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Innate sensing of HIV-infected cells.

Lepelley A, Louis S, Sourisseau M, Law HK, Pothlichet J, Schilte C, Chaperot L, Plumas J, Randall RE, Si-Tahar M, Mammano F, Albert ML, Schwartz O - PLoS Pathog. (2011)

Bottom Line: In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing.In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells.Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Virus and Immunity Unit, URA CNRS 3015, Paris, France.

ABSTRACT
Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

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Sensing of HIV-infected lymphocytes by 293T-derived epithelial cells.a. Role of CD4 and CXCR4 in 293T cells. Activity of the IFNβ promoter in parental 293T cells, and in 293T cells expressing CXCR4 (293T CXCR4), or CD4 and CXCR4 (293T-4X4). These cells were transfected with IFNβ-luciferase reporter plasmid, and cocultivated for 16 h with MT4C5 cells expressing wild-type, ΔEnv, or F552Y HIV. The fold induction of luciferase activity, compared to non-stimulated cells is shown. The paramyxovirus Sendai virus (SeV) was used as a positive control (right panel). b. Role of IRF3. 293T-4X4 cells were transfected with an irrelevant (CTRL) or an anti IRF3 siRNA. 48 h later, cells were cocultivated with HIV-infected MT4C5 cells and assayed for IFN-β promoter activity. Left panel: IRF3 and NFkB levels, assessed by western blot, in control (CTRL) and silenced 293T-4X4 cells. Right panel: IFN-β promoter activity is expressed as a percentage of the signal obtained with control cells (CTRL). SeV, which signals through IRF3, was used as a control. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). c–d. Automated quantification of IRF3 nuclear translocation. 293T-4X4 cells were cocultivated with non-infected (NI) or HIV-infected Far-red dye stained MT4C5 cells, exposed to SeV, or left non-stimulated (NS) for 16 h. Cells were then stained with anti-IRF3 Abs and nuclei were visualized with DAPI. Automated quantification of IRF3 nuclear translocation on gated 293T-4X4 (Far-red dye negative) cells was performed using the ImageStream technology. c. Representative images of Bright field, Dapi (blue) IRF3 (green), DAPI/IRF3 composite images, for cells with either a diffuse intracellular (non translocated, low similiarity value) or a nuclear localization (translocated, high similarity value) are shown. d. 293T-4X4 cells with nuclear-localized IRF3 have higher similarity values, because the aspect of IRF3 and DAPI staining is similar. The percentage of cells with similarity values above an arbitrary value of 1.5 (where most, if not all of the cells fall in the translocated category) is indicated on each histogram. Data are representative of 4 independent experiments.
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ppat-1001284-g005: Sensing of HIV-infected lymphocytes by 293T-derived epithelial cells.a. Role of CD4 and CXCR4 in 293T cells. Activity of the IFNβ promoter in parental 293T cells, and in 293T cells expressing CXCR4 (293T CXCR4), or CD4 and CXCR4 (293T-4X4). These cells were transfected with IFNβ-luciferase reporter plasmid, and cocultivated for 16 h with MT4C5 cells expressing wild-type, ΔEnv, or F552Y HIV. The fold induction of luciferase activity, compared to non-stimulated cells is shown. The paramyxovirus Sendai virus (SeV) was used as a positive control (right panel). b. Role of IRF3. 293T-4X4 cells were transfected with an irrelevant (CTRL) or an anti IRF3 siRNA. 48 h later, cells were cocultivated with HIV-infected MT4C5 cells and assayed for IFN-β promoter activity. Left panel: IRF3 and NFkB levels, assessed by western blot, in control (CTRL) and silenced 293T-4X4 cells. Right panel: IFN-β promoter activity is expressed as a percentage of the signal obtained with control cells (CTRL). SeV, which signals through IRF3, was used as a control. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). c–d. Automated quantification of IRF3 nuclear translocation. 293T-4X4 cells were cocultivated with non-infected (NI) or HIV-infected Far-red dye stained MT4C5 cells, exposed to SeV, or left non-stimulated (NS) for 16 h. Cells were then stained with anti-IRF3 Abs and nuclei were visualized with DAPI. Automated quantification of IRF3 nuclear translocation on gated 293T-4X4 (Far-red dye negative) cells was performed using the ImageStream technology. c. Representative images of Bright field, Dapi (blue) IRF3 (green), DAPI/IRF3 composite images, for cells with either a diffuse intracellular (non translocated, low similiarity value) or a nuclear localization (translocated, high similarity value) are shown. d. 293T-4X4 cells with nuclear-localized IRF3 have higher similarity values, because the aspect of IRF3 and DAPI staining is similar. The percentage of cells with similarity values above an arbitrary value of 1.5 (where most, if not all of the cells fall in the translocated category) is indicated on each histogram. Data are representative of 4 independent experiments.

