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Lactose-driven Enterococcus expansion linked to unfavorable prognosis in GvHD

Jan 28, 2020

Patients undergoing allogenic hematopoietic stem cell transplant (allo-HSCT) have been shown to display a reduced diversity and marