Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease

Eiko Hayase¹, Tomo Hayase², Akash Mukherjee³, Stuart C Stinson³, Mohamed A Jamal², Miriam R Ortega², Christopher A Sanchez², Saira S Ahmed², Jennifer L Karmouch², Chia-Chi Chang², Ivonne I Flores², Lauren K McDaniel², Alexandria N Brown², Rawan K El-Himri², Valerie A Chapa², Lin Tan⁴, Bao Q Tran⁴, Yao Xiao⁵, Christopher Fan², Dung Pham², Taylor M Halsey², Yimei Jin², Wen-Bin Tsai², Rishika Prasad², Israel K Glover², Altai Enkhbayar², Aqsa Mohammed², Maren Schmiester², Katherine Y King⁶, Robert A Britton⁷, Pavan Reddy⁸, Matthew C Wong², Nadim J Ajami², Jennifer A Wargo², Samuel Shelburne⁹, Pablo C Okhuysen¹⁰, Chen Liu¹¹, Stephanie W Fowler¹², Margaret E Conner¹³, Zoe Katsamakis¹⁴, Natalie Smith¹⁴, Marina Burgos da Silva¹⁴, Doris M Ponce¹⁴, Jonathan U Peled¹⁵, Marcel R M van den Brink¹⁵, Christine B Peterson¹⁶, Gabriela Rondon³, Jeffrey J Molldrem¹⁷, Richard E Champlin³, Elizabeth J Shpall³, Philip L Lorenzi⁴, Rohtesh S Mehta³, Eric C Martens⁵, Amin M Alousi³, Robert R Jenq¹⁸

Affiliations

¹ Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. Electronic address: ekhayase@gmail.com.
² Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA.
³ Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁴ Metabolomics Core Facility, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA.
⁵ Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
⁶ Center for Cell and Gene Therapy and Department of Pediatrics, Division of Infectious Diseases, Baylor College of Medicine, Houston, TX 77030, USA.
⁷ Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
⁸ Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
⁹ Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA; Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁰ Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹¹ Department of Pathology, Yale School of Medicine, New Haven, CT 06520, USA.
¹² Department of Molecular Virology and Microbiology and Department of Education, Innovation, and Technology, Baylor College of Medicine, Houston, TX 77030, USA; Center for Comparative Medicine and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
¹³ Department of Molecular Virology and Microbiology and Department of Education, Innovation, and Technology, Baylor College of Medicine, Houston, TX 77030, USA.
¹⁴ Adult Bone Marrow Transplantation Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
¹⁵ Adult Bone Marrow Transplantation Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Weill Cornell Medical College, New York, NY 10021, USA.
¹⁶ Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁷ Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Hematopoietic Biology & Malignancy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁸ Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA; Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; CPRIT Scholar in Cancer Research, Houston, TX, USA. Electronic address: rjenq@coh.org.

PMID: 39214085
PMCID: PMC11441101
DOI: 10.1016/j.chom.2024.08.004

Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease

Eiko Hayase et al. Cell Host Microbe. 2024.

. 2024 Sep 11;32(9):1621-1636.e6.

doi: 10.1016/j.chom.2024.08.004. Epub 2024 Aug 29.

Authors

Affiliations

¹ Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. Electronic address: ekhayase@gmail.com.
² Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA.
³ Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
⁴ Metabolomics Core Facility, Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA.
⁵ Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
⁶ Center for Cell and Gene Therapy and Department of Pediatrics, Division of Infectious Diseases, Baylor College of Medicine, Houston, TX 77030, USA.
⁷ Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
⁸ Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
⁹ Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA; Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁰ Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹¹ Department of Pathology, Yale School of Medicine, New Haven, CT 06520, USA.
¹² Department of Molecular Virology and Microbiology and Department of Education, Innovation, and Technology, Baylor College of Medicine, Houston, TX 77030, USA; Center for Comparative Medicine and Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
¹³ Department of Molecular Virology and Microbiology and Department of Education, Innovation, and Technology, Baylor College of Medicine, Houston, TX 77030, USA.
¹⁴ Adult Bone Marrow Transplantation Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
¹⁵ Adult Bone Marrow Transplantation Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Weill Cornell Medical College, New York, NY 10021, USA.
¹⁶ Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁷ Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Hematopoietic Biology & Malignancy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
¹⁸ Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA; Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; CPRIT Scholar in Cancer Research, Houston, TX, USA. Electronic address: rjenq@coh.org.

