Defense Date

6-29-2023

Graduation Date

Summer 8-5-2023

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

Committee Chair

Philip E. Auron

Committee Member

Jill A. Dembowski

Committee Member

Jan E. Janecka

Committee Member

Deborah L. Galson

Keywords

Spi1, PU.1, HIF1, HIF2, EPAS1, Innate immune memory, Hypoxia, inflammation, transcription, TAF

Abstract

Interleukin 1β (IL-1β), an important cytokine in both health and disease, functions as a major proinflammatory mediator of pathological challenges, including involvement in the cytokine storm associated with Macrophage Activation Syndrome, Hemophagocytic Lymphohistiocytosis and the severe secondary effects of SARS-CoV-2 infection. Therapeutic approaches to limiting the effects of IL-1β protein have focused either on specific protein neutralization before release from activated macrophages (by general metabolic inhibitors and inflammasome inhibition) or after release by ligand and receptor obstruction (e.g., Canakinumab, Anakinra and Rilonacept). Although these approaches are efficacious, they inhibit steps after gene expression. Since a single gene expresses thousands of molecules, direct transcription inhibition would have greater effectiveness. Unfortunately, most genes use universal mechanisms, with inhibition resulting in pleiotropic effects. Our investigation of IL1B gene induction has demonstrated unique molecular transcription mechanisms, generating a form of innate immune memory with LPS intolerance, suggesting the potential for selective gene drug-ability. We previously reported that LPS/TLR4 initiates hypoxia in mice, with HIF-1a binding the murine Il1b gene, suggesting that TLR4 directly activates Trained Innate Immune Memory (TIIM)1. To address whether human monocyte IL1B gene regulation parallels that of mouse, mRNA and chromatin immunoprecipitation (ChIP) kinetics, with small molecule inhibitors under either normoxia or hypoxia, were evaluated. IL1B transcription kinetics in Lipopolysaccharide (LPS)-treated human THP-1 monocytes revealed an Immediate-early (IE) phase of IL1B gene induction, requiring Spi1, NF-κB and C/EBPβ transcription factors (TF), followed by a late-phase of lower-level continuous non-tolerized TIIM expression that depends upon hypoxia-inducible TFs. We observed that Spi1 is required for IL1B gene transcription, performing a critical role in inducing the promoter for IE expression via a TAF1-independent mechanism. The unique promoter structure of IL1B and other IE genes we identify, have poorly conserved core promoter motifs, instead having prebound Spi1, which replaces TAF1. Late HIF-2a-dependent IL1B expression is due to LPS pseudo-hypoxic stabilization of the labile HIF factor. In contrast to IE activation, late-phase transcription revealed minimal dependence on NF‑κB due to human-specific competition for an overlapping NF‑κB/HIF enhancer element. Our studies reveal that IL1B in naïve THP-1 human monocytes binds HIF2a, not HIF1a, while PMA treatment morphologically polarizes THP-1 macrophages to bind both HIF isoforms at IL1B upon PMA+LPS treatment.

Language

English

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