Article Figures & Data
Figures
- FIGURE 1.
Stable antiapoptosis gene modulation in HIV-1 monocytes ex vivo. a, Tree view of 38 apoptosis genes exhibiting stable differential expression between HIV-1 (P) and control (C) donors. Gene symbols are shown for the 38 genes. Genes down-regulated in P are in green and genes up-regulated in P are in red. C01–C12 are in blue, C01–C04 repeats are in green, P01–P13 are in red, and P01–P04 repeats are in black. Log values for viral load in HIV-1 individuals are indicated. For visual enhancement, expression levels of each gene were converted to z scores. b, Cluster dendrogram of P and C samples for the 38 apoptosis-related genes exhibiting stable expression.
- FIGURE 2.
HIV-1+ monocytes exhibit resistance to apoptosis in contrast to HIV-1+ CD4 T cells ex vivo. HIV-1 CD4 T cells exhibit greater constitutive apoptosis (b) than do control (HIV-1−) CD4 T cells (a). HIV-1 CD4 T cells exhibit greater CdCl2-induced apoptosis (d) than do control (HIV-1−) CD4 T cells (c). Results represent the median ± SD for percentage of annexin V CD4 T cells in the unstimulated (e) and CdCl2-stimulated (f) conditions in eight control donors and six viremic donors. ∗, p ≤ 0.05.
- FIGURE 3.
HIV-1+ monocytes exhibit resistance to apoptosis in contrast to HIV-1+ CD4 T cells ex vivo. In contrast to CD4 T cells, no difference was found in constitutive apoptosis between control (HIV-1−) (a) and HIV-1+ (b) monocytes, but a higher CdCl2-induced apoptosis was found in HIV-1− (c) than in HIV-1+ (d) monocytes. Results represent the median ± SD for percentage of caspase-3 monocytes for constitutive (e) and for CdCl2-stimulated (f) conditions in 10 control and 9 viremic HIV-1 donors. Following FasL stimulation, no difference was found in constitutive apoptosis between control (HIV-1−) (g) and HIV-1+ (h) monocytes, but a higher FasL-induced apoptosis was noted in HIV-1− (i) than in HIV-1+ (j) monocytes. Results represent the median ± SD for percentage of caspase-3 monocytes for constitutive (k) and for FasL-stimulated (l) conditions in seven control and six viremic HIV-1 donors. Independent confirmation of results further show no difference in constitutive apoptosis between control (HIV-1−) (m) and HIV-1+ (n) myeloid cells but a higher CdCl2-induced apoptosis in control (HIV-1−) (o) than in HIV-1+ (p) myeloid cells. Results represent the median ± SD for percentage of caspase-3 in constitutive (q) and in CdCl2-stimulated (r) conditions for 10 controls and 9 viremic HIV-1 donors. ∗, p ≤ 0.05.
- FIGURE 4.
HIV-1 binding via CCR5 induces resistance in in vitro MDM cultures. Constitutive apoptosis in MDMs is shown in a and f. MDM binding by R5 tropic virus mediates MDM apoptosis resistance in vitro. CdCl2 induces cultured monocyte apoptosis (b) that is decreased upon preincubation with infectious HIV-1 BAL (c). Results represent the median ± SD for percentage of caspase-3 (d) and percentage of live (e) cells of three donors that were untreated (C), treated with CdCl2, and preincubated with HIV-1 BAL (HIV-1 BAL + CdCl2). CdCl2 induces cultured monocyte apoptosis (g) that is decreased upon preincubation with noninfectious AT2R5 (h). CCR5 binding inhibitor M657 reverses AT2R5-mediated protection (i). Results represent the median ± SD for percentage of live (j) cells in 5 donors that were untreated (C), treated with CdCl2, preincubated with M657 (Inh + CdCl2), preincubated with AT2R5 (AT2R5 + CdCl2), and preincubated with M657 and AT2R5 (AT2R5 + Inh + CdCl2). ∗, p ≤ 0.05.
- FIGURE 5.
HIV-1 binding via CCR5 induces resistance in in vitro MDM cultures. MDM binding by CCR5 chemokines mediates MDM apoptosis resistance in vitro. Indicated is the median ± SD for percentage of caspase-3 cells in five donors that were untreated (C), treated with CdCl2, and preincubated with MIP-1α (MIP1α + CdCl2), MIP-1β (MIP1β + CdCl2), and RANTES (RANTES + CdCl2). ∗, p ≤ 0.05.
- FIGURE 6.
Lower ex vivo surface CCR5 expression in HIV-1 monocytes versus control monocytes. Surface CCR5 expression was significantly lower in HIV-1 monocytes vs controls as indicated by lower CCR5 mean fluorescence intensity in HIV-1 monocytes (a) vs control monocytes (b) and lower percentage of CCR5+CD14+ monocytes in HIV-1 vs control groups (c). A total of six HIV-1 and six control donors were tested for CCR5 expression. ∗, p ≤ 0.05.
Tables
Donor ID Experiment CD4 Count (Cells/Cubic mm) Viral Load RNA (Copies/ml) Sex Age (Years) 1 Microarray 352 22,170 F 48 1ab Repeat microarray 333 100,550 F 49 2 Microarray 458 23,262 M 46 2ab Repeat microarray 435 18,196 M 46 3 Microarray 300 10,611 M 40 3ab Repeat microarray 201 44,650 M 41 4 Microarray 353 24,075 M 54 4ab Repeat microarray 408 36,561 M 55 5 Microarray 332 29,587 F 46 6 Microarray 306 3,283 F 55 7 Microarray 504 61,470 M 36 8 Microarray 201 56,643 M 35 9 Microarray 1128 9,810 F 35 10 Microarray 416 74,296 M 38 11 Microarray 896 18,088 F 39 12 Microarray 604 22,150 F 20 13 Microarray 168 61,432 M 33 14 Protein assays 525 3,104 F 40 15 Protein assays 264 30,640 F 43 16 Protein assays 701 32,168 F 42 17 Protein assays 290 24,849 M 54 18 Protein assays 266 24,795 F 52 19 Protein assays 293 14,832 F 52 20 Protein assays 203 65,329 F 42 a Nos. 1–13 served as donors for microarray experiments; the four individuals resampled after 7 mo are indicated as 1a, 2a, 3a, and 4a. Nos. 14–20 served as donors for protein assays. Donors were asymptomatic and had a history of viremia with >10,000 copies of virus/ml of plasma and were not on therapy. Viral load and CD4 T cell count measured on the day of blood drawing are indicated.
b Second sample that was taken 7 mo after the initial samples.
Gene Symbol Forward Primer 5′–3′ Reverse Primer 5′–3′ MT-1G CAA CTG CTC CTG TGC C AGC TGC ACT TCT CCG A MT-1H CCT CTT CTC TTC TCG CTT GG GCA AAT GAG TCG GAG TTG TAG PAI2 GCT TCC AGA TGA AAT TGC CGA GCT TCA GTG CCC TCC TCA TTC CCL2 ATC AAT GCC CCA GTC ACC AGT CTT CGG AGT TTG GG IER3 CCA GCA TCT CAA CTC CGT CTG T CAC CCT AAA GGC GAC TTC AAG A IL6 CAA TCT GGA TTC AAT GAG GAG AC CTC TGG CTT GTT CCT CAC TAC TC MBD4 CAC ATC TCT CCA GTC TGC CGA CGT AAA GCC TTT AAG AA HIVEP1 GCA CAC ATT CCA GGT CTC CA TGA GTT CAG GCT TGG GCT T p21 TTA GCA GCG GAA CAA GGA GT CAG TAC AGG GTG TGG TCC CT SIVA ACA TGG CAA AAC CCT GTC TC TCT TCT CGA AGA CCT CCT GC CD153 CTC CTG GAG ACA CAG CC GGT GCT TGT ATC TAT GTA CT PPBP GGT TGT CTT TAT ACA CAT GCA G ATG AGC CTC AGA CTT GAT AC SELL 5′CTC ATC ATG GGT TGG ATT AG CTG CAA GTG ACA TCT CTT T BIGM103 AGT GTG GTA TCT CTA CAG G CAA GGC TTG TCG AGT G CCL20 CTG CTT TGA TGT CAG TGC TGC TCA CCC AAG TCT GTT TTG G LPL CCG AGA GTG AGA ACA TCC CAT TCA CCT TTC TGC AAA TGA GAC ACT TTC TC a Gene symbols and forward and reverse primer sequences for differentially expressed genes that were validated by real-time PCR are indicated.
