Alterations in tryptophan metabolism and de novo NAD+ biosynthesis within the microbiota-gut-brain axis in chronic intestinal inflammation

Abstract

Background: Inflammatory bowel disease is an incurable and idiopathic disease characterized by recurrent gastrointestinal tract inflammation. Tryptophan metabolism in mammalian cells and some gut microbes comprise intricate chemical networks facilitated by catalytic enzymes that affect the downstream metabolic pathways of de novo nicotinamide adenine dinucleotide (NAD⁺) synthesis. It is hypothesized that a correlation exists between tryptophan de novo NAD⁺ synthesis and chronic intestinal inflammation.

Methods: Transcriptome analysis was performed using high-throughput sequencing of mRNA extracted from the distal colon and brain tissue of Winnie mice with spontaneous chronic colitis and C57BL/6 littermates. Metabolites were assessed using ultra-fast liquid chromatography to determine differences in concentrations of tryptophan metabolites. To evaluate the relative abundance of gut microbial genera involved in tryptophan and nicotinamide metabolism, we performed 16S rRNA gene amplicon sequencing of fecal samples from C57BL/6 and Winnie mice.

Results: Tryptophan and nicotinamide metabolism-associated gene expression was altered in distal colons and brains of Winnie mice with chronic intestinal inflammation. Changes in these metabolic pathways were reflected by increases in colon tryptophan metabolites and decreases in brain tryptophan metabolites in Winnie mice. Furthermore, dysbiosis of gut microbiota involved in tryptophan and nicotinamide metabolism was evident in fecal samples from Winnie mice. Our findings shed light on the physiological alterations in tryptophan metabolism, specifically, its diversion from the serotonergic pathway toward the kynurenine pathway and consequential effects on de novo NAD⁺ synthesis in chronic intestinal inflammation.

Conclusion: The results of this study reveal differential expression of tryptophan and nicotinamide metabolism-associated genes in the distal colon and brain in Winnie mice with chronic intestinal inflammation. These data provide evidence supporting the role of tryptophan metabolism and de novo NAD⁺ synthesis in IBD pathophysiology.

Keywords: chronic intestinal inflammation; colitis; gut-brain axis; inflammatory bowel disease; microbiota; nicotinamide metabolism; tryptophan metabolism.

Figure 1

Heatmap representation of DEGs associated with tryptophan and nicotinamide metabolism in the Winnie mouse colon Heat map representations of tryptophan (A) and nicotinamide (B) metabolism-associated DEGs in colons from Winnie mice (n = 6) compared to colons from C57BL/6 mice (n = 8). Individual colon samples from C57BL/6 and Winnie mice are visualized in columns and DEGs are represented in rows. Plotted data presented as Z-score distributions (−2 to +2 across samples) of the counts per million values indicate gene expression values from DEGs analysis. Red represents upregulated gene expression, while blue represents downregulated gene expression.

Figure 2

Functional analysis identifies upregulation and downregulation of key elements of tryptophan and nicotinamide metabolism in the Winnie mouse colon. Tryptophan (A) and nicotinamide (B) metabolism KEGG pathway analysis of distal colon samples from Winnie mice (n = 6) vs. C57BL/6 littermates (n = 8). DEGs of the tryptophan and nicotinamide metabolism KEGG pathways are colored according to their sign Log₂FC × Log₁₀P value changes. Rectangular nodes indicate gene expression data by RNA-seq gene expression data. Positive values (red) indicate genes that are upregulated (tryptophan—EC:1.4.3.4, Mao-b; EC:1.13.11.52, Ido1; EC:3.7.1.3, Kynu; EC:2.3.1.9, Acat1, nicotinamide—EC:2.4.99.20, Cd38; EC:3.2.2.6, Cd38; EC:3.2.2.5, Art2a; EC:2.4.2.12, Nampt1; EC:3.1.3.5, Cd73; EC:2.4.2.1, Pnp) and negative values (green) indicate genes that are downregulated [tryptophan—(EC):3.5.1.9, Afmid; EC:4.1.1.28 and EC:4.1.1.105, Ddc; EC:2.1.1.49, SAMe; EC:1.4.3.22, Aoc1; EC:1.2.1.3, Aldh2; OADH, Dhtkd1; EC:1.3.8.6, Gcdh, nicotinamide—EC:2.3.1.286, Sirt2; EC:2.7.7.1, Nmnat2; EC:2.7.7.18, Nmnat3; EC:2.7.1.22, Nmrk1; EC:2.7.1.173, Nmrk2; EC:6.3.5.1, Nadsyn1; EC:6.3.4.21, Naprt] in the Winnie mouse colon relative to colons from C57BL/6 mice.

