Patient characteristics
For this study, five pediatric UC patients and five age- and sex-matched controls were recruited for RNA-Seq analyses. The control children were free of fevers and diarrhea, exhibited normal white blood cell (WBC) counts, C-reactive protein (CRP) levels, and erythrocyte values, and had normal colonic histology, colonoscopy, and abdominal imaging results. The UC patients showed bloody stool, elevated WBC, CRP, and/or ESR levels, and evidence of intestinal ulceration and inflammation observed by colonoscopy and had been diagnosed in accordance with the ECCO guidelines [10]. The Pediatric Ulcerative Colitis Activity Index (PUCAI) values for these patients ranged from 20 to 60, consistent with mild-to-moderate disease. None of the patients had undergone specific treatment before sample collection.
Transcriptomic analyses
To explore UC-related changes in the transcriptomic landscape of the intestinal mucosa, total RNA was isolated from mucosal samples from the 10 patients and sequenced. After the removal of low-quality and ambiguous reads, each sample yielded 44–57 million clean paired reads (Additional file 2: Table S2). Over 85% of these read pairs were successfully aligned with the human reference genome. Standardized transcript levels were analyzed using FPKM values, and correlation matrices showed that the results were highly consistent within the two patient groups (R2 ≥ 0.822; Fig. 1A). Samples were clustered using principal component analysis (PCA), showing clear separation of the clusters of the two groups (Fig. 1B).
DEG identification and GO annotation
In total, 2,290 DEGs were identified when comparing the UC and control groups, of which 1,258 and 1,032 were up- and downregulated, respectively (Fig. 2A and Additional file 3: Table S3). Clustering analyses of the DEGs are shown in Fig. 2B. The 20 genes that were most highly upregulated in UC were SPINK4, CCL11, LRP8, SLC6A20, VWF, MMP10, SERPINB5, ARNTL2, MMP3, LIPG, PDPN, AQP9, SELP, TNC, LOXL2, VSIG1, ADAMTS12, STRIP2, LPIN1, and TREM1.
To begin examining the biological roles of the DEGs, GO annotation analyses were conducted to assess their enrichment in specific biological process (BP), cellular component (CC) and molecular function (MF), classifications, as shown in Fig. 3A where the number of mRNAs associated with each term is shown along the horizontal axis. In total, 437 significant GO terms were identified (P.adj ≤ 0.01), and the 30 most significantly GO distributions of upregulated genes between the UC and control samples are listed in Additional file 4: Table S4. A directed acyclic graph was used to represent the relationships among identified enriched CC terms (Fig. 3B). Overall, the identified DEGs were enriched in several extracellular matrix (ECM)-related GO terms including extracellular structure organization (BP GO:0043062), extracellular matrix organization (BP GO:0030198), extracellular matrix (CC GO:0031012), extracellular matrix component (CC GO:0044420), complex of collagen trimers (CC GO:0098644), and extracellular matrix structural constituent (MF GO:0005201).
KEGG pathway enrichment analysis
Next, DEGs were subjected to a KEGG pathway enrichment analysis that identified 296 enriched pathways. 25 significantly enriched pathways associated with 530 up-regulated genes (P.adj ≤ 0.05) were identified (Additional file 5: Table S5), including the Cytokine-cytokine receptor interaction, ECM-receptor interaction, PI3K-Akt signaling pathway, Inflammatory bowel disease (IBD), etc.
Based on a combination of GO results, KEGG pathways, and genes of interest, 8 DEGs were identified including PIK3CD, IL1β, IL1α, TIMP1, MMP1, MMP12, COL6A3, and HLADRB5, of which 7 were associated with intestinal inflammation and fibrosis in UC, and involved in KEGG pathways associated with the ECM-receptor interaction and inflammatory bowel disease (Additional file 6: Table S6). This finding suggests the coexistence of intestinal inflammation and fibrosis even during the early stages of pediatric UC.
To confirm these findings, qPCR was used to assess the expression of these 8 DEGs in intestinal mucosal tissue samples from children with UC and controls (Additional file 7: Table S7) using the primers (Biotree, Shanghai, China) listed in Additional file 8: Table S8. All of these genes were significantly differentially expressed in UC patient samples relative to controls (Fig. 4), confirming the above transcriptomic results. However, while PIK3CD was upregulated in UC samples relative to controls in the RNA-Seq analyses, it was found to be downregulated on qPCR verification (Fig. 4). Nevertheless, both up- and downregulation are indicative of altered immune functionality [21,22,23].
Intestinal inflammation and fibrosis in pediatric UC
The intestines contain high levels of bacteria, including many Gram-negative species that have cell walls composed primarily of lipopolysaccharide (LPS), which can stimulate an innate immune response in host cells and often plays a major role in the pathogenesis of many bacterial infections.
To simulate the effect of intestinal bacteria, Caco2 intestinal epithelial cells were cultured in the presence of LPS (10 μg/ml), resulting in HLA-DRB5 upregulation at the mRNA level. Peak HLA-DRB5 expression was observed at 3 h post-LPS treatment, whereas IL-1α expression peaked at 8 h post-LPS treatment. The highest HLA-DRB5 and IL-1α protein levels in these cells were detected at 12 h following stimulation (Fig. 5A–F).
An HLA-DRB5 expression construct was next prepared and transfected into Caco2 cells, resulting in a significant rise in HLA-DRB5 after 24 h (Fig. 6A). IL-1α levels also rose significantly at 24 h after HLA-DRB5 overexpression (Fig. 6B). HLA-DRB5 and IL-1α expression levels increased significantly following LPS treatment after HLA-DRB5 overexpression (Fig. 6C, D), suggesting that HLA-DRB5 serves as a key mediator of IL-1α induction.
Next, HLA-DRB5 was knocked out by generating an sgRNA construct targeting the exonic sequence of this gene as recorded in the NCBI database using appropriate CRISPR software (http://crisper.mit.edu/). Plasmids expressing sgRNA and Cas9 were then transfected into Caco2 cells to knock out HLA-DRB5, which was confirmed by Western immunoblotting (Fig. 6E). While LPS increased IL-1α expression in control cells after 8 h, there was no change in its expression after HLA-DRB5 knockout (Fig. 6F). These results strongly suggested that HLA-DRB5 upregulation in response to LPS is required for IL-1α induction.
LPS induced HLA-DRB5 expression and HLA-DRB5, in turn, promoted IL-1α production. In addition, IL-1α was able to promote the intestinal epithelial-mesenchymal transition, contributing to higher levels of COL6A3 and MMP12 expression (Fig. 7A, B).
Together these data suggest that in response to intestinal bacteria, intestinal epithelial cells upregulate HLA-DRB5, thereby promoting IL-1α secretion. This cytokine, in turn, promotes intestinal inflammation, the epithelial-mesenchymal transition, and COL6A3 and MMP12 expression. COL6A3 can support microfibril formation and ECM deposition, contributing to intestinal fibrosis, whereas MMP12 can break down components of the ECM to protect against fibrosis. When the balance between these mechanisms is disrupted, intestinal fibrosis can occur (Fig. 7C).
Other pro- and anti-fibrotic factors including TIMP-1 and MMP1 may also play roles in this context. While LPS stimulation and HLA-DRB5 transfection had no effect on the expression of IL-1β in intestinal epithelial cells (data not shown), IL-1β is a key cytokine necessary for intestinal inflammatory response induction, and other pathways may thus govern IL-1β expression and secretion in this context.