Date: Monday, November 6, 2017
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Epigenetic changes in systemic lupus erythematosus (SLE) offer a potential explanation for the chronicity of disease. We previously found that interferon regulatory factor-1(IRF1) binding sites were highly enriched in histone H4 acetylation (H4ac) peaks in SLE monocytes. IRF1 directly interacts with histone-modifying enzymes. IRF1 is a downstream immune response mediator that plays an important role in the interferon (IFN) pathway. IRF1 is highly inducible by prolactin (PRL), a hormone implicated in the pathogenesis of SLE. PRL is involved in immune response modulation through the JAK/STAT/IRF1 pathway. Our studies extend this by defining a pivotal role for PRL in induction of histone modifying enzymes that alter the balance of histone acetylation and IRF1 activation and downstream chromatin effects.
Methods: Primary monocytes and THP-1 cells were treated with recombinant human PRL. Flow cytometry for H4ac were run on the Accuri C6 with isotype controls. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) evaluated the effects of PRL stimulation on expression of HATs/HDACs and interferon-response genes. Western blot, immunoprecipitation and ChIP assays defined PRL effects on HAT/HDAC protein expression and interactions with IRF1. IRF1 knockdown experiments were performed using a lentivirus system.
Flow cytometry found significantly increased total H4ac, H4K5, H4K8, H4K12, and H4K16 acetylation in primary monocytes stimulated by PRL.
qRT-PCR studies of HAT/HDAC expression patterns showed increases in PCAF, CBP, P300, GCN5 and ATF2 expression in THP-1 cells stimulated by prolactin that was time-dependent.
Western blot assays found that nuclear levels of IRF1, PCAF, p300, CBP, ATF2, TIP60, MOF and HBO1 increased with PRL stimulation; HDAC3 decreased with PRL stimulation.
IRF1 co-immunoprecipitation assays found IRF1-binding with PCAF, P300, CBP and ATF2 in both unstimulated and stimulated states. IRF1 interactions with p300, CBP and ATF2 increased at 1-2 hrs of PRL stimulation. ChIP assays revealed increased IRF1 binding to PRL-response genes TGFb and ID1; increased H4ac was seen at the promoter of ID1 with PRL stimulation.
IRF1 knock-down experiments validated that PRL effects on HATs are IRF1-dependent. PRL-induced increases in the gene expression of PCAF, CBP, ATF2 and TIP60 were abrogated in the IRF1 knock-down cells as compared to controls.
PRL effects on the IFN signature genes were defined by qRT-PCR. CXCL10, IFIT1, IFIT3, B2M, OAS1 and CD40 expression levels were significantly increased by 4-hrs of PRL stimulation. Longer exposure to PRL stimulation also found further significant increases in the expression of IFIT1, IFIT3, CXCL10, MX1, B2M, CD40, OAS1, OASL and NOD2 after 2 wks.
Conclusion: Prolactin has long been associated with autoimmunity. These studies provide key insights into the mechanisms. These data demonstrate that prolactin stimulation induces IRF1 activation and a pattern of acetylated H4 that corresponds to the changes seen in SLE. We found dominant effects at the level of epigenetics, an area not previously explored with respect to prolactin effects in autoimmunity.
To cite this abstract in AMA style:Leung YT, Shi L, Maurer K, Song L, Sullivan KE. Prolactin Induces an Interferon Signature in Monocytes and Drives IRF1-HAT Interactions [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/prolactin-induces-an-interferon-signature-in-monocytes-and-drives-irf1-hat-interactions/. Accessed July 11, 2020.
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