Sphingosine-1-Phosphate Signaling and Metabolism Gene Signature in Pediatric Inflammatory Bowel Disease: A Matched-case Control Pilot Study

Abstract

Goal: The aim of this study was to investigate gene expression levels of proteins involved in sphingosine-1-phosphate (S1P) metabolism and signaling in a pediatric inflammatory bowel disease (IBD) patient population.

Background: IBD is a debilitating disease affecting 0.4% of the US population. The incidence of IBD in childhood is rising. Identifying effective targeted therapies that can be used safely in young patients and developing tools for selecting specific candidates for targeted therapies are important goals. Clinical IBD trials now underway target S1PR1, a receptor for the pro-inflammatory sphingolipid S1P. However, circulating and tissue sphingolipid levels and S1P-related gene expression have not been characterized in pediatric IBD.

Methods: Pediatric IBD patients and controls were recruited in a four-site study. Patients received a clinical score using PUCAI or PCDAI evaluation. Colon biopsies were collected during endoscopy. Gene expression was measured by qRT-PCR. Plasma and gut tissue sphingolipids were measured by LC-MS/MS.

Results: Genes of S1P synthesis (SPHK1, SPHK2), degradation (SGPL1), and signaling (S1PR1, S1PR2, and S1PR4) were significantly upregulated in colon biopsies of IBD patients with moderate/severe symptoms compared with controls or patients in remission. Tissue ceramide, dihydroceramide, and ceramide-1-phosphate (C1P) levels were significantly elevated in IBD patients compared with controls.

Conclusions: A signature of elevated S1P-related gene expression in colon tissues of pediatric IBD patients correlates with active disease and normalizes in remission. Biopsied gut tissue from symptomatic IBD patients contains high levels of pro-apoptotic and pro-inflammatory sphingolipids. A combined analysis of gut tissue sphingolipid profiles with this S1P-related gene signature may be useful for monitoring response to conventional therapy.

Figure 1.

Inflammatory bowel disease (IBD) patients with moderate/severe disease severity express higher SPHK1 and SPHK2 expression in ascending colon. Gene expression levels of SPHK1, SPHK2, and ORMDL3 in biopsied ascending colon samples were quantified relative to HPRT1 gene expression by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), using the 2^-ΔΔCT method. Results were plotted using a Tukey box plot. Whiskers in the Tukey box plot show the full range of values, with box depicting 25–75 interquartile ranges. The line within the box shows the group median. Grubbs test was used to identify outliers shown as individual values. Generalized estimated equation models were used to determine statistical differences between control (N = 17), IBD (UC+CD) patients in remission (N = 16), those with mild disease (N = 6), and those with moderate/severe disease (moderate/severe; N = 8). Results show that SPHK1 and SPHK2 expression levels were significantly (P = 0.0001) higher in pediatric IBD patients with moderate/severe symptoms as compared with non-IBD controls. To compare the expression levels of non-IBD controls with ulcerative colitis (UC) and Crohn’s Disease (CD) patients, median values from symptomatic (mild+moderate/severe) UC and CD patient were compared. Differences between control, UC, and CD were analyzed by nonparametric analysis of variance (Kruskal-Willis) and post hoc tests were used to adjust for multiple comparisons. Although median expression levels of SPHK1 and SPHK2 were highest among UC patients, these differences were not statistically significant.

Figure 2.

Significant increase in SGPL1 expression is associated with moderate/severe inflammatory bowel disease (IBD) and represents the major pathway for sphingosine-1-phosphate (S1P) removal. S1P can either be irreversibly degraded by sphingosine phosphate lyase (SPL), which is encoded by SGPL1 (A) or reversibly dephosphorylated by sphingosine phosphate phosphatases (Sgpp1 or Sgpp2) (B). Results show a significant 15-fold increase in median SGPL1 expression among pediatric IBD patients in moderate/severe disease category (N = 8; P = 0.002) relative to non-IBD controls (N = 17; Panel A). SGPL1 levels among IBD patients in remission (N = 16) or with mild disease (N = 7) remained similar to control values (A). In contrast to SGPL1, no significant differences in SGPP1 gene expression were noted (B). SGPP2 expression trended higher among ulcerative colitis (UC) patients (N = 7) relative to controls (N = 17; P = 0.07). Levels in Crohn’s Disease (CD) patients (N = 8) remained unchanged relative to controls.

Figure 3.

