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P761 Gene expression differences between Crohn's disease aphthous ulcers and healthy Peyer's patches highlights potential treatment strategies

O'Brien C.*1,2, Patel H.3, Freeman T.4, Zhang K.5, Peng K.5, Pavli P.1,2

1Australian National University, Medical School, Canberra, ACT, Australia 2Canberra Hospital, Gastroenterology and Hepatology Unit, Canberra, ACT, Australia 3Australian National University, John Curtin School of Medical Research, Canberra, ACT, Australia 4University of Edinburgh, The Roslin Institute and Royal (Dick) School of Veterinary Studies, Edinburgh, United Kingdom 5Australian National University, Biological Research Facility, Canberra, ACT, Australia

Background

The earliest macroscopic lesion in Crohn's disease (CD) is the aphthous ulcer, which overlies Peyer's patches and lymphoid follicles. Our aim was to characterize differences in gene expression of aphthous ulcers and Peyer's patches.

Methods

Biopsies were obtained from the terminal ileum of 12 patients (6 with CD, 6 healthy controls). Aphthous ulcers and adjacent unaffected mucosa were obtained from CD patients, and Peyer's patches and adjacent mucosa from the controls. All patients were medication-free, except one. RNA was extracted using Qiagen kits. NextSeq 500 libraries were constructed using NextSeq 500/550 High output kits (Illumina) in a 150 bp paired-end format. Transcripts were assessed for quality using FASTQC, trimmed using Trimmomatic, and aligned to the human reference genome using subread mapper. Fragment counts were obtained using featureCount, and expression values normalized using the trimmed mean of M-values normalization method (TMM). Differential gene expression analyses were performed using generalized linear models in edgeR. Cell-specific gene expression was determined using ImSig.

Results

We obtained 36 million tags per sample, 87% were retained for downstream processing, and 93% of these mapped to the human genome. 685 genes were significantly differentially expressed between aphthous ulcers and Peyer's patches, all were upregulated in aphthous ulcers. Differential gene expression analysis revealed 34 pathways that were upregulated in aphthous ulcers relative to Peyer's patches. Receptors for the constant region of immunoglobulin (Ig) G were represented in 13 of these pathways. Expression of the high affinity FCGR1A (CD64), and low affinity FCGR3A (CD16), FCGR2A and FCGR2C (CD32), was significantly upregulated in aphthous ulcers (p=1.14E-12), but not the inhibitory FCGR2B (CD32B). Other pathways that were highly upregulated in aphthous ulcers included those involved in responding to bacteria, leukocyte chemotaxis, inflammatory response, and creation of C2 and C4 activators. ImSig, which is capable of using transcriptome data to indicate cell types and their activation state, revealed that core marker genes for plasma cells were overrepresented in aphthous ulcers relative to Peyer's patches and unaffected/normal mucosa.

Conclusion

The most significantly upregulated Fc gamma receptors in aphthous ulcers are the major receptors expressed on monocytes/macrophages, which are involved in regulating innate and adaptive immune responses, as well as plasma cell survival (FCGR2B), making them an attractive target for immunotherapy.