Introduction Even in the absence of inflammation, persistent symptoms in patients with Crohn’s disease (CD) are prevalent and worsen quality of life. We previously demonstrated enrichment in sulfidogenic microbes in quiescent Crohn’s disease patients with (qCD+S) vs. without persistent GI symptoms (qCD-S). Thus, we hypothesized that sulfur metabolic pathways would be enriched in stool while differentially abundant microbes would be associated with important sulfur-metabolic pathways in qCD+S.

Methods We performed a multi-center observational study nested within SPARC IBD. Quiescent inflammation was defined by fecal calprotectin level <150 mcg/g. Persistent symptoms were defined by CD-PRO2. Active CD (aCD) and non-IBD diarrhea-predominant irritable bowel syndrome (IBS-D) were included as controls.

Results Thirty-nine patients with qCD+S, 274 qCD-S, 21 aCD, and 40 IBS-D underwent paired shotgun metagenomic sequencing and untargeted metabolomic profiling. The fecal metabolome in qCD+S was significantly different relative to qCD-S and IBS-D but not aCD. Patients with qCD+S were enriched in sulfur-containing amino acid pathways, including cysteine and methionine, as well as serine, glycine, and threonine. Glutathione and nicotinate/nicotinamide pathways were also enriched in qCD+S relative to qCD-S, suggestive of mitochondrial dysfunction, a downstream target of H2S signaling. Multi-omic integration demonstrated that enriched microbes in qCD+S were associated with important sulfur-metabolic pathways. Bacterial sulfur-metabolic genes, including CTH, isfD, sarD, and asrC, were dysregulated in qCD+S. Finally, sulfur metabolites with and without sulfidogenic microbes showed good accuracy in predicting presence of qCD+S.

Discussion Microbial-derived sulfur pathways and downstream mitochondrial function are perturbed in qCD+S, which implicate H2S signaling in the pathogenesis of this condition. Future studies will determine whether targeting H2S pathways results in improved quality of life in qCD+S.

Key Messages What is Already Known

Even in the absence of inflammation, persistent gastrointestinal symptoms are common in Crohn’s disease.

The microbiome is altered in quiescent Crohn’s disease patients with persistent symptoms, but the functional significance of these changes is unknown.

What is New Here

Sulfur metabolites and sulfur metabolic pathways were enriched in stool in quiescent Crohn’s disease patients with persistent symptoms.

Multi-omic integration showed enriched microbes were associated with important sulfur metabolic pathways in quiescent Crohn’s disease patients with persistent symptoms.

How Can This Study Help Patient Care

KR is supported in part from an investigator-initiated grant from Merck & Co, Inc.; he has consulted for Seres Therapeutics, Inc., Rebiotix, Inc. and Summit Therapeutics, Inc. VBY has consulted for Vendanta Biosciences, Debiopharm and conducted a speaking engagement sponsored by Aimunne. AAL has consulted for GlaxoSmithKline and Atmo Biosciences. The other authors declare no conflict of interest exist.

The results published here are in part based on data obtained from the IBD Plexus program of the Crohns & Colitis Foundation. This study was also supported by grants from The Leona M. and Harry B. Helmsley Charitable Trust (to AAL) and the National Institutes of Health grants DK124567 and DK139095 (to AAL); HS027431 (to KR); T32-DK062708, R01-DK125687, R01-DK118154 (to PDHR); AI182787 (to VBY); and DK123403 (to SB).

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The institutional review board at the University of Michigan gave ethical approval for this work.

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Conflict of Interest: KR is supported in part from an investigator-initiated grant from Merck & Co, Inc.; he has consulted for Seres Therapeutics, Inc., Rebiotix, Inc. and Summit Therapeutics, Inc. VBY has consulted for Vendanta Biosciences, Debiopharm and conducted a speaking engagement sponsored by Aimunne. AAL has consulted for GlaxoSmithKline and Atmo Biosciences. The other authors declare no conflict of interest exist.

Grant Support: The results published here are in part based on data obtained from the IBD Plexus program of the Crohn’s & Colitis Foundation. This study was also supported by grants from The Leona M. and Harry B. Helmsley Charitable Trust (to AAL) and the National Institutes of Health grants DK124567 and DK139095 (to AAL); HS027431 (to KR); T32-DK062708, R01-DK125687, R01-DK118154 (to PDHR); AI182787 (to VBY); and DK123403 (to SB).

Data Availability: Sequencing and metabolomic data are available by request through the IBD Plexus (please see https://www.crohnscolitisfoundation.org/research/plexus/academic-request-for-proposal for full details).

Summary: Microbial-derived sulfur pathways and downstream mitochondrial function are perturbed in quiescent Crohn’s disease patients with persistent GI symptoms, which implicate H2S signaling in the pathogenesis of this condition.