Lactose drives Enterococcus expansion to promote graft-versus-host disease

Abstract

Disruption of intestinal microbial communities appears to underlie many human illnesses, but the mechanisms that promote this dysbiosis and its adverse consequences are poorly understood. In patients who received allogeneic hematopoietic cell transplantation (allo-HCT), we describe a high incidence of enterococcal expansion, which was associated with graft-versus-host disease (GVHD) and mortality. We found that Enterococcus also expands in the mouse gastrointestinal tract after allo-HCT and exacerbates disease severity in gnotobiotic models. Enterococcus growth is dependent on the disaccharide lactose, and dietary lactose depletion attenuates Enterococcus outgrowth and reduces the severity of GVHD in mice. Allo-HCT patients carrying lactose-nonabsorber genotypes showed compromised clearance of postantibiotic Enterococcus domination. We report lactose as a common nutrient that drives expansion of a commensal bacterium that exacerbates an intestinal and systemic inflammatory disease.

Fig. 1.. Enterococcus domination occurs globally and increases risk of GVHD and mortality after allo-HCT.

Fecal microbiota were profiled using 16S rRNA gene sequencing of 1,325 adult allo-HCT recipients. The patients attended one of four HCT centers in different countries: MSKCC (USA), Duke University (USA), Hokkaido University (Japan) and University Hospital Regensburg (Germany). (A) Left panel, cumulative incidence of patients who experienced at least one instance of genus Enterococcus domination of the gut microbiota (domination defined as a genus relative abundance of ≥0.3 (on a unitless scale from 0 to 1) over the course of allo-HCT (day −20 to +24 relative to HCT; using 7-day sliding windows) at different transplant centers; right panel, fraction of fecal specimens with enterococcal domination of the gut microbiota. Domination is defined as the relative genus abundance ≥0.3 in at least one sample between indicated days. (B) Relative abundance of different Enterococcus spp. in the microbiota of allo-HCT patients from the MSKCC and multicenter-validation cohort over the course of HCT determined by 16S rRNA gene sequencing of fecal samples; each point represents a fecal sample, color indicates the different Enterococcus spp.; the red dotted-line indicates the threshold for domination set at a relative abundance ≥0.3. (C) Overall survival and cumulative incidence of GVHD-related mortality in the T-cell replete graft recipients in the MSKCC patient cohort (see table S3), stratified into non-dominated and Enterococcus-dominated groups (domination is defined as the relative genus abundance ≥0.3 in at least one sample between day 0 and +21). (D) Overall survival and cumulative incidence of GVHD-related mortality in Enterococcus-dominated (at genus level) vs. non-dominated allo-HCT patients in the combined multicenter-validation cohort (table S3). Clinical outcomes in C and D were analyzed using the R packages survival and cmprsk. Wald p values <0.05 signify higher risks (hazard ratios, HR) of mortality among patients with Enterococcus domination as compared with those without domination.

Fig. 2.. Enterococcus dominates mouse gut microbiota after HCT and can exacerbate GVHD.

(A) High-density sampling and 16S rRNA gene sequencing of fecal microbiota from 129S1/Sv mice (1 box = 1 mouse) receiving bone marrow (BM; upper row) or T cell-replete BM (BM+T; 2×10⁶ T cells; lower row); right panel, BM+ T cell transplanted mice develop lethal GVHD as shown by survival analysis. (B) Left panel, relative abundance of the genus Enterococcus of BALB/c host mice transplanted with C57BL/6 BM or BM+T (1×10⁶ T cells) at different time points relative to HCT; colony forming units (CFUs) of enterococci in fecal samples and mesenteric lymph nodes (mLN). Scatter plot data show mean ± S.E.M; right panel, survival of BALB/c recipient mice after HCT (BM vs. BM + 1×10⁶ T cells). (C) Schematic showing HCT of LP/J BM vs. BM+T (4×10⁶ T cells) into C57BL/6 mice after chemotherapy conditioning; relative abundance of the genus Enterococcus in the feces of transplanted mice at different time points relative to HCT; right, comparison of overall survival between BM and BM+T mice. (D) Schematic showing colonization of germ-free C57BL/6 mice with a 6-strain minimal microbiota with (+EF) or without (-EF) E. faecalis OG1RF (2×10⁷ CFUs per mouse); after 14 days, colonized mice received chemo conditioning with busulfan/cyclophosphamide and, subsequently, an HCT of LP/J BM vs. BM+T (4×10⁶ T cells). Middle panel, comparison of overall survival; right, relative abundances of E. faecalis spiked to the minimal microbiota in the EF+ group with samples collected at the day of HCT (day 0) and 7 days later (n=4–11/group; p = 0.09, paired testing of relative abundances of enterococci of day 0 vs BM+T day 7). Scatter plot data are presented as mean ±S.E.M. **p<0.01, ***p<0.001 (independent T test for BM vs. BM+T); survival data were statistically analyzed by Mantel-Cox log-rank test.

