Specific disruption of NFAT5 or NF-κB binding sites in the LTR of HIV-1 subtype B.

<p>(A) Mutations introduced into a HIV-1_Lai/Bal-env infectious molecular clone. NF-κB and NFAT5 binding site mutations were introduced into the LTR of the HIV-1_Lai/Bal-env-wild type (WT) infectious molecular clone. The NF-κB binding site mutants (HIV-1_Lai/Bal-env-κB I-Mut, HIV-1_Lai/Bal-env-κB II-Mut, HIV-1_Lai/Bal-env-κB I+II-Mut), and NFAT5 binding site mutant (HIV-1_Lai/Bal-env-N5-Mut) are shown along with the HIV-1_Lai/Bal-env-WT sequence. Mutations were introduced into the 3′ LTR of the HIV-1_Lai/Bal-env–WT proviral sequence. (B) Specific mutation of the NFAT5 binding site abolishes NFAT5 binding to the viral LTR but does not affect NF-κB p50/p65 binding to the overlapping NF-κB binding site. Quantitative DNaseI footprinting analysis is shown using HIV-1 LTR fragments (-262 to +4 nt relative to the transcription start site) from HIV-1_Lai/Bal-env-WT and HIV-1_Lai/Bal-env-N5-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng), or NFAT5 (10 ng, 50 ng, and 250 ng). The regions that are protected from DNase I cleavage by the binding of NF-κB and NFAT5 are indicated with a bars. (C) Specific disruption of the HIV-1 subtype B NF-κB binding sites effectively abrogates recombinant p50/p65 binding. Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1_Lai/Bal-env-WT, HIV-1_Lai/Bal-env-κB I-Mut, HIV-1_Lai/Bal-env-κB II-Mut, and HIV-1_Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NF-κB (p50/p65) (25 ng, 100 ng, and 500 ng). The regions that are protected from DNase I cleavage by the binding of recombinant NF-κB are indicated with a bars. (D) Specific disruption of the HIV-1 subtype B NF-κB binding site does not inhibit but enhances NFAT5 binding to this region. Quantitative DNaseI footprinting analysis is shown of nucleotides −262 to +4 from HIV-1_Lai/Bal-env-WT, HIV-1_Lai/Bal-env-κB I-Mut, HIV-1_Lai/Bal-env-κB II-Mut, and HIV-1_Lai/Bal-env-κB I+II-Mut and increasing concentrations of recombinant NFAT5 (10 ng, 50 ng, and 250 ng). The regions that are protected from DNase I cleavage by the binding of recombinant NFAT5 are indicated with a bars.</p

MTb induces NFAT5 gene expression and knockdown of NFAT5 impairs HIV-1 replication during MTb co-infection.

<p>(A) MTb but not HIV-1 increases NFAT5 mRNA synthesis in human macrophages. MDM were isolated from PBMC obtained from four normal donors. The cells were incubated for 24 or 48 hours with heat-inactivated or live R5-tropic HIV-1 subtype B, C, or E isolates, live MTb strain CDC1551 (1∶1 MDM∶bacilli), or left untreated. Quantitative real-time PCR analysis of NFAT5 mRNA expression levels revealed that the low, constitutively present level of NFAT5 mRNA expressed in untreated cells was not affected by exposure to live or inactivated HIV-1, but was significantly increased at both 24 (*, p<0.05) and 48 (**, p<0.01) hours following infection with MTb. (B) NFAT5-specific siRNA effectively suppresses NFAT5 expression in MDM in both the absence and presence of MTb co-infection. MDM (1×10⁶ cells/well in a 6-well plate) from four normal donors were transfected with siRNA specific for NFAT5 or, as a control, GFP, and were then infected with MTb (1∶1 MDM∶bacilli) or left uninfected. In the cells transfected with control siRNA, MTb infection significantly increased NFAT5 expression in comparison to uninfected cells (open bars). When MDM were transfected with NFAT5-specific siRNA, both constitutively expressed (p = 0.048) and MTb-induced (p = 0.021) NFAT5 mRNA were significantly suppressed (grey bars). (C) HIV-1 subtype B replication is significantly suppressed in the presence of MTb co-infection when NFAT5 expression is abrogated in human MDM. MDM from four normal donors were transfected with NFAT5-specific and control siRNAs as described in 2B. Cells were then infected with HIV-1_Lai/Bal-env followed by co-infection with MTb CDC1551. Virus replication was measured at days 6, 9, and 12 post-infection. At day 12, virus replication was significantly reduced in cells transfected with NFAT5-specific siRNA in comparison to cells transfected with control siRNA (*, p<0.05). We monitored the morphology of cells within the infected cultures and did not observe noticeable macrophage necrosis even at the final timepoint of viral harvest.</p

NFAT5 interaction with the LTR is important for MTb-induced HIV-1 transcription.

