Consequences of collagen induced inflammatory arthritis on circadian regulation of the gut microbiome

Abstract

The gut microbiota is important for host health and immune system function. Moreover autoimmune diseases, such as rheumatoid arthritis, are associated with significant gut microbiota dysbiosis, although the causes and consequences of this are not fully understood. It has become clear that the composition and metabolic outputs of the microbiome exhibit robust 24 h oscillations, a result of daily variation in timing of food intake as well as rhythmic circadian clock function in the gut. Here, we report that experimental inflammatory arthritis leads to a re-organization of circadian rhythmicity in both the gut and associated microbiome. Mice with collagen induced arthritis exhibited extensive changes in rhythmic gene expression in the colon, and reduced barrier integrity. Re-modeling of the host gut circadian transcriptome was accompanied by significant alteration of the microbiota, including widespread loss of rhythmicity in symbiont species of Lactobacillus, and alteration in circulating microbial derived factors, such as tryptophan metabolites, which are associated with maintenance of barrier function and immune cell populations within the gut. These findings highlight that altered circadian rhythmicity during inflammatory disease contributes to dysregulation of gut integrity and microbiome function.

Keywords: arthritis; circadian; colon; lactobacillus; microbiota.

FIGURE 1

Temporal gene expression in colonic tissue from CIA and naïve animals. (A) Expression of core clock genes in naïve and arthritic colonic tissue measured during RNA sequencing, n = 5/time point, values are mean ± SD. Compare rhythms was used to define rhythmicity where * denotes significantly rhythmic transcripts and △ denotes a change in amplitude or phase between treatments (naïve versus CIA). (B and C) Heatmaps illustrating the temporal organization of transcripts across the 24 h day (orange: ZT0, ZT4 and ZT8 and black: ZT12, ZT16, ZT20), n = 5/time point. Transcripts are grouped as same (no change in rhythmicity); change (remains rhythmic but change in peak or phase); rhythmic in naïve (but not in CIA); and rhythmic in CIA (but not in naïve). Pathway analysis (KEGG mouse 2019, padj < .01) of transcripts that are rhythmic in (D) both naïve and arthritic colon (same) and (E) naïve tissue only.

FIGURE 2

Rhythmic IgA production in the setting of arthritis. (A) Volcano plot illustrating differentially expressed (DE) transcripts (FDR <0.05) in colonic tissue from naïve (n = 30) and arthritic (n = 30) mice. (B) Pathway analysis (KEGG mouse 2019, padj < .05) of transcripts that are down in CIA compared to naïve tissue. Temporal expression of (C) differentially expressed (DE) genes and (D) all genes in the IgA pathway in naïve and arthritic colon, n = 5/time point, values are mean ± SD. (E) IgA levels in fecal pellets collected longitudinally over 24 h from naïve and arthritic mice (5 mice per treatment group), values are mean ± SEM, * denotes significant 24 h rhythms (assessed using Compare Rhythms). Two‐way ANOVA revealed a significant effect of time (P < 0.005) and disease (P < 0.05) on fecal IgA levels.

FIGURE 3

CIA alters colonic expression of tight junction genes and barrier permeability. (A) Heat maps illustrating the temporal organization of transcripts from the tight junction pathway across the 24 h day (orange: ZT0, 4 and 8 and black: ZT12, ZT16, ZT20), n = 5/time point. Transcripts which exhibited significant differential expression between treatment groups are highlighted in the upper portion. Temporal expression of (B) differentially expressed (DE) genes and (C) all genes in the tight junction genes pathway in naïve and arthritic colon, n = 5/time point, values are mean ± SD. Temporal expression of (D) Claudin 4 and (E) Jam3 in naïve and arthritic colon, n = 5/time point, values are mean ± SD, * denotes significant 24 h rhythms (assessed using Compare Rhythms). (F) Albumin levels in fecal pellets collected longitudinally over 24 h from naïve and arthritic mice (6 mice per treatment group), values are mean ± SEM, * denotes significant 24 h rhythms (assessed using Compare Rhythms). Two‐way ANOVA revealed a significant effect of time (P < 0.005) on fecal albumin levels.

FIGURE 4

CIA is associated with altered daily rhythms in the gut microbiota in a taxa specific manner. (A) Abundance and rhythmicity (JTK_Cycle analysis) of the OTUs detected in each condition. OTUs which have are significantly rhythmic in naïve animals (padj < .05) are highlighted in pink in both graphs. (B) Numbers of significantly rhythmic (padj < .05) OTUs in each treatment group. (C) Abundance of the two dominant phyla bacteroidetes and firmicutes across the circadian day in naïve and CIA animals (n = 5/time point), values are mean ± SEM, * denotes significant 24 h rhythms (padj < .05) (assessed using JTK_Cycle analysis). (D) Abundance of 4 bacterial genera across the circadian day in naïve and CIA animals (n = 5/time point), values are mean ± SEM, * denotes significant 24 h rhythms (padj < .05) (assessed using JTK_Cycle analysis). (E) Abundance of 6 species of lactobacillus across the circadian day in naïve and CIA animals (n = 5/time point), values are mean ± SEM, * denotes significant 24 h rhythms (padj < .05) (assessed using JTK_Cycle analysis).

FIGURE 5

Circadian profiles of microbial metabolites are altered in inflammatory arthritis. (A) Metabolomic analysis of short chain fatty acids (SCFA) in caecal samples from naïve and CIA mice over the 24 h day, n = 5/time point, values are mean ± SEM, asterix denotes significant 24 h rhythms (assessed using JTK_Cycle) whereby *padj < .05, **padj < .01 and ***padj < .005. Metabolomic analysis of (B) tryptophan metabolites and (C) bile acids in plasma samples from naïve and CIA mice over the 24 h day, n = 5/time point, values are mean ± SEM asterix denotes significant 24 h rhythms (assessed using JTK_Cycle) whereby *padj < .05, **padj < .01 and ***padj < .005.