Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Abstract

Background: The etiology of the inflammatory bowel diseases, including ulcerative colitis (UC), remains incompletely explained. We hypothesized that an analysis of the UC colon proteome could reveal novel insights into the disease etiology.

Methods: Mucosal colon biopsies were taken by endoscopy from noninflamed tissue of 10 patients with UC and 10 controls. The biopsies were either snap-frozen for protein analysis or prepared for histology. The protein content of the biopsies was characterized by high-throughput gel-free quantitative proteomics, and biopsy histology was analyzed by light microscopy and confocal microscopy.

Results: We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed.

Conclusions: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate immunity are all increased in abundance in the morphologically normal colon mucosa from patients with UC. The increased abundance of these antimicrobial compounds points to the stimulation of the innate immune system in the etiology of UC.

FIGURE 1

Biopsy histology analysis. A, Overview of the human small and large intestine. B, Cross section of a human colon. Representative H&E-stained colon biopsy slices from (C) control Ctrl_10 and (D) patient UC_05. The crypt architecture is in both slices preserved. However, an increased number of stained cell nuclei surrounding the crypts (red arrows) are apparent in the UC biopsies compared with the control biopsies (scale bars 100 μm).

FIGURE 2

Representative 2-dimensional plot of the liquid chromatography-mass spectrometry analysis of patient UC_9 illustrating the complexity of the analyzed peptide material. On the x-axis, the intensities of the peptide ion signals of the 19,549 acquired full MS scans are plotted, with a color gradient from white to green. The time of analysis is given on the y-axis. Each full MS scan was succeeded by up to 12 MS/MS scans of ions detected on the full MS scan and each cycle time was approximately 1 second; + indicates that the given ion signal was selected for MS/MS analysis, which came to 119,560 MS/MS scans in this 1 replicate.

FIGURE 3

Proteomics data verification. A, Representative scatter plot of the triplicate biopsy analysis of UC_1 of the 5711 quantified proteins. The log2 transformed protein abundances of all proteins in replicates 1, 2, and 3 are plotted against one another on the x- and y-axes, respectively. Ideally, the measurements should yield identical protein abundances, represented by a Pearson's correlation coefficients (R) of 1. B, PCA scores plot of principle components 1 and 2 of the protein abundances as measured in the UC replicates (+) and control replicates (□). Technical replicates (encircled) group together in all cases, demonstrating a high degree of technical repeatability of the method. The study cohort subject ID is given as numbers (e.g., red 3 at [+] equals UC_3) (Table 2).

FIGURE 4

Gene ontology characterization of the 5711 quantified proteins in terms of (A) biological processes performed by the proteins, (B) cellular component to which the proteins are associated, and (C) the primary molecular functions of the proteins.

FIGURE 5

Correlation analysis of inflammatory proteins. Colon inflammation grade scores and the protein abundance in the colon tissue of the inflammatory proteins lactotransferrin (●), S100-A8 (■), and S100-A9 (▲) (S100-A8 and S100-A9 protein constitute calprotectin).

FIGURE 6

Fold change of statistically significantly changing NET-associated proteins between biopsies from the patients with UC and controls. The 11 proteins are on average 42.2 times (P < 0.0005) more abundant in the biopsies from the UC group compared with the biopsies from the control group. Center line shows the median, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, crosses represent sample means, and the circle represents the fold change of lactotransferrin that lies outside the whiskers.

FIGURE 7

Confocal microscopic images of TO-PRO-3–stained biopsy slices (scale bars 100 μm): (A) Ctrl_5 and (B) UC_3. An increased number of stained cell nuclei surrounding the intestinal crypts are in general apparent in the UC biopsies compared with the control biopsies.

FIGURE 8

Confocal microscopy images of TO-PRO-3–stained biopsy slices from the UC group (scale bars 100 μm): (A) UC_6 with an intact neutrophil, with segmented multiobulated-shaped nucleus; (B) UC_4 with NET formation by excretion of the neutrophil DNA.