<p>(A) MTb stimulation increases activity of LTRs derived from HIV-1 subtypes B, C, and E. HIV-1 LTRs (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates were cloned into plasmid pGL3. THP-1 cells (0.8×10⁶/ml) were transfected with each reporter plasmid (0.3 µg/ml) plus the <i>Renilla</i> luciferase control plasmid pRL-TK (0.03 µg/ml) and incubated at 37°C for 16 hours. Cells were then either left untreated or treated with 10 µg/ml MTb lysate for 8 hours before termination of the cultures. In the histogram, open bars represent individual LTR activities in untreated cells. Light grey bars represent mean values of LTR activities from each subtype in untreated cells. Black bars represent individual LTR activities in MTb lysate-treated cells, and dark grey bars represent mean values of LTR activities from each subtype in cells treated with MTb lysate. LTR transcriptional activity for all of the representative LTRs tested was significantly increased in cultures treated with MTb lysate in comparison to untreated cultures. Results are from three independent experiments performed in duplicate (*, p<0.05; **, p<0.01 as compared to unstimulated cultures). (B) Specific disruption of the NFAT5 binding site significantly reduces LTR-reporter gene activity in monocytic cells in response to MTb lysate treatment. THP-1 cells were transfected with luciferase expression vectors encoding nucleotides 208 to +64 of the wild-type HIV-1_Lai LTR and an HIV-1_Lai LTR containing the NFAT5 binding site mutations (N5-Mut). After 16 hours, the cells were left untreated or exposed to 10 µg/ml MTb lysate for 8 hours at 37°C. Disruption of NFAT5 binding to the enhancer region significantly suppressed LTR-driven reporter gene expression in comparison to the wild-type LTR when cells were treated with MTb lysate (p<0.01). LTR activity was also suppressed in the untreated cells but to a lesser extent (p<0.05). Results are from three independent experiments performed in duplicate and adjusted to <i>Renilla</i> luciferase control expression (*, p<0.05; **, p<0.01). Nucleotide sequences representing the wild-type and NFAT5 binding site-mutated HIV-1_Lai LTRs are shown at the bottom of the figure. (C) MTb lysate increases NFAT5 protein levels in monocytic cells. THP-1 cells were left untreated (control) or exposed to 10 µg/ml MTb lysate for 8 or 24 hours at 37°C. Whole cell extracts were collected and analyzed by western blot with anti-NFAT5 antibody. An antibody directed against Lamin-B1 was used as a loading control. The histogram shows densitometric analysis of the NFAT5 bands from the western blot autoradiograph displayed and values represent mean band intensities at 0, 8, and 24 hours post-stimulation with MTb lysate.</p

The MyD88-mediated signaling cascade is important for MTb induction of NFAT5 expression in human monocytes.

<p>(A) Engagement of TLR2 significantly enhances NFAT5 mRNA synthesis in monocytes. THP-1 cells were incubated with the TLR2-specific ligand Pam3Cys (5 µg/ml), infected with MTb strain CDC1551 (1∶1 cells∶bacilli), or left untreated. After 16 hours, NFAT5 mRNA levels were measured. NFAT5 mRNA expression was significantly increased when the cells were incubated with Pam3Cys (*, p<0.05) or infected with MTb (**, p<0.01). (B) MyD88 is important for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered shRNA targeting MyD88 or control shRNA targeting GFP were constructed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002620#s2" target="_blank">Materials and Methods</a>. Cells were infected with MTb strain CDC1551 (1∶1 cells∶bacilli) or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 mRNA levels were significantly inhibited in the cells transduced with MyD88-specific shRNA (black bars) compared with the cells transduced with control shRNA (open bars) in both, the absence or presence of MTb infection. TNF and CD86 mRNAs, the expression of which are MyD88-dependent and -independent, respectively, during MTb infection, were measured as controls. (C, D) IRAK1 and TRAF6 are required for MTb induction of NFAT5 gene expression in human monocytes. THP-1 cells that constitutively express lentivirally-delivered IRAK1- or TRAF6-specific shRNA were constructed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002620#s2" target="_blank">Materials and Methods</a>. THP-1 cells expressing control shRNA were used as described in 7B. Cells were infected with MTb strain CDC1551 or left uninfected for 16 hours at 37°C, and RNA was analyzed. NFAT5 expression was significantly inhibited in the MTb-infected cells expressing either IRAK1 (C) or TRAF6 (D) shRNA (black bars) compared to cells expressing control shRNA (open bars). TNF and CD86 mRNAs, both of which require IRAK1 and TRAF6 to be induced by PRR activation, were measured as controls. (*, p<0.05; **, p<0.01).</p

MTb-induced HIV-1 replication in PBMC depends on binding of NFAT5 and NF-κB to the LTR.

