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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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Pathways of recognition of HIV-infected cells by hematopoïetic cells.a. Effect of drugs targeting endosomal TLR signaling or HIV reverse transcription in PBMCs. PBMCs were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 12 h, in the absence or presence of the indicated drugs. NVP (25 µM) is a reverse transcriptase inhibitor. Bafilomycin A1 (Bafilo, 125 nM) is an inhibitor of vesicular acidification. A151 (5 µg/mL) is a TLR antagonist. Nevirapin (NVP 25 µM) is a reverse transcriptase inhibitor. IFN levels are expressed as the % of the signal obtained without drugs. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). b-d. Viral proteins required for the recognition of HIV-expressing cells by PBMCs. b. Schematic representation of the experimental system. HeLa cells are transfected with various HIV mutants, cocultivated with PBMCs, and levels of IFN released in supernatants are measured 22 h later. c. Defective viruses trigger IFN production by PBMCs. HeLa cells were transfected with the indicated proviral mutants. Supernatants were harvested and analyzed for the presence of infectious virus, after normalization for Gag p24 levels. Env-deleted (ΔEnv), non-fusogenic Env (HIV F522Y), reverse transcriptase (RT-), integrase (IN), protease (PR), Rnase H (RH), and nucleocapsid (NC44) mutants are not or poorly infectious (left panel). Levels of IFN in supernatants, after coculture of tranfected HeLa cells with PBMCs. IFN levels are expressed as a percentage of the signal obtained with WT HIV (right panel). d. Expression of Env alone does not trigger IFN production. HeLa-Env cells (stably expressing a functional Env glycoprotein complex) were either not infected (NI) or infected with ΔEnv or WT viruses (pseudotyped with VSV), then processed and analyzed as in c. c-d: Mean+sd of 2-3 independent experiments is shown. *p<0.05 (Kruskal-Wallis).
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ppat-1001284-g002: Pathways of recognition of HIV-infected cells by hematopoïetic cells.a. Effect of drugs targeting endosomal TLR signaling or HIV reverse transcription in PBMCs. PBMCs were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 12 h, in the absence or presence of the indicated drugs. NVP (25 µM) is a reverse transcriptase inhibitor. Bafilomycin A1 (Bafilo, 125 nM) is an inhibitor of vesicular acidification. A151 (5 µg/mL) is a TLR antagonist. Nevirapin (NVP 25 µM) is a reverse transcriptase inhibitor. IFN levels are expressed as the % of the signal obtained without drugs. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). b-d. Viral proteins required for the recognition of HIV-expressing cells by PBMCs. b. Schematic representation of the experimental system. HeLa cells are transfected with various HIV mutants, cocultivated with PBMCs, and levels of IFN released in supernatants are measured 22 h later. c. Defective viruses trigger IFN production by PBMCs. HeLa cells were transfected with the indicated proviral mutants. Supernatants were harvested and analyzed for the presence of infectious virus, after normalization for Gag p24 levels. Env-deleted (ΔEnv), non-fusogenic Env (HIV F522Y), reverse transcriptase (RT-), integrase (IN), protease (PR), Rnase H (RH), and nucleocapsid (NC44) mutants are not or poorly infectious (left panel). Levels of IFN in supernatants, after coculture of tranfected HeLa cells with PBMCs. IFN levels are expressed as a percentage of the signal obtained with WT HIV (right panel). d. Expression of Env alone does not trigger IFN production. HeLa-Env cells (stably expressing a functional Env glycoprotein complex) were either not infected (NI) or infected with ΔEnv or WT viruses (pseudotyped with VSV), then processed and analyzed as in c. c-d: Mean+sd of 2-3 independent experiments is shown. *p<0.05 (Kruskal-Wallis).

Mentions: To determine the mechanisms by which PBMCs sense HIV-infected lymphocytes, we first evaluated the effect of Bafilomycin A1, an inhibitor of vesicular acidification. As controls we utilized FLUAV and CpG, stimuli for TLR7 and TLR9, respectively [35] [41]. Bafilomycin A1 (125 nM, a concentration that did not detectably affected cell viability at 24 h, not shown) inhibited HIV recognition (Fig. 2a), consistently with the requirement for an acidic endosome and viral degradation in order to achieve TLR signaling [15], [42], [43]. Of note, Bafilomycin A1 at 25 nM also inhibited HIV recognition (not shown). FLUAV and CpG stimulation were strongly inhibited by Bafilomycin A1, as expected. We also tested the effect of A151, an oligonucleotide described as a TLR antagonist, (inhibiting TLR7 and, to a lower extent, TLR9) [15], [43]. A151 (5 µg/mL) inhibited IFN production by PBMCs cocultivated with HIV-infected MT4C5 cells (Fig. 2a). At 5 µg/ml, A151 inihibited FLUAV, but not CpG stimulation of PBMCs. At a higher concentration (20 µg/ml), A151 partly inhibited CpG stimulation (not shown), confirming that this compound may antagonize both TLR7 and TLR9. Altogether, these results suggest that detection of HIV-infected cells by pDCs and by PBMCs requires an acidic environment and is in large part mediated by endosomal TLRs.


