Assessing Cellular and Transcriptional diversity of IIeal Mucosa amongst Treatment Naïve and Treated Crohn’s disease

Background and Aims Crohn’s disease is a life-long disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn’s. Methods Ileal biopsies were obtained from (i) controls (n=6), (ii) treatment-naïve (n=7), and (iii) established (n=14) Crohn’s patients along with remission (n=3) and refractory (n=11) treatment groups. The biopsies were processed using 10x Genomics single cell 5’ yielded 139,906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell-type proportions, differential expression analysis and gene ontology enrichment were carried out for each cell type. Results We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between ctrl, treatment-naive and established groups, with the significant changes in the epithelial subtypes of the treatment-naive patients, including microfold, tuft, goblet, enterocytes and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed, however gene expression in the epithelial and immune compartment was different between Crohn’s phenotypes indicating changes in cellular activity. Conclusions Our study identified cellular and transcriptional signatures associated with treatment-naive that collectively points to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity amongst patients within the same disease phenotype, shinning new light on personalized treatment responses and strategies. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by The Leona M. and Harry B. Helmsley Charitable Trust (G-2007-04028 and G-2204-05197), as well as the National Science Foundation (CCF2007029). P.Q. is an ISAC Marylou Ingram Scholar and a Wallace H. Coulter Distinguished Faculty Fellow. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Emory University gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors * (CD) : Crohn’s Disease (Es_CD) : Established CD (GEMs) : Gel Bead in Emulsion (GO) : Gene Ontology (GWAS) : Genome wide associations Studies (CyTOF) : Mass Cytometry (M cells) : Microfold cells (RPCA) : Reciprocal Principal Component Analysis (SESCD) : Simple Endoscopic Score for Crohn Disease (scRNA-seq) : Single cell RNA sequencing (TN_CD) : Treatment Naïve CD (UMAP) : Uniform Manifold Approximation and Projection