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Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells.

Parikh BA, Piersma SJ, Pak-Wittel MA, Yang L, Schreiber RD, Yokoyama WM - PLoS Pathog. (2015)

Bottom Line: The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice.While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood.These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

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Related in: MedlinePlus

IFN-I or IL-12 are required for GzmB and Prf1 induction in NK cells in a cell intrinsic manner during MCMV infection.(A and B) Mice deficient in IFNAR1 and/or IL-12p40 were infected with WT MCMV at 5000 PFU/mouse. After 3 days, splenocytes were harvested and analyzed for GzmB and Prf1 expression as indicated. Representative contour plots of individual mice are shown in (A). The median fluorescence intensity (MFI) is indicated for each plot and the cumulative data for one experiment is shown in (B). (C) GzmB levels of WT mice infected with 1x105 PFU WT or Δm157 MCMV 2 days after infection. (D) 40x106 WT or IFNAR1-/-xIL-12Rβ2-/- splenocytes were adoptively transferred to congenic host that was simultaneously infected with 1x105 PFU MCMV/mouse. GzmB levels 2 days post-infection in host and transferred NK cells is depicted. Data are representative of three to four independent experiments with individual points representing a single mouse.
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ppat.1005323.g005: IFN-I or IL-12 are required for GzmB and Prf1 induction in NK cells in a cell intrinsic manner during MCMV infection.(A and B) Mice deficient in IFNAR1 and/or IL-12p40 were infected with WT MCMV at 5000 PFU/mouse. After 3 days, splenocytes were harvested and analyzed for GzmB and Prf1 expression as indicated. Representative contour plots of individual mice are shown in (A). The median fluorescence intensity (MFI) is indicated for each plot and the cumulative data for one experiment is shown in (B). (C) GzmB levels of WT mice infected with 1x105 PFU WT or Δm157 MCMV 2 days after infection. (D) 40x106 WT or IFNAR1-/-xIL-12Rβ2-/- splenocytes were adoptively transferred to congenic host that was simultaneously infected with 1x105 PFU MCMV/mouse. GzmB levels 2 days post-infection in host and transferred NK cells is depicted. Data are representative of three to four independent experiments with individual points representing a single mouse.

Mentions: Interestingly, at three days following MCMV infection, we found that levels of GzmB and Prf1 in splenic NK cells examined ex vivo were significantly elevated in mice with deficiency in either IFNAR1 or IL-12p40 (Fig 5A and 5B). Upregulation of GzmB appeared to be equivalent in both Ly49H+ and Ly49H- cells and did not require the presence of m157-expressing virus at 40 hours after infection (Fig 5C). IFNAR1–/–mice demonstrated a less robust increase in the expression of GzmB, as compared to WT and IL-12p40-deficient mice which displayed equivalent levels. Prf1 production was increased in IFNAR1–/–mice while IL-12p40–/–mice displayed decreased levels of Prf1 compared to WT, suggesting that GzmB and Prf1 are differentially regulated. Importantly, double cytokine-deficient mice did not display enhanced GzmB nor Prf1 protein levels following infection (Fig 5A and 5B). Indeed, the GzmB levels were even below those observed in uninfected WT mice, suggesting that both IL-12 and IFN-I contribute to production of small amounts of GzmB in naïve mice. For Prf1 this effect was not clearly observable, which may be due to the less robust Prf1 changes or detection. WT MCMV induced elevated levels of GzmB in Ly49H+ NK cells compared to their Ly49H- counterparts, similar to what was observed in vitro (S2 Fig). Importantly, this effect was also observed for Δm157 MCMV (S2B Fig), further indicating that signaling through Ly49H is not required for enhanced GzmB production in Ly49H+ NK cells during MCMV infection. Regardless, both IL-12 and IFN-I redundantly contribute to enhanced GzmB and Prf1 protein expression during MCMV infection.


Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells.

