HIV-1 Infected Peripheral Blood Mononuclear Cells Modulate the Fibrogenic Activity of Hepatic Stellate Cells through Secreted TGF-β and JNK Signaling

<div><p>Background and Aims</p><p>Patients with liver disease infected with the human immunodeficiency virus (HIV) exhibit accelerated progression of hepatic fibrosis and liver cirrhosis compared to uninfected individuals. We studied the effects of soluble factors secreted by HIV-infected peripheral blood mononuclear cells (PBMCs) on hepatic stellate cells (HSCs), which are central mediators of liver fibrosis.</p><p>Methods</p><p>An <i>in vitro</i> model was used in which a HSC line, LX2, was treated with culture supernatants of human PBMCs infected with macrophage tropic (R5) or T cell tropic (X4) strains of HIV-1. Quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to assess the expression of fibrogenic and proinflammatory markers; LX2 proliferation and intracellular signaling pathways were also studied. A qRT-PCR based miRNome array was used for comparative miRNA profiling of LX2 cells treated with infected PBMC culture supernatants.</p><p>Results</p><p>Pro-fibrogenic, angiogenic and proinflammatory markers, and proliferation of LX2 cells were increased following exposure to culture supernatants from HIV-1 infected PBMCs. The profiling of miRNAs in LX2 cells treated with culture supernatants from HIV-1 R5- or X4-infected PBMCs showed 66 and 22 miRNAs respectively, to be significantly altered compared to mock-treated LX2 cells. While different sets of miRNAs were altered in the two cases, bioinformatics analyses predicted these to be associated with common pathways, including TGF-β signaling and extracellular matrix receptor interaction pathways.</p><p>Conclusions</p><p>HIV infection creates a favorable milieu for the activation of hepatic stellate cells and increased hepatic fibrosis. We identify some regulatory molecules important for these effects.</p></div

Soluble factors from R5- or X4 tropic HIV-1 infected PBMCs activate human hepatic stellate cells.

<p>LX2 cells were treated with virus-free culture supernatants from HIV-1 infected (R5- or X4- tropic) human PBMCs for 72 hr. Mock (M) represents control group that was treated with supernatants from uninfected PBMCs. (A) Total RNA was isolated from LX2 cells and used for the expression analysis of various pro-fibrogenic and pro-inflammatory marker genes using qRT-PCR, as described in Methods. Data represents mean ± S.E. of three independent experiments. *p<0.05 and **p<0.01 vs mock. (B) LX2 cells were treated with culture supernatants from HIV-1 infected PBMCs for 24 hr as described in Methods. Total cell lysates were analyzed for TIMP-1 expression by western blotting. GAPDH was used as a loading control. Data representative of one of the three experiments is shown. (C) LX2 cells were plated in a 96-well plate at passage 4 and serum starved for 24 hr. The cells were then incubated for 72 hr with culture supernatants from X4-, R5- or mock- infected PBMCs. Following treatment, cells were assayed for proliferation and cell survival using the MTT assay. Data represents mean ± S.E. of three independent experiments.</p

Differentially regulated miRNAs in LX2 cells.

<p>qRT-PCR profiling array result showing fold upregulation or downregulation of the indicated miRNAs in LX2 cells treated with supernatants from (A) R5-tropic or (B) X4-tropic HIV-1 infected PBMCs as compared to mock-infected PBMCs. Fold changes were determined using comparative C_T method. Data represents mean of the three independent experiments. Only those miRNAs that were significantly modulated (p<0.05) in R5- or X4- supernatant treated LX2 cells, as compared to mock-treated cells, are shown.</p

Pathway analysis and validation in treated LX2 cells.

<p>Heat map showing predicted pathways for the miRNAs that were differentially modulated in LX2 cells treated with supernatants from (A) R5-tropic or (B) X4-tropic HIV-1 infected PBMCs. DIANA miRPath v2.0 was used to predict the pathways with default p-value threshold of 0.05 and microT threshold of 0.8. This clustering is based on significance analysis. All significantly targeted pathways are marked with deep red. On the vertical axis, miRNAs are clustered together by exhibiting similar pathway targeting patterns.</p

miRNA expression profiling of LX2 cells.

