Two-dimensional electrophoresis of fecal protein samples of Galphai2^-/- mice, Galphai2^+/- mice, and wild-type littermates.

<p>Samples were run on gels with broad coverage of isoelectric points (3‒11) and molecular weights. A few spots of high intensity, that markedly differed from their heterozygous and wild-type littermates, predominated in the gels from Galphai2^-/- mice.</p

Immunoblots of peptidase D.

<p>A new set of fecal protein samples from Galphai2^-/- mice and healthy littermates were subjected to immunoblotting for detection of peptidase D. Equal amounts of fecal protein were loaded from five Galphai2^-/- mice at different stages of colitis. The first two lanes (from left) were from 4-week-old pre-symptomatic mice whereas lanes 3‒5 were from 7-, 10-, and 7-week-old mice with manifest colitis. Healthy mice were all 4 weeks old at the time of sample collection. The boxplot show densitometric quantitation of blots where peptidase D levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates. a.u. = arbitrary units.</p

Elevated fecal peptidase D at onset of colitis in Galphai2^-/- mice, a mouse model of IBD

<div><p>Background</p><p>The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms.</p><p>Methods</p><p>Fecal samples were collected at onset of inflammation in Galphai2^-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice.</p><p>Results</p><p>As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2^-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2^-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2^-/- mice at different stages of disease.</p><p>Conclusions</p><p>These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2.</p></div

Top fecal proteins that were identified to be upregulated or downregulated in feces of Galphai2^-/- mice.

<p>Top fecal proteins that were identified to be upregulated or downregulated in feces of Galphai2^-/- mice.</p

Subcellular distribution of proteins identified in feces from Galphai2^-/- mice and healthy mice.

<p>Subcellular distribution of proteins identified in feces from Galphai2^-/- mice and healthy mice.</p

Immunoblots of trypsin-2.

<p>Fecal protein samples from Galphai2^-/- mice, Galphai2^+/- mice, and wild-type littermates were subjected to immunoblotting for detection of trypsin-2. (A) Equal amounts of fecal protein were loaded from Galphai2^-/- mice with early signs of colitis, along with samples from Galphai2^+/- mice and wild-type littermates of similar ages. (B) Fecal protein samples from Galphai2^-/- mice at different stages of colitis showed a tendency towards higher trypsin levels with advanced disease. The boxplots show densitometric quantitation of blots where trypsin-2 levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates (A) or the pre-symptomatic Galphai2^-/- mice (B). a.u. = arbitrary units.</p

PAR2 immunoblots of homogenates from colonic and pancreatic tissue of Galphai2^-/- mice and healthy littermates.

<p>To examine the possible effect of increased amounts of luminal proteases on PAR2 levels, all Galphai2^-/- mice samples loaded on gels were from mice of different stages of clinically observable colitis (> 5 weeks). <b>A.</b> PAR2 in Galphai2^-/- and wild-type mice colonic homogenates. The band of slightly lower molecular weight was a cross-reacting protein of the anti-mouse secondary antibody and was excluded from the densitometric quantitation. <b>B.</b> PAR2 in Galphai2^-/- and wild-type mice pancreatic homogenates. Relative to Galphai2^-/- the PAR2 signal was weak in wild-type mice. The boxplots show densitometric quantitation of blots where PAR2 levels of Galphai2^-/- mice were depicted relative to the mean value of the healthy wild-type littermates. a.u. = arbitrary units.</p

Subphenotypes of inflammatory bowel disease are characterized by specific serum protein profiles

<div><p>Objective</p><p>Genetic and immunological data indicate that inflammatory bowel disease (IBD) are characterized by specific inflammatory protein profiles. However, the serum proteome of IBD is still to be defined. We aimed to characterize the inflammatory serum protein profiles of Crohn’s disease (CD) and ulcerative colitis (UC), using the novel proximity extension assay.</p><p>Methods</p><p>A panel of 91 inflammatory proteins were quantified in a discovery cohort of CD (n = 54), UC patients (n = 54), and healthy controls (HCs; n = 54). We performed univariate analyses by t-test, with false discovery rate correction. A sparse partial least-squares (sPLS) approach was used to identify additional discriminative proteins. The results were validated in a replication cohort.</p><p>Results</p><p>By univariate analysis, 17 proteins were identified with significantly different abundances in CD and HCs, and 12 when comparing UC and HCs. Additionally, 64 and 45 discriminant candidate proteins, respectively, were identified with the multivariate approach. Correspondingly, significant cross-validation error rates of 0.12 and 0.19 were observed in the discovery cohort. Only FGF-19 was identified from univariate comparisons of CD and UC, but 37 additional discriminant candidates were identified using the multivariate approach. The observed cross-validation error rate for CD <i>vs</i>. UC remained significant when restricting the analyses to patients in clinical remission. Using univariate comparisons, 16 of 17 CD-associated proteins and 8 of 12 UC-associated proteins were validated in the replication cohort. The area under the curve for CD and UC was 0.96 and 0.92, respectively, when the sPLS model from the discovery cohort was applied to the replication cohort.</p><p>Conclusions</p><p>By using the novel PEA method and a panel of inflammatory proteins, we identified proteins with significantly different quantities in CD patients and UC patients compared to HCs. Our data highlight the potential of the serum IBD proteome as a source for identification of future diagnostic biomarkers.</p></div

sPLS plot showing the distribution of patients with ileal CD, colonic CD, and UC in the discovery cohort, based on the 20 most important proteins from the multivariate analysis.

<p>sPLS plot showing the distribution of patients with ileal CD, colonic CD, and UC in the discovery cohort, based on the 20 most important proteins from the multivariate analysis.</p

Error rates of the sparse partial least-squares model for discrimination of subgroups generated from the discovery cohort, when applied to the replication cohort.

<p>Error rates of the sparse partial least-squares model for discrimination of subgroups generated from the discovery cohort, when applied to the replication cohort.</p