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. 2016 May 18;8(339):339ra71.
doi: 10.1126/scitranslmed.aaf2311.

Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice

Yusuke Shono  1 , Melissa D Docampo  1 , Jonathan U Peled  1   2   3 , Suelen M Perobelli  1 , Enrico Velardi  1   4 , Jennifer J Tsai  1 , Ann E Slingerland  1 , Odette M Smith  1 , Lauren F Young  1 , Jyotsna Gupta  1 , Sophia R Lieberman  1 , Hillary V Jay  1 , Katya F Ahr  1 , Kori A Porosnicu Rodriguez  1 , Ke Xu  1 , Marco Calarfiore  1 , Hendrik Poeck  1 , Silvia Caballero  1 , Sean M Devlin  5 , Franck Rapaport  6 , Jarrod A Dudakov  1   7 , Alan M Hanash  2   3 , Boglarka Gyurkocza  2   3 , George F Murphy  8 , Camilla Gomes  8 , Chen Liu  9 , Eli L Moss  10 , Shannon B Falconer  10 , Ami S Bhatt  10 , Ying Taur  2   11 , Eric G Pamer  1   2   11 , Marcel R M van den Brink #  1   2   3 , Robert R Jenq #  2   3
Affiliations

Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice

Yusuke Shono et al. Sci Transl Med. .

Abstract

Intestinal bacteria may modulate the risk of infection and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Allo-HSCT recipients often develop neutropenic fever, which is treated with antibiotics that may target anaerobic bacteria in the gut. We retrospectively examined 857 allo-HSCT recipients and found that treatment of neutropenic fever with imipenem-cilastatin and piperacillin-tazobactam antibiotics was associated with increased GVHD-related mortality at 5 years (21.5% for imipenem-cilastatin-treated patients versus 13.1% for untreated patients, P = 0.025; 19.8% for piperacillin-tazobactam-treated patients versus 11.9% for untreated patients, P = 0.007). However, two other antibiotics also used to treat neutropenic fever, aztreonam and cefepime, were not associated with GVHD-related mortality (P = 0.78 and P = 0.98, respectively). Analysis of stool specimens from allo-HSCT recipients showed that piperacillin-tazobactam administration was associated with perturbation of gut microbial composition. Studies in mice demonstrated aggravated GVHD mortality with imipenem-cilastatin or piperacillin-tazobactam compared to aztreonam (P < 0.01 and P < 0.05, respectively). We found pathological evidence for increased GVHD in the colon of imipenem-cilastatin-treated mice (P < 0.05), but no difference in the concentration of short-chain fatty acids or numbers of regulatory T cells. Notably, imipenem-cilastatin treatment of mice with GVHD led to loss of the protective mucus lining of the colon (P < 0.01) and the compromising of intestinal barrier function (P < 0.05). Sequencing of mouse stool specimens showed an increase in Akkermansia muciniphila (P < 0.001), a commensal bacterium with mucus-degrading capabilities, raising the possibility that mucus degradation may contribute to murine GVHD. We demonstrate an underappreciated risk for the treatment of allo-HSCT recipients with antibiotics that may exacerbate GVHD in the colon.

