Differential effects of Tat proteins derived from HIV-1 subtypes B and recombinant CRF02_AG on human brain microvascular endothelial cells: implications for blood-brain barrier dysfunction

Abstract

HIV-1 genetic differences influence viral replication and progression to AIDS. HIV-1 circulating recombinant form (CRF)02_AG is the predominant viral subtype infecting humans in West and Central Africa, but its effects on HIV neuropathogenesis are not known. In the present study, we investigated the effects of Tat proteins from HIV-1 subtype B (Tat.B) and HIV-1 CRF02_AG (Tat.AG) on primary human brain microvascular endothelial cells (HBMEC), the major component of the blood-brain barrier (BBB). Using Affymetrix GeneChip Human Gene 1.0.ST arrays, we showed that Tat.AG had minimal effects while Tat.B induced transcriptional upregulation of 90 genes in HBMEC, including proinflammatory chemokines, complement components C3, C7, and complement factor B, matrix metalloproteinases (MMP)-3, MMP-10, and MMP-12. These results were confirmed by real-time PCR. Compared with Tat.AG, Tat.B significantly increased MMP-3, MMP-10, and MMP-12 activities in HBMEC, and the MMPs tissue inhibitor of metalloproteinase-2 blocked Tat-induced increase in MMPs activity. Western blot analyses also showed that Tat increased the expression of C3 and its cleaved fragment C3b in HBMEC. These data suggest that genetic differences between HIV-1 subtypes B and CRF02_AG influence the effects of Tat proteins from these two clades on HBMEC, including molecular and cellular functions, and canonical pathways, which would affect BBB dysfunction and viral neuropathogenesis.

Figure 1

Assessment of microarray data quality. MvA plots of the probe-set signal estimates after Robust Multi-array Analysis model fit (GC Robust Multi-array Average background correction plus quantile normalization). None of the samples reveal any special pattern; data quality, probe intensity, and probe-set signals were consistent across arrays and across donor replicate samples. Y axis (M value)=log2 geometric mean intensity for condition 2−log2 geometric mean intensity for condition 1; X axis (A value)=(log2 geometric mean intensity for condition 2+log2 geometric mean intensity for condition 1)/2. Lines are drawn after the Loess fitting of M values and correspond to the zero level of M values. Sample IDs are as follows: Controls: untreated human brain microvascular endothelial cells (HBMEC), TAT.AG: HBMEC treated with HIV-1 Tat.AG; TAT.B: HBMEC treated with HIV-1 Tat.B; Heat.TAT.AG: HBMEC treated with heat-inactivated Tat.AG. All Tat proteins were used at 100 ng/mL, and all cells were treated for 48 hours. Each analysis was performed using replicate data from all three donors.

Figure 2

Hierarchical clustering of differentially expressed genes. Cluster images show the expression profiles of transcripts with at least 1.9-fold change and control of false discovery rate (FDR) at 0.05 when comparing human brain microvascular endothelial cells (HBMEC) treated with Tat-AG with all other treatment conditions: cells treated with Tat-B, heat-inactivated Tat-AG (TAT.AG-HI), and untreated control. All gene expression was estimated using untreated controls as a reference, and differential expression analysis was performed using the linear model for microarray data. Hierarchical analysis performed as described in Materials and methods. Expression levels are represented on a continuum from green (low abundance) to red (high abundance). Sample donors' IDs are as follows: D1: donor 1; D2: donor 2; D3: donor 3.

Figure 3

Real-time PCR validation of differentially expressed enzymes. Data confirmed that compared with Tat.AG, Tat.B upregulates zinc endopeptidases matrix metalloproteinase (MMP)-3 (A), MMP-10 (B), MMP-12 (C), the enzymes transglutaminase-2 (TGM2) (D), and chitinase-3-like protein 2 (CHI3L2) (E) in human brain microvascular endothelial cells (HBMEC). Controls are untreated HBMEC; Tat.AG represents HBMEC treated with Tat.AG; Tat.B represents HBMEC treated with Tat.B; Tat.AG-HI represents HBMEC treated with heat-inactivated Tat.AG. All Tat proteins were used at 100 ng/mL, and all cells were treated for 48 hours. Data using HBMEC from three different human donors are shown, and each donor was tested in triplicate. (***P<0.001, compared with cells treated with Tat.AG, cells treated with heat-inactivated Tat, or untreated controls). Full names of genes are provided in Table 1.

Figure 4

Real-time PCR validation of differentially expressed complement and chemokines. Data confirmed that compared with Tat.AG, Tat.B upregulates C3 (A) and chemokines (C-C motif) ligand 5 (CCL5) (B) and (C-X-C motif) ligand 6 (CXCL6) (C) in human brain microvascular endothelial cells (HBMEC). Controls are untreated HBMEC; Tat.AG represents HBMEC treated with Tat.AG; Tat.B represents HBMEC treated with Tat.B; Tat.AG-HI represents HBMEC treated with heat-inactivated Tat.AG. All Tat proteins were used at 100 ng/mL, and all cells were treated for 48 hours. Data using HBMEC from three different human donors are shown, and each donor was tested in triplicate (***P<0.001, compared with cells treated with Tat.AG, cells treated with heat-inactivated Tat, or untreated controls). Full names of genes are provided in Table 1.

Figure 5

Increased matrix metalloproteinase (MMP)-3, MMP-10, and MMP-12 activities in human brain microvascular endothelial cells (HBMEC) treated with Tat.B compared with cells treated with Tat.AG. HBMEC were treated with Tat.B or Tat.AG at 1 to 1,000 ng/mL for 48 hours and MMPs activity in culture supernatants quantified by fluorometric assay as described in Materials and methods. Controls are untreated HBMEC, Tat.AG represents HBMEC treated with Tat.AG; Tat.B represents HBMEC treated with Tat.B; Tat.B-HI represents HBMEC treated for 48 hours with heat-inactivated Tat.B. HBMEC treated for 48 hours with lipopolysaccharide (LPS) (50 μg/mL) were used as positive controls. Data using HBMEC from human donor 1 (A, E, and I), donor 2 (B, F, and J), and donor 3 (C, G, and K) are shown; and for each donor all experimental conditions were tested in triplicate. Tat.B treatment significantly increased MMP-3 (A–D), MMP-10 (E–H), and MMP-12 (I–L) activities, in comparison with cells treated with Tat.AG, cells treated with heat-inactivated Tat.B, or untreated controls (*P<0.05, **P<0.01, ***P<0.001). P values for Tat.AG-treated cells are in comparison with untreated controls or cells treated with heat-inactivated Tat.B. P value for LPS-treated cells is in comparison with untreated controls, cells treated with Tat.AG, or cells treated with heat-inactivated Tat.B. The MMPs inhibitor tissue inhibitor of metalloproteinase-2 (TIMP2) diminished Tat-induced MMP-3 (D), MMP-10 (H), and MMP-12 (L) activities.

Figure 6

HIV-1 Tat proteins increases C3 and C3b expression in human brain microvascular endothelial cells (HBMEC). (A) Analysis of primary cells from three different human donors showed that both Tat.B and Tat.AG increased C3 and C3b protein levels in HBMEC, compared with untreated controls. Treatment with heat-inactivated Tat.B (Tat.B-HI) or Tat.AG (Tat.AG-HI) at 100 ng/mL also increased C3 and C3b expression but to a lesser extent than cells treated with similar concentrations of Tat.B or Tat.AG. Lipopolysaccharide (LPS) did not have much effect on C3 or C3b levels. (B, C) Western blot analyses using HIV-1 Tat monoclonal antibody #1974 (B), and HIV-1 Tat monoclonal antibody #4138 (C) confirmed the presence of Tat proteins in purified Tat.AG solutions. M1 and M2 are molecular weight markers.