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. 2003 Jun 13;17(9):1269-77.
doi: 10.1097/00002030-200306130-00001.

Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus

Eric D Miller  1 , Jonathan A Smith, Monika Lichtinger, Liping Wang, Lishan Su
Affiliations

Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus

Eric D Miller et al. AIDS. .

Abstract

Objective: To identify HIV-induced host factors in the severe combined immunodeficient (SCID)-hu Thy/Liv mouse that may contribute to HIV pathogenesis in the thymus.

Design: To identify genes specifically altered by HIV-1 infection using the cDNA microarray assay, SCID-hu Thy/Liv organs derived from the same donors were used. Therefore, no genetic variations existed between HIV and mock-infected samples. In addition, the 12-14 day post-infection timepoint was chosen because no significant thymocyte depletion was detected in HIV-infected Thy/Liv organs, so mRNA from the same cell types could be compared.

Methods: Using SCID-hu Thy/Liv mice constructed from the same donor tissues, we analysed the expression of 9183 host genes in response to HIV infection with cDNA microarrays. Expression of selected genes with more than threefold induction was confirmed by measuring RNA (reverse transcriptase-polymerase chain reaction; RT-PCR) and proteins.

Results: HIV-1 (JD or NL4-3) infection of the SCID-hu Thy/Liv mouse led to more than threefold induction of 19 genes, 12 of which were IFN-inducible and six were unknown EST clones. We confirmed induction by RT-PCR and protein blots. Both signal transducer and activator of transcription (STAT)1 and STAT2 proteins were induced, and STAT1 was also activated by phosphorylation at the Tyr701 and Ser727 sites in human thymus infected with HIV-JD or NL4-3. Treatment of human fetal thymus organ culture or human thymocytes with recombinant HIV-1 gp120 proteins also led to induction or activation of STAT1.

Conclusion: HIV-1 infection of the thymus led to activation of the STAT1 signaling pathway in thymocytes, which may contribute to HIV-1 pathogenesis in the thymus.