PMID: 39214085
PMCID: PMC11441101
DOI: 10.1016/j.chom.2024.08.004

Abstract

Acute lower gastrointestinal GVHD (aLGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation. Although the intestinal microbiota is associated with the incidence of aLGI-GVHD, how the intestinal microbiota impacts treatment responses in aLGI-GVHD has not been thoroughly studied. In a cohort of patients with aLGI-GVHD (n = 37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and a disrupted fecal microbiome characterized by reduced abundances of Bacteroides ovatus. In a murine GVHD model aggravated by carbapenem antibiotics, introducing B. ovatus reduced GVHD severity and improved survival. These beneficial effects of Bacteroides ovatus were linked to its ability to metabolize dietary polysaccharides into monosaccharides, which suppressed the mucus-degrading capabilities of colonic mucus degraders such as Bacteroides thetaiotaomicron and Akkermansia muciniphila, thus reducing GVHD-related mortality. Collectively, these findings reveal the importance of microbiota in aLGI-GVHD and therapeutic potential of B. ovatus.

Keywords: Akkermansia muciniphila; Bacteroides ovatus; Bacteroides thetaiotaomicron; allogeneic hematopoietic stem cell transplantation; graft-versus-host disease; intestinal microbiome; mucus layer; polysaccharide utilization loci; polysaccharides; xylose.

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Conflict of interest statement

Declaration of interests R.R.J. has served as a consultant or advisory board member for Postbiotics Plus, Merck, Microbiome DX, Karius, MaaT Pharma, LISCure, Seres, Kaleido, and Prolacta and has received patent license fee or stock options from Seres, Kaleido, and Postbiotics Plus. E.J.S. has served as a consultant or advisory board member for Adaptimmune, Axio, Navan, Fibroblasts, and FibroBiologics, NY Blood Center, and Celaid Therapeutics and has received patent license fee from Takeda and Affimed. J.U.P. reports research funding, intellectual property fees, and travel reimbursement from Seres Therapeutics, and consulting fees from DaVolterra, CSL Behring, Crestone Inc, and from MaaT Pharma. J.U.P. serves on an advisory board of and holds equity in Postbiotics Plus Research. J.U.P. has filed intellectual property applications related to the microbiome (reference numbers #62/843,849, #62/977,908, and #15/756,845). Memorial Sloan Kettering Cancer Center (MSK) has financial interests relative to Seres Therapeutics. E.H., M.A.J., J.L.K., and R.R.J. are inventors on a patent application by The University of Texas MD Anderson Cancer Center supported by results of the current study entitled, “Methods and Compositions for Treating Cancer therapy-induced Neutropenic Fever and/or GVHD.”

Figures

**Figure 1.. The intestinal microbiome of steroid-refractory aLGI-GVHD patients shows significantly more dysbiosis than that of steroid-responsive aLGI-GVHD patients.**
(**A-D, G-M**) The intestinal microbiome analyzed by 16S rRNA sequencing in patient stool samples collected at presentation with aLGI-GVHD. (A) Cluster dendrogram analyzed using hierarchical clustering of weighted UniFrac distances. (B) The microbiome composition shown as stacked bar graphs. (C) PCoA of fecal samples collected from healthy volunteers and two clusters of aLGI-GVHD patients. (D) Distances from healthy volunteers quantified by weighted UniFrac. (E) Numbers of patients with steroid-responsive and -refractory GVHD. (F) Proportions of patients with steroid-responsive and -refractory GVHD. (G) Volcano plot of differentially abundant genera between clusters 1 and 2. (H) Alpha diversity quantified by Shannon index. (I) Principal coordinates analysis (PCoA) of fecal samples collected from healthy volunteers, steroid-responsive, and steroid-refractory patients. (J) Distances from healthy volunteers quantified by weighted UniFrac. (K) The composition of the intestinal microbiome. (L) Volcano plot of differentially abundant genera. (M) Relative abundances of genera that were significantly different between steroid-responsive and -refractory aLGI-GVHD.

