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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE233119 | GEO | 2023/08/08

REPOSITORIES: GEO

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Project description:Background: Epigenetic mechanisms are implicated in chronic inflammatory pathogenesis. Bromodomain Extra-Terminal (BET) proteins are druggable epi-reader proteins which bind acetylated histones and transcription factors regulating gene expression, including inflammatory genes. Objectives: To evaluate the role of specific histone acetylation events and BET protein interactions in promoting keratinocyte inflammatory and transcriptional responses, to determine the effects of pharmacological inhibition of BET proteins and thereby test the hypothesis that BET proteins represent an effective therapeutic target to treat cutaneous inflammation. Methods: Primary human keratinocytes were stimulated with tumour necrosis factor alpha and interleukin 17 (TNFalpha + IL-17), +/- BET inhibitor (I-BET151). Cytokine induced interleukin 6 and 8 (IL-6 /-8) responses were determined by qPCR and ELISA. Chromatin immunoprecipitation (ChIP) assays were undertaken to characterise epigenetic changes at the IL-6 and IL-8 gene promoters, while the effect of I-BET151 treatment on keratinocyte transcriptional response was determined by Illumina array. Results: TNFalpha + IL-17 induced gene-specific epigenetic changes, including: histone hyperacetylation, recruitment of BET proteins (BRD2/3/4) to the promoter regions of IL-6/-8 and activation of RNA polymerase II (PolII(S2P)), correlating with increased IL-6/-8 expression. I-BET151 reduced recruitment of BRD4 and activation of PolII(S2P), at the IL-6 promoter, and BRD3/4 and PolII(S2P), at the IL-8 promoter; which is consistent with I-BET151 inhibitory effects on TNFalpha + IL-17-induced IL-6/-8 expression. A ~10-fold enrichment of BRD4/p65 was observed at the IL-6 promoter compared to the IL-8 promoter. Transcriptomics analysis showed that I-BET151 modulated expression of genes involved in cell cycle, cell proliferation and the innate immune response in keratinocytes stimulated with TNFalpha + IL-17. Conclusions: In human keratinocytes, disease-relevant stimuli induced dynamic epigenetic changes at inflammatory gene loci. BET proteins played a critical role in keratinocyte innate immune responses and displayed differential effects; for example BRD4/p65 was particularly enriched at the IL-6 promoter, indicating that BRD4 may be a critical factor in integrating inflammatory stimuli via NF-B in chronic skin inflammation. Inhibition of these responses by I-BET151 highlights BET proteins as potential therapeutic targets in inflammatory skin disease.

Project description:The hallmark of Kaposi’s sarcoma (KS), the most common cancer in HIV-infected patients caused by Kaposi’s sarcoma-associated herpesvirus (KSHV) infection, is hyperinflammation. However, the role of inflammation and the mechanism of induction of inflammation in KSHV-induced cancer remain unclear. In a previous high-throughput screening against KSHV-induced oncogenesis, over half of the identified candidates were anti-inflammatory agents. Among them, dexamethasone, a common anti-inflammatory corticosteroid, functions through binding to and activating glucocorticoid receptor (GR). In this study, we examined the mechanism mediating KSHV-induced inflammation. We found that numerous inflammatory pathways were activated in KSHV-transformed cells. Particularly, the interleukin-1 alpha (IL-1α) and IL-1 receptor antagonist (IL-1Ra) from the IL-1 family, implicated in a wide variety of inflammatory diseases, were the most induced and suppressed cytokines in KSHV-transformed KMM cells compared to the matched primary MM cells, respectively. By using reverse genetics, we showed that KSHV-encoded miRNAs mediated IL-1α induction while both miRNAs and vFLIP mediated IL-1Ra suppression. Furthermore, the GR signaling was inhibited in KSHV-transformed cells, which was mediated by vFLIP and vCyclin. Importantly, dexamethasone treatment inhibited cell proliferation, and colony formation in softagar of KMM cells but had a minimal effect on MM cells. Consequently, dexamethasone suppressed the initiation and growth of KS-like KMM tumors in mice. Mechanistically, dexamethasone suppressed IL-1α expression but induced IL-1Ra expression. Treatment with recombinant IL-1α protein was sufficient to rescue the inhibitory effect of dexamethasone while overexpression of IL-1Ra alone caused a weak growth inhibition of KMM cells. Furthermore, dexamethasone induced IκBα expression resulting in the inhibition of the classical and alternative NF-kB pathways and IL-1α expression. Taken together, these results revealed the important role of IL-1 signaling pathway in KSHV-induced inflammation and oncogenesis, which can be inhibited by dexamethasone-activated GR signaling. This study determined the mechanism of inflammation in KSHV-induced oncogenesis and identified IL-1-mediated inflammation as a potential therapeutic target for KSHV-induced malignancies.

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

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