Breen‐Lyles, Maggie

Background Immortalized cell lines are conventionally used to examine bacterial virulence and host responses. These models lack heterogeneity, complexity and do not allow interrogation of host specific characteristics. Human intestinal organoids provide a model of human epithelium and allow determination of whether bacterial infections elicit host dependent responses. Objective Determine how gender and stress influence responses to Campylobacter infection using human colonic organoids. Methods Colonic biopsies were collected from 20 volunteers (10/sex), with Hospital Anxiety and Depression Score of <4 (low stress) or >14 (high stress). Crypts were isolated from biopsies. Monolayers were generated, experimentally infected with 3 different clinical isolates of C. jejuni (OD 600 1) and averaged. Bacterial virulence and transepithelial resistance (TER) was measured throughout infection. Proteomics was done 24h post‐infection, with analysis via 1D‐LC‐MS using an Orbitrap Eclipse. Data was processed with MS2 ID pipeline MyriMatch‐IDPicker and proteins FDR<0.1 considered significant. Results Colonoid cultures were generated from 75% of volunteers. C. jejuni strains attachmed and invaded all colonoids. Greater bacterial attachment was seen on high stress volunteer colonoids compared low stress (0.08 ± 0.11 vs. 0.03 ± 0.08 CFU/starting inoculum for males, 0.015 ± 0.036 vs. 0.014 ± 0.014 for females, p<0.05 for both) and on high stress males compared to females (p<0.001). Colonoids from high stress volunteers had a significant drop in TER 24h post‐infection compared to non‐infected controls (42.1 ± 30.7 vs 125.2 ± 62.4, % baseline, p<0.05). Proteomic analysis showed minimal differences between high and low stress or between female and male colonoids at baseline (4 and 9 proteins respectively). Upon infection 195 proteins were differentially abundant with 161 increased in stressed organoids (FDR<0.1). Increased apoptotic pathway activation and cellular reorganization; pro‐apoptotic death‐associated protein‐kinase 3 (DAPK3), and DNA damage response serine/threonine‐protein kinase protein (TAOK3) were seen. Gene set enrichment analysis (GSEA) showed increased interferon alpha and beta signaling, IL‐1 and IL‐17 responses and MHC1/2 antigen processing in high stress infected monolayers (FDR<0.1). No differences between male high and low stress organoids post infection. In contrast, 1289 proteins were differentially abundant between high and low stress females with 84% up in stressed organoids. IL‐1 and IL‐12 signaling was up in high stress female organoids (FDR<0.1). Conclusions Colonoid monolayers can be developed from human volunteers and used to evaluate host pathogen interactions. Colonoid cultures highlight variability in C. jejuni infectivity and host responses; with robust C. jejuni virulence seen with high stress male individuals and the greatest change in proteomic profile in response to infection in high stress females. These findings demonstrate utility of colonoids and provide a platform to study personalized host responses to infection.