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TLR2 and TLR4 triggering exerts contrasting effects with regard to HIV-1 infection of human dendritic cells and subsequent virus transfer to CD4+ T cells.

Thibault S, Fromentin R, Tardif MR, Tremblay MJ - Retrovirology (2009)

Bottom Line: Recognition of microbial products through Toll-like receptors (TLRs) initiates inflammatory responses orchestrated by innate immune cells such as dendritic cells (DCs).No noticeable and consistent effect was observed following engagement of TLR5, 7 and 9.Additional studies indicated that both HIV-1 infection of DCs and DC-mediated virus transmission to CD4+ T cells were reduced upon TLR4 triggering due to secretion of type-I interferons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculté de Médecine, Université Laval, Québec, Canada. sandra.thibault@crchul.ulaval.ca

ABSTRACT

Background: Recognition of microbial products through Toll-like receptors (TLRs) initiates inflammatory responses orchestrated by innate immune cells such as dendritic cells (DCs). As these cells are patrolling mucosal surfaces, a portal of entry for various pathogens including human immunodeficiency virus type-1 (HIV-1), we investigated the impact of TLR stimulation on productive HIV-1 infection of DCs and viral spreading to CD4+ T cells.

Results: We report here that engagement of TLR2 on DCs increases HIV-1 transmission toward CD4+ T cells by primarily affecting de novo virus production by DCs. No noticeable and consistent effect was observed following engagement of TLR5, 7 and 9. Additional studies indicated that both HIV-1 infection of DCs and DC-mediated virus transmission to CD4+ T cells were reduced upon TLR4 triggering due to secretion of type-I interferons.

Conclusion: It can thus be proposed that exposure of DCs to TLR2-binding bacterial constituents derived, for example, from pathogens causing sexually transmissible infections, might influence the process of DC-mediated viral dissemination, a phenomenon that might contribute to a more rapid disease progression.

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Related in: MedlinePlus

TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4+ T cells. A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Balenv and co-cultured with autologous CD4+ T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P < 0.01; ***: P < 0.001). B) IM-MDDCs were initially either left untreated or treated with EFV. Thereafter, cells were either left untreated or treated with Pam3Csk4. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 8 distinct donors. C) A similar experimental approach was used except that transfer studies were carried out with the X4-tropic strain NL4-3. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 3 different donors.
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Figure 1: TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4+ T cells. A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Balenv and co-cultured with autologous CD4+ T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P < 0.01; ***: P < 0.001). B) IM-MDDCs were initially either left untreated or treated with EFV. Thereafter, cells were either left untreated or treated with Pam3Csk4. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 8 distinct donors. C) A similar experimental approach was used except that transfer studies were carried out with the X4-tropic strain NL4-3. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 3 different donors.

Mentions: To define whether TLR stimulation can affect HIV-1 transfer, IM-MDDCs were first treated for only 2 hours with one of the tested TLR agonists before pulsing with the R5-using HIV-1 strain NL4-3/Balenv. Thereafter, the cell-virus mixture was co-cultured with autologous CD4+ T cells and cell-free supernatants were harvested at 2, 3 and 6 days post-co-culture (dpcc) to measure virus transfer. As depicted in Fig. 1A (left panel), transmission of HIV-1 was markedly increased upon TLR2 stimulation at 2 dpcc, whereas a diminution was seen following LPS-mediated engagement of TLR4. The kinetics of virus production in the co-cultures revealed that TLR2 and 4 triggering affects an early step(s) in the process of virus transfer since the modulatory effects were disappearing over time (small insert in the left panel). Engagement of TLR5, 7 and 9 did not affect the DC-mediated propagation of HIV-1. Similar patterns of HIV-1 transfer were obtained when experiments were conducted with multiple independent donors (Fig. 1A, right panel). Next, we evaluated whether the observed modulation of virus transfer could be attributable to de novo virus production by IM-MDDCS (i.e. late transfer). This issue was solved by adding the inhibitor of reverse transcription Efavirenz (EFV) before pulsing IM-MDDCs with virions. Results illustrated in Fig. 1B indicate that the TLR2-mediated signal transduction pathway was affecting primarily direct productive infection of IM-MDDCs (i.e. late transfer due to newly formed viral entities) since the Pam3Csk4-dependent augmentation in virus transfer was almost totally abrogated upon treatment with EFV. Although it is well accepted that primary HIV-1 infection is caused by R5-tropic viruses, some experiments were also carried out with an X4-using isolate of HIV-1 (i.e. NL4-3). The TLR2-mediated enhancement in virus transfer was also seen with the X4-tropic variant as well as the reduction of HIV-1 propagation by the TLR4 agonist (Fig. 1C). The effect of the studied TLR agonists on cell viability was also monitored using the fluorescent cytotoxic MTS assay. Cell viability was not affected by the studied TLR ligands used at concentrations known to modulate the DC-mediated transfer of HIV-1 (data not shown).


TLR2 and TLR4 triggering exerts contrasting effects with regard to HIV-1 infection of human dendritic cells and subsequent virus transfer to CD4+ T cells.