Parikh BA, Piersma SJ, Pak-Wittel MA, Yang L, Schreiber RD, Yokoyama WM - PLoS Pathog. (2015)

IFN-I or IL-12 are required for GzmB and Prf1 induction in NK cells in a cell intrinsic manner during MCMV infection.(A and B) Mice deficient in IFNAR1 and/or IL-12p40 were infected with WT MCMV at 5000 PFU/mouse. After 3 days, splenocytes were harvested and analyzed for GzmB and Prf1 expression as indicated. Representative contour plots of individual mice are shown in (A). The median fluorescence intensity (MFI) is indicated for each plot and the cumulative data for one experiment is shown in (B). (C) GzmB levels of WT mice infected with 1x105 PFU WT or Δm157 MCMV 2 days after infection. (D) 40x106 WT or IFNAR1-/-xIL-12Rβ2-/- splenocytes were adoptively transferred to congenic host that was simultaneously infected with 1x105 PFU MCMV/mouse. GzmB levels 2 days post-infection in host and transferred NK cells is depicted. Data are representative of three to four independent experiments with individual points representing a single mouse.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4697817&req=5

ppat.1005323.g005: IFN-I or IL-12 are required for GzmB and Prf1 induction in NK cells in a cell intrinsic manner during MCMV infection.(A and B) Mice deficient in IFNAR1 and/or IL-12p40 were infected with WT MCMV at 5000 PFU/mouse. After 3 days, splenocytes were harvested and analyzed for GzmB and Prf1 expression as indicated. Representative contour plots of individual mice are shown in (A). The median fluorescence intensity (MFI) is indicated for each plot and the cumulative data for one experiment is shown in (B). (C) GzmB levels of WT mice infected with 1x105 PFU WT or Δm157 MCMV 2 days after infection. (D) 40x106 WT or IFNAR1-/-xIL-12Rβ2-/- splenocytes were adoptively transferred to congenic host that was simultaneously infected with 1x105 PFU MCMV/mouse. GzmB levels 2 days post-infection in host and transferred NK cells is depicted. Data are representative of three to four independent experiments with individual points representing a single mouse.
Mentions: Interestingly, at three days following MCMV infection, we found that levels of GzmB and Prf1 in splenic NK cells examined ex vivo were significantly elevated in mice with deficiency in either IFNAR1 or IL-12p40 (Fig 5A and 5B). Upregulation of GzmB appeared to be equivalent in both Ly49H+ and Ly49H- cells and did not require the presence of m157-expressing virus at 40 hours after infection (Fig 5C). IFNAR1–/–mice demonstrated a less robust increase in the expression of GzmB, as compared to WT and IL-12p40-deficient mice which displayed equivalent levels. Prf1 production was increased in IFNAR1–/–mice while IL-12p40–/–mice displayed decreased levels of Prf1 compared to WT, suggesting that GzmB and Prf1 are differentially regulated. Importantly, double cytokine-deficient mice did not display enhanced GzmB nor Prf1 protein levels following infection (Fig 5A and 5B). Indeed, the GzmB levels were even below those observed in uninfected WT mice, suggesting that both IL-12 and IFN-I contribute to production of small amounts of GzmB in naïve mice. For Prf1 this effect was not clearly observable, which may be due to the less robust Prf1 changes or detection. WT MCMV induced elevated levels of GzmB in Ly49H+ NK cells compared to their Ly49H- counterparts, similar to what was observed in vitro (S2 Fig). Importantly, this effect was also observed for Δm157 MCMV (S2B Fig), further indicating that signaling through Ly49H is not required for enhanced GzmB production in Ly49H+ NK cells during MCMV infection. Regardless, both IL-12 and IFN-I redundantly contribute to enhanced GzmB and Prf1 protein expression during MCMV infection.

Bottom Line: The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice.While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood.These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

ABSTRACT
Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

Show MeSH
Related in: MedlinePlus