Induction of IL-17 and nonclassical T-cell activation by HIV-Tat protein

Abstract

Chronic immune activation is a major complication of antiretroviral therapy (ART) for HIV infection and can cause a devastating immune reconstitution inflammatory syndrome (IRIS) in the brain. The mechanism of T-cell activation in this population is not well understood. We found HIV-Tat protein and IL-17-expressing mononuclear cells in the brain of an individual with IRIS. Tat was also present in the CSF of individuals virologically controlled on ART. Hence we examined if Tat protein could directly activate T cells. Tat transcriptionally dysregulated 94 genes and induced secretion of 11 cytokines particularly activation of IL-17 signaling pathways supporting the development of a proinflammatory state. Tat increased IL-17 transcription and secretion in T cells. Tat entered the T cells rapidly by clathrin-mediated endocytosis and localized to both the cytoplasm and the nucleus. Tat activated T cells through a nonclassical pathway dependent upon vascular endothelial growth factor receptor-2 and downstream secondary signaling pathways but independent of the T-cell receptor. However, Tat stimulation of T cells did not induce T-cell proliferation but increased viral infectivity. This study demonstrates Tat's role as a virulence factor, by driving T-cell activation and contributing to IRIS pathophysiology. This supports the necessity of an anti-Tat therapy in conjunction with ART and identifies multiple targetable pathways to prevent Tat-mediated T-cell activation.

Keywords: CNS inflammation; central nervous system; chromatin; chronic inflammation; lymphocyte.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Detection of Tat in CSF and characterization of immune infiltrates and HIV antigens in CNS-IRIS. (A and B) Immunohistochemical analysis of brain tissue from an individual with CNS-IRIS showed infiltrates of CD3 + T cells which were predominantly CD8+ with few CD4+ cells and occasional IL-17+ cells (arrow). Images shown are 40×. (Scale bar: 10 µm.) (B) Cell counts were performed on three 20× fields. (C) There was no detectable p24 antigen but robust cytoplasmic production of Tat was seen in infiltrating mononuclear cells (arrows). (D) Three of eight CSF samples from HIV-infected individuals controlled on ART were positive for Tat by ELISA. (E and F) Tat was consistently produced by HIV-infected PBMC despite treatment with darunavir (E), whereas viral production was inhibited as measured by product enhanced reverse transcriptase assay (F). Data represent mean ± SD from three independent experiments, *P < 0.05 by paired Student t test.

Fig. 2.

Tat induces proinflammatory gene expression changes and induces a Th17 phenotype. (A) Heat map depicting expression differences between primary T cells exposed to Tat for 24 h versus 0 h for six separate pairs of cells (y axis) for 94 genes (x axis). Data are fold change units, with magnitude and direction described using color. Each of the 94 genes has an absolute difference in means ≥ 1.25-fold and a P < 0.05 by paired Student t test. (B) Treatment of primary human T cells for 72 h with Tat showed a dose-dependent release of IL-17 secretion (n = 3). (C) T cells were treated with Tat for 6 h and IL-17 transcripts were quantified by PCR. *P < 0.05 by paired Student t test (n = 8). (D) IL-17–expressing T cells are increased after Tat treatment, *P < 0.05 by two-way paired Student t test (n = 5). (E) Tat released by Jurkat–Tat cells is capable of activating T cells. Primary T cells were cocultured with Jurkat or Jurkat–Tat cells in transwells for 72 h in the presence or absence of anti-Tat antibody (Ab) or irrelevant isotype Ab. Significantly increased levels of granzyme B were released when cocultured with Jurkat–Tat cells, which was blocked by anti-Tat Ab but not isotype Ab. Data represent mean ± SD from three independent experiments and were analyzed by ANOVA and Bonferroni’s multiple comparison test, *P < 0.05, **P < 0.001.

Fig. 3.

T-cell stimulation by Tat is mediated by endocytosis and is independent of antigen recognition. (A) T cells were incubated with Tat-venus (green) for various time periods; nuclei were stained with DAPI (red) and analyzed by ImageStream. Data shown include bright field (BF), Tat-Venus, DAPI, and merged images. One representative image from each time point demonstrates the uptake and localization of Tat. (B) Graph illustrates the percentage of Tat present in the cytoplasm versus the nucleus for each time point. (C) T-cell stimulation by Tat as measured by IL-17 in culture supernatants was completely blocked by chlorpromazine and amiloride but not by filipin. (D) Pretreatment with NDZ to block TCR signaling had no effect on Tat-mediated T-cell stimulation. Data in C and D are mean ± SD from three to five independent experiments compared by ANOVA with Bonferroni’s multiple comparison,*P < 0.05, **P < 0.001; ns, not significant.

Fig. 4.

T-cell activation by Tat is dependent upon VEGFR2 and downstream signals. (A) SU 1498, a VEGFR2 inhibitor, inhibited Tat mediated T-cell stimulation. (B) T cells were transfected without RNA (mock) nonspecific siRNA (si-control) or siRNA to VEGFR2 (si-VEGFR2) for 24 h and then exposed to Tat for 24 h. (C) Pretreatment of T cells with TPCK (NFκB inhibitor), genistein (Gen; TK inhibitor), LY294002 (PI3 kinase inhibitor), SNS-032 (CDK 2, 7, and 9 inhibitor) or with rapamycin (Rapa; mTOR inhibitor) inhibited Tat-mediated T-cell stimulation. (D) Pretreatment of T cells with RO-32-0432 (PKC inhibitor) or margatoxin (MgTX; Kv1.3 inhibitor) had no effect on Tat-mediated T-cell stimulation. Data are mean ± SD from three to five independent experiments compared by ANOVA with Bonferroni’s multiple comparison, *P < 0.05, **P < 0.001; ns, not significant.

Fig. 5.

T-cell stimulation by Tat enhances histone acetylation and viral infection. (A) Tat stimulation of T cells for 24 h increases acetylated histone H3 in nuclear extracts. Data are mean ± SD from three independent experiments compared by two-way paired T test, *P < 0.05. (B) Immunoblots on the same extracts confirm a corresponding increase in acetylated histone H3 (H3-ac) with unchanged total H3 levels. (C) T cells were left unstimulated (control) or treated for 5 d with either Tat or PHA. For the last 18 h, cells were pulsed with [3H]thymidine. No significant (ns) uptake was seen in the Tat-treated cells. Data represent mean ± SD from three independent experiments and were analyzed by ANOVA with Bonferroni’s multiple comparison test, **P < 0.001. (D) T cells were stimulated with PHA or Tat in the presence or absence of anti-Tat antibody and infected with HIV, and p24 antigen was measured in culture supernatants 1 wk later. Tat-stimulated T cells showed increased HIV replication, which was blocked by anti-Tat antibody. Data are mean ± SD from seven independent experiments compared by ANOVA with Bonferroni’s multiple comparison test, **P < 0.001.