Citrullination of CAMP exacerbating mucosal inflammation in inflammatory bowel disease

Abstract

Background: Cathelicidin (CAMP), plays important roles in pathogen defense, immune regulation, and epithelial barrier maintenance. While previous studies have highlighted its protective function, the post-translational modifications and downstream immune-metabolic effects of CAMP in the pathogenesis of inflammatory bowel disease remain unclear.

Methods: A dextran sodium sulfate (DSS)-induced colitis mouse model was employed to assess the role of CAMP and its citrullination mediated by peptidyl arginine deiminase 4 (PAD4). Proteomic and metaproteomic analyses were performed to investigate microbiota composition and functional shifts. We generated gene-deficient mouse models, CAMP knockout (KO) and PAD4-KO mice, to dissect molecular mechanisms. Epithelial integrity, inflammatory markers, and immune responses have been evaluated at both the protein and mRNA levels. Bone marrow-derived dendritic cells and primary CD4⁺ T cells were co-cultured to examine the effects of CAMP-related metabolites on antigen presentation and Th17 differentiation. Furthermore, we evaluated the impact of CAMP peptide supplementation and the effects of CAMP-KO mice on DSS-induced colitis.

Results: CAMP citrullination was significantly elevated in DSS-induced colitis mice but restored by PAD4 deletion. Citrullination was found to reduce CAMP protein levels without affecting its transcriptional expression. The absence of CAMP exacerbated intestinal inflammation in DSS-treated mice. Metaproteomic analysis identified 70 differentially expressed proteins and 15 altered microbiota families associated with CAMP deficiency. Elevated levels of arginase-1 and its metabolites, particularly polyamines, enhanced dendritic cell maturation and increased Th17 polarization in CAMP-KO mice.

Conclusions: Our findings highlight that the protein level of CAMP decreased after PAD4-mediated citrullination, thus playing a vital role in regulating taxonomic community structure, restricting arginine metabolism, and regulating dendritic cell-Th17 immune responses in IBD.

Keywords

cathelicidin / peptidyl arginine deiminase 4 / citrulliantion / microbiota / inflammatory bowel disease

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant Nos. 82100587 and 82400605), the China National Postdoctoral Program for Innovative Talents (grant No. BX20220288), the China Postdoctoral Science Foundation (grant No. 2022M720138), the Clinical Research Special Project of Shanghai Municipal Health Commission (grant No. 20244Y0209), Capability Enhancement Project for Clinical Research Physician of the First Affiliated Hospital of Naval Medical University (grant No. 2024LYC01), the Basic Medical Research Project of the First Affiliated Hospital of Naval Medical University (grant No. 2023PY06), the Basic Medical Research Projects of Naval Medical University (grant Nos. 2024MS08 and 2024QN026), the “Yuanhang” Talent Program of Naval Medical University, the “Changjian” Talent Program of Changhai Hospital of Naval Medical University, the “Changying” Talent Program of Changhai Hospital of Naval Medical University and Youth Project of Shanghai Key Laboratory (grant Nos. 2025QN01 and 2025QN09).

Author contributions

X.C., S.W., Y.B., and Z.L. conceived and designed the research and supervised the studies. X.C., H.W., Y.S., F.H., Y.Z., H.S., and Y.J. performed the experiments, conducted the statistical analyses and interpreted the data. X.C. and H.W. wrote the manuscript. S.W., T.X., and Y.B. revised the manuscript. All authors read and approved the final manuscript.

Supplementary data

Supplementary data is available at PCMEDI Journal online.

Conflict of interest

All the authors declare that they have no conflict of interest.

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Changhai Hospital (Approval No. CHEC [A.E]2025-064). Animal experiment protocols complied with the Guide for the Care and Use of Laboratory Animals. Written informed consent to participate was obtained from all participants prior to inclusion in the study.

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