Thibault S, Fromentin R, Tardif MR, Tremblay MJ - Retrovirology (2009)

TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4+ T cells. A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Balenv and co-cultured with autologous CD4+ T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P < 0.01; ***: P < 0.001). B) IM-MDDCs were initially either left untreated or treated with EFV. Thereafter, cells were either left untreated or treated with Pam3Csk4. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 8 distinct donors. C) A similar experimental approach was used except that transfer studies were carried out with the X4-tropic strain NL4-3. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 3 different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: TLR2 and 4 triggering modulates HIV-1 transfer between IM-MDDCs and CD4+ T cells. A) IM-MDDCs were either left untreated (mock) or stimulated for 2 hours with the following TLR agonists: Pam3Csk4 (5 μg/ml), LPS (0.1 μg/ml), flagellin (5 μg/ml), R837 (5 μg/ml) and unmethylated DNA (5 μg/ml). Cells were then pulsed with NL4-3/Balenv and co-cultured with autologous CD4+ T cells. Finally cell-free supernatants were harvested at 2, 3 and 6 days post-coculture (dpcc) and the viral content was assessed by a p24 assay. Data depicted in the left panel represent the mean ± standard deviations of quadruplicate samples from a representative single donor at 2 dpcc, whereas the kinetics of virus production for the same donor are displayed in the small insert. Results from multiple different donors are illustrated in the right panel (2 dpcc) (**: P < 0.01; ***: P < 0.001). B) IM-MDDCs were initially either left untreated or treated with EFV. Thereafter, cells were either left untreated or treated with Pam3Csk4. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 8 distinct donors. C) A similar experimental approach was used except that transfer studies were carried out with the X4-tropic strain NL4-3. Data shown represent the mean ± standard deviations of quadruplicate samples from a single donor at 2 dpcc and are representative of 3 different donors.
Mentions: To define whether TLR stimulation can affect HIV-1 transfer, IM-MDDCs were first treated for only 2 hours with one of the tested TLR agonists before pulsing with the R5-using HIV-1 strain NL4-3/Balenv. Thereafter, the cell-virus mixture was co-cultured with autologous CD4+ T cells and cell-free supernatants were harvested at 2, 3 and 6 days post-co-culture (dpcc) to measure virus transfer. As depicted in Fig. 1A (left panel), transmission of HIV-1 was markedly increased upon TLR2 stimulation at 2 dpcc, whereas a diminution was seen following LPS-mediated engagement of TLR4. The kinetics of virus production in the co-cultures revealed that TLR2 and 4 triggering affects an early step(s) in the process of virus transfer since the modulatory effects were disappearing over time (small insert in the left panel). Engagement of TLR5, 7 and 9 did not affect the DC-mediated propagation of HIV-1. Similar patterns of HIV-1 transfer were obtained when experiments were conducted with multiple independent donors (Fig. 1A, right panel). Next, we evaluated whether the observed modulation of virus transfer could be attributable to de novo virus production by IM-MDDCS (i.e. late transfer). This issue was solved by adding the inhibitor of reverse transcription Efavirenz (EFV) before pulsing IM-MDDCs with virions. Results illustrated in Fig. 1B indicate that the TLR2-mediated signal transduction pathway was affecting primarily direct productive infection of IM-MDDCs (i.e. late transfer due to newly formed viral entities) since the Pam3Csk4-dependent augmentation in virus transfer was almost totally abrogated upon treatment with EFV. Although it is well accepted that primary HIV-1 infection is caused by R5-tropic viruses, some experiments were also carried out with an X4-using isolate of HIV-1 (i.e. NL4-3). The TLR2-mediated enhancement in virus transfer was also seen with the X4-tropic variant as well as the reduction of HIV-1 propagation by the TLR4 agonist (Fig. 1C). The effect of the studied TLR agonists on cell viability was also monitored using the fluorescent cytotoxic MTS assay. Cell viability was not affected by the studied TLR ligands used at concentrations known to modulate the DC-mediated transfer of HIV-1 (data not shown).

Bottom Line: Recognition of microbial products through Toll-like receptors (TLRs) initiates inflammatory responses orchestrated by innate immune cells such as dendritic cells (DCs).No noticeable and consistent effect was observed following engagement of TLR5, 7 and 9.Additional studies indicated that both HIV-1 infection of DCs and DC-mediated virus transmission to CD4+ T cells were reduced upon TLR4 triggering due to secretion of type-I interferons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Faculté de Médecine, Université Laval, Québec, Canada. sandra.thibault@crchul.ulaval.ca

ABSTRACT

Background: Recognition of microbial products through Toll-like receptors (TLRs) initiates inflammatory responses orchestrated by innate immune cells such as dendritic cells (DCs). As these cells are patrolling mucosal surfaces, a portal of entry for various pathogens including human immunodeficiency virus type-1 (HIV-1), we investigated the impact of TLR stimulation on productive HIV-1 infection of DCs and viral spreading to CD4+ T cells.

Results: We report here that engagement of TLR2 on DCs increases HIV-1 transmission toward CD4+ T cells by primarily affecting de novo virus production by DCs. No noticeable and consistent effect was observed following engagement of TLR5, 7 and 9. Additional studies indicated that both HIV-1 infection of DCs and DC-mediated virus transmission to CD4+ T cells were reduced upon TLR4 triggering due to secretion of type-I interferons.

Conclusion: It can thus be proposed that exposure of DCs to TLR2-binding bacterial constituents derived, for example, from pathogens causing sexually transmissible infections, might influence the process of DC-mediated viral dissemination, a phenomenon that might contribute to a more rapid disease progression.

Show MeSH
Related in: MedlinePlus