<p>LX2 cells were treated with virus-free culture supernatants from mock, R5- or X4-tropic HIV-1 infected PBMCs for 72 hr. Total RNA was isolated and subjected to miRNA profiling as described in Methods. (A) Percent distribution of C_T values for the 1008 miRNAs across the samples. Any C_T value above 35 was taken as 35. Entire profiling was done in set of three and data represents mean ± S.E. of three independent experiments. (B) Heat map depicting relative expression levels of miRNAs that showed significant changes across all the biological replicates. The heat map shows hierarchical clustering of miRNAs. Each row represents miRNA and each column represents samples tested. Red and green represent miRNAs with expression levels above and below the mean, respectively.</p

Culture supernatants from HIV-infected PBMCs activate JNK and NF-κB pathway in LX2 cells.

<p>Serum starved LX2 cells were treated with virus-free culture supernatants from HIV-infected (R5- or X4-tropic) human PBMCs for the indicated times. Mock (M) represents control group that was treated with supernatants from uninfected PBMCs. (A) LX2 cell lysates were analyzed for phosphorylation of JNK, Erk and Akt; total levels of these proteins served as the respective loading controls. Data representative of one of three experiments is shown. (B) LX2 cell lysates were analyzed for phospho-c-Jun, c-Fos and phospho-ATF-2 levels; total c-Jun and β-actin served as the loading controls. Data representative of one of three experiments is shown. Values below the lanes show fold-change relative to the mock lanes after normalizing to total c-Jun or β-actin levels. (C) Serum-starved LX2 cells were treated with 25 µM SP600125 (JNK inhibitor) for 2 hr; DMSO was used as control. Culture supernatants from HIV infected PBMCs (mock, R5- or X4-tropic) were diluted 1∶3 with fresh culture medium and added to LX2 cells for 72 hr; the inhibitor was replenished every 24 hr. RNA was isolated from treated LX2 cells and the expression levels of TGF-β and MMP-2 were evaluated by qRT-PCR. Data was normalized using β-actin levels and represents mean ± S.E. of three independent experiments. Values above the bars show p value for comparison of X4/R5/DMSO to either Mock/DMSO or X4/R5/SP6. (D) LX2 cell lysates were analyzed for NF-κB p65 phosphorylation; total levels of p65 served as the loading control. Data representative of one of three experiments is shown. Values below the lanes show fold-change relative to the mock lanes after normalizing to total p65 levels.</p

Profibrogenic effects of HIV-1 Vpu expressing monocytes on human hepatic stellate LX2 cells.

<p>The LX2 cells were cocultured with (A) U937-VpuGFP and U937-GFP cells or (B) U1-Scrambled and U1-VpuShRNA cells, as described in Methods. The expression levels of the indicated mRNAs in the LX2 cells were then estimated by qRT-PCR. All expression values were normalized to those in the control cells, n = 3. (C) Confocal microscopic analysis of intracellular αSMA-1 expression in LX2 cells cocultured with either U937-GFP or U937-VpuGFP cells. The bar plot on the right represents the mean intensity of αSMA-1 in the LX2 cells, n = 32. Flow cytometric analysis of intracellular COL-1 levels in LX2 cells cocultured with (D) U937-VpuGFP and U937-GFP cells or (E) U1-Scrambled and U1-VpuShRNA cells. The bar plot on the right represents the mean fluorescence intensity (MFI) of COL-1 in the LX2 cells, n = 3. The LX2 cells were treated with culture supernatants from (F) U937-VpuGFP and U937-GFP cells or (G) U1-Scrambled and U1-VpuShRNA cells, as described in Methods. The expression levels of the indicated mRNAs in the LX2 cells were then estimated by qRT-PCR. All expression values were normalized to those in the control cells, n = 3. Error bars represent mean ± SD (*, p<0.05).</p

TGF-β secreted from Vpu-expressing monocytic cells is responsible for the profibrogenic effects on LX2 stellate cells.

