J Proteomics. 2015 Jan 1;112:166-79. doi: 10.1016/j.jprot.2014.09.002.

Longitudinal study of circulating protein biomarkers in inflammatory bowel disease

Emilie Viennois*1,2,3, Mark T. Baker1,2, Bo Xiao1,2, Lixin Wang1,2,3, Hamed Laroui1,2 and Didier Merlin1,2,3

1 Institute for Biomedical Sciences, 2 Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, USA

3 Veterans Affairs Medical Center, Decatur, GA, USA

*email address: eviennois@gsu.edu



Inflammatory bowel diseases (IBD) are chronic and progressive inflammatory disorders of the gastrointestinal tract. In IBD, protein serological biomarkers could be relevant tools for assessing disease activity, performing early-stage diagnosis and managing the treatment. Using the interleukin-10 knockout (IL-10-/-) mouse, a model that develops a time-dependent IBD-like disorder that predominates in the colon; we performed longitudinal studies of circulating protein biomarkers in IBD. Circulating protein profiles in serum samples collected from 30-, 93-, and 135-day-old IL-10-/- mice were investigated using two-dimensional differential gel electrophoresis and MALDI TOF/TOF tandem mass spectrometry. A total of 15 different proteins were identified and confirmed by ELISA and Western blot to be differentially accumulated in serum samples from mid- to late-stage IL-10-/- mice compared to early non-inflamed IL-10-/- mice. The use of another model of colitis and an extra-intestinal inflammation model validated this biomarker panel and demonstrated that comprised some global inflammatory markers, some intestinal inflammation-specific markers and some chronic intestinal inflammation markers. Statistical analyses using misclassification error rate charts validated the use of these identified proteins as powerful biomarkers of colitis. Unlike standard biomarker screening studies, our analyses identified a panel of proteins that allowed the definition of protein signatures that reflect colitis status.

KEYWORDS: 2D-DIGE; Diagnostics; IBD; Serological biomarkers; Therapeutics

PMID: 25230104



Inflammatory bowel diseases (IBD), principally comprising ulcerative colitis (UC) and Crohn’s disease (CD), are a group of chronic inflammatory disorders of the gastrointestinal tract. Crohn’s and Colitic Foundation of America (CCFA) reports that 1.6 million Americans suffer from IBD with 70,000 new cases diagnosed every year [1]. In Europe a recent study reported a combined estimate of about 250,000 new cases each year for a total of nearly 4 million people affected [2]. While clinical diagnosis of IBD is generally achieved through colonoscopy, longstanding effort in developing serologic markers are in progress to facilitate early diagnosis, differentiate disease subtype, and provide reliable indicators of disease activity. These markers include levels of antibodies directed toward specific microbial products, such as anti-OmpC antibody, as well as more general inflammatory markers such as serum C-reactive protein (CRP) and calprotectin [3, 4]. The formers are helpful in confirming diagnosis but are not specific to the intestine and reflect any inflammatory condition, thus limiting their value as an early marker of IBD [5, 6]. Consequently, there remained an urgent need for sensitive and specific serum markers for diagnosis of IBD, predicting its development, indicating subtype, and monitoring disease activity. The concept driving our study was that deregulated proteins identified in serum could be incorporated as part of a panel to yield a biomarker signature capable of discriminating between healthy and colitis, as well as between different stages of colitis. Biomarkers were identified using well-defined tractable colitis models in which the timing of disease development and its underlying causes are well characterized. In a longitudinal study, the well-established IL-10-/- mouse model of IBD was employed in conjunction with a proteomic approach, two-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry, to identify mouse serum protein biomarkers that could facilitate the prediction and monitoring of the degree of intestinal inflammation during IBD progression. In serum samples from mid- (day 93) to late-stage (day 135) colitis IL-10-/- mice, we identified 15 proteins (Haptoglobin (HP), Hemopexin (HPX), Kininogen 1, Serpin peptidase inhibitor, clade A, member 1, Transferrin, Thrombospondin 1, Collagen, Type I, Alpha 1 (COL1A1), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Pregnancy Zone protein (PZP), Peroxiredoxin 2 (PRDX2), alpha-1-B glycoprotein (A1BG), Hemoglobin, alpha 1, Contrapsin, histocompatibility 2, Q region locus 10 and Complement component 3 (C3)) with altered accumulation levels compared to non colitic IL10-/- mice (day 30). Another model of intestinal inflammation (DSS-induced colitis) and an extra intestinal inflammation model (arthritis) were used to validate respectively the colitis subtype and the intestinal specificities of the newly identified panel. Among the previously identified proteins, some were specific for intestinal inflammation development (PZP, COL1A1, PRDX2), the IL10−/− model (PZP, PRDX2), development of arthritis (Serum amyloid P-component and Transthyretin) or displayed altered expression under all inflammatory conditions (HP, ITIH4, HPX, C3 and A1BG) (Figure 1).


Figure 1


In an effort to statistically define a protein signature effective in distinguishing non-colitis mice from their mild and severe colitis counterparts, we analyzed the differences in protein accumulation profiles between the sera of severe and mild colitis versus non-colitis mice. To this end, we analyzed the six proteins that were deregulated on both days 135 and 93 (HP, A1BG, PZP, HBA1, SERPINA1 and COL1A1). A heatmap representation of relative protein abundance is shown in Figure 2. The data demonstrate a separate clustering for mild colitis (Day 93), severe colitis (Day 135), and non-colitis mice. The mild colitis profile was intermediate between non-colitis and severe colitis groups.

The power and innovation of our study lies in the use of a combination of several biomarkers to define a protein signature for IBD. Data obtained with two other models of inflammation (DSS and Collagen Antibody-induced arthritis) showed that the biomarker panel identified for IBD comprises global inflammatory markers, intestinal inflammation-specific markers and chronic intestinal inflammation markers. This combination of specific and non-specific markers serves as a unique signature for IBD. Compared to the biomarkers currently used in the clinic, biomarker panels offer increased sensitivity and specificity of IBD detection.





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