Accession No. Gene Symbol Gene Name p Value for Comparison of HIV-1-Infected Individuals vs Controls (Quantitative PCR) Ratio of Signal between HIV-1-Infected Individuals and Controls N98591 IL6 IL-6 0.0004 166.40 W92812 PPBP Proplatelet basic protein (chemokine (C-X-C motif) ligand 7) 0.0010 3.58 AA425102 CCL2 Chemokine (C-C motif) ligand 2 0.0001 12.85 NM_002984 CCL4 Chemokine (C-C motif) ligand 4 0.0050 5.99 AA457705 IER3 Immediate early response 3 0.0071 3.06 H77597 MT1H Metallothionein 1H 0.0002 8.58 H53340 MT1G Metallothionein 1G 0.0008 5.09 T49159 PAI2 Serine (or cysteine) proteinase inhibitor, clade B (OVA), member 2 0.0043 6.88 W02699 CD153 CD30L 0.0004 0.38 AA167728 SIVA CD27-binding (Siva) protein 0.0180 0.48 AI952615 p21 CDKN1A/Waf1/Cip1/Sdi1 0.0220 1.54 a Differentially expressed apoptosis genes validated by real-time PCR are shown. The accessions, gene symbols, gene names, p values, and fold ratios for apoptosis genes differentially expressed between HIV-1 and control monocytes (all but CCL4 and p21 were also identified as significant by microarray analysis) were confirmed for significance by real-time PCR analysis and are indicated.
- Table IV.
21 Stable apoptosis genes in vivo are previously reported in HIV-1-exposed/infected M/Ma
Accession No. Gene Symbol Gene Name Reference to Previous HIV-1 Report(s) N63940 ACHE Acetylcholinesterase (YT blood group) (38 ) AA683578 ADA Adenosine deaminase (38 ) AA455941 APEH N-acylaminoacyl-peptide hydrolase (38 ) AA425102 CCL2 Chemokine (C-C motif) ligand 2 (18 37 38 39 77 ) AA101971 ETS1 V-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (38 ) AA410375 GMPR Guanosine monophosphate reductase (38 ) H50114 GRIN2C Glutamate receptor, ionotropic, N-methyl-d-aspartate 2C (38 ) T80846 HAAO 3-Hydroxyanthranilate-3,4-dioxygenase (38 ) AA457705 IER3 Immediate early response 3 (39 ) AI870374 LDOC1 Leucine zipper, down-regulated in cancer 1 (38 ) R83837 LYN V-yes-1 Yamaguchi sarcoma viral-related oncogene homolog (24 39 ) H86558 MAD MAX dimerization protein 1 (38 ) NM_012219 MRAS Muscle RAS oncogene homolog (38 ) AA872383 MT1E Metallothionein 1E (functional) (38 ) N55459 MT1F Metallothionein 1F (functional) (38 ) H77597 MT1H Metallothionein 1H (38 ) AA446246 NRAS Neuroblastoma RAS viral (v-ras) oncogene homolog (39 ) AI952615 p21 CDKN1A/Waf1/Cip1/Sdi1 (39 ) AA258001 RELB V-rel reticuloendotheliosis viral oncogene homolog B, nuclear factor of κ light polypeptide gene enhancer in B cells 3 (avian) (38 ) AI473336 WISP1 WNT1 inducible signaling pathway protein 1 (37 ) AA425900 UNG2 Uracil-DNA glycosylase 2 (41 42 ) a Indicated are the accession numbers, gene symbols, and gene names for 21 genes from the stable apoptosis signature that we identify in in vivo HIV-1 monocytes that have also been previously reported as modulated in in vitro macrophages by HIV-1. The references for previous studies that have reported HIV-1-associated modulation of these genes are additionally provided.
Data Supplement
Files in this Data Supplement:
- Supplementary Figure Legend (PDF, 7.02 Kb)
- Supplementary Figure 1 (PDF, 85.3 Kb) - Multi-Dimentional Scaling Plots showing composite gene clusters for circulating monocytes exhibit from HIV-infected and control subjects.
- Supplementary Table I (PDF, 104 Kb) - 376 constitutively modulated genes in circulating monocytes from HIV-infected subjects as compared to uninfected controls.
In this issue
