Session: 113. Hemoglobinopathies, Excluding Thalassemia: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science
METHODS: The study was approved by Howard University IRB and IACUC committees. Townes SCD mice carrying human normal α (hα) and sickle hemoglobin beta (βS) genes and control Townes mice carrying human normal hα and βA genes were used in the study. EcoHIV virus obtained from David Volsky (Albert Einstein University) was constructed in pNL4-3 vector with the substitution of gp120 with the envelope-coding gp80 region from ectopic MLV. Whole blood samples were obtained from 9 SCD patients and 9 healthy individuals. RNA Seq analysis was conducted on Illumina® NextSeq 500 and the data were mapped and analyzed using Dragen software. Normalized gene counts were used for gene set enrichment analysis (GSEA) and Gene ontology (GO) analysis. RNA expression was measured by quantitative RT-PCR (Roche). Protein expression in mouse splenocytes was determined by ELISA.
RESULTS: Infection of control mice with EcoHIV virus, which contained MLV envelope and was able to infect mouse cells, led to robust viral replication in spleen at 7 days post infection, as detected by the expression of HIV-1 Nef mRNA (Fig. 1) and Gag mRNA. We also detected HIV-1 p24 in splenic tissue. Comparing to the control, SCD mice showed significantly reduced EcoHIV replication both at the levels of RNA expression (Figs 1) and p24 production.
GSEA analysis against the 72 genes that included all known HIV-1 restriction factors revealed strong enrichment of these genes in the human activated SCD PBMCs compared to the activated control PBMCs (Fig.2). These genes included APOBEC3A, HMOX1, CH25H, IFIT3, CDKN1A, APOBEC3B, IFITM3, SAMHD1 and TSPO. GSEA analysis also showed upregulation of several pathogen-associated molecular patterns sensing genes including NOD1, NLRC4, TRL7 and TLR9 in human PBMCS. In SCD mice splenocytes, we detected overexpression of FPN, and transferrin receptor (Tfr) genes indicating upregulation of iron metabolism similar to previously observed in human SCD PBMCs. In mouse splenocytes we detected upregulation of CDKN1A, HMOX1, IFITM3, CH25H and TSPO mRNAs. Protein expression analysis showed upregulation of FPN, TFR, HO-1, APOBEC, CH25H, IFIT3 and TSPO in SCD mouse splenocytes.
CONCLUSION: Together, these data reveal, for the first time, that HIV-1 infection is suppressed in vivo in SCD mouse infected with the mouse-adapted EcoHIV. SCD mouse splenocytes expressed higher levels of FPN and TFR, and exhibited higher expression of antiviral factor, which were also observed in human SCD PBMCs. The observed upregulation of similar sets of antiviral factors in SCD PBCMs and SCD mice splenocytes suggests that these factors upregulation might be driven by the SCD-related processes such as hemolysis.
Taken together, our findings point to a novel mechanism of upregulation of antiviral factors mediated by SCD that included induction of antiviral, heme- and iron- regulatory pathways.
ACKNOWLEDGEMENTS: We thank Dr. David Volsky for the gift of EcoHIV and the George Washington University SPH Genomics Core for library preparation and sequencing. This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685.
Disclosures: Taylor: Baush Health: Other: expert witness; Pfizer: Other: Expert witness; University of Pittsburg: Research Funding; NHLBI, NIH: Research Funding; Forma: Other: Expert witness; ironwood: Other: Expert witness.
See more of: Oral and Poster Abstracts