Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection.

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression

Bak expression is increased and correlates with CD4+ T cell counts and CD95-induced apoptosis in HIV-1-infected individuals.

<p>(A) Mean fluorescence intensity (MFI) of Bak, Bax and Bim expression <i>ex vivo</i> shown in CD4+ T cells and CD8+ T cells from HIV-1+ patients and healthy controls. Each dot depicts a donor, the lines indicate 10% and 90% and the boxes depict 25%, median and 75% quantiles. The P values were calculated by using the nonparametric Wilcoxon signed rank test for unpaired samples. (B) Spearman's rho correlation shown between <i>ex vivo</i> Bak (n = 25), Bax (n = 24) and Bim (n = 19) expression in CD4+ T cells and CD4+ T counts in HIV-1-infected individuals. (C) Spearman's rho or Pearson's r correlations shown between <i>ex vivo</i> Bak expression and the percentage of spontaneous apoptosis, AICD and CD95/Fas-mediate apoptosis in CD4+ and CD8+ T cells from HIV-1-infected individuals (n = 10).</p

<i>Ex vivo</i> pro- and anti-apoptotic molecule expression in HIV-1-infected patient T cells.

∧<p>ΔMFI ± standard error.</p>*<p>P<0.05,</p>**<p>P<0.02,</p>***<p>P<0.005 compared to healthy controls.</p

Type I IFN increases Bak expression in T cells from healthy donors and sensitizes them to CD95/Fas-mediated apoptosis.

<p>(A) Bak expression shown in CD4+ T cells and CD8+ T cells from healthy individuals after PBMC were untreated or treated with Type I IFN (1000 U/ml) for 72 hours. Each symbol depicts one donor (n = 6). Lines indicate 10% and 90% and the box depicts 25%, median and 75% quantiles. (B) Apoptosis sensitivity of T cells shown following a 72 hour Type I IFN treatment. Cells were stimulated with media alone (spontaneous apoptosis), plate-bound anti-CD3 antibody (AICD) or plate bound anti-CD95 antibody for 14 hours after pre-stimulation with Type I IFN as described in A and apoptosis was measured (n = 5). Bars depict mean ± standard error. P values shown in Panel A and B graphs were calculated by using the Student's t-test for paired samples.</p

Contrasting relationships between viral loads and Type I IFN levels during acute and chronic SIV infection.

<p>(A) Plasma IFNα during pathogenic SIV infection as measured by a modified Luminex assay. (B) Plasma SIV viral load was determined by PCR (dashed line indicates assay detection limit) (C) CD4+ T cell counts during the acute and chronic stages of SIV infection. Spearman's rho correlations shown between plasma IFNα levels and viral loads during (D) acute and (E) chronic SIV infection of rhesus macaques. (F) Spearman's rho correlations shown between IFNα expression and CD4+ T cell counts in chronic SIV infection. Measurements are shown for SIV-infected rhesus macaques (n = 10).</p

Pro- and anti-apoptotic protein expression in health donor T cells untreated or treated with Type I IFN.

∧<p>ΔMFI ± standard error.</p>*<p>P = 0.03 compared to untreated control.</p

IFNα-induced gene expression is increased in HIV-1 infection and correlates with Bak expression, CD95/Fas-induced T cell apoptosis and CD4+ T cell loss.

<p>(A) <i>Ex vivo IFI6-16</i> and (B) <i>ISG56</i> levels as measured by real-time RT PCR are significantly increased in HIV-1-infected patients (n = 7–14) compared to healthy donors (n = 6–8). P values were calculated by using the Wilcoxon signed rank test for unpaired samples. Spearman's rho correlations shown between <i>IFI6-16</i> expression in PBMC and (C) <i>ex vivo</i> T cell Bak expression (n = 11), (D) <i>ex vivo</i> T cell Bax expression (n = 11) (E) <i>ex vivo</i> T cell Bim expression (n = 7) (F) <i>ex vivo</i> CD95/Fas-mediated T cell apoptosis sensitivity (n = 12) and (G) CD4+ T cell counts (n = 10) in ART-naive HIV-1-infected individuals. Correlations are shown for ART-naïve HIV-1-infected patients (n = 7) between plasma viral load and (H) IFI6-16 mRNA levels, (I) ISG56 mRNA levels. Spearman's rho correlations depicted between CD95/Fas-specific apoptosis of CD4+ T cells and (J) <i>ISG56</i> expression, (K) <i>IFI6-16</i> expression and (L) plasma viral load in ART-naïve HIV-1 patients.</p

Type I IFN increases the sensitivity of T cells from HIV-1-infected individuals to CD95/Fas-induced death, but not TRAIL- and TNFα-mediated apoptosis.

