Analysis of the interferon-γ-induced secretome of intestinal endothelial cells: putative impact on epithelial barrier dysfunction in IBD

Abstract

The development of inflammatory bowel diseases (IBD) involves the breakdown of two barriers: the epithelial barrier and the gut-vascular barrier (GVB). The destabilization of each barrier can promote initiation and progression of the disease. Interestingly, first evidence is available that both barriers are communicating through secreted factors that may accordingly serve as targets for therapeutic modulation of barrier functions. Interferon (IFN)-γ is among the major pathogenesis factors in IBD and can severely impair both barriers. In order to identify factors transmitting signals from the GVB to the epithelial cell barrier, we analyzed the secretome of IFN-γ-treated human intestinal endothelial cells (HIEC). To this goal, HIEC were isolated in high purity from normal colon tissues. HIEC were either untreated or stimulated with IFN-γ (10 U/mL). After 48 h, conditioned media (CM) were harvested and subjected to comparative hyper reaction monitoring mass spectrometry (HRM™ MS). In total, 1,084 human proteins were detected in the HIEC-CM. Among these, 43 proteins were present in significantly different concentrations between the CM of IFN-γ- and control-stimulated HIEC. Several of these proteins were also differentially expressed in various murine colitis models as compared to healthy animals supporting the relevance of these proteins secreted by inflammatory activated HIEC in the inter-barrier communication in IBD. The angiocrine pathogenic impact of these differentially secreted HIEC proteins on the epithelial cell barrier and their perspectives as targets to treat IBD by modulation of trans-barrier communication is discussed in detail.

Keywords: IBD-inflammatory bowel disease; angiocrine; barrier; cytokines; endothelial; interferon; paracrine; secretion.

FIGURE 1

The secretome of IFN-γ-treated human intestinal endothelial cells. (A) Cultivated human intestinal endothelial cells (HIEC) uniformly express the endothelial cell-specific CD31 antigen whereas the epithelial colorectal cancer cell line DLD1 is negative. (B) No difference in the cell phenotype is detected in untreated and IFN-γ-treated HIEC. (C) IFN-γ treatment (10 U/mL, 48 h) induces expression of GBP-1 in all HIEC cultures as determined by RT-qPCR. (D) Volcano blot of the secretome of IFN-γ-treated HIEC. Proteins present in significantly different concentrations in the cell culture supernatants of IFN-γ-treated and untreated HIEC are indicated in red. (E) Box blots showing differential secretion of the different factors in all HIEC cultures (n = 5) in response to IFN-γ. p-values were calculated with the one sample t-test (μ = 0) and were corrected for overall FDR using the q-value approach (Storey and Tibshirani, 2003). (F) IFN-γ treatment (10 U/mL, 48 h) induces secretion of GBP-1 in all HIEC cultures as determined by GBP-1-specific ELISA. (G) Expression of genes encoding the top ten secreted proteins from IFN-γ-treated HIEC in different experimentally induced murine colitis models. Expression relative to healthy control mice is indicated by color code. Numbers are representing adjusted p-values of statistical differences. (A,B) Scale bars correspond to 500 µm. (C,F) p values: *** = p <0.001, ** = p <0.01, and * = p <0.05, paired t-test.