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Figures

Fig. 1
Fig. 1. Microarray assay of gene expression in HIV-1-infected Thy/Liv organs
(a) Quantitative measurement of differential gene expression. RNA from six different yeast-specific genes was mixed with the HIV or mock-infected RNA samples in the microarray assay. RNA from each yeast gene was added at a known ratio (3, 10 or 25) in the Cy3 (HIV) or Cy5 (mock) sample (Cy3/Cy5 added). The six yeast genes were arrayed on the slides in quadruplets. Positive values indicate threefold, 10-fold or 25-fold more RNA were used in the Cy3 (HIV) sample. Negative values (−3, −10 or −25) indicate that threefold, 10-fold or 25-fold more RNA were included in the Cy5 (mock) sample. An equal amount of RNA in Cy3 and Cy5 samples is indicated as 1 (+1 or −1). The X axis shows the added ratios and the Y axis indicates Cy3/Cy5 readout data from the assay. Error bars indicate standard deviations. (b) RNA sample and reaction control. RNA samples from HIVJD (Cy3 labeled) or mock (Cy5 labeled) infected severe combined immunodeficient (SCID)-hu Thy/Liv mice were analysed using the microarray assay. Relative expression levels of 23 000 Mr highly basic protein (HBP), ribosomal subunit S9 (S9 rRNA) and α-tubulin were quantified in quadruplets. Three sets of human genes known to express in multiple tissues (complex target 1–3) were also included as controls. Positive values (Cy3 signal/Cy5 signal) indicate more RNA in the Cy3 (HIV) sample, thus induction of expression by HIV. Negative values (Cy5 signal/Cy3 signal) indicate more RNA in the Cy5 (mock) sample, thus suppressed expression by HIV. Error bars indicate standard deviations from the quadruplet arrays. The relative expression of three T cell markers (TCRβ, CD8 and Thy-1) from the microarray assay was also shown. Comparison of HIV (JD or NL4-3) and mock infected samples from different donor tissues showed similar results. (c) Alteration of gene expression in SCID-hu Thy/Liv mice after HIV-1 infection. A small number (< 0.2%) of genes were induced (> threefold) in HIV-1-infected Thy/Liv organs over mock infected samples. The majority of genes were in the +2.5 to −2.5 range (99.5%). Error bars indicate standard deviations from three experiments (two with JD and one with NL4-3) with different donor tissues.
Fig. 2
Fig. 2. Induction of the IFN-induced gene cluster and of the signal transducer and activator of transcription 1 protein in HIV-1-infected Thy/Liv organs
(a) Confirmation of gene induction by reverse trancriptase-polymerase chain reaction (RT-PCR). Severe combined immunodeficient (SCID)-hu Thy/Liv mice infected with mock (−) or with HIV-JD (+) were harvested at 2 weeks post-infection. Total thymocyte RNA from mock or HIV-infected Thy/Liv organs were analysed by RT–PCR with primers specific for the selected genes. 1 × and 5 × indicate 25 ng and 125 ng of total RNA used in the RT–PCR reaction. Expression of actin was used to monitor cellular RNA. No significant thymocyte depletion was detected in mock or HIV-infected samples. Two independent experiments with different donor tissues were performed with similar results. (b) Induction of signal transducer and activator of transcription (STAT)1 and STAT2. SCID-hu Thy/Liv mice infected with mock (−) or with HIV-JD (+) were harvested at 2 weeks post-infection. Total thymocyte proteins (50 μg) from two representative mock or two HIV-infected Thy/Liv organs were analysed by Western blot with the anti-p91 (STAT1) monoclonal antibody or anti-STAT2 polyclonal antibody. Anti-β-tubulin antibody was used to monitor cellular proteins. No significant thymocyte depletion was detected in mock or HIV-infected samples. Four independent experiments (infected with JD or NL4-3) with different donor tissues were performed with similar results.
Fig. 3
Fig. 3. Activation of the signal transducer and activator of transcription 1 protein in HIV-1-infected Thy/Liv organs
Total thymocyte proteins were blotted with polyclonal anti-signal transducer and activator of transcription (STAT)1 (p84/p91) antibody or with antibodies specifically reactive with STAT1 phosphorylated at Ser727 (pSTAT1-Ser) or Tyr701 (pSTAT1-Tyr). Anti-pSTAT1 (Y701) detects both forms of phospho-STAT1 (p91 and p84) and anti-pSTAT1 (Ser727) is specific to phospho-STAT1-p91. Two representative mock or HIV-(JD)-infected Thy/Liv samples were presented. Proteins from IFNα–treated Hela or IFNγ–treated 293T cells were used as positive control for the anti-P-STAT1 (Y701) antibody. Three independent experiments (infected with JD or NL4-3) with different donor tissues were performed with similar results.
Fig. 4
Fig. 4. Activation of signal transducer and activator of transcription 1 in human fetal thymus organ culture or thymocytes by HIV infection or recombinant HIV-1 gp120
(a) Induction and activation of signal transducer and activator of transcription (STAT)1 in human fetal thymus organ culture (HF-TOC) by HIV-1 infection and recombinant gp120. HF-TOC infected with mock (M) or HIV-JD, or treated with recombinant gp120 (IIIB, 100 ng/ml) or IFNγ were analysed for STAT1 protein expression (STAT1 p84/p91) and activation (pSTAT1Y701) as described above. NL4-3 infection also led to STAT1 activation (not shown). (b) Activation of STAT1 in human thymocytes by recombinant gp120. Human fetal thymocytes were cultured with medium (mock), with 5 ng/ml (lanes 1) or 50 ng/ml (lanes 2) of recombinant HIV-1 gp120 (IIIB or BaL) preparation. Thymocytes were harvested at 12 h (o/n) post culture and analysed for total STAT1 proteins (p84/p91) and Tyr701 phosphorylated STAT1 (Y701). Human thymocytes treated with IFNγ or SDF1α were used as controls.
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