P0225 Histone Serotonylation (H3Q5ser): A Novel Epigenetic Signature in Crohn’s Disease Pathogenesis
Authors: Li J , Overberg K , Metselaar P , te Velde A , Löwenberg M , D'Haens G , Derikx J , de Jonge W , Ghiboub M 
Published: January 2025

Background

Serotonin, predominantly produced in the gut, regulates key processes like intestinal inflammation. Elevated serotonin levels in intestinal mucosa are linked to increased severity of Crohn’s disease (CD) (1). Despite existing serotonin-targeting drugs for CD, their efficacy is limited due to gaps in understanding serotonin's roles (2). Serotonin can covalently bind to glutamine at histone H3 position 5, forming histone serotonylation (H3Q5ser) in vitro in peripheral blood mononuclear cells (PBMCs) (3). The enzyme TGM2, crucial for this binding, is highly expressed in inflamed CD mucosa (4). Suggesting a potential H3Q5ser enrichment CD patients. Here, we aim to investigate the association between this novel epigenetic signature and CD pathogenesis.

Methods

To establish a causative link between serotonin and H3Q5ser in vitro set up, PBMCs isolated from buffy coats were treated with increasing concentrations of serotonin, followed by Western blot (WB) analysis using an H3Q5ser antibody. Serotonin and H3Q5ser levels in the mucosa of CD tissue and HCs (Non-CD) were assessed using immunofluorescence staining (IF) alongside with CD45 to mark immune cells. Flow cytometry (FACS) and WB were performed to assess H3Q5ser in PBMCs and compare its levels between CD patients (n=14) and HCs (n=7). FACS was also used to investigate the distribution of H3Q5ser in PBMC subtypes comparing active CD (n=5), CD in remission (n=9) and HCs. The percentage of H3Q5ser+ cells and mean fluorescence intensity was quantified in CD14+ monocytes, CD19+ B cells and CD3+ T cells.

Results

PBMCs treated with serotonin exhibited a dose-dependent increase in H3Q5ser. IF revealed elevated serotonin levels in CD mucosal tissues, particularly in intestinal immune and epithelial cells, with nuclear localization in some cells, suggesting a potential enrichment of H3Q5ser. H3Q5ser levels were higher in CD mucosa, with abundant H3Q5ser+CD45+ cells in inflamed mucosa. FACS and WB analyses showed high abundance of H3Q5ser in CD45+ in PBMCs, with CD14+ monocytes exhibiting significantly higher H3Q5ser levels than other PBMC subsets. The percentage of H3Q5ser+CD45+ and H3Q5ser+CD14+ cells was markedly higher in CD patients. CD patients, both active and quiescent disease, demonstrated elevated H3Q5ser levels compared to HCs.

Conclusion

These data suggest a causative link between serotonin and H3Q5ser levels, demonstrating its higher levels in the mucosa and PBMCs of CD patients. H3Q5ser was enriched in CD14+ monocytes, both in active and quiescent CD, suggesting its contribution to immune dysregulation in CD. Future studies are warranted to map the genomic locations of H3Q5ser and to investigate its impact on transcriptional regulation explore potential therapeutic targets for CD.

References

1-Ghiboub M et al. Sustained Diet-Induced Remission in Pediatric Crohn's Disease Is Associated With Kynurenine and Serotonin Pathways. Inflamm Bowel Dis. 2023 Jan 13:izac262. doi: 10.1093/ibd/izac262. Epub ahead of print. PMID: 36637175.

2-Hatamnejad MR, Baradaran Ghavami S, Shirvani M, Asghari Ahmadabad M, Shahrokh S, Farmani M, Sherkat G, Asadzadeh Aghdaei H, Zali MR. Selective serotonin reuptake inhibitors and inflammatory bowel disease; Beneficial or malpractice. Front Immunol. 2022 Oct 6;13:980189. doi: 10.3389/fimmu.2022.980189. PMID: 36275739; PMCID: PMC9583131.

3-Farrelly LA et al. Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature. 2019 Mar;567(7749):535-539. doi: 10.1038/s41586-019-1024-7. Epub 2019 Mar 13. PMID: 30867594; PMCID: PMC6557285.

4-Jeong EM, Son YH, Choi Y, Kim JH, Lee JH, Cho SY, Kim IG. Transglutaminase 2 is dispensable but required for the survival of mice in dextran sulfate sodium-induced colitis. Exp Mol Med. 2016 Nov 4;48(11):e267. doi: 10.1038/emm.2016.95. PMID: 27811936; PMCID: PMC5133373.