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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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Role of cell contacts and of TLR7 on recognition of HIV-infected cells by Gen2.2 cells.a. Activation of Gen2.2 requires a direct contact with HIV-infected cells. Shaking cocultures of Gen2.2 and HIV-1-infected MT4C5 (left panel), or separating infected MT4C5 cells and target Gen2.2 in a transwell chamber (right panel) impairs IFN production, whereas recognition of FLUAV by Gen2.2 is not affected. Mean+sd of 3 independent experiments is shown b. Inhibition of IFN production by Bafilomycin A1. Gen2.2 cells were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 24 h, in the absence or presence of Bafilomycin A1 (Bafilo 125 nM). IFN levels are expressed as the % of the signal obtained without drugs. c. Role of TLR7. Gen2.2 cells were transduced with lentiviral vectors expressing shRNAs against TLR7 (shTLR7) or an irrelevant target (shCTRL). Levels of TLR7 mRNA in transduced cells were measured by RT-PCR. Data are normalized to GAPDH mRNA and expressed as relative levels of mRNA compared to shCTRL cells. Mean+sd of 3 independent experiments are shown (left panel). IFN production in TLR7-silenced and control Gen2.2 cells, cocultivated with HIV-infected MT4C5 or stimulated with FLUAV or CpG for 40 h. IFN levels are expressed as a percentage of the signal obtained with shCTRL Gen2.2 cells.
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ppat-1001284-g004: Role of cell contacts and of TLR7 on recognition of HIV-infected cells by Gen2.2 cells.a. Activation of Gen2.2 requires a direct contact with HIV-infected cells. Shaking cocultures of Gen2.2 and HIV-1-infected MT4C5 (left panel), or separating infected MT4C5 cells and target Gen2.2 in a transwell chamber (right panel) impairs IFN production, whereas recognition of FLUAV by Gen2.2 is not affected. Mean+sd of 3 independent experiments is shown b. Inhibition of IFN production by Bafilomycin A1. Gen2.2 cells were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 24 h, in the absence or presence of Bafilomycin A1 (Bafilo 125 nM). IFN levels are expressed as the % of the signal obtained without drugs. c. Role of TLR7. Gen2.2 cells were transduced with lentiviral vectors expressing shRNAs against TLR7 (shTLR7) or an irrelevant target (shCTRL). Levels of TLR7 mRNA in transduced cells were measured by RT-PCR. Data are normalized to GAPDH mRNA and expressed as relative levels of mRNA compared to shCTRL cells. Mean+sd of 3 independent experiments are shown (left panel). IFN production in TLR7-silenced and control Gen2.2 cells, cocultivated with HIV-infected MT4C5 or stimulated with FLUAV or CpG for 40 h. IFN levels are expressed as a percentage of the signal obtained with shCTRL Gen2.2 cells.

Mentions: Next, we examined how Gen2.2 cells reacted when encountering HIV-infected T cells. To this end, infected MT4C5 were mixed with Gen2.2, and IFN levels were measured at 12 or 24 h of coculture. IFN was barely detected at 12 h (not shown). At 24 h, about 25 U/ml IFN were released in supernatants (Fig. 3c), which is 10–30 fold less than in primary pDCs. FLUAV potently activated Gen2.2 cells, whereas cell-free HIV particles (500 ng/ml Gag p24) elicited no IFN release (Fig. 3c). We reported that a gentle shaking of cocultures of infected cells and target cells inhibits direct cell-to-cell viral spread [49]. Shaking cocultures of HIV-infected MT4C5 cells and Gen 2.2 cells strongly decreased IFN production, whereas induction by FLUAV was not affected (Fig. 4a). Furthermore, IFN levels were below detection levels when infected donors cells and target Gen2.2 recipients were separated by a Transwell chamber (Fig. 4a). This strongly suggested that a direct contact between HIV-infected lymphocytes and Gen 2.2 cells is required to mediate recognition. Confirming our observation made with primary pDCs, optimal recognition of infected cells required a functional Env, as evidenced by the low levels of IFN (<1 U/ml) induced by ΔEnv- and F522Y-infected MT4C5 cells. HIV capture by Gen2.2 cells was assessed by flow cytometry after 12 h of coculture (Fig. 3c). WT and, to a slightly lower extent, F522Y HIV were efficiently captured, whereas very few Gen2.2 cells were Gag+ after contact with ΔEnv-expressing lymphocytes. These differences in HIV capture by Gen2.2 cells were already visible after 1 h of coculture, as evidenced by immunofluorescence staining (Fig. 3d). WT and F522Y-infected cells formed virological synapses or polysynapse-like structures [50] with Gen2.2 cells, and patches of viral (Gag+) material were readily transferred to targets. Of note, coculture of Gen2.2 with HIV-1 infected primary CD4+ lymphocytes activated Gen2.2 as efficiently as MT4C5 cells (Fig. 3c).


