-NKG2D antibodies 24 and 6 h prior to infection with VV (VV+anti-NKG2D). 48 h after infection, splenic NK cells were analyzed for IFN-γ and Granzyme B production. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is indicated (n  =  4 per group) (B). The ovaries of female mice were harvested for measurement of viral load. Data represents the mean viral titer ± SD as pfu per ovary (n  =  4 per group) (C). (D) 48 h after infection, splenocytes from WT mice were assayed for NK cell lytic activity on VV-infected L929 cells in the presence of anti-NKG2D antibodies (+anti-NKG2D) or NKp46-Fc chimera (+NKp46-Fc), for 4 hr at different effector∶target ratios. The mean percentage ± SD of specific lysis is indicated (n  =  4 per group). Data shown is representative of two independent experiments.
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Related In: Results  -  Collection

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ppat-1000811-g006: The NKG2D pathway is required for efficient NK activation and function in response to VV infection.(A) WT or TLR2−/− DX5+CD3− NK cells were co-cultured with WT CD11c+ DCs and stimulated with VV alone (+VV), VV in the presence of anti-NKG2D (VV+anti-NKG2D) or NKp46-Fc chimera (VV+NKp46-Fc), or left uninfected (Uninfected). 24 hr later, NK cells were assayed for intracellular IFN-γ and Granzyme B. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is shown. (B–C) WT or TLR2−/− mice were infected with VV (+VV) or left uninfected (Naïve). Some mice were pre-treated with anti-NKG2D antibodies 24 and 6 h prior to infection with VV (VV+anti-NKG2D). 48 h after infection, splenic NK cells were analyzed for IFN-γ and Granzyme B production. The mean percentage ± SD of IFN-γ or Granzyme B positive cells among DX5+CD3− cells is indicated (n  =  4 per group) (B). The ovaries of female mice were harvested for measurement of viral load. Data represents the mean viral titer ± SD as pfu per ovary (n  =  4 per group) (C). (D) 48 h after infection, splenocytes from WT mice were assayed for NK cell lytic activity on VV-infected L929 cells in the presence of anti-NKG2D antibodies (+anti-NKG2D) or NKp46-Fc chimera (+NKp46-Fc), for 4 hr at different effector∶target ratios. The mean percentage ± SD of specific lysis is indicated (n  =  4 per group). Data shown is representative of two independent experiments.
Mentions: Previous studies have shown that NKG2D is partially involved in NK cell-mediated control of mousepox virus in vivo [27], and that recognition of VV-infected cells by human NK cells is, in part, mediated by natural cytotoxicity receptors, NKp30, NKp44 and NKp46 [26]. Among natural cytotoxicity receptors, only NKp46 is expressed in mice. Thus, we investigated whether NKG2D or NKp46 contributed to TLR2-independent NK cell activation in response to VV infection. We first tested this in vitro with the NK-DC co-culture system. Purified NK cells from WT or TLR2−/− mice were co-cultured in vitro with WT CD11c+ DCs in the presence of a blocking anti-NKG2D antibody or a blocking NKp46-Fc fusion protein, followed by infection with VV, the activation of NK cells was analyzed 24 h later. The production of IFN-γ and granzyme B by WT NK cells was significantly (p <0.01) decreased in the presence of anti-NKG2D compared to the control without anti-NKG2D (Figure 6A). In addition, the production of IFN-γ and granzyme B by TLR2−/− NK cells was completely abolished with the NKG2D blocking (Figure 6A). However, blocking with NKp46-Fc had no effect on activation of WT or TLR2−/− NK cells (Figure 6A). These results indicate that NKG2D, but not NKp46, is also involved in NK cell activation upon VV infection.

Bottom Line: However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood.In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets.We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Duke University Medical Center, Durham, North Carolina, United States of America.

ABSTRACT
Natural killer (NK) cells play an essential role in innate immune control of poxviral infections in vivo. However, the mechanism(s) underlying NK cell activation and function in response to poxviruses remains poorly understood. In a mouse model of infection with vaccinia virus (VV), the most studied member of the poxvirus family, we identified that the Toll-like receptor (TLR) 2-myeloid differentiating factor 88 (MyD88) pathway was critical for the activation of NK cells and the control of VV infection in vivo. We further showed that TLR2 signaling on NK cells, but not on accessory cells such as dendritic cells (DCs), was necessary for NK cell activation and that this intrinsic TLR2-MyD88 signaling pathway was required for NK cell activation and played a critical role in the control of VV infection in vivo. In addition, we showed that the activating receptor NKG2D was also important for efficient NK activation and function, as well as recognition of VV-infected targets. We further demonstrated that VV could directly activate NK cells via TLR2 in the presence of cytokines in vitro and TLR2-MyD88-dependent activation of NK cells by VV was mediated through the phosphatidylinositol 3-kinase (PI3K)-extracellular signal-regulated kinase (ERK) pathway. Taken together, these results represent the first evidence that intrinsic TLR signaling is critical for NK cell activation and function in the control of a viral infection in vivo, indicate that multiple pathways are required for efficient NK cell activation and function in response to VV infection, and may provide important insights into the design of effective strategies to combat poxviral infections.

Show MeSH
Related in: MedlinePlus