<p>(A) LX2 cells were cocultured with either U937-GFP or U937-VpuGFP cells together with either a pantropic anti-TGFβ antibody or an isotype control antibody as described in Methods. Quantitative RT-PCR was then carried out to estimate the expression levels of mRNAs for COL-1, αSMA-1, PCT-III, MMP2 and VEGF. All expression levels are shown as fold changes normalized to the U937-GFP control. (B) LX2 cells were cocultured with U1-scrambled cells together with either a pantropic anti-TGFβ antibody or an isotype control antibody, and flow cytometry was used to estimate the expression levels of intracellular COL-1. (C) LX2 cells were cocultured with either U937-GFP or U937-VpuGFP cells together with the TGF-β receptor inhibitor SB431452 (or DMSO as control) as described in Methods. Quantitative RT-PCR was then carried out to estimate the expression levels of mRNAs for COL-1, MMP2, PCT-III, and VEGF. All expression levels are shown as fold changes normalized to the U937-GFP control. (D) LX2 cells were cocultured with U1-scrambled cells in the presence of either SB431452 or DMSO, and flow cytometry was used to estimate the expression levels of intracellular COL-1. Error bars represent mean ± SD from three independent experiments; *p<0.05.</p

The Expression of HIV-1 Vpu in Monocytes Causes Increased Secretion of TGF-β that Activates Profibrogenic Genes in Hepatic Stellate Cells

<div><p>There is faster progression to fibrosis in persons with liver injury who are also infected with HIV. Other reports have suggested that HIV can directly infect and activate stellate cells, and the viral Tat and gp160 proteins also induce profibrogenic factors from peripheral blood mononuclear cells (PBMCs). We tested the role of HIV-1 Vpu accessory protein in promoting profibrogenic activation of hepatic stellate cells. Human stellate LX2 cells were cocultured with human monocytic U937 cells stably expressing the Vpu protein or latently infected U1 cells knocked down for Vpu expression, LX2 cells were also cultured with the supernatants from these cells. The expression of profibrogenic markers was evaluated in LX2 cells usingquantitative reverse transcription polymerase chain reaction (qRT-PCR),western blotting, immunofluorescence,flow cytometry and ELISA were used to confirm and quantitate protein expression. Monocytic cells expressing Vpu increased the expression of profibrogenic markers in LX2 cells. The culture supernatants of these cells contained increased levels of transforming growth factor beta (TGF-β), which correlated with increased activity of the AP-1 transcription factor. Antibodies against TGF-β or a TGF-β receptor inhibitor (SB431452) reversed Vpu-mediated profibrogenic activation of LX2 cells, suggesting that TGF-β mediated these effects. The cytokine macrophage migration inhibitory factor (MIF) attenuated Vpu-mediated TGF-β secretion and profibrogenic effects on LX2 cells. Besides its other roles in pathogenesis, Vpu is likely to contribute to hepatic fibrosis through this hitherto unknown mechanism.</p></div

MIF treatment reduces TGF-β in monocytes and ameliorates the profibrogenic effects of Vpu expressing monocytes.

<p>(A) Quantitative RT-PCR estimation of TGF-β mRNA levels in U937-GFP and U937-VpuGFP cells treated with MIF. The expression levels are shown as fold changes normalized to the untreated controls. (B) Quantitation of TGF-β in the culture supernatants of the U937-VpuGFP and U937-GFP cultures treated with MIF or untreated controls. (C) LX2 cells were cocultured with either U937-GFP or U937-VpuGFP cells together with MIF as described in Methods. Quantitative RT-PCR was then carried out to estimate the expression levels of mRNAs for COL-1, MMP2, PCT-III and TGF-β in LX2 cells. All expression levels are shown as fold changes normalized to the U937-GFP control. (D) LX2 cells were cocultured with U1-scrambled cells together with MIF, and quantitative RT-PCR was used to estimate the expression levels of COL-1 and αSMA-1 mRNAs; the values are expressed as fold changes normalized to the untreated culture condition. The data are representative of three biological replicates. Error bars represent mean ± SD; *p<0.05.</p