**Figure 2.. Higher abundances of *Bacteroides ovatus* and *B. ovatus*-derived pathways are associated with steroid-responsive GVHD.**
(A) Graphical summary of antibiotics used in individual patients between hematopoietic stem cell transplant (HSCT) and onset of GVHD. (B) Proportions of patients with antibiotic exposures between HSCT and onset of GVHD. (C) Univariate logistic regression analysis for associations between antibiotic exposures and steroid-refractory GVHD. (**D-F**) Data analyzed by DNA shotgun sequencing of fecal samples collected from aLGI-GVHD patients (steroid-responsive; n=11, steroid-refractory; n=12). (D) Volcano plot of differentially abundant species between steroid-responsive and -refractory GVHD. (E) Volcano plot of differentially abundant pathways of the genus *Bacteroides*. **(F)** The top 50 subclasses of differentially abundant pathways of the genus *Bacteroides*.

**Figure 3.. Oral introduction of *Bacteroides ovatus* reduces GVHD-related mortality in mice with meropenem-aggravated colonic GVHD.**
(A) Experimental schema of murine GVHD model using meropenem treatment followed by oral gavage of 20 million colony-forming units of *B. ovatus* daily for 3 days. (B) Overall survival after allo-HSCT. Data are combined from two independent experiments. (C) Bacterial densities of mouse stool samples collected on day 21. Bacterial densities were measured by 16S rRNA gene qPCR. (D) Alpha diversity, measured by the Shannon index, was quantified in fecal samples. (E) Principal coordinates analysis (PCoA) of fecal samples. (F) Bacterial genera composition of fecal samples. (G) Volcano plot of differentially abundant zero-diameter operational taxonomic units (ZOTUs). (H) Relative abundances of *B. ovatus* (left), *B. theta* (middle) and *A. muciniphila* (right). (I) Absolute abundances of *B. ovatus* (left), *B. theta* (middle) and *A. muciniphila* (right). (J) Relative abundances of *B. ovatus* in mouse stool samples collected on days 21 and 28. (K) Periodic acid-Schiff (PAS) staining of histological distal colon sections collected on day 23. Bar, 100 μm. The areas inside dotted lines indicate the inner dense colonic mucus layer. (L) Mucus thickness on day 23. Data are shown from one representative experiment. (M) PAS staining of histological proximal colon sections collected on day 21. Areas in the yellow squares are magnified and shown to the bottom of the original images. Bar, 100 μm. (N) Numbers of goblet cells per crypt in the proximal or distal colon. (O) GVHD histology scores of the colon harvested on day 28. GVHD histology scores were quantified by a blinded pathologist. (P) Numbers of bacterial CFUs cultivated from mesenteric lymph nodes (MLNs) on day 21. Combined data from three independent experiments are shown.

Figure 4.. Expression of predicted mucus-degrading enzymes by *Bacteroides thetaiotaomicron* and *Akkermansia muciniphila* is suppressed in meropenem-treated mice after administration of Bacteroides ovatus.
(A) Heatmap showing scaled relative expression levels of polysaccharide utilization loci (PUL) genes in *B. theta* RNA transcripts sequenced from stool collected from meropenem-treated allo-HSCT mice with or without administration of *B. ovatus* on day 21. Right: Significantly altered PUL genes and their substrates. (B) Heatmap showing scaled relative expression of carbohydrate-active enzymes (CAZymes) by *A. muciniphila* in stool collected from meropenem-treated allo-HSCT mice with or without administration of *B. ovatus* on day 21. (C) Relative concentrations of monosaccharides of supernatants from colonic luminal content collected from meropenem-treated allo-HSCT mice with or without administration of *B. ovatus* on day 23 measured by ion chromatography-mass spectrometry (IC-MS). Combined data from two independent experiments are shown as means ± SEM. (D) Relative abundances of short-chain fatty acids of supernatants from colonic luminal content collected from meropenem-treated allo-HSCT mice with or without administration of *B. ovatus* on day 23 measured by IC-MS.

