P126 Differential granulocyte populations as therapeutic targets in fistula

Abstract

Abstract Background Despite the recent increase in therapeutic options in IBD care, treatment of perianal disease and in particular fistula remains highly challenging. Specific targeted medical therapy is not currently available, and a majority of patients fails to respond to surgical intervention. In contrast, cryptoglandular fistula show much higher response rates with closure in up to 80-90% of patients. Interestingly, the most present cell type in fistula, the granulocyte, is also the least studied due to their fragile nature. To fill this void, we evaluated granulocytes in perianal fistula and those present in the blood. Methods Curettage material of 18 fistula (Crohn n=15 and cryptoglandular n=5) was obtained and processed by mass cytometry. Two additional samples were obtained (1 Crohn, 1 cryptoglandular) together with matching blood samples and assessed by single cell RNASeq. Results As expected, granulocytes formed the majority of all cells in all fistula (mean 64%). Cryptoglandular fistula displayed significantly more granulocytes than Crohn’s related fistula. Single cell analysis showed 4 subsets: Subset 1 was the main subset in blood, and expressed high levels of LAMTOR4. Interestingly, although found both in blood and fistula, abundance was much higher in blood (80 vs 10%) and signaling cascades differed. In blood, activity was associated with degranulation, IL8 activity and migration. In fistula, a very strong IFNa/b signature was present. Subset 2 expressed high MMP9, and showed degranulating activity. Subsets 3 and 4 were almost exclusively found in the fistula tracts, and were defined by expression of PI3 and LRG1 respectively. Pathway analysis showed the PI3+ population to have strong cytokine signaling but little degranulating or migratory activity. Conversely, the LRG1+ population showed little cytokine activity, but stronger degranulating activity and integrin signaling. The two populations also expressed distinctive chemokine receptors, with PI3+ cells expressing high levels of CXCR4, while LRG1+ express CXC1 and CXCR2, suggesting differntial recruiment and potential modulation. In that light, it is interesting to note the proportion of PI3+ and LRG1+ cells differed between patients, with the majority of Crohn derived cells being PI3+ while the cryptoglandular fistula mainly contained LRG1+ cells. Conclusion Granulocytes form the main cellular component of fistula tracts in both Crohn and cryptoglandular fistula. Four different subsets of granulocytes could be identified, of which the two main subtypes in fistula tracts appear to differ significantly both in phenotype, activity and method of recruitment. This data may be a starting point for specific interventions in this highly abundant but often overlooked cell type.