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)

Role of cell contacts and of TLR7 on recognition of HIV-infected cells by Gen2.2 cells.a. Activation of Gen2.2 requires a direct contact with HIV-infected cells. Shaking cocultures of Gen2.2 and HIV-1-infected MT4C5 (left panel), or separating infected MT4C5 cells and target Gen2.2 in a transwell chamber (right panel) impairs IFN production, whereas recognition of FLUAV by Gen2.2 is not affected. Mean+sd of 3 independent experiments is shown b. Inhibition of IFN production by Bafilomycin A1. Gen2.2 cells were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 24 h, in the absence or presence of Bafilomycin A1 (Bafilo 125 nM). IFN levels are expressed as the % of the signal obtained without drugs. c. Role of TLR7. Gen2.2 cells were transduced with lentiviral vectors expressing shRNAs against TLR7 (shTLR7) or an irrelevant target (shCTRL). Levels of TLR7 mRNA in transduced cells were measured by RT-PCR. Data are normalized to GAPDH mRNA and expressed as relative levels of mRNA compared to shCTRL cells. Mean+sd of 3 independent experiments are shown (left panel). IFN production in TLR7-silenced and control Gen2.2 cells, cocultivated with HIV-infected MT4C5 or stimulated with FLUAV or CpG for 40 h. IFN levels are expressed as a percentage of the signal obtained with shCTRL Gen2.2 cells.
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ppat-1001284-g004: Role of cell contacts and of TLR7 on recognition of HIV-infected cells by Gen2.2 cells.a. Activation of Gen2.2 requires a direct contact with HIV-infected cells. Shaking cocultures of Gen2.2 and HIV-1-infected MT4C5 (left panel), or separating infected MT4C5 cells and target Gen2.2 in a transwell chamber (right panel) impairs IFN production, whereas recognition of FLUAV by Gen2.2 is not affected. Mean+sd of 3 independent experiments is shown b. Inhibition of IFN production by Bafilomycin A1. Gen2.2 cells were cocultivated with HIV-infected MT4C5 cells, exposed to FLUAV, or to CpG (a TLR9 agonist) during 24 h, in the absence or presence of Bafilomycin A1 (Bafilo 125 nM). IFN levels are expressed as the % of the signal obtained without drugs. c. Role of TLR7. Gen2.2 cells were transduced with lentiviral vectors expressing shRNAs against TLR7 (shTLR7) or an irrelevant target (shCTRL). Levels of TLR7 mRNA in transduced cells were measured by RT-PCR. Data are normalized to GAPDH mRNA and expressed as relative levels of mRNA compared to shCTRL cells. Mean+sd of 3 independent experiments are shown (left panel). IFN production in TLR7-silenced and control Gen2.2 cells, cocultivated with HIV-infected MT4C5 or stimulated with FLUAV or CpG for 40 h. IFN levels are expressed as a percentage of the signal obtained with shCTRL Gen2.2 cells.
Mentions: Next, we examined how Gen2.2 cells reacted when encountering HIV-infected T cells. To this end, infected MT4C5 were mixed with Gen2.2, and IFN levels were measured at 12 or 24 h of coculture. IFN was barely detected at 12 h (not shown). At 24 h, about 25 U/ml IFN were released in supernatants (Fig. 3c), which is 10–30 fold less than in primary pDCs. FLUAV potently activated Gen2.2 cells, whereas cell-free HIV particles (500 ng/ml Gag p24) elicited no IFN release (Fig. 3c). We reported that a gentle shaking of cocultures of infected cells and target cells inhibits direct cell-to-cell viral spread [49]. Shaking cocultures of HIV-infected MT4C5 cells and Gen 2.2 cells strongly decreased IFN production, whereas induction by FLUAV was not affected (Fig. 4a). Furthermore, IFN levels were below detection levels when infected donors cells and target Gen2.2 recipients were separated by a Transwell chamber (Fig. 4a). This strongly suggested that a direct contact between HIV-infected lymphocytes and Gen 2.2 cells is required to mediate recognition. Confirming our observation made with primary pDCs, optimal recognition of infected cells required a functional Env, as evidenced by the low levels of IFN (<1 U/ml) induced by ΔEnv- and F522Y-infected MT4C5 cells. HIV capture by Gen2.2 cells was assessed by flow cytometry after 12 h of coculture (Fig. 3c). WT and, to a slightly lower extent, F522Y HIV were efficiently captured, whereas very few Gen2.2 cells were Gag+ after contact with ΔEnv-expressing lymphocytes. These differences in HIV capture by Gen2.2 cells were already visible after 1 h of coculture, as evidenced by immunofluorescence staining (Fig. 3d). WT and F522Y-infected cells formed virological synapses or polysynapse-like structures [50] with Gen2.2 cells, and patches of viral (Gag+) material were readily transferred to targets. Of note, coculture of Gen2.2 with HIV-1 infected primary CD4+ lymphocytes activated Gen2.2 as efficiently as MT4C5 cells (Fig. 3c).

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