Failure of TLR4-driven NF-kappa B activation to stimulate virus replication in models of HIV type 1 activation

Abstract

The interaction of HIV-1 with Toll-like receptors (TLR) on host target cells is incompletely understood. Data from several in vivo and in vitro model systems suggest that TLR2, TLR4, and TLR9 remain functional and if stimulated, cause an upregulation of viral replication. In the present studies employing two different chronically HIV-1-infected cell lines and highly purified TLR agonists, we found ligation of TLR2 and TLR9, but not TLR4, resulted in significant upregulation of HIV-1 production. This result was not due to a lack of TLR4 expression or impaired NF-kappa B activation. Using HEK293 cells transfected with individual TLRs and an HIV-1 LTR reporter confirmed that TLR4 signaling does not directly activate the HIV-1 LTR. Finally, ultrapurified LPS did not enhance production of IL-1 beta or IL-6 in chronically infected U1 cells, whereas significant cytokine production was observed in uninfected U937 cells. These results confirm the biological activity of ultrapurified LPS and raise the possibility that TLR4 signaling pathways may be altered during chronic HIV-1 infection. Collectively, these studies suggest that although several TLR can upregulate NF-kappaB in HIV-1-infected cells, upregulation of NF-kappaB alone is insufficient to activate the viral LTR. Further dissection of the TLR signaling pathways is necessary to determine how TLR stimulation leads to LTR activation and whether HIV-1 infection can alter signaling through TLR4.

Figure 1. Unpurified LPS activates TLR2, 4 and 9

HEK 293T cells stably transfected with (A) TLR2, (B) TLR4/MD2 or (C) TLR9 gene constructs were transfected with an NF-κB- SEAP reporter and treated with 500 ng/ml Pam₃CSK₄, 5 μg/ml ultra-purified LPS, 5 μg/ml WT LPS, 1 μM B class CpG-ODN or B class control ODN. Cell culture supernatant was collected 24 h post-treatment and assayed for SEAP. Data are expressed as fold-increase over unstimulated cells within each respective cell line and are the mean and SEM of three separate experiments.

Figure 2. Activation of TLR4 does not significantly enhance HIV production

(A) 1 × 10⁶ U1 cells were treated with TLR agonists as described in Figure 1. Cell culture supernatants were collected 48 h post-treatment and assayed for HIV p24 by ELISA. Data represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of U1 cells with the specified agonists resulted in a significant increase in HIV p24 production over that of untreated cells. (B) 5 × 10⁵ THP_896GFP cells were seeded into wells of a 96 well plate and stimulated with TLR agonists as described in Figure 1. Cells were photographed 48 h post-TLR activation using a fluorescent microscope under 100 x magnification. (C) EGFP from TLR-stimulated THP_896GFP cells was quantified using an IVIS imaging system. Data are expressed as relative fluorescent units (RFU) after subtraction of background from untreated controls and represent the mean and SEM of 3 separate experiments. (D) Cell culture supernatants from 1 × 10⁶ THP_896GFP cells were collected 48 h post-TLR activation and assayed for HIV p24 by ELISA. Data represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of THP_896GFP cells with the specified agonists resulted in a significant increase in HIV p24 production over that of untreated cells.

Figure 2. Activation of TLR4 does not significantly enhance HIV production

(A) 1 × 10⁶ U1 cells were treated with TLR agonists as described in Figure 1. Cell culture supernatants were collected 48 h post-treatment and assayed for HIV p24 by ELISA. Data represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of U1 cells with the specified agonists resulted in a significant increase in HIV p24 production over that of untreated cells. (B) 5 × 10⁵ THP_896GFP cells were seeded into wells of a 96 well plate and stimulated with TLR agonists as described in Figure 1. Cells were photographed 48 h post-TLR activation using a fluorescent microscope under 100 x magnification. (C) EGFP from TLR-stimulated THP_896GFP cells was quantified using an IVIS imaging system. Data are expressed as relative fluorescent units (RFU) after subtraction of background from untreated controls and represent the mean and SEM of 3 separate experiments. (D) Cell culture supernatants from 1 × 10⁶ THP_896GFP cells were collected 48 h post-TLR activation and assayed for HIV p24 by ELISA. Data represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of THP_896GFP cells with the specified agonists resulted in a significant increase in HIV p24 production over that of untreated cells.

Figure 2. Activation of TLR4 does not significantly enhance HIV production

(A) 1 × 10⁶ U1 cells were treated with TLR agonists as described in Figure 1. Cell culture supernatants were collected 48 h post-treatment and assayed for HIV p24 by ELISA. Data represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of U1 cells with the specified agonists resulted in a significant increase in HIV p24 production over that of untreated cells. (B) 5 × 10⁵ THP_896GFP cells were seeded into wells of a 96 well plate and stimulated with TLR agonists as described in Figure 1. Cells were photographed 48 h post-TLR activation using a fluorescent microscope under 100 x magnification. (C) EGFP from TLR-stimulated THP_896GFP cells was quantified using an IVIS imaging system. Data are expressed as relative fluorescent units (RFU) after subtraction of background from untreated controls and represent the mean and SEM of 3 separate experiments. (D) Cell culture supernatants from 1 × 10⁶ THP_896GFP cells were collected 48 h post-TLR activation and assayed for HIV p24 by ELISA. Data represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of THP_896GFP cells with the specified agonists resulted in a significant increase in HIV p24 production over that of untreated cells.

Figure 3. Constitutive expression of TLR2, TLR4 and TLR9 is not altered by chronic HIV infection

Flow cytometric histograms representative of 3 separate experiments in which U937 and U1 cells were stained for surface expression of (A) TLR2 and (B) TLR4, and intracellular expression of (C) TLR9.

Figure 4. HIV infected cells down-regulate TLR2 and TLR4 in response to specific agonists

Flow cytometric histograms representative of 3 separate experiments in which U937 and U1 cells were either left untreated or stimulated with 500 ng Pam₃CSK₄ or 5 μg/ml ultra-purified LPS and analyzed for (A) TLR2 and (B) TLR4 expression 8 hours later.

Figure 5. TLR agonists activate NF-κB in infected cells

(A) 1 × 10⁶ U937 and U1 cells were transfected with an NF-κB-SEAP reporter and exposed to 500 ng Pam₃CSK₄, 5 μg/ml ultra-purified LPS, 5 μg/ml WT LPS or 1 μM CpG DNA. Cell culture supernatant was collected 48 h post-treatment and assayed for SEAP activity. Data represent the mean and SD of 3 separate experiments. (*) p < 0.05, treatment of U937 cells with the specified TLR ligand resulted in a significantly higher induction of activated NF-κB when compared with U1 cells. (B–D) 1 × 10⁶ U937 and U1 cells were treated as described above. Cell culture supernatant was collected 24 h post-treatment and assayed for (B) IL-1β, (C) TNFα and (D) IL-6 using Luminex fluorescent bead technology. (*) p < 0.05, treatment with the specified agonists resulted in a significant difference in cytokine secretion between U937 and U1 cells.

Figure 6. TLR4 engagement does not activate the HIV-LTR

1 × 10⁶ TLR2, TLR4/MD2 or TLR9 293T cells were transfected with an attenuated NL4-luciferase reporter and stimulated for 12 h as described in Figure 1. Data represent the fold-increase in NL4 activity relative to un-stimulated cells and represent the mean and SEM of 3 separate experiments. (*) p < 0.05, treatment of cell lines with the specified agonists resulted in a significant increase in NL4-lucifierase activity compared with the respective un-stimulated cells.