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. 2021 Feb 8;6(3):e136841.
doi: 10.1172/jci.insight.136841.

A single strain of Bacteroides fragilis protects gut integrity and reduces GVHD

M Hanief Sofi  1 , Yongxia Wu  1 , Taylor Ticer  1 , Steven Schutt  1 , David Bastian  1 , Hee-Jin Choi  1 , Linlu Tian  1 , Corey Mealer  1 , Chen Liu  2 , Caroline Westwater  1   3 , Kent E Armeson  4 , Alexander V Alekseyenko  3   4 , Xue-Zhong Yu  1
Affiliations

A single strain of Bacteroides fragilis protects gut integrity and reduces GVHD

M Hanief Sofi et al. JCI Insight. .

Abstract

Graft-versus-host disease (GVHD) is a pathological process caused by an exaggerated donor lymphocyte response to host antigens after allogeneic hematopoietic cell transplantation (allo-HCT). Donor T cells undergo extensive clonal expansion and differentiation, which culminate in damage to recipient target organs. Damage to the gastrointestinal tract is a main contributor to morbidity and mortality. The loss of diversity among intestinal bacteria caused by pretransplant conditioning regimens leads to an outgrowth of opportunistic pathogens and exacerbated GVHD after allo-HCT. Using murine models of allo-HCT, we found that an increase of Bacteroides in the intestinal microbiota of the recipients was associated with reduced GVHD in mice given fecal microbial transplantation. Administration of Bacteroides fragilis through oral gavage increased gut microbiota diversity and beneficial commensal bacteria and significantly ameliorated acute and chronic GVHD development. Preservation of gut integrity following B. fragilis exposure was likely attributed to increased short chain fatty acids, IL-22, and regulatory T cells, which in turn improved gut tight junction integrity and reduced inflammatory cytokine production of pathogenic T cells. The current study provides a proof of concept that a single strain of commensal bacteria can be a safe and effective means to protect gut integrity and ameliorate GVHD after allo-HCT VSports手机版. .

Keywords: Adaptive immunity; Bone marrow transplantation; Cancer immunotherapy; Immunology; Transplantation V体育安卓版. .

