Abstract
Abstract Background Neutrophils are key players in intestinal damage in Crohn’s disease (CD) and ulcerative colitis (UC) but their immunological role in disease heterogeneity is largely overlooked. In both diseases, mature circulating neutrophils enter the intestinal tissue to clear invading bacteria but cause irreversible tissue damage. However, neutrophil function can be altered by pathogenic Th1/17 CD4+ memory T cell (Tm) responses even before the cells enter the tissue. We hypothesize that altered neutrophil function differs between, and within, CD and UC and that interpatient differences in pathogenic Tm responses change transcriptional profiles in circulating neutrophils, impacting their function in the inflamed mucosa. Methods We analyzed clinically well-characterized therapy-naive pediatric CD (n=33) and UC (n=12) patients and IBD-negative controls (IBDneg; n=11) from the PIBD-SETQ cohort and performed RNAseq on purified circulating neutrophils, neutrophil flow cytometry and plasma proteomics using Proximity Extension Assay technology. Results Comparison of gene expression profiles at diagnosis revealed 263 differentially expressed genes (DEG) in neutrophils from CD vs IBDneg, and 421 DEG in UC vs IBDneg, with 137 genes common to both comparisons and 137 DEG in CD vs UC. CD and UC neutrophil transcriptomes were enriched for granule biology pathways (MMP8, S100A8/9), while IFN-related pathways were only present in CD neutrophils. As granule proteins are expressed in neutrophils during maturation in bone marrow but not in circulation, we assessed the maturity of circulating neutrophils by flow cytometry. Neutrophils from UC, but not CD patients had phenotypic features of immaturity (lower surface expression of CD10, CD16 and CXCR2). As predicted, neutrophil gene expression profiles showed high interpatient heterogeneity within CD and UC. Within both CD and UC, hierarchical clustering on the basis of neutrophil DEG identified two patient clusters. In UC1 patients, neutrophils had higher expression of genes for granule proteins which related to higher plasma concentrations of IL-17A, CCL20 and MMP10 compared to UC2. In CD2 patients, neutrophils had increased gene expression for IFN-signaling and extracellular matrix re-organization relating to higher plasma IFNg, CDCP1 and CXCL9 compared to CD1. Conclusion We demonstrate that at time of diagnosis circulating neutrophils have acquired changes in transcriptional and maturity profiles that differ between and within CD and UC. In individual patients, transcriptomic changes correlate to pathogenic Th1/17 CD4+ Tm profiles. These changes may functionally alter neutrophil-mediated bacterial clearance and tissue damage in the inflamed mucosa and help decipher disease pathogenesis in individual patients.
Published Version
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