Background/Purpose: Ankylosing spondylitis (AS) is a complex genetic disease with strong association to the human leukocyte antigen (HLA)-B27. Several observations including frequent, occult histologic evidence for inflammation, clinical overlap with Crohn’s disease, and microbial dysbiosis point to the gut as a potential primary cause of spondyloarthritis. However, the role of HLA-B27 associated gut epithelial biology or host-microbe interaction is not clear in spondyloarthritis pathogenesis. While immortalized cell-lines transfected with HLA-B27 can be studied, they cannot mimic the complexity of the epithelial tissue. In addition, animal and cell line models cannot recapitulate the interindividual variability of the human population. To counter these issues, we have developed human colonic organoids and epithelial monolayers from colon biopsies of HLA-B27 positive and negative healthy individuals to study the effect of HLA-B27 on host-microbe interactions and epithelial cell biology.

Methods: To generate colonic organoids, frozen biopsies from healthy individuals undergoing elective colonoscopies were used. These biopsies were minced and the tissue digested on a shaker to release the crypts. The crypts were washed and resuspended in undiluted Cultrex reduced-growth-factor basement membrane extract. The organoids were expanded after ~1 week by mechanical disruption and splitting. After 2-5 passages, these organoids were frozen during exponential growth phase for optimal recovery. To generate epithelial monolayers, organoids were washed and mechanically disrupted and cells were plated over Matrigel matrix coated transwells to form monolayers.

Results: We have developed colonic organoids and epithelial monolayers from multiple HLA-B27 positive and negative individuals and are in the process of generating colonic organoids from AS patients. We have shown that organoids maintain their stemness after being frozen, as confirmed by the presence of spheroids (early-stage organoids), and their ability to differentiate into mature (lobular) organoids and epithelial monolayers. These organoids have been characterized for the presence of goblet cells, and experiments using various proliferation markers to confirm recapitulation of key aspects of in vivo sample are underway. Monolayers will be used to study HLA-B27 associated alteration of epithelial cell biology. In addition, organoids/monolayers will be cultured with either microbial products (e.g., lipopolysaccharide) or HLA-B27 associated microbes as determined in our previous studies, and the loss of barrier function will be analyzed by global gene expression, tight-junction proteins, and production of antimicrobial peptides.

Conclusion: To our knowledge, we are amongst the first groups to use human colonic organoids to study the role of intestinal epithelial cells in barrier function and host-microbe interaction in any rheumatic disease. Using organoids developed from HLA-B27 positive AS patients and healthy individuals and HLA-B27 negative controls, we will be able to functionally assay whether HLA-B27 is associated with altered barrier function and host-microbe interactions.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-novel-technology-to-study-the-role-of-intestinal-biology-in-spondyloarthritis-pathogenesis-human-colonic-organoids-and-epithelial-monolayers/