Abstract
Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.
Highlights
Sepsis is caused by a deregulated host immune response to an infection and a major cause of death in hospitalized patients [1, 2]
Human monocytic THP1 cells with stable overexpression of MD2, CD14 and a nuclear factor-kB (NF-kB) secreted embryonic alkaline phosphatase (SEAP) reporter construct (THP1MD2-CD14 cells) and primary human monocytes harvested from healthy donors responded with an upregulation of HIVEP1 mRNA upon stimulation with PAM3CSK4 (TLR2 ligand) or LPS (TLR4 ligand) (Figures 1C, D)
These data indicate that human monocytes increase HIVEP1 mRNA upon exposure to bacterial agonists in a NF-kB dependent manner and that THP1-MD2-CD14 cells are a suitable model to study the functional role of HIVEP1
Summary
Sepsis is caused by a deregulated host immune response to an infection and a major cause of death in hospitalized patients [1, 2]. HIVEP1 was originally isolated from a human B cell cDNA expression library that was screened for proteins binding to a DNA fragment derived from the MHC class I gene enhancer sequence 5’-TGGGGATTCCCA-3’ [10]. This ĸB motif is present in the enhancer elements of other mammalian promoters, such as those of the IL-2 receptor and IFNB1 genes, as well as in viral promoters, including those of human immunodeficiency virus (HIV)-1 and cytomegalovirus [9]. The primary objective of the current study was to investigate the functional role of HIVEP1 in the response of monocytes to bacterial agonists in vitro and during experimental sepsis in vivo
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