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)

Pathways of recognition of HIV-infected cells by hematopoïetic cells.a. Effect of drugs targeting endosomal TLR signaling or HIV reverse transcription in PBMCs. PBMCs were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 12 h, in the absence or presence of the indicated drugs. NVP (25 µM) is a reverse transcriptase inhibitor. Bafilomycin A1 (Bafilo, 125 nM) is an inhibitor of vesicular acidification. A151 (5 µg/mL) is a TLR antagonist. Nevirapin (NVP 25 µM) is a reverse transcriptase inhibitor. IFN levels are expressed as the % of the signal obtained without drugs. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). b-d. Viral proteins required for the recognition of HIV-expressing cells by PBMCs. b. Schematic representation of the experimental system. HeLa cells are transfected with various HIV mutants, cocultivated with PBMCs, and levels of IFN released in supernatants are measured 22 h later. c. Defective viruses trigger IFN production by PBMCs. HeLa cells were transfected with the indicated proviral mutants. Supernatants were harvested and analyzed for the presence of infectious virus, after normalization for Gag p24 levels. Env-deleted (ΔEnv), non-fusogenic Env (HIV F522Y), reverse transcriptase (RT-), integrase (IN), protease (PR), Rnase H (RH), and nucleocapsid (NC44) mutants are not or poorly infectious (left panel). Levels of IFN in supernatants, after coculture of tranfected HeLa cells with PBMCs. IFN levels are expressed as a percentage of the signal obtained with WT HIV (right panel). d. Expression of Env alone does not trigger IFN production. HeLa-Env cells (stably expressing a functional Env glycoprotein complex) were either not infected (NI) or infected with ΔEnv or WT viruses (pseudotyped with VSV), then processed and analyzed as in c. c-d: Mean+sd of 2-3 independent experiments is shown. *p<0.05 (Kruskal-Wallis).
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Related In: Results  -  Collection

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ppat-1001284-g002: Pathways of recognition of HIV-infected cells by hematopoïetic cells.a. Effect of drugs targeting endosomal TLR signaling or HIV reverse transcription in PBMCs. PBMCs were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 12 h, in the absence or presence of the indicated drugs. NVP (25 µM) is a reverse transcriptase inhibitor. Bafilomycin A1 (Bafilo, 125 nM) is an inhibitor of vesicular acidification. A151 (5 µg/mL) is a TLR antagonist. Nevirapin (NVP 25 µM) is a reverse transcriptase inhibitor. IFN levels are expressed as the % of the signal obtained without drugs. Mean+sd of 3 independent experiments is shown. *p<0.05 (Kruskal-Wallis). b-d. Viral proteins required for the recognition of HIV-expressing cells by PBMCs. b. Schematic representation of the experimental system. HeLa cells are transfected with various HIV mutants, cocultivated with PBMCs, and levels of IFN released in supernatants are measured 22 h later. c. Defective viruses trigger IFN production by PBMCs. HeLa cells were transfected with the indicated proviral mutants. Supernatants were harvested and analyzed for the presence of infectious virus, after normalization for Gag p24 levels. Env-deleted (ΔEnv), non-fusogenic Env (HIV F522Y), reverse transcriptase (RT-), integrase (IN), protease (PR), Rnase H (RH), and nucleocapsid (NC44) mutants are not or poorly infectious (left panel). Levels of IFN in supernatants, after coculture of tranfected HeLa cells with PBMCs. IFN levels are expressed as a percentage of the signal obtained with WT HIV (right panel). d. Expression of Env alone does not trigger IFN production. HeLa-Env cells (stably expressing a functional Env glycoprotein complex) were either not infected (NI) or infected with ΔEnv or WT viruses (pseudotyped with VSV), then processed and analyzed as in c. c-d: Mean+sd of 2-3 independent experiments is shown. *p<0.05 (Kruskal-Wallis).
Mentions: To determine the mechanisms by which PBMCs sense HIV-infected lymphocytes, we first evaluated the effect of Bafilomycin A1, an inhibitor of vesicular acidification. As controls we utilized FLUAV and CpG, stimuli for TLR7 and TLR9, respectively [35] [41]. Bafilomycin A1 (125 nM, a concentration that did not detectably affected cell viability at 24 h, not shown) inhibited HIV recognition (Fig. 2a), consistently with the requirement for an acidic endosome and viral degradation in order to achieve TLR signaling [15], [42], [43]. Of note, Bafilomycin A1 at 25 nM also inhibited HIV recognition (not shown). FLUAV and CpG stimulation were strongly inhibited by Bafilomycin A1, as expected. We also tested the effect of A151, an oligonucleotide described as a TLR antagonist, (inhibiting TLR7 and, to a lower extent, TLR9) [15], [43]. A151 (5 µg/mL) inhibited IFN production by PBMCs cocultivated with HIV-infected MT4C5 cells (Fig. 2a). At 5 µg/ml, A151 inihibited FLUAV, but not CpG stimulation of PBMCs. At a higher concentration (20 µg/ml), A151 partly inhibited CpG stimulation (not shown), confirming that this compound may antagonize both TLR7 and TLR9. Altogether, these results suggest that detection of HIV-infected cells by pDCs and by PBMCs requires an acidic environment and is in large part mediated by endosomal TLRs.

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