Background: The polymorphic form of ATG16L1 autophagy protein (T300A; rs2241880) impairs clearance of intracellular pathogens and increases the risk of Crohn’s disease (CD). How defective autophagy affects intestinal epithelial subtypes and their response to cytokine and bacterial ligands is not fully understood. We have applied a novel patient-derived organoid (PDO) model combined with multiplexed single-cell measurement of cell-type, cell-state and protein signalling to investigate epithelial cell responses in response to TNF-α and lipopolysaccharide (LPS) stimulation.

Hypothesis: The polymorphism ATG16L1 T300A, associated with CD, exhibits distinct effects on various intestinal epithelial subtypes following innate immune stimulation.

Methods: Endoscopic pinch biopsy-derived stem cells from adult CD (n=2), ulcerative colitis (UC) (n=4) and healthy control (HC) (n=1) patients, with and without ATG16L1 T300A homozygosity carriage, were used to generate normal (AG) and autophagy-defective (GG) PDOs. PDO phenotypes in expansion and differentiation conditions were validated by immunofluorescence, quantitative polymerase chain reaction and immunoblot. PDOs were stimulated with or without TNF-α and bacterial LPS and fixed in 96-well format using a novel application of thiol-reactive barcoding in situ (TOBis) and multiplexed mass cytometry (MC) [1] to measure 1.2 million cells across 7 PDO lines for 23 post-translational modification (PTM) signals, cell subtypes (enterocyte, Paneth, stem, goblet, enteroendocrine) and cell state (apoptotic, G0, G1, S, G2, M). High-dimensional biomarker data was quantified by the Earth Mover’s Distance (EMD) metric and single-cell data visualised by the dimensionality-reduction method PHATE [2].

Results: PDOs generated from pinch biopsies were shown to be successfully analysed at the single-cell level using TOBis/MC. Of the 7 PDOs, GG UC PDOs exhibited an increased autophagy (SQSTM1) and inflammatory (NF-κB) response upon stimulation, as compared to AG UC, CD and HC (Figure 1). Single-cell analysis of PDOs indicated a tendency towards increased SQSTM1 expression in the Paneth cells of GG PDOs compared to AG PDOs, although this remained highly conserved (Figure 2).

Conclusions: The novel application of a multiplexed single-cell protein signalling assay to an IBD and HC PDO model is able to assess cell subtype-specific signalling responses under ligand stimulation. Further work will be focused on optimising signalling intensity to better understand the effect of immune cell or bacterial co-culture on the PDOs.

References: [1] Sufi, J et al. Nat Protoc. 2021 Oct;16(10):4897-4918. doi: 10.1038/s41596-021-00603-4. [2] Moon, KR et al. Nat Biotechnol. 2019 Dec;37(12):1482-1492. doi: 10.1038/s41587-019-0336-3.

Charles Murray

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