Background/Purpose: Epigenetic changes have been described in systemic lupus erythematosus (SLE) and offer a potential explanation for the chronicity of disease and missing heritability. Therapies targeting the epigenome will require a greater understanding of the cellular pathways regulating the epigenetic changes. We previously found a global increase in histone H4 acetylation (H4ac) in monocytes of SLE patients as compared to controls using tiling arrays. Transcription factor motif analysis found 63% of genes with increased H4ac had potential interferon regulatory factor (IRF) 1 binding sites within the 5kb upstream region. IRF1 is highly inducible by prolactin, a hormone implicated in the pathogenesis of SLE: hyperprolactinemia has been reported in 15-33% of SLE patients as compared to 0.4-3% of controls. Prolactin upregulation of IRF1 can lead to H4ac in Nb2 T cells, representing a potential pathological pathway in SLE. Prolactin-induced IRF1 activation in MonoMac-6 (MM6) cells was examined, with the aim to identify IRF1 interactions with specific histone acetyltransferases (HATs) and histone deacetylases (HDAC) leading to pathological H4ac in SLE.

Methods: Flow cytometry for acetylated H4 lysines: K5, K8, K12, K16 were run on the Accuri C6 with isotype controls. H4ac was defined in monocytes from 20 controls and 24 SLE patients, as well as in unstimulated and prolactin-stimulated MM6 cells. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) evaluated HAT/HDAC expression in monocytes from 10 SLE patients and 4 controls. IRF1 activation by prolactin in MM6 cells was studied by immunofluorescence microscopy and western blot analysis.

Results: Flow cytometry data found significantly increased H4K5, H4K8, H4K12, and H4K16 acetylation (p = 0.03, 0.01, 0.004, 0.02) in SLE monocytes compared to controls. H4K5 and H4K16 acetylation were also increased in MM6 cells stimulated by prolactin concentrations comparable to pregnancy (200 ng/mL) and prolactinoma levels (2000 ng/mL).     

Immunofluorescence microscopy showed translocation of IRF1 from the cytoplasm to the nucleus in prolactin-stimulated MM6 cells by 1 hr, demonstrating IRF1 activation. This was supported by western blots of MM6 nuclear extracts showing IRF1 increase at 1-2 hrs of prolactin stimulation.  

Potential mechanisms were examined. qRT-PCR studies of HAT/HDAC expression patterns found decreased HDAC3 and HDAC11 expression in SLE monocytes (p = 0.19 and 0.16). HDAC3 deacetylates all H4 lysine acetyl groups, preferentially H4K5 and H4K12. PCAF, a HAT that places H4K5, H4K8, and H4K16 acetylation marks, had significantly (p = 0.001) higher expression in SLE monocytes than in controls. IRF1 was overexpressed and H4ac at target genes defined by ChIP assays. Overexpression led to increased H4ac at known IRF1 targets.

Conclusion: These data demonstrate that prolactin stimulation of monocytes induces IRF1 activation and a pattern of acetylated H4 that corresponds to the changes seen in monocytes of SLE patients. This helps to explain the known association of prolactinomas with SLE. The identification of candidate HATs that associate with IRF1 in the context of prolactin stimulation may provide for potential therapeutic targets in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/prolactin-induced-interferon-regulatory-factor-1-activation-and-histone-h4-hyperacetylation-in-monomac-6-cells-correlating-with-changes-seen-in-monocytes-from-systemic-lupus-erythematosus-patients/