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Figures

Fig. 1
Fig. 1
Clinical use of imipenem-cilastatin and piperacillin-tazobactam is associated with increased GVHD-related mortality. (A to D) A retrospective cohort of 857 adult patients was identified who received non-T cell depleted allo-HSCT at our center from 1992 to 2015. (A) GVHD-related mortality in patients exposed to aztreonam, cefepime, imipenem-cilastatin or piperacillin-tazobactam. (B) Analyses of overall grade 2-4 GVHD are shown. (C) Analyses of upper GI grade 1 GVHD are shown. (D) Analyses of lower GI grade 1–4 GVHD are shown. (E) A subset of patients who had been treated for neutropenic fever was stratified according to whether they received first-line treatment with imipenem-cilastatin or piperacillin-tazobactam, versus aztreonam or cefepime. Outcomes indicated were depicted by Kaplan-Meier plots and curves compared by the logrank test. *, P < 0.05; **, P < 0.01 in A–E. (F) Intestinal microbiota composition analysis using 16S rRNA sequencing prior to and after beginning treatment with the indicated antibiotics in allo-HSCT recipients. *, P < 0.05 after multiple comparison with Holm-Sidak correction.
Fig. 2
Fig. 2
Imipenem-cilastatin treatment, compared to aztreonam treatment, suppresses anaerobic commensals and elevates GVHD severity in mice. (A) Intestinal microbiota composition analysis using 16S rRNA sequencing prior to and after treatment with the indicated antibiotics in healthy C57BL/6 mice. Mice were treated with subcutaneous (SC) injections of each antibiotic twice a day for two days (100 mg/kg) and stool specimens were collected the following day. Values represent mean ± SEM (n = 6–7). *, P < 0.05; **, P < 0.01 by multiple comparisons corrected by Holm-Sidak test. Data are combined from two independent experiments. (B to D) Lethally irradiated 129S1 recipients were transplanted with MHC-matched C57BL/6 T-cell depleted bone marrow (“BM” in the figure) cells and 1 × 106 C57BL/6 T cells (“T” in the figure). Recipients were treated with aztreonam, cefepime, imipenem-cilastatin or piperacillin-tazobactam (pip-tazo) (100 mg/kg, SC, 3 times a week from day 10 to day 24 following allo-HSCT). (B) Comparison of overall survival, with combined data from two independent experiments (n = 17–18). *, P < 0.05; **, P < 0.01 by Mantel-Cox logrank test. (C) Comparison with control mice with T-cell depleted BM only (no GVHD). Overall survival, with combined data from three independent experiments (n = 15–43). ****, P < 0.0001 by Mantel-Cox logrank test. Tx indicates the period of antibiotics treatment in B and C. (D) Mice with GVHD treated with antibiotics were sacrificed on day 21 and GVHD histology scores in target organs were quantified by a blinded pathologist. Data are combined from three independent experiments (n = 5–20). *, P < 0.05 by Mann-Whitney U test.
Fig. 3
Fig. 3
Treatment with imipenem-cilastatin in GVHD mice results in elevated numbers of donor CD4+ T cells, and increased production of IL-23 in the colon. (A to F) Lethally irradiated 129S1 recipients were transplanted with C57BL/6 T-cell depleted bone marrow cells with 1× 106 C57BL/6 T cells. Recipients were treated with imipenem-cilastatin or aztreonam as described in Fig. 2. (A) Colonic lamina propria-infiltrating leukocytes from recipients were analyzed on day 21 by flow cytometry. Data are combined from two independent experiments. Values represent mean ± SEM (n = 10–14). *, P < 0.05; **, P < 0.01 by Mann-Whitney U test. (B) Concentration of IL-23 in serum, whole small intestine homogenate, and whole colon homogenate. Data are combined from two independent experiments. Values represent mean ± SEM (n = 10–12). *, P < 0.05 by Mann-Whitney U test. The individual plots in the graphs indicate individual animals sacrificed at the time of the analysis in A and B. (C) Lamina propria-infiltrating leukocytes from the colon on day 21 were enriched for CD11b and CD11c simultaneously using a mixture of magnetic beads; IL-23 transcripts were quantified by real time PCR. Data are representative of two independent experiments. Values represent mean ± SEM (n = 3). *, P < 0.05 by Mann-Whitney U test. (D) Immunofluorescence staining was used to quantify pSTAT3, CD3, and DAPI-positive cells in colonic tissue collected on day 21 (the white bar indicates 500 μm). (E) RNA sequencing analysis of the distal colon on day 16 after allo-HSCT. The top 50 regulated genes are shown in the heatmap panel. (F) Immunofluorescence staining was used to quantify CD11b, B220, and DAPI-positive cells in colonic tissue collected on day 21 (the white bar indicates 50 μm). Data are representative of two independent experiments. Values represent mean ± SEM (n = 7–8). **, P < 0.01; ***, P < 0.001; ****; P < 0.0001 in D and F by Mann-Whitney U test.
Fig. 4
Fig. 4
Mice treated with imipenem-cilastatin in the setting of GVHD show increased abundance of Akkermansia. (A) Stool specimens obtained from mice treated with imipenem-cilastatin or aztreonam were collected on day 21 and analyzed by 16S rRNA gene sequencing (as in Fig. 2), followed by principal coordinate analysis of weighted and normalized UniFrac distances. Proportion of variance accounted for by each principal component is indicated. (B and C) Differential taxonomic abundance between aztreonam treated and imipenem-cilastatin treated recipients was analyzed by (B) linear discriminate analysis coupled with effect size measurements (LEfSe) and (C) by LEfSe projected as a cladogram. Data are representative of more than five independent experiments in A to C. (D and E) Shown are comparisons of bacterial abundance at the phylogenetic levels of (D) order, and (E) genus. Data are combined from 6 independent experiments (n = 32–36). ***, P < 0.001 by multiple comparisons, corrected by Holm-Sidak test. (F to H) Stool specimens collected from mice with GVHD treated with antibiotics were collected on day 21, and evaluated by metagenomic shotgun sequence analysis. (F) Comparison of bacterial species abundance determined by taxonomy. Numbers 1 through 6 along the x-axis represent the individual subjects. (G) Principal component analysis of quantification of sequence reads from KEGG gene orthologs comparing specimens from mice treated with aztreonam and imipenem-cilastatin. (H) Quantification of gene sequences by homology was performed on stool specimens collected on day 21. Amuc_0953, a sulfatase, and Amuc_2164 a glycosyl hydrolase, are two predicted secreted mucolytic genes found in the genome of Akkermansia muciniphila ATCC BAA-835, isolated from human feces. **, P < 0.01 by Mann-Whitney U test.
Fig. 5
Fig. 5
Mice treated with imipenem-cilastatin in the setting of GVHD result in the loss of the colonic mucus layer, and impaired intestinal barrier function. (A to C) Colon tissues from mouse recipients were fixed by water-free Methanol-Carnoy's fixative on day 21, stained with Periodic acid-Schiff (PAS) stain and visualized by light microscopy. Orange triangles in A indicate the location of the inner mucus layer. Quantification of mucus layer thickness is shown in B. Number of goblet cells are shown in C. Data are representative of two independent experiments. Values represent mean ± SEM (n = 10). **, P < 0.01 by Mann-Whitney U test. (D) Immunostaining for Muc2 (green) of the colon sections with general bacterial 16S rRNA gene FISH probe EUB338 (red) counterstained with Hoechst (blue). Data are representative of two independent experiments (n = 10). Orange arrowheads indicate inner mucus layer; red arrowheads indicate bacteria penetrating beyond the mucus layer and colonic epithelium. The white scale bar indicates 10 μm. (E) Allo-HSCT recipients of bone marrow and T cells treated with aztreonam or imipenen-cilastatin were challenged with oral gavage of FITC-dextran on day 15 post-transplant. The graph shows plasma FITC-dextran concentrations. Data are representative of two independent experiments. Values represent mean ± SEM (n = 6–8). *, P < 0.05 by Mann-Whitney U test.
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