Prolonged Kaposi's Sarcoma-associated Herpesvirus vIL-6 Exposure Enhances Inflammatory Responses by Epigenetic Reprogramming (SLAM-seq)

Kaposi sarcoma-associated herpesvirus (KSHV) inflammatory cytokine syndrome (KICS) is a newly described chronic inflammatory disease condition caused by KSHV infection and is characterized by high KSHV viral load and sustained elevations of serum KSHV-encoded IL-6 (vIL-6) and human IL-6 (hIL-6). KICS has significant immortality and possesses greater risks of having other complications, which include malignancies. Although prolonged inflammatory vIL-6 exposure by persistent KSHV infection is expected to have key roles in subsequent disease development, the biological effects of prolonged vIL-6 exposure remain elusive. Using thiol(SH)-Linked Alkylation for the Metabolic Sequencing (SLAM-seq) and Cleavage Under Target & Release Using Nuclease (CUT&RUN) analysis, we studied the effect of prolonged vIL-6 exposure in chromatin landscape and resulting cytokine production. The studies showed that prolonged vIL-6 exposure increased Bromodomain containing 4 (BRD4) and histone H3 lysine 27 acetylation (H3K27Ac) co-occupancies on chromatin, and the recruitment sites were frequently co-localized with poised RNA polymerase II with associated enzymes. Increased BRD4 recruitment on promoters was associated with increased and prolonged p65 (RelA) binding after the LPS stimulation. The p65 binding resulted in quicker and sustained transcription bursts from the promoters; this mechanism increased total amounts of hIL-6 and IL-10 in tissue culture. Pretreatment with the BRD4 inhibitor, OTX015, eliminated the enhanced inflammatory cytokine production. These findings suggest that persistent vIL-6 exposure may establish a chromatin landscape favorable for the reactivation of inflammatory responses in monocytes. This epigenetic memory may explain the greater risk of chronic inflammatory disease development in KSHV-infected individuals. Overall design: The goal of these studies was to utilize thiol(SH )-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) in order to define the direct transcriptional effects in response to cytokine stimulation. This was performed in the context of the parental THP-1 and vIL-6/THP-1 cell line models. Replicate cultures from each cell line were treated with either vehicle control, viral IL-6 (vIL-6), human IL-6 (hIL-6), transforming growth factor-B (TGF-B), the lipopolysaccharide (LPS). Additionally, sequential treatments of vIL-6 -> vIL-6 restimulation, vIL-6 -> hIL-6 stimulation, and vIL-6 -> TGF-B stimulation were performed. Subsequently, the cells were incubated for an additional 1 hour in the presence of 4-Thiouridine (s4U; 300 uM) in order to label nascent transcripts. Following treatment, the cells were harvested for isolation of total RNA (containing s4Uracil-labeled transcripts) and followed by library preparation and next-generation sequencing (NGS).