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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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TLR2-dependent activation of NK cells by VV is mediated by PI3K and ERK.(A) DX5+CD3− NK cells were infected with VV (+VV), VV in the presence of the PI3K inhibitor, LY294002 (+LY294002) or the ERK inhibitor, PD98059 (+PD98059) in vitro. 48 h after infection, 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. Data shown is representative of three independent experiments. (B–C) DX5+CD3− NK cells were stimulated in vitro with VV for the indicated time periods. After stimulation for 2, 4, and 6 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt), which served as a loading control (B). After stimulation for 12, 18, and 24 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK) (C). (D) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), or left unstimulated (Control) for 4 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt). (E) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), VV and the PI3K inhibitor LY294002 (+LY294002), or left unstimulated (Control) for 18 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK). Data shown is a representative blot of five independent experiments.
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ppat-1000811-g008: TLR2-dependent activation of NK cells by VV is mediated by PI3K and ERK.(A) DX5+CD3− NK cells were infected with VV (+VV), VV in the presence of the PI3K inhibitor, LY294002 (+LY294002) or the ERK inhibitor, PD98059 (+PD98059) in vitro. 48 h after infection, 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. Data shown is representative of three independent experiments. (B–C) DX5+CD3− NK cells were stimulated in vitro with VV for the indicated time periods. After stimulation for 2, 4, and 6 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt), which served as a loading control (B). After stimulation for 12, 18, and 24 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK) (C). (D) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), or left unstimulated (Control) for 4 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt). (E) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), VV and the PI3K inhibitor LY294002 (+LY294002), or left unstimulated (Control) for 18 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK). Data shown is a representative blot of five independent experiments.

Mentions: How does stimulation of TLR2-Myd88 pathway on NK cells by VV lead to NK cell activation? Recent studies have shown that in T cells, MyD88 can interact with PI3K and activate the PI3K pathway, leading to enhanced T cell activation and survival upon TLR stimulation [32], and PI3K is the common signaling mediator downstream of activating NK receptors, such as ITAM-bearing NK receptors and NKG2D [17]. Furthermore, the PI3K-ERK pathway has been shown to play an important role in NK cell activation and cytotoxicity [33],[34]. Thus, we hypothesized that TLR2-MyD88 signaling on NK cells activated the downstream PI3K-ERK pathway, leading to activation of NK cells upon VV infection. To test this hypothesis, we first examined if activation of NK cells by VV was mediated by PI3K and ERK. FACS-purified NK cells were stimulated with VV in the presence of the PI3K inhibitor, LY294002 or the ERK1/2 inhibitor, PD98059 and analyzed for their activation 48 h later. Indeed, the production of IFN-γ and granzyme B by NK cells was significantly (p <0.001) reduced in the presence of LY294002 or PD98059, compared to the control without inhibitors (Figure 8A), suggesting that both PI3K and ERK are involved in NK cell activation by VV.


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)

TLR2-dependent activation of NK cells by VV is mediated by PI3K and ERK.(A) DX5+CD3− NK cells were infected with VV (+VV), VV in the presence of the PI3K inhibitor, LY294002 (+LY294002) or the ERK inhibitor, PD98059 (+PD98059) in vitro. 48 h after infection, 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. Data shown is representative of three independent experiments. (B–C) DX5+CD3− NK cells were stimulated in vitro with VV for the indicated time periods. After stimulation for 2, 4, and 6 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt), which served as a loading control (B). After stimulation for 12, 18, and 24 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK) (C). (D) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), or left unstimulated (Control) for 4 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt). (E) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), VV and the PI3K inhibitor LY294002 (+LY294002), or left unstimulated (Control) for 18 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK). Data shown is a representative blot of five independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837413&req=5

ppat-1000811-g008: TLR2-dependent activation of NK cells by VV is mediated by PI3K and ERK.(A) DX5+CD3− NK cells were infected with VV (+VV), VV in the presence of the PI3K inhibitor, LY294002 (+LY294002) or the ERK inhibitor, PD98059 (+PD98059) in vitro. 48 h after infection, 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. Data shown is representative of three independent experiments. (B–C) DX5+CD3− NK cells were stimulated in vitro with VV for the indicated time periods. After stimulation for 2, 4, and 6 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt), which served as a loading control (B). After stimulation for 12, 18, and 24 h, NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK) (C). (D) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), or left unstimulated (Control) for 4 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated Akt (pAkt) as well as total Akt (Total Akt). (E) WT, TLR2−/−, and MyD88−/− DX5+CD3− NK cells were stimulated in vitro with VV (+VV), VV and the PI3K inhibitor LY294002 (+LY294002), or left unstimulated (Control) for 18 h. NK cells were removed from culture and total cell lysates were collected for Western blot analysis of phosphorylated ERK1/2 (pERK1/2) as well as total ERK (Total ERK). Data shown is a representative blot of five independent experiments.
Mentions: How does stimulation of TLR2-Myd88 pathway on NK cells by VV lead to NK cell activation? Recent studies have shown that in T cells, MyD88 can interact with PI3K and activate the PI3K pathway, leading to enhanced T cell activation and survival upon TLR stimulation [32], and PI3K is the common signaling mediator downstream of activating NK receptors, such as ITAM-bearing NK receptors and NKG2D [17]. Furthermore, the PI3K-ERK pathway has been shown to play an important role in NK cell activation and cytotoxicity [33],[34]. Thus, we hypothesized that TLR2-MyD88 signaling on NK cells activated the downstream PI3K-ERK pathway, leading to activation of NK cells upon VV infection. To test this hypothesis, we first examined if activation of NK cells by VV was mediated by PI3K and ERK. FACS-purified NK cells were stimulated with VV in the presence of the PI3K inhibitor, LY294002 or the ERK1/2 inhibitor, PD98059 and analyzed for their activation 48 h later. Indeed, the production of IFN-γ and granzyme B by NK cells was significantly (p <0.001) reduced in the presence of LY294002 or PD98059, compared to the control without inhibitors (Figure 8A), suggesting that both PI3K and ERK are involved in NK cell activation by VV.

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