Figure 2.
Figure 2. Altered iron metabolism in SCD-derived PBMCs is critical for HIV-1 inhibition. (A) Heat map showing expression of selected iron and HIV-1 regulatory genes in SCD PBMCs. RNA was extracted from PBMCs obtained from 3 SCD patients and 3 controls. RNA was reverse transcribed and analyzed by RT-PCR using primers for BACH1, CDK2, Cyclin A, Cyclin E, EGR1, ferroportin, hepcidin, HIF-1α, HO-1, IKBα, p21, p27, PP1α, and SAMHD1. The 18S RNA was used as a housekeeping control gene. Heat map was constructed using Morpheus software. P values were determined using paired Student t test. (B) Validation of candidate genes expression. Expression of IKBα, HO-1, p21, HIF-1α, and ferroportin mRNA was analyzed from additional 3 controls and 7 SCD PBMCs. SAMHD1 expression was analyzed in 3 controls and 6 SCD PBMCs. RNA was isolated, reverse transcribed, and analyzed by real time. 18S rRNA was used as a reference for ΔΔCt analysis. The means ± SE and P value calculated with Student t test are shown (C) IPA of the iron and HIV-1 regulatory genes. Ingenuity software analysis of genes shown in panel A identified a protein network that connected iron and HIV-1 regulatory genes through virus replication, anemia, and cell cycle progression networks (D). Upregulated genes are colored in red and downregulated genes are colored in green. (D) IPA of viral and pathogen restricting genes in SCD. Ingenuity software analysis of 250 upregulated and downregulated genes determined by meta-analysis of Geoset GSE53441 (data from PBMCs of 24 SCD patients and 10 controls) identified a protein network of 12 genes involved in antiviral response and viral replication. (E) Hepcidin restores HIV-1 replication in SCD PBMCs. Activated PBMCs from 3 SCD patients and 3 control individuals were infected with HIV-1-LUC-G virus and treated with 0.9 μM hepcidin, 20 μM ferric ammonium citrate (iron), or 10 μM PPYeT iron chelator. At 48 hours PI, luciferase activity was measured. The means and P values determined by Student t test are shown. (F) Hepcidin in plasma from SCD patients. Hepcidin was measured in plasma obtained from 13 SCD and 12 healthy subjects by HR/SIM method. The means and P values determined by Student t test are shown.

Altered iron metabolism in SCD-derived PBMCs is critical for HIV-1 inhibition. (A) Heat map showing expression of selected iron and HIV-1 regulatory genes in SCD PBMCs. RNA was extracted from PBMCs obtained from 3 SCD patients and 3 controls. RNA was reverse transcribed and analyzed by RT-PCR using primers for BACH1, CDK2, Cyclin A, Cyclin E, EGR1, ferroportin, hepcidin, HIF-1α, HO-1, IKBα, p21, p27, PP1α, and SAMHD1. The 18S RNA was used as a housekeeping control gene. Heat map was constructed using Morpheus software. P values were determined using paired Student t test. (B) Validation of candidate genes expression. Expression of IKBα, HO-1, p21, HIF-1α, and ferroportin mRNA was analyzed from additional 3 controls and 7 SCD PBMCs. SAMHD1 expression was analyzed in 3 controls and 6 SCD PBMCs. RNA was isolated, reverse transcribed, and analyzed by real time. 18S rRNA was used as a reference for ΔΔCt analysis. The means ± SE and P value calculated with Student t test are shown (C) IPA of the iron and HIV-1 regulatory genes. Ingenuity software analysis of genes shown in panel A identified a protein network that connected iron and HIV-1 regulatory genes through virus replication, anemia, and cell cycle progression networks (D). Upregulated genes are colored in red and downregulated genes are colored in green. (D) IPA of viral and pathogen restricting genes in SCD. Ingenuity software analysis of 250 upregulated and downregulated genes determined by meta-analysis of Geoset GSE53441 (data from PBMCs of 24 SCD patients and 10 controls) identified a protein network of 12 genes involved in antiviral response and viral replication. (E) Hepcidin restores HIV-1 replication in SCD PBMCs. Activated PBMCs from 3 SCD patients and 3 control individuals were infected with HIV-1-LUC-G virus and treated with 0.9 μM hepcidin, 20 μM ferric ammonium citrate (iron), or 10 μM PPYeT iron chelator. At 48 hours PI, luciferase activity was measured. The means and P values determined by Student t test are shown. (F) Hepcidin in plasma from SCD patients. Hepcidin was measured in plasma obtained from 13 SCD and 12 healthy subjects by HR/SIM method. The means and P values determined by Student t test are shown.

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