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. FMT from Taconic donor mice reduces GVHD.
BALB/c or BD2F1 mice were treated with busulfan at 20 mg/kg and cyclophosphamide at 120 mg/kg daily for 6 days and then injected with 15 × 106/mouse T cell–depleted BM (TCD-BM) alone or plus 15 × 106 total splenocytes for BALB/c and 30 × 106/mouse splenocytes for BD2F1 from normal B6 mice. The bedding of recipient cages was replaced with bedding without (control) or with fecal pellets from Taconic B6 mice. The FMT procedure was done thrice a week starting at 2 weeks prior to BMT and then weekly for a month post-BMT. Recipients were monitored for clinical score (A and C) and survival (B and D) (n = 9–15 per group). Data shown as mean ± SEM were pooled from 2 replicate experiments. Statistical tests: (A and C) 1-way ANOVA, (B and D) log-rank (Mantel-Cox). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. FMT effects on T cell activation and differentiation.
B6 BALB/c BMT was initiated and FMT was administered as described in Figure 1. Three weeks after BMT, spleens and livers were collected from the recipients, and mononuclear cells were isolated and subjected to cell counting and FACS staining for surface H2Kb (donor MHC), CD4, CD8, and intracellular IFN-γ and Foxp3. CD4 and CD8 expression was shown on gated live H2Kb+ donor cells (A and B). IFN-γ and Foxp3 (A and B) expression were shown on gated live H2Kb+CD4+ donor cells. Data represent 1 of 3–5 mice in each group of recipients. The percentage of IFN-γ+ or Foxp3+ donor (H2Kb+Ly5.1–) CD4+ (C and E), CD8+, and IFN-γ+ cells (D and F) in recipient spleen and liver are shown, respectively. Data shown as mean ± SEM are from 1 of 2 representative experiments. Significance was determined by Student’s t test. Asterisks indicate statistical significance *P < 0.05, **P < 0.01.
Figure 3
Figure 3. B. fragilis ameliorates GVHD.
BALB/c (A and B) or BD2F1 (C and D) mice were preconditioned and BMT performed as described in Figure 1. The recipients were administered thrice a week through oral gavage with approximately 109 live B. fragilis CFU (WT B. fragilis) from 2 weeks before BMT and then weekly until up to a month after BMT. Recipients were monitored for clinical score (A–C) and survival (B–D) until 80 days post-BMT (n = 10 per group). Data shown were pooled from 2 replicate experiments. In a similar setting, liver, lung, SI, colon, and skin were collected from the recipients 3 weeks post-BMT. Pathological score means ± SD of GVHD target organs are depicted (E). Data shown as mean ± SEM were pooled from 3 replicate experiments. Asterisks indicate statistical significance *P < 0.05, **P < 0.01.
Figure 4
Figure 4. B. fragilis affects T cell activation and differentiation.
B6 BALB/c BMT was initiated and B. fragilis was administered as described in Figure 3. Three weeks after BMT, spleen, liver, and gut (small and large intestine together) were collected from the recipients, and mononuclear cells were isolated and subjected to cell counting and FACS staining for surface H2Kb (donor MHC), CD4, CD8, and intracellular IFN-γ and Foxp3. CD4 and CD8 expression were shown on gated live H2Kb+ donor cells (A–C). IFN-γ, Foxp3, and IL-17 expression were shown on gated live H2Kb+CD4+ donor cells and IFN-γ on gated live H2Kb+CD8+ donor cells. The percentage of IFN-γ+, Foxp3+ from (H2Kb+Ly5.1–) CD4+ (D and F) and IFN-γ+, Foxp3+, and IL-17+ from (H2Kb+Ly5.1–) CD4+ T cells shown in recipient spleen, liver, and (H) gut, respectively. IFN-γ from CD8+ T cells shown in recipient spleen and liver, respectively (E and G). Percentage CXCR3+ cells is summarized on donor-derived CD4+ and CD8+ cells in recipient spleen (I). Data shown as mean ± SEM are from 1 of 2 representative experiments. Significance was determined by Student’s t test. Asterisks indicate statistical significance *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5. Impact of FMT and B. fragilis on recipient microbiota after allogenic BMT.
B6 BALB/c was initiated and FMT and B. fragilis were administered as described in Figure 1 and Figure 3, respectively. Thirty days after BMT, ileums were collected from the recipients and extracted for total DNA, which was used for 16S rRNA sequencing. (A) Bacterial composition of abundantly expressed (>1% abundance in at least 1 sample) genus-level taxa is depicted, and nonparametric 1-way Kruskal-Wallis ANOVA identifies 12 genera to be statistically different after false discovery correction (q < 0.1). (B) Diversity among the groups is shown by Shannon diversity index, which demonstrates a statistically significant linear trend (P < 0.007).
Figure 6
Figure 6. B. fragilis increases SCFAs in the recipient intestine after BMT.
B6 BALB/c BMT was initiated and B. fragilis was administered as described in Figure 3. BALB/c recipients were administered FITC-dextran by oral gavage and deprived of food and water for 4 hours on day 7 post-BMT. Peripheral blood was collected from the recipients and concentrations of FITC-dextran were measured (A). Data shown are from 2 representative experiments with each mouse sample in triplicates of 4–8 mice from each group. Three weeks after BMT, recipient ileums were collected and snap-frozen in dry ice. SCFAs were measured in intestinal tissues. The expression of different SCFAs are shown (B). The data shown here are from 1 of the 2 representative experiments with 3–4 mice from each group. The quantitative expression of different genes is shown (C). Data from 1 of the 2 representative experiments with each mouse sample in triplicates of 3–4 mice from each group. Significance is determined by 1-way ANOVA (using multiple comparison test). Asterisks indicate statistical significance *P < 0.05, **P < 0.01, **P < 0.001.
Figure 7
Figure 7. Reshaping commensal microbiota using B. fragilis reduces GVHD.
Recipient BALB/c mice were treated with a cocktail of broad-spectrum antibiotics for 21 days followed by 2 days of rest. A group of these recipients were administered through oral gavage with either vehicle or live B. fragilis as described in Figure 1. Recipients were monitored for clinical score (A) and survival (B) until 60 days post-BMT (n = 10 per group). Data shown are from 2 combined experiments. (C) Fecal pellets were collected from the recipients on day 30 after BMT and extracted for total DNA, which was used for 16S rRNA sequencing. Data shown are from 1 representative experiment. One-way ANOVA analysis of center log ratio–transformed abundances of major genera (present at >1% in at least 1 sample) is shown (*P < 0.05). (D) PCoA of the Bray-Curtis distances indicates visual and statistically significant separation according to Wd* test (P < 0.007).
Figure 8
Figure 8. B. fragilis effects on GVL response.
B6→BALB/c BMT was initiated and recipients were treated as shown in Figure 3 with or without BC-CML cells at a dose of 1 × 106 BC-CML cells per mouse. Recipients were monitored for clinical score (A), survival (B), and (C) tumor mortality until 80 days post-BMT (n = 10 per group). Peripheral blood from recipients was collected periodically starting 21 days after transplant and analyzed via flow cytometry for expression of GFP+ BC-CML cells. Percentage of GFP+ cells is shown in recipient blood (D and E). Data shown as mean ± SEM were pooled from 2 replicate experiments. Significance is determined by 1-way ANOVA (using multiple comparison test). Asterisks indicate statistical significance *P < 0.05, ***P < 0.001.
Figure 9
Figure 9. B. fragilis is effective in preventing cGVHD.
BALB/c mice were lethally irradiated and transplanted with 5 × 106/mouse TCD-BM (Ly5.1+) alone or plus purified splenocytes (Ly5.2+) (0.5 × 106/mouse) from B6 mice. The recipients were administered live B. fragilis 3 times a week through oral gavage with approximately 109 live B. fragilis CFU for 1 week before BMT, then once a week until up to 1 month after BMT. Recipients were monitored for body weight (A) and clinical score (B) until 60 days post-BMT (n = 10 per group, 1-way ANOVA). Absolute numbers and phenotypes of recipient thymocytes were determined. The percentages of CD4+/CD8+ double-positive thymocytes (C) and total thymocytes (D) are shown. The percentages of IFN-γ+ and IL-17+ on donor CD4+ and IFN-γ+ on CD8+ cells are shown in recipient lymph nodes (MLNs) (E). Percentage IFN-γ+ and IL-17+ on gated donor CD4+ cells is shown in recipient MLNs (F). (G) Expression of Foxp3+ cells on gated donor CD4+ and follicular regulatory cells and (H) percentage of regulatory T cells, follicular regulatory T cells (Foxp3+CXCR5+PD-1+), and follicular helper T cells (Foxp3–CXCR5+PD-1+). (I and J) Splenic B cells were analyzed for B220+ and B220−CD138+, respectively. Data shown as mean ± SEM are from 1 of the 2 replicate experiments. Asterisks indicate statistical significance *P < 0.05, **P < 0.01.
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