Sphingosine phosphate lyase (SPL) protein expression patterns in control colon and pediatric IBD colon (A–E). Colon tissue samples stained with hematoxylin & eosin (H&E). A, normal control pediatric colon biopsy. B, crypt architectural distortion, documenting chronic inflammation and regeneration with aberrant crypts. C, active cryptitis, neutrophils (arrows) infiltrate crypt epithelium. D, crypt abscess (center), neutrophils fill and expand the crypt lumen with disruption of the epithelial continuity. E, quiescent colitis, changes include mild crypt drop out, edematous lamina propria and patchy increased plasma cell infiltrates without active colitis. Serial sections of the same areas shown above stained with anti-SPL antibody (F–J) followed by secondary antibody and avidin-biotin linked hydrogen peroxide IHC. Chronic inflammatory cells, lymphocytes and plasma cells also express SPL, and this is increased in the disease tissues compared with control. Scale bar in panel A applies to all images (A-J). (K) Intensity of SPL staining based on quantitative image analysis. Straight red peak to the right of the image represents zero staining, and signals shifted leftward indicate increasing levels of staining intensity. Blue line is control. Red line is IBD (all categories). L, intensity of SPL staining separated by IBD disease category. Blue line is control. Red lines is CD. Green line is UC. Straight green peak to the right of the image represents zero staining.

Figure 4.

S1PR1, S1PR2, and S1PR4 gene expression levels are increased in colons of moderate/severe inflammatory bowel disease (IBD) patients. Panel A shows significant median S1PR1 gene expression increased by approximately 3.3-fold (P = 0.05) and 6.5-fold (P = 0.002) in pediatric moderate/severe IBD as compared with non-IBD controls and IBD patients in remission, respectively. Median expression of S1PR2 was also significantly upregulated in moderate/severe IBD by 235-fold (P = 0.01) and 360-fold (P = 0.02) relative to control and IBD patients in remission, respectively (B). Panel C shows no significant changes in S1PR3 expression across all groups examined. Panel D shows that median expression of S1PR4 in moderate/severe pediatric IBD was upregulated by 256-fold (P = 0.001) and 92-fold (P = 0.01) relative to controls and IBD patients in remission. These results suggest that upregulation of S1PR2 and S1PR4 are dominant signatures of severe IBD.

Figure 5.

Intestinal dihydroceramide (DHCer), ceramide, and ceramide-1-phosphate (C1P) levels are significantly associated with worsening IBD symptoms. Tissue levels of DHCer increased as a function of worsening IBD severity (A). Box plot shows the median, range and 25th–75th percentiles. Median DHCer concentrations increased in stepwise manner from control values of 0.28 nmol/mg protein (N = 10) to 0.36 nmol/mg protein, 0.50 nmol/mg protein (P = 0.004) and to 0.63 nmol/mg protein (P = 0.004), in the remission, mild, and moderate/severe IBD groups, respectively (A). DHcer formed are further desaturated by DHCer desaturases (DES1 and DES1) to form ceramides. B, tissue ceramide content also increased with worsening IBD severity. Tissue ceramides increased from control values of 0.9 nmol/mg protein to 4.0 ± 0.6 (P = 0.1), 5.4 (P = 0.003), and 7.3 nmol/mg protein (P = 0.003) in IBD patients in remission, mild, and moderate/severe groups, respectively. In contrast to the increase in ceramides, tissue levels of sphingosine were similar across all groups. C, tissue levels of S1P trended lower in the moderate/severe pediatric IBD patients (0.006 nmol/mg tissue) as compared with controls (0.01 nmol/mg tissue; P = 0.07).

Figure 6.

Alterations in dihydroceramide (DHcer), ceramide, and ceramide-1-phosphate (C1P) metabolites organized by increasing acyl-chain length and disease severity. Specific changes in DHCer, ceramide, and C1P species of varying acyl-chain lengths are displayed in the heatmap. Each of these metabolites is organized in rows. Columns represent each tissue specimen analyzed. Tissues values were first normalized by median values across all samples and expressed as a fold-change over group median as indicated by color intensity shown in the legend. Among IBD patients with moderate/severe disease severity, significant increases were observed in C14:0 DHCers (2.9-fold increase relative to controls; P < 0.001) and C16:0 DHCers (2.4-fold increase relative to controls; P < 0.001). C24:0 DHCers decreased by ~25% relative to controls (P < 0.001). C20:0 and C22:0 DHCer species were significantly elevated by ~2-fold (P < 0.05). Among the ceramide species, C14:0 and 16:0 ceramides were the most significantly increased by 14.7- and 7.0-fold relative to controls (P < 0.05), respectively. Other ceramide species (C18:1, C20:0, C20:4, and C24:0) were increased by >2-fold in IBD patients (mild+moderate/severe) relative to controls. C16:0 C1P levels increased in the moderate/severe IBD patients (3.3-fold; P < 0.001) and C24:0 C1P decreased by 50% (P < 0.001).