<p>Specific disruption of the NF-κB or NFAT5 binding sites in the HIV-1 subtype B LTR inhibits R5-tropic virus replication in human PBMC after co-infection with MTb. PBMC from four normal donors were infected with 1000 TCID₅₀ HIV-1_Lai/Bal-env-WT or the mutants HIV-1_Lai/Bal-env-κB I-Mut, HIV-1_Lai/Bal-env-κB II-Mut, HIV-1_Lai/Bal-env-κB I+II-Mut, or HIV-1_Lai/Bal-env-N5-Mut and either; (A) left untreated, or (B) co-infected with MTb isolate CDC1551 (10∶1 cells∶bacilli). Viral p24 levels in culture supernatants were measured at days 4, 7 and 12 post-infection. (C) Histograms show viral p24 levels at day 12 in the MTb uninfected (grey bars) and MTb co-infected (black bars) PBMC cultures. Replication of the mutant viruses was compared to wild-type virus replication under the same experimental conditions (without and with MTb co-infection, respectively). *, p<0.05; **, p<0.01, as compared to HIV-1_Lai/Bal-Env-WT.</p

Disruption of NF-κB or NFAT5 sites in the LTR inhibits MTb-induced HIV-1 replication in MDM.

<p>MDM from four normal donors were infected with 1000 TCID₅₀ of HIV-1_Lai/Bal-env–WT or the mutants HIV-1_Lai/Bal-env-κB I-Mut, HIV-1_Lai/Bal-env-κB II-Mut, HIV-1_Lai/Bal-env-κB I+II-Mut, or HIV-1_Lai/Bal-env-N5-Mut, and were either (A) left infected with virus alone or (B) co-infected with MTb CDC1551 (1∶1 MDM∶bacilli). Viral p24 levels in the culture supernatants were measured at days 4, 7 and 12 post-infection. Presentation of viral p24 levels at day 12 in cultures infected with (C) HIV-1 alone or (D) co-infected with MTb are shown as histograms. (*, p<0.05; **, p<0.01, as compared to HIV-1_Lai/Bal-env–WT).</p

Distinct dynamics of cytokine mRNA expression in the lungs of MTb-infected NFATp^−/− and WT mice.

<p>NFATp^−/− and WT mice were infected with MTb and total RNA was purified from the lungs at 2, 4, 6, 8 (8.4), and 10 weeks post-infection. (<b>A</b>) IFN-γ, (<b>B</b>) TNF, and (<b>C</b>) IL-4 mRNA was measured by real-time PCR and values were normalized to ubiquitin mRNA. Error bars indicate mean of 3 samples ±SD.</p

Correlation of serum TNF protein levels with MTb disease progression in WT and NFATp^−/− mice.

<p>WT and NFATp^−/− mice were infected with the aerosolized clinical MTb strain HN878 at ∼100 CFU/mouse as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041427#pone.0041427-Barczak1" target="_blank">[50]</a>. Four random mice were bled at four-week intervals after initial infection and serum levels of TNF were measured by ELISA.</p

Varying NFATp requirement for cytokine expression in different primary cell populations.

<p><b>A and B.</b> TNF expression in BM-derived dendritic cells is not NFATp-dependent. Primary BM-DC were derived from WT and NFATp^−/− mice. After 7 days cells were stimulated with MTb lysates. At indicated time points the cells were lysed to prepare total RNA. (<b>A</b>) TNF mRNA was measured by real-time PCR. (<b>B</b>) TNF protein levels in the supernatants were measured by ELISA. <b>C and D.</b> TNF and IFN-γ expression in murine primary T cells is NFATp-dependent. Purified primary total CD4⁺ and naïve (CD4⁺CD62L⁺CD45RB^high) T cells from uninfected WT and NFATp^−/− mice were stimulated immediately with anti-CD3/CD28 antibody and then left in culture in neutral conditions for another six days and re-stimulated with anti-CD3/CD28 antibodies. Aliquots of cells were harvested at different time points post-induction as indicated. Purified total RNA was analyzed by real-time PCR with TNF and IFN-γ-specific primers, and cellular supernatants were used to measure TNF and IFN-γ protein levels by ELISA.</p

Increased susceptibility of NFATp^−/− mice to aerosolized <i>M. tuberculosis</i>.

<p><b>A.</b> Survival of BALB/c (WT) vs. NFATp^−/− mice after infection with MTb. 20 WT and 16 NFATp^−/− mice were infected with the aerosolized clinical MTb strain HN878 (∼149 CFU) and monitored for survival. <b>B</b> and <b>C.</b> Lung pathology. (<b>B</b>) NFATp^−/− and (<b>C</b>) WT mice were infected with MTb, and lungs taken from premortal NFATp^−/− mice and from the WT mice sacrificed at the same time were fixed in 10% neutral buffered Formalin. Fixed tissues were embedded in paraffin, and 6–10 µm sections were H&E or Ziehl-Neelsen acid-fast stained as indicated.</p