**Figure 5.. Byproducts of xylose-containing polysaccharides metabolized by *Bacteroides ovatus* suppress mucus-degrading functionality in *Bacteroides thetaiotaomicron*.**
(A) Experimental schema of in vitro bacterial culture assay using *B. ovatus* (MDA-HVS BO001) cultured in minimum nutrition medium with each polysaccharide and *B. theta* (MDA-JAX BT001) cultured in BYEM10 with porcine gastric mucin. (B) Concentrations of porcine gastric mucin in the culture supernatant without (left) or with *B. theta* (right) were determined using a PAS-based colorimetric assay. Combined data from two independent experiments are shown as means ± SEM. (C) Relative concentrations of monosaccharides of the *B. ovatus* culture supernatant with each polysaccharide measured by ion chromatography-mass spectrometry (IC-MS). (D) Experimental schema of gnotobiotic model using introduction of 20 million colony-forming units of *B. ovatus* (MDA-HVS BO001). (E) Heatmap showing scaled relative expression of polysaccharide utilization loci (PUL) genes by *B. theta* from *B. theta* (ATCC 29148)-colonized gnotobiotic mice with or without co-administration of *B. ovatus.* Expression was evaluated on day 14 after bacterial introduction to germ-free mice. Right: Significantly altered PUL genes and their substrates. (F) Experimental schema of gnotobiotic model using introduction of 20 million colony-forming units of wild-type *B. ovatus* (ATCC8483 with gene deletion of thymidine kinase) or xylan-PUL deficient *B. ovatus*. (G) Relative concentrations of xylose in supernatants from colonic luminal contents collected from gnotobiotic mice with administration of wild-type *B. ovatus* (ATCC8483 with gene deletion of thymidine kinase) or xylan-PUL deficient *B. ovatus* on day 14 measured by ion chromatography-mass spectrometry (IC-MS). (H) Experimental schema of murine GVHD model using meropenem treatment followed by oral gavage of 20 million colony-forming units of wild-type *B. ovatus* (ATCC8483 with gene deletion of thymidine kinase) or xylan-PUL deficient *B. ovatus* daily for 3 days. (I) Overall survival after allo-HSCT. Data are combined from three independent experiments.

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Update of

Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease.
Hayase E, Hayase T, Mukherjee A, Stinson SC, Jamal MA, Ortega MR, Sanchez CA, Ahmed SS, Karmouch JL, Chang CC, Flores II, McDaniel LK, Brown AN, El-Himri RK, Chapa VA, Tan L, Tran BQ, Pham D, Halsey TM, Jin Y, Tsai WB, Prasad R, Glover IK, Ajami NJ, Wargo JA, Shelburne S, Okhuysen PC, Liu C, Fowler SW, Conner ME, Peterson CB, Rondon G, Molldrem JJ, Champlin RE, Shpall EJ, Lorenzi PL, Mehta RS, Martens EC, Alousi AM, Jenq RR. Hayase E, et al. Res Sq [Preprint]. 2023 Jan 31:rs.3.rs-2460097. doi: 10.21203/rs.3.rs-2460097/v1. Res Sq. 2023. Update in: Cell Host Microbe. 2024 Sep 11;32(9):1621-1636.e6. doi: 10.1016/j.chom.2024.08.004. PMID: 36778495 Free PMC article. Updated. Preprint.

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Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease

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Bacteroides ovatus alleviates dysbiotic microbiota-induced graft-versus-host disease

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