P-172 Despite an Overall Inflammatory Phenotype, IBD Patient Plasma Drives a Regulatory Signature in a Novel Transcriptional Bioassay

Abstract

Significant advances have been made in the understanding of the role of genetics and environmental factors involved in the pathogenesis of inflammatory bowel disease (IBD). But little is understood how these factors influence the immune system to collectively shape the inflammatory state. Factors such as chemokines, cytokines, and inflammatory lipids act as communicators for immune cells and are key elements in IBD pathogenesis. There is significant literature describing cytokine levels in IBD. These studies have yielded inconsistent results. The complex milieu of inflammatory mediators associated with autoimmune and chronic inflammatory diseases is often too dilute to directly measure in the periphery. Moreover cytokines and cytokines do not act in isolation. The disease phenotype is, in part, a consequence of the combinatorial effect of the many mediators produced by inflammatory and non-inflammatory cells. Our approach relies on the sensitivity of a well-controlled healthy peripheral blood mononuclear cell (PBMC) population to transcriptionally respond to mediators in patient serum or plasma during a co-culture. This response is comprehensively measured with a microarray. The purpose of this study is to evaluate the ability of this method in advancing our understanding of inflammatory processes in IBD. Serum was obtained from treatment-naive Crohn's disease (CD) and ulcerative colitis (UC) patients at Children's Hospital of Wisconsin. For controls, we obtained serum from unrelated healthy volunteers lacking a family history of autoimmune disease. We co-cultured highly characterized healthy control PBMC with patient or control serum. The transcriptional responses to the various mediators in the serum were measured using an Affymetrix GeneChip human genome U133 plus 2.0. Genes were considered differentially expressed if they met the following criteria- FDR < 1%, Fold change >1.4, ANOVA P < 0.05. Ontological analysis was performed using the DAVID annotation tool (http://david.abcc.ncifcrf.gov/). We included 37 IBD (22 CD & 15 UC) patients and 5 controls. We identified total of 1,224 genes that are differentially expressed between patient versus control serum. Of these 656 genes were common to both UC and CD; 329 were unique to CD and 239 were unique to UC. Of these 75% (919/1224) were down-regulated by patient compared to control serum. Top most (>10 fold) genes down-regulated by IBD patients serum compared to controls were IL-1α, IL-6, Gap junction protein β2, CXCL-3, IL-1β, IL-8, integrin β8 and TNF alpha-induced protein 6. Significantly regulated biologic pathways were related to cytokine-cytokine receptor interaction and included, NOD-like receptor signaling, TLR signaling, apoptosis and Jak-STAT signaling pathways. The Ingenuity Pathway Analysis identified IL-10 and TGFβ as significant upstream regulators. IBD patient serum induces distinct signature compared to healthy controls. Serum signature induced by CD patients differs from those induced by UC patient's serum. Despite possessing an overall inflammatory phenotype, the sera of IBD patients contains factors that predominantly suppress pro-inflammatory pathways in healthy cells. This result may indicate that IBD patients' inflammatory cells are inherently unresponsive to regulatory signals in vivo.