Plasma-induced signatures reveal an extracellular milieu possessing an immunoregulatory bias in treatment-naive paediatric inflammatory bowel disease

Abstract

SummaryThe inflammatory state associated with Crohn's disease (CD) and ulcerative colitis (UC) remains incompletely defined. To understand more clearly the extracellular milieu associated with inflammatory bowel disease (IBD), we employed a bioassay whereby plasma of treatment naive paediatric IBD patients (n 5 22 CD, n 5 15 UC) and unrelated healthy controls (uHC, n 5 10) were used to induce transcriptional responses in a healthy leucocyte population. After culture, gene expression was measured comprehensively with microarrays and analysed. Relative to uHC, plasma of CD and UC patients induced distinct responses consisting, respectively, of 985 and 895 regulated transcripts [|log 2 ratio| ! 0Á5 (1Á4-fold); false discovery rates (FDR) 0Á01]. The CD:uHC and UC:uHC signatures shared a nonrandom, commonly regulated, intersection of 656 transcripts (v 2 5 P < 0Á001) and were highly correlative [Pearson's correlation coefficient 5 0Á96, 95% confidence interval (CI) 5 0.96, 0.97]. Despite sharing common genetic susceptibility loci, the IBD signature correlated negatively with that driven by plasma of type 1 diabetes (T1D) patients (Pearson's correlation coefficient 5 -0Á51). Ontological analyses revealed the presence of an immunoregulatory plasma milieu in IBD, as transcripts for cytokines/chemokines, receptors and signalling molecules consistent with immune activation were under-expressed relative to uHC and T1D plasma. Multiplex enzyme-linked immunosorbent assay (ELISA) and receptor blockade studies confirmed transforming growth factor (TGF)-b and interleukin (IL)-10 as contributors to the IBD signature. Analysis of CD patient signatures detected a subset of transcripts associated with responsiveness to 6-mercaptopurine treatment. Through plasma-induced signature analysis, we have defined a unique, partially TGF-b/IL-10-dependent immunoregulatory signature associated with IBD that may prove useful in predicting therapeutic responsiveness.

Identification of a serum-induced transcriptional signature associated with metastatic cervical cancer

“…However, miRNAs are very stable biomarkers and have been shown to vary little after long-term storage or with mode of storage, which is why we explored these serum factors further in this study [ 31 ]. Moreover, while we cannot easily test the effects of storage time on a single sample, we do know from prior studies done at our laboratory that samples collected a decade apart in patients with type 1 diabetes and stored at -80° Celsius produce highly correlative transcriptional signatures [ 4 , 5 ].…”

“…Gene expression was induced by culturing commercially cryopreserved PBMCs from a single healthy donor (UPN727; Cellular Technology Ltd, Shaker Heights, OH) for 9 h at 37°C in 5% CO 2 with media supplemented with sera of cancer patients from each of the 2 groups (local or metastatic), as previously described by Hessner et al [ 4 , 5 ]. The composition of the UPN727 cell by a standard flow panel was described in a previous publication and was found to be in the “normal range” [ 6 ].…”

“…The composition of the UPN727 cell by a standard flow panel was described in a previous publication and was found to be in the “normal range” [ 6 ]. The post-thaw viability of UPN727 is >90%, and multiparameter flow cytometry has shown that the relative abundance of NK cells, CD8+ T cells, CD4+ T cells, B cells, and monocytes does not differ significantly from the normal ranges expected for fresh PBMCs [ 5 ]. The cultures were prepared in a Costar 24-well plate (Corning, St Louis, MO), using 12 wells per condition (500,000 cells/well in 300 μl of RPMI 1640 medium plus 200 μl of patient-derived serum).…”

“…PBMCs contain a breadth of surface receptors due to their varied cell populations of T and B lymphocytes, monocytes, NK cells, and dendritic cells. This assay was previously shown to be sensitive and accurate for detecting inflammatory biomarkers that predict type 1 diabetes as well as characterizing inflammation associated with inflammatory bowel disease and disorders characterized by airway infection and inflammation [ 4 , 5 ]. Using sera from patients with squamous cell carcinoma (SCC) of the cervix, we identified a gene expression signature for metastatic cervical cancer.…”

Evaluation of Pathway Activation for a Single Sample Toward Inflammatory Bowel Disease Classification

“…We downloaded two pediatric datasets and three adult datasets from the Gene Expression Omnibus (GEO) (Edgar et al, 2002), namely GSE9686 (Carey et al, 2007), GSE3365 (Burczynski et al, 2006), GSE36807 (Montero-Meléndez et al, 2013), GSE71730 (Gurram et al, 2016), and GSE16879 (Arijs et al, 2009). All of them contain UC, DC, and normal samples.…”