Circulating S100A8/A9 is potentially a biomarker that could reflect the severity of experimental colitis in rats

Abstract

Aims: The clinical significance of circulating S100A8/A9 (calprotectin) in patients with ulcerative colitis (UC) is poorly understood. We examined whether serum S100A8/A9 is a good biomarker for UC, and whether the serum level is a useful index for the severity of the disease.

Main methods: Experimental animal (rats) were used to verify clinical significance of serum S100A8/A9 as a biomarker. Rats treated with 5% dextran sulfate sodium (DSS) alone (UCR) or with 5%DSS plus tacrolimus (TMR) were subjected to the experiment. The serum concentrations of rat S100A8/A9 (r-S100A8/A9) and other inflammatory biomarkers, such as C-reactive protein (CRP) and inflammatory cytokines, in the both groups were measured using enzyme-linked immunosorbent assays (ELISAs). The tissue damage in the large intestinal tract was visualized by hematoxylin-eosin staining. The relationship between the serum concetrations of these inflammatory biomarkers and the histological scores of the rectal tissue was statistically analyzed.

Principle findings: As determined by the ELISAs, the serum concentration of r-S100A8/A9 in the UCR hardly correlated with those of not only CRP but also some inflammatory cytokines. The deterioration of the rectal tissue, mainly epithelium structure of a large intestine, in the UCR was clearly observed, but was not so severe as that in the TMR. The histological scores of the rectal tissue in the UCR significantly correlated with the serum level of r-S100A8/A9, but not with other inflammatory biomarkers. Furthermore, macrophages actively produced r-S100A8/A9 in response to stimulation with lipopolysaccharide and quickly secreted it in circulation. Therefore, the serum level of r-S100A8/A9 suggestively changes in accordance with the severity of experimental UC.

Conclusion: Circulating r-S100A8/A9 is a useful biomarker for experimental UC, and its serum level correlates with the disease severity as judged by histological score.

Keywords: Biochemistry; Biological sciences; Biomarker; C-reactive protein; Cell biology; Gastrointestinal system; Immunology; Inflammatory cytokines; Molecular biology; Physiology; Proteins; S100A8/A9; Ulcerative colitis.

Figure 1

Protocol of animal experiments. Wistar rats (male, 10-week-old, 220–250 g/rat) were treated with 5% DSS alone (UCR) and 5% DSS plus tacrolimus (TMR) during the experimental period, in which tacrolimus (3 mg/rat/day) was orally administrated to the TMR every day as indicated by vertical arrows. Blood samples were drawn from rats in the UCR and TMR. Five rats were sacrificed (n = 5 per day) every two days as indicated by vertical arrows.

Figure 2

Changes in the body weight and the length of a large colon of rats. The body weight was measured every day. The length of a large colon of the UCR and TMR were measured every two days for the period as indicated by vertical arrows. In A, the y-axis indicates the percentage of decrease in the body weight of the UCR and TMR after the start of experiment. In B, the y-axis shows the length of a large colon of rats in both the two groups after their sacrifice. In A and B, the x-axis indicates the number of days after the start of experiment. Data are the mean ± standard deviation (±SD) (see Methods). ∗P < 0.05.

Figure 3

Changes in the serum levels of r-S100A8/A9, CRP, and inflammatory cytokines in the rats treated with 5% DSS alone or 5% DSS plus tacrolimus. The concentrations of r-S100A8/A9, CRP, TNF-α, IL-6, and IL-1β in the serum of the UCR and TMR were measured by ELISAs for each protein according to manufacturer's instructions. In A, the y-axis indicates the absorbance at 490 nm on behalf of the concentration of r-S100A8/A9 in the serum of rats because it was difficult to prepare the heterodimer as a standard. In B, the y-axis indicates the serum concentration of CRP (μg/L) in the UCR and TMR. In C, D, and E, the y-axis indicates the concentrations of TNF-α, IL-6, and IL-1β (pg/mL) in the serum of rats, respectively. In A to E, the x-axis indicates the number of days after the start of experiment. Data are the mean ± standard deviation (±SD) (see Methods). ∗P < 0.05.

Figure 4

Histological evaluation of large colons of rats in the UCR and TMR by H&E staining. H&E staining was performed to observe the histological changes in the rectal tissues of the UCR and TMR. Panels A and B show the microscopic images of the rectal tissues in the UCR and TMR, respectively. Panels A (A1-A4) and B (B1–B4) indicate the results of H&E staining of the rectal tissues in the UCR and TMR on each day 4, 6, 8, and 10 after the start of experiment. All microscopic images were observed by BIOREVO BZ-9000 (KEYENCE Co. Ltd., Osaka, Japan). Magnification of all panels is high power field (× 400). In C and D, HS was performed using the rectal tissues of the UCR and TMR, respectively, based on HS criteria (Table 1). Data are the mean ± standard deviation (±SD) (see Methods).

Figure 5

The correlation between histological scores of the rectal tissues of rats and the serum concentrations of r-S100A8/A9, CRP, or inflammatory cytokines in the UCR and TMR. In A, the x-axis indicates the absorbance at 490 nm on behalf of the serum concentration of r-S100A8/A9 because it is very difficult to purify native r-S100A8/A9 or to prepare its recombinant protein with high purity as a standard. In B, the x-axis indicates the serum concentration (μg/L) of CRP. In C, D, and E, the x-axis indicates the serum concentrations of TNF-α, IL-6, and IL-1β (pg/mL), respectively. In A-E, the y-axis indicates the values of HS. The correlations between HS and r-S100A8/A9, CRP, or inflammatory cytokines were assessed by Spearman analysis. R-value between 0.5 and 1.0 is significant statistically.

Figure 6

Microscopic observation of intracellular localization of r-S100A8/A9 in macrophages upon stimulation by LPS and the concentration of the heterodimer secreted from the cells. Peritoneal macrophages from WT were stimulated with LPS (1.0 μg/mL) for 1 h and 2 h in 5% CO₂ at 37 °C. In A, fluorescent immunochemical staining was carried out using the macrophages. The procedures are summarized in Materials and Methods. Briefly, r-S100A8 was detected with mAb2H6-biotin and STA-TR, while r-S100A9 with mAb10D11 and anti-mouse IgG (horse) IgG-FITC. r-S100A8 and r-S100A9 were indicated by red and green, respectively, in which the color of yellow indicates microscopic images merged. Nucleus was counter-stained with DAPI (blue). All microscopic images were observed by BIOREVO BZ-9000 (KEYENCE Co. Ltd., Osaka, Japan). Magnification of all panels is high power field (× 2000). In B and C, the x-axis indicates the time points of stimulation with LPS, in which DW on behalf of LPS was used as negative control. The y-axis indicates the absorbance at 490 nm on behalf of the concentration of r-S100A8/A9. Data are the mean ± standard deviation (±SD) (see Methods). ∗P < 0.05.