Figure 3

Homology of tryptophan and nicotinamide gene expression in colon samples from Winnie mice and IBD patients. Heat map representations of homology of tryptophan (A) and nicotinamide (B) metabolism associated genes in Winnie mice and IBD patients. Upregulated gene expression levels are marked red, low expression levels are marked green, and neutral expression levels are marked black.

Figure 4

Changes in tryptophan metabolite concentrations in the Winnie mouse colon. Quantification of tryptophan metabolites (μg/ml) in Winnie mice distal colons compared to colons from C57BL/6 littermates (n = 6–8) as measured by UFLC: (A) Tryptophan, (B) 5-HIAA, (C) Kynurenine, (D) KYNA, (E) QUIN, (F) Nicotinamide, and (G) PIC. Data are expressed as mean ± SEM. *p < 0.05, ***p < 0.001 when compared to C57BL/6 mice.

Figure 5

Changes to tryptophan and nicotinamide metabolism-associated microbiota at the genus level in fecal samples from Winnie mice. Microbiota communities were profiled from C57BL/6 and Winnie mice (n = 11/group). (A) Chao's richness estimate, (B) community diversity assessed by calculating the Shannon diversity, and (C) evenness by Simpson index based on abundance data. (D) Heat map representation of the percent relative abundance of tryptophan and nicotinamide metabolism-associated microbiota at the genus level in fecal samples from Winnie compared to C57BL/6 mice. Individual samples from C57BL/6 and Winnie mice are visualized in columns and genus level microbiota involved in tryptophan metabolism are represented by rows. Percent relative abundance was calculated by the number of ASVs by taxonomic classification at the genus level relative to the total number of ASVs per sample. Asterisks mark significant differences in the mean percent relative abundance between groups. *p<0.05, **p<0.01, ***p<0.001 when compared to C57BL/6 mice.

Figure 6

DEGs associated with tryptophan metabolism in the Winnie mouse brain. Pathview plot for the tryptophan metabolism KEGG pathway analysis of brain samples from Winnie mice (n = 6) vs. C57BL/6 littermates (n = 8). DEGs of the tryptophan metabolism KEGG pathways are colored according to their sign Log₂FC × Log₁₀P value changes. Rectangular nodes indicate gene expression data determined by RNA-seq. Positive values (red) indicate genes that are upregulated (tryptophan—EC:3.5.1.9, Afmid; EC:2.1.1.49, SAMe; EC:1.4.3.22, Aoc1; EC:1.14.16.4, TpH1; EC:1.4.3.2, Laao; EC:1.2.3.1, Aox; OADH, Dhtkd1; and EC:1.14.14.1, Cyp1a1, nicotinamide—EC:2.7.1.22 and EC:2.7.1.173, Nmrk1, and EC:1.2.3.1, Aox) and negative values (green) indicate genes that are downregulated (tryptophan—EC:1.13.11.52, Ido1, EC:1.2.1.3, Aldh2; EC:1.1.1.35, Hadh, nicotinamide—EC:2.4.2.1 Pnp; EC:3.2.2.6 and EC:249920, Cd38) in the Winnie mouse brain relative to brains from C57BL/6 mice. Circular nodes indicate the relative expression of tryptophan metabolism compounds.

Figure 7

DEGs associated with nicotinamide metabolism in the Winnie mouse brain. Pathview plot for the nicotinate and nicotinamide metabolism KEGG pathway analysis of brain samples from Winnie mice (n = 6) vs. C57BL/6 littermates (n = 8). DEGs of the nicotinamide metabolism pathways are colored according to their sign Log₂FC × Log₁₀P value changes. Rectangular nodes indicate gene expression data determined by RNA-seq. Red represents upregulated gene expressions and green indicates genes that are downregulated in the Winnie mouse brain relative to brains from C57BL/6 mice. Circular nodes indicate the relative expression of nicotinamide metabolism compounds.

Figure 8

Changes in tryptophan metabolite concentrations in Winnie mouse brains. Quantification of tryptophan metabolites (μg/ml) measured by UFLC in Winnie mouse brains compared to brains from C57BL/6 mice (n = 5–8 for both). (A) Tryptophan, (B) 5-HIAA, (C) Kynurenine, (D) KYNA, (E) QUIN, (F) Nicotinamide, and (G) PIC. Data is expressed as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 when compared to C57BL/6 mice.