Upregulation of Antiviral Factors That Inhibit HIV-1 Infection in Sickle Cell Disease

Introduction Patients with Sickle cell disease (SCD) have lower risk for HIV-1 infection. We showed that ex vivo HIV-1 replication is blocked in SCD PBMCs in part because of the increased expression of ferroportin (FPN) and activation of SAMHD1, a host antiviral restriction factor. We hypothesized that rupture of sickling red blood cells releases sickle cell hemoglobin (HbS) that is phagocyted by macrophages leading to upregulation of innate antiviral response and inhibition of HIV-1 replication. We accessed changes in antiviral gene expression in PBMCs obtained from SCD patients compared to healthy controls. We also analyzed antiviral gene expression in macrophages treated with HbS compared to the HbA treatment. Methods The study was approved by Howard University review board (IRB) and all subjects consented to sample collection. Whole blood was collected from 9 SCA patients and 9 age and gender- matched healthy controls. PBMCs were activated with PHA (0.5 μg/ml) for 24-48 hrs followed by IL-2 (10 U/ml) for 24 hrs. Human THP-1 cells were differentiated into macrophages with PMA (25nM) for 72 hrs and treated with purified HbS or HbA (5µM). RNA strand-specific libraries were constructed using TruSeq Stranded Total RNA Gold kit (Illumina) and sequenced on an Illumina NextSeq 500 using 75 bp paired-end sequencing on two v2.5 150 cycle High-Output kits, generating 40-50 million paired-end reads per sample. The sequencing data were mapped using Dragen RNA and compared using Dragen differential expression software (Illumina). Ingenuity Pathway analysis (IPA, Qiagen) was used for pathway analysis. Results In activated SCD PBMCs compared to control PBMCs, 40432 genes were detected including 2230 differentially expressed genes (5.5%, 1.5-fold difference, 287 down and 1943 up) at 5% false discovery rate. In non-activated SCD PBMCs compared to control PBMCs, 33119 genes were detected including 5299 differentially expressed genes (16%, 923 down and 4376 up). In THP-1-differentiated macrophages treated with HbS versus HbA, 28362 genes were detected including 322 differentially expressed genes (1.1%, 187 down and 135 up). We focused our analysis on 61 genes including viral restriction factors and iron regulatory genes. In activated SCD PBMCs, four genes had highest upregulation: APOBEC3A (23-fold, p=2 x 10 -5), CH25H (11-fold, p=4 x 10 -5), heme oxygenase-1 (HMOX1, 13-fold, p=1.5 x 10 -12) and FPN (SLC40A1, 5-fold, p = 9 x 10 -8) (Fig.1). Several additional genes were u