<p>(A) Treatment-specific apoptosis shown for CD4+ T cells (n = 10) and CD8+ (n = 10) T cells pre-treated with IFNα (1000 U/ml) for 72 hours prior to a 14 hour stimulation with plate-bound anti-CD95/Fas antibody, TRAIL (10 ng/ml) or TNFα (10 ng/ml). n.s. = not significant. (B) Representative flow cytometry (left panel) and pooled data (right panel) showing apoptosis sensitivity of HIV-specific CD8+ T cells (n = 7) and CMV/EBV-specific CD8+ T cells (n = 5) after pre-incubation of PBMC with IFNα or IFNβ (1000 U/ml) for 72 hours and subsequent stimulation with plate-bound anti-CD95/Fas antibody for 14 hours. Bars in graphs indicate mean ± standard error. P values shown in graphs were calculated by using the parametric Student's t-test or nonparametric Wilcoxon signed rank test for paired samples.</p

Bak silencing markedly reduces the sensitivity of T cells in chronic HIV-1 infection to CD95/Fas-induced apoptosis.

<p>(A) Efficiency of siRNA uptake in T cells transfected with a pool of siRNAs and siGlo fluorescent oligonucleotides (to monitor transfection) compared to an electroporated negative control (no RNA or siGlo). Silencing of (B) Bak and (C) Bax (off-target control) by siRNA sequences targeting Bak in primary PBMC from a representative HIV+ patient measured by quantitative PCR. Expression (mean, SEM) normalized to 18S rRNA is presented as fold change relative to non-targeting control siRNA. (D) CD95/Fas-specific apoptosis of CD4+ T cells and CD8+ T cells in chronic HIV-1 infection following transfection with a pool of siRNAs directed against Bak, relative to a pool of non-targeting siRNAs (negative control). (E) Percent inhibition of CD95/Fas-specific cell death in T cells from chronically HIV-1-infected individuals following transfection with a Bak siRNA pool. Results were obtained from 3 independent experiments with 3 different HIV-1-infected patients.</p

CD95/Fas-induced apoptosis of T cells exposed to HIV-1 is Type I IFN-dependent.

<p>(A) TLR7/9 inhibitors inhibit HIV-1_Ba-L-induced production of IFNα in infected PBMC from healthy donors. PBMC were exposed to 10⁵ TCID₅₀/ml HIV-1_Ba-L in the presence or absence of a TLR7/9 antagonist for 24 hours (n = 13) and IFNα in supernatants was measured by ELISA. Bars indicate mean ± standard error. P values were calculated by using the Wilcoxon signed rank test for paired samples. (B) HIV-1_Ba-L exposure increases CD95/Fas apoptosis of T cells, which is inhibited by a TLR7/9 antagonist and anti-IFNα/β receptor blocking antibodies. Sensitivity to CD95/Fas-induced apoptosis shown for CD4+ T cells and CD8+ T cells from healthy donors following a 72 hour exposure to HIV-1_Ba-L in the presence or absence of anti-IFNα/β receptor blocking antibodies or a TLR7/9 antagonist (n = 5). Bars depict mean ± standard error. P values were calculated by using the Student's t-test for paired samples. (C) Representative flow cytometry plots from one out of three healthy donors depicting the sensitivity of CD4+ T cells to CD95/Fas-mediated apoptosis following exposure to non-infectious (AT-2 treated) HIV-1_Ba-L for 72 hours in the presence or absence of an isotype control antibody, anti-IFNα/β receptor blocking antibody, and a TLR7/9-specific antagonist. Numbers indicate the percentage of apoptosis.</p