Age-of-diagnosis dependent ileal immune intensification and reduced alpha-defensin in older versus younger pediatric Crohn Disease patients despite already established dysbiosis

Abstract

Age-of-diagnosis associated variation in disease location and antimicrobial sero-reactivity has suggested fundamental differences in pediatric Crohn Disease (CD) pathogenesis. This variation may be related to pubertal peak incidence of ileal involvement and Peyer's patches maturation, represented by IFNγ-expressing Th1 cells. However, direct mucosal evidence is lacking. We characterize the global pattern of ileal gene expression and microbial communities in 525 treatment-naive pediatric CD patients and controls (Ctl), stratifying samples by their age-of-diagnosis. We show a robust ileal gene signature notable for higher expression of specific immune genes including GM-CSF and INFγ, and reduced expression of antimicrobial Paneth cell α-defensins, in older compared to younger patients. Reduced α-defensin expression in older patients was associated with higher IFNγ expression. By comparison, the CD-associated ileal dysbiosis, characterized by expansion of Enterobacteriaceae and contraction of Lachnospiraceae and Ruminococcaceae, was already established within the younger group and did not vary systematically with increasing age-of-diagnosis. Multivariate analysis considering individual taxa, however did demonstrate negative associations between Lachnospiraceae and IFNγ, and positive associations between Bacteroides and α-defensin expression. These data provide evidence for maturation of mucosal Th1 immune responses and loss of epithelial antimicrobial α-defensins which are associated with specific taxa with increasing age-of-diagnosis in pediatric CD.

Figure 1.. Decreased epithelial Paneth cell α-defensins signature in pediatric iCD ≥ 10 years at diagnosis.

(A) Venn diagram shows an overlap between the previously reported 1340 core iCD gene signature and the 365 genes that were differentially expressed in the ileum between older (A1b) and younger (A1a) iCD (FDR correction [0.05], fold-change≥1.5). 124 of these 365 genes including α-defensins and REG3A were not included in the core iCD gene list. (B) Unsupervised hierarchical clustered heat map of the 365 genes differentially expressed genes between older and younger iCD with up-regulated genes in red and down-regulated genes in blue. Above the heat map, younger Ctl (light blue), older Ctl (dark blue), younger CD (orange), and older CD (red) samples are indicated. (C) TPM ileal gene expression is shown for the indicated genes for the indicated groups stratified by age-of-diagnosis with Kruskal-Wallis test with Dunn's multiple comparisons. (D) Immunohistochemistry in representative CD patients and non-IBD controls. Relatively high DEFA5 staining is shown for older non-IBD control (i) and younger CD (ii) that correlated with transcripts per million (TPM) values of >10,000 and 6143 for gene expression by RNASeq, respectively, and relatively low DEFA5 staining in older CD that correlated with transcripts per million (TPM) values of 1239 for gene expression by RNASeq. Images were captured using an Olympus BX51 light microscope and digitally recorded at 20x magnification. (E) Averaged and unsupervised hierarchical clustering heat map of 121 of 365 (top third differentially expressed genes between older and younger iCD) stratified by yearly age-of-diagnosis as indicated. Number of patients for each age group is indicated adjacent to the heat map.

Figure 2.. Enhanced ileal innate and adaptive Th1 immune responses in pediatric iCD ≥10 years at diagnosis.

(A) Top functional annotation enrichment analyses using Toppgene/ToppCluster platforms of the 171 up-regulated and differentially expressed genes between older and younger iCD and Cytoscape was used for visualization. (B) Top functional annotation enrichment analyses using Toppgene/ToppCluster platforms using the 194 down-regulated and differentially expressed genes between older and younger iCD and Cytoscape was used for visualization.

Figure 3.. Decreased epithelial Paneth cell signature in pediatric CD ≥10 years at diagnosis is associated with clinical ileal inflammation.

(A) Unsupervised averaged hierarchical clustered genes heat map of 365 genes differentially expressed between older and younger iCD is shown for each clinical sub-group [Ctl, cCD and iCD divided to <10 and ≥10 years]. (B & C) DEFA5 TPM ileal gene expression is shown for the indicated younger and older CD age-of-diagnosis groups as indicated, stratified by their ATG16L1 (B) or NOD2 (C) genotype. R; risk allele (homozygote or heterozygotes), nn; no risk allele, nR; heterozygote for the risk allele. RR; homozygote for the risk allele.

Figure 4.. Age-associated shifts in the ileal microbial community composition detected in non-IBD controls are not present within CD.

(A) PCoA with Bray−Curtis distance comparing microbial community diversity in samples from CD patients (n=272) and Ctl (n=178). Left panel, samples are colored by the Chao1 diversity index. Right panel, samples are colored by <10 and ≥10 years. Triangular shape indicates Ctl, filled circles indicate CD samples. (B) Mean and standard deviation of Chao1 α-diversity is shown for Ctl and CD divided in younger <10 and older ≥10 years. *p < 0.01, a two-sided t-test was used. (C) The bar graph shows fold change (mean older A1b Ctl/mean younger Ctl) for significant associations between the indicated taxa as determined by MaAsLin while taking Paris age, gender, and body mass index (BMI) into account.

Figure 5.. Co-variation of the ileal microbial community structure with host gene expression.

(A) The bar graph shows fold change (mean CD/mean Ctl) for significant associations between the indicated taxa and clinical phenotype (Ctl, CD) as determined by MaAsLin while taking Paris age, gender, body mass index (BMI, as a measure of nutritional status), endoscopic severity (deep ulcers in ileum), clinical severity (Pediatric Crohn’s Disease Activity Index, PCDAI), antibiotics, ileal gene expression of CSF2, CXCR1, IFNG, MMP3, DEFA5, GSTA1, and LCT, and NOD2, and ATG16L1 IBD risk allele carriage into account. (B) Scatter plots are shown for significant associations between the indicated taxa (y-axis) and host gene expression (x-axis) based on the multivariate statistical analysis described in A.

Figure 6.. Summarizing cartoon.

Using high throughput mRNA and 16S rRNA amplicon sequencing of treatment naïve newly diagnosed ileal biopsies from CD cases we identify 365 genes that were robustly differentially expressed between older iCD (10–16 years) and younger iCD (<10 years). Those differentially expressed genes showed increased Th1-related IFNγ profile associated with amplified innate myeloid inflammatory activation, and enhanced extra cellular matrix and collagen signatures in older iCD cases. Remarkably, these signatures for enhanced immune activation was associated with a specific reduction in epithelial Paneth cell α-defensin expression in older iCD, and was not associated with systematic changes in the local mucosal microbial communities.