Fig. 3.. Metagenomic and metabolomic analyses of Enterococcus-dominated fecal specimens in human HCT patients and mice

(A) Left, differential abundances of shotgun-sequenced and HUMAnN2-annotated bacterial metabolic pathways between paired pre- and post-HCT fecal samples from MSKCC patients who received allo-HCT for acute myeloid leukemia (AML) analyzed by linear discriminant analysis (LDA) coupled with effect size measurements (LEfSe). (definitions: pre: day −8 to −1; post: day 3–25 after allo-HCT). Right panel, pie chart showing mean relative abundances of bacterial genera (analyzed by MetaPhlAn2) found in patient fecal samples pre- and post-HCT; data are aggregated across all patients. (B) LEfSe analysis of bacterial metabolic pathway abundances in HCT day +7 fecal samples of 129S1/SV mice transplanted with C57BL/6 BM vs BM+T (2×10⁶ T cells) (see Fig. 2A). (C) Left panel, pie chart with metabolic pathway abundances determined by whole genome sequencing of E. faecalis (isolated from feces of a BALB/c GVHD mouse, day +7 after HCT) and E. faecium (human allo-HCT fecal isolate; only pathways with an abundance ≥ 2% are shown). (D) Left panel, in vitro growth of E. faecalis (mouse GVHD isolate; upper panel) and E. faecium (human allo-HCT isolate; lower panel) in non-treated BHI broth or in BHI broth pretreated with lactase. Right panel, E. faecalis or E. faecium incubated in lactase-pretreated BHI were put into regular BHI broth after 8h to assess growth dynamics in regular BHI (grey symbol); 4 experiments combined, values represent mean ±S.E.M. (E) Left panel, fecal butyrate concentrations (mean ±S.E.M.) from pre- and post-transplant fecal samples from AML patients from MSKCC who received allo-HCT and were selected on a highly diverse pre-HCT microbiota and a post-transplant E. faecium domination (by 16S rRNA gene sequencing; 6 (out of 8) patients are presented in Fig. 3A; right panel), correlation of butyrate concentrations with relative abundances of the genus Enterococcus (n=139 patients; 8 patients from Fig. 3E in the left panel), and 131 allo-HCT patients from a dataset published by Haak et al. (29); statistical analysis was performed using Kendall’s tau rank correlation coefficient. (F) Stool samples were collected at the time of engraftment (~24 days after allo-HCT). Data show Kendall’s tau rank correlation of relative abundances of the genera Clostridium and Enterococcus from the data-set of Haak et al. (G) Butyrate concentration (mean ±S.E.M.) in cecal contents of BALB/c mice transplanted with C57BL/6 BM or BM+T (1×10⁶ T cells) at day 7 after HCT. Statistical analysis: *p<0.05 (paired T-test (E) or independent T-test for (F)).

Fig. 4.. Lactose-free diet reduces experimental GVHD and lactase genotypes associated with microbiota dynamics after allo-HCT in humans.

(A) Schematic showing that BALB/c recipient mice received C57BL/6 BM or BM + T (5×10⁵ T cells) and were fed control chow (ctr) vs. lactose-free chow (LF) from day −7 to 14 relative to transplant; comparison of survival between BM and BM+T mice (middle panel) and relative abundance of the genus Enterococcus in mouse feces (right panel). Scatter plot data presented as mean ±S.E.M; *p<0.05 (independent T test). (B) Schematic showing HCT of LP/J BM vs. BM+T (4×10⁶ T cells) into C57BL/6 mice after chemotherapy conditioning; comparison of survival between BM and BM+T mice (middle panel) and relative abundance of the genus Enterococcus at different time points relative to HCT (right panel). Scatter plot data presented as mean ±S.E.M; *p<0.05 (independent T test) (C) Left, relative abundance (log10) of Enterococcus (genus) by days relative to the day of antibiotic cessation (broad-spectrum antibiotics for neutropenic fever: either piperacillin-tazobactam i.v., imipenem-cilastatin i.v., or meropenem iv); box plot-inserts display the median relative abundances of the genus Enterococcus of time binned in the indicated day ranges relative to antibiotic cessation; whiskers represent maximum and minimum. Statistical analysis of box plot data: *p<0.05, ***p<0.001 (Wilcoxon rank test). (D) Cumulative incidence of acute GVHD grade 2–4 in rs4988235 SNP-genotyped MSKCC patients (T cell-depleted grafts excluded; graft source: BM/PBSC unmodified = 213 patients; cord blood = 102 patients; C/C = 175, T/C+T/T = 140). The cumulative incidence of Grade 2–4 acute GVHD was compared between genotype groups using the R package cmprsk. HR, hazard ratio.