Mentions: The experiments with fusion-defective viruses described above implicated cytosolic receptors in HIV recognition. We asked whether HIV-infected lymphocytes can be detected by additional, TLR7 independent cellular pathways. To this end, we performed cocultures with 293T cells as targets, as these latter do not express TLR7, TLR9 and TLR3 [51] and are widely used to dissect pathways of IFN signaling. We confirmed that 293T cells do not express detectable levels of TLR7 mRNA by real-time PCR, and that they are refractory to stimulation by the TLR7/8 agonist Gardiquimod (not shown). Next, we evaluated whether HIV-1 infected lymphocytes could activate in 293T cells a luciferase reporter that is under the control of the IFNβ promoter (IFNβ-luc). This technique is widely used to assess the IFNβ pathway in 293T cells [52], [53], [54], [55], because the levels of IFN released in the supernatants are below detection limits (not shown). Importantly, there was no induction of luciferase over background levels, upon coculture of HIV-infected MT4C5 cells with IFNβ-luc transfected 293T cells (Fig. 5a). 293T cells lack HIV receptors CXCR4 and CD4. We generated 293T cells expressing either CXCR4 (293T-CXCR4) or both CD4 and CXCR4 (293T-4X4 cells). In contrast to parental cells and to 293T-CXCR4 cells, 293T-4X4 cells activated the IFNβ promoter, when mixed with MT4C5 cells infected with wild-type HIV (7 fold increase, when compared to non-infected cells). Noteworthy, MT4C5 cells expressing either ΔEnv or the non-fusogenic F522Y strain failed to elicit luciferase activity (Fig. 5a). As a positive control, we used Sendai virus (SeV), a parainfluenza virus that activates IFNβ in 293T cells through the RIG-I/IRF3 pathway [52], [53], [54]. Moreover, free HIV virions (even at high concentrations) did not activate 293T-4X4 (not shown). Altogether, these results indicated that viral fusion activates the IFNβ promoter in the absence of TLR7. Detection of HIV-infected cells is more efficient than that of virions also in this experimental system.


Innate sensing of HIV-infected cells.

Lepelley A, Louis S, Sourisseau M, Law HK, Pothlichet J, Schilte C, Chaperot L, Plumas J, Randall RE, Si-Tahar M, Mammano F, Albert ML, Schwartz O - PLoS Pathog. (2011)

Sensing of HIV-infected lymphocytes by 293T-derived epithelial cells.a. Role of CD4 and CXCR4 in 293T cells. Activity of the IFNβ promoter in parental 293T cells, and in 293T cells expressing CXCR4 (293T CXCR4), or CD4 and CXCR4 (293T-4X4). These cells were transfected with IFNβ-luciferase reporter plasmid, and cocultivated for 16 h with MT4C5 cells expressing wild-type, ΔEnv, or F552Y HIV. The fold induction of luciferase activity, compared to non-stimulated cells is shown. The paramyxovirus Sendai virus (SeV) was used as a positive control (right panel). b. Role of IRF3. 293T-4X4 cells were transfected with an irrelevant (CTRL) or an anti IRF3 siRNA. 48 h later, cells were cocultivated with HIV-infected MT4C5 cells and assayed for IFN-β promoter activity. Left panel: IRF3 and NFkB levels, assessed by western blot, in control (CTRL) and silenced 293T-4X4 cells. Right panel: IFN-β promoter activity is expressed as a percentage of the signal obtained with control cells (CTRL). SeV, which signals through IRF3, was used as a control. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). c–d. Automated quantification of IRF3 nuclear translocation. 293T-4X4 cells were cocultivated with non-infected (NI) or HIV-infected Far-red dye stained MT4C5 cells, exposed to SeV, or left non-stimulated (NS) for 16 h. Cells were then stained with anti-IRF3 Abs and nuclei were visualized with DAPI. Automated quantification of IRF3 nuclear translocation on gated 293T-4X4 (Far-red dye negative) cells was performed using the ImageStream technology. c. Representative images of Bright field, Dapi (blue) IRF3 (green), DAPI/IRF3 composite images, for cells with either a diffuse intracellular (non translocated, low similiarity value) or a nuclear localization (translocated, high similarity value) are shown. d. 293T-4X4 cells with nuclear-localized IRF3 have higher similarity values, because the aspect of IRF3 and DAPI staining is similar. The percentage of cells with similarity values above an arbitrary value of 1.5 (where most, if not all of the cells fall in the translocated category) is indicated on each histogram. Data are representative of 4 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040675&req=5

ppat-1001284-g005: Sensing of HIV-infected lymphocytes by 293T-derived epithelial cells.a. Role of CD4 and CXCR4 in 293T cells. Activity of the IFNβ promoter in parental 293T cells, and in 293T cells expressing CXCR4 (293T CXCR4), or CD4 and CXCR4 (293T-4X4). These cells were transfected with IFNβ-luciferase reporter plasmid, and cocultivated for 16 h with MT4C5 cells expressing wild-type, ΔEnv, or F552Y HIV. The fold induction of luciferase activity, compared to non-stimulated cells is shown. The paramyxovirus Sendai virus (SeV) was used as a positive control (right panel). b. Role of IRF3. 293T-4X4 cells were transfected with an irrelevant (CTRL) or an anti IRF3 siRNA. 48 h later, cells were cocultivated with HIV-infected MT4C5 cells and assayed for IFN-β promoter activity. Left panel: IRF3 and NFkB levels, assessed by western blot, in control (CTRL) and silenced 293T-4X4 cells. Right panel: IFN-β promoter activity is expressed as a percentage of the signal obtained with control cells (CTRL). SeV, which signals through IRF3, was used as a control. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). c–d. Automated quantification of IRF3 nuclear translocation. 293T-4X4 cells were cocultivated with non-infected (NI) or HIV-infected Far-red dye stained MT4C5 cells, exposed to SeV, or left non-stimulated (NS) for 16 h. Cells were then stained with anti-IRF3 Abs and nuclei were visualized with DAPI. Automated quantification of IRF3 nuclear translocation on gated 293T-4X4 (Far-red dye negative) cells was performed using the ImageStream technology. c. Representative images of Bright field, Dapi (blue) IRF3 (green), DAPI/IRF3 composite images, for cells with either a diffuse intracellular (non translocated, low similiarity value) or a nuclear localization (translocated, high similarity value) are shown. d. 293T-4X4 cells with nuclear-localized IRF3 have higher similarity values, because the aspect of IRF3 and DAPI staining is similar. The percentage of cells with similarity values above an arbitrary value of 1.5 (where most, if not all of the cells fall in the translocated category) is indicated on each histogram. Data are representative of 4 independent experiments.
Mentions: The experiments with fusion-defective viruses described above implicated cytosolic receptors in HIV recognition. We asked whether HIV-infected lymphocytes can be detected by additional, TLR7 independent cellular pathways. To this end, we performed cocultures with 293T cells as targets, as these latter do not express TLR7, TLR9 and TLR3 [51] and are widely used to dissect pathways of IFN signaling. We confirmed that 293T cells do not express detectable levels of TLR7 mRNA by real-time PCR, and that they are refractory to stimulation by the TLR7/8 agonist Gardiquimod (not shown). Next, we evaluated whether HIV-1 infected lymphocytes could activate in 293T cells a luciferase reporter that is under the control of the IFNβ promoter (IFNβ-luc). This technique is widely used to assess the IFNβ pathway in 293T cells [52], [53], [54], [55], because the levels of IFN released in the supernatants are below detection limits (not shown). Importantly, there was no induction of luciferase over background levels, upon coculture of HIV-infected MT4C5 cells with IFNβ-luc transfected 293T cells (Fig. 5a). 293T cells lack HIV receptors CXCR4 and CD4. We generated 293T cells expressing either CXCR4 (293T-CXCR4) or both CD4 and CXCR4 (293T-4X4 cells). In contrast to parental cells and to 293T-CXCR4 cells, 293T-4X4 cells activated the IFNβ promoter, when mixed with MT4C5 cells infected with wild-type HIV (7 fold increase, when compared to non-infected cells). Noteworthy, MT4C5 cells expressing either ΔEnv or the non-fusogenic F522Y strain failed to elicit luciferase activity (Fig. 5a). As a positive control, we used Sendai virus (SeV), a parainfluenza virus that activates IFNβ in 293T cells through the RIG-I/IRF3 pathway [52], [53], [54]. Moreover, free HIV virions (even at high concentrations) did not activate 293T-4X4 (not shown). Altogether, these results indicated that viral fusion activates the IFNβ promoter in the absence of TLR7. Detection of HIV-infected cells is more efficient than that of virions also in this experimental system.

Bottom Line: In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing.In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells.Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Virus and Immunity Unit, URA CNRS 3015, Paris, France.

ABSTRACT
Cell-free HIV-1 virions are poor stimulators of type I interferon (IFN) production. We examined here how HIV-infected cells are recognized by plasmacytoid dendritic cells (pDCs) and by other cells. We show that infected lymphocytes are more potent inducers of IFN than virions. There are target cell-type differences in the recognition of infected lymphocytes. In primary pDCs and pDC-like cells, recognition occurs in large part through TLR7, as demonstrated by the use of inhibitors and by TLR7 silencing. Donor cells expressing replication-defective viruses, carrying mutated reverse transcriptase, integrase or nucleocapsid proteins induced IFN production by target cells as potently as wild-type virus. In contrast, Env-deleted or fusion defective HIV-1 mutants were less efficient, suggesting that in addition to TLR7, cytoplasmic cellular sensors may also mediate sensing of infected cells. Furthermore, in a model of TLR7-negative cells, we demonstrate that the IRF3 pathway, through a process requiring access of incoming viral material to the cytoplasm, allows sensing of HIV-infected lymphocytes. Therefore, detection of HIV-infected lymphocytes occurs through both endosomal and cytoplasmic pathways. Characterization of the mechanisms of innate recognition of HIV-infected cells allows a better understanding of the pathogenic and exacerbated immunologic events associated with HIV infection.

Show MeSH
Related in: MedlinePlus