Properdin, transcortin and HGFAC are novel plasma biomarkers in canine chronic inflammatory enteropathies from active disease to remission

Abstract

Canine chronic inflammatory enteropathies (CCIE) is a group of intestinal inflammatory conditions causing chronic or relapsing gastrointestinal symptoms. Accurate diagnosis, treatment and monitoring remain challenging, necessitating novel diagnostic tools. This study aims to investigate the plasma proteome of ten dogs with histologically confirmed CCIE during active disease and clinical remission compared to ten healthy controls. We utilized surplus lithium-heparin plasma and performed label-free quantification mass spectrometry. A significant upregulation of complement factor properdin (CFP) during disease was noted, pointing toward microbial-driven intestinal inflammation. During remission, CFP levels remained elevated compared to controls, indicating persistent subclinical inflammation. We report hepatocyte growth factor activator (HGFAC) as a novel canine plasma protein associated with decreased risk for CCIE and as a potential therapeutic target, similarly, as reported in humans. Linear regression analysis revealed that disease severity was negatively correlated to transcortin/SERPINA6, as negative acute phase protein. Proteins involved in inflammation and tissue repair, such as inter-alpha-trypsin inhibitor heavy chain 4, (ITIH4), and anti-inflammatory molecules like apolipoprotein A-IV (APOA4), were significantly upregulated in remission. Conversely, proteins related to DNA remodeling and methylation, including histone H2B and carboxypeptidase N subunit 2 (CPN2), were downregulated during remission. Gene ontology analysis revealed altered pathways linked to immune response and coagulation. In CCIE patients we identified for the first time markers such as properdin for intestinal inflammation, while transcortin and HGFAC may serve as markers for remission. Future studies with larger cohorts are needed to validate the use of these proteins for monitoring disease progression and remission and refine their clinical applicability.

Fig. 1

Boxplot demonstrating the statistically significant difference in the canine chronic enteropathy activity index (CCECAI) of the dogs in the CCIE patients upon enrollment and after reaching clinical remission.

Fig. 2

Boxplots illustrating the statistically significant differences in the clinicopathological parameters of active disease (CCIE) and remission.

Fig. 3

Abundance rank of the identified canine plasma proteins amongst all samples (iBAQ). Different levels of abundances are represented by the labelled proteins. ALB = Albumin, APOA1 = Apolipoprotein 1, HP = Haptoglobin, HBB = Hemoglobin subunit beta, SERPIND1 = Plasminogen activator inhibitor-1, CP = Ceruloplasmin, SHBG = Sex hormone-binding globulin, H4C9 = H4 Clustered Histone 9, FCN2 = Ficolin 2, PKIB = CAMP-Dependent Protein Kinase Inhibitor Beta, ATRN = Attractin.

Fig. 4

Principal Component Analysis of the plasma proteome of patients with CCIE during active disease (CCIE) and clinical remission (Remission) compared to healthy controls (Control). There is no statistically significant separation between the three groups in terms of PCA Clustering.

Fig. 5

Volcano plots (− log p-values versus log2 fold-change) of the intensity of plasma proteins of from the three conditions in this study (A–C). Proteins with a minimum 2-fold intensity change compared to the control (log2 fold-change ≥ 1 or log2 fold-change ≤ − 1) and a q-value ≤ 0.05 were considered significantly abundant. Black dots represent non-significant differentially abundant proteins, red dots show the significantly higher abundance proteins in the CCIE group, green dots the significantly higher abundance proteins in the control group and the blue ones represent the significantly higher abundance proteins in the remission group.

Fig. 6

(A, B). (A) Gene Ontology enrichment analysis of the differentially abundant plasma proteins from dogs with CCIE during enrollment and after reaching clinical remission. Only genes that had a functional category described are represented. (B)The string network figure illustrates the interactions between various plasma proteins of the dogs with chronic enteropathies during both sampling points (enrollment and remission). The significant FDR-adjusted p-value was used as including criterion with regard of the expression level. Each node represents a unique protein, and the edges connecting nodes indicate known or predicted interactions. Node color indicates the type of interaction evidence, ranging from experimental data to curated databases and text mining.

Fig. 7

(A, B, C). Linear regression analysis of the log2 intensity of the glutamate dehydrogenase 1 (GLUD1) (A), transcortin (SERPINA6) (B), and hepatocyte growth factor activator (HGFAC) (C) and the canine chronic enteropathy activity index (CCECAI). There is a significant negative correlation between GLUD1, HGFAC and transcortin levels and disease severity reflected by the CCECAI, as GLUD1, HGFAC and SERPINA6 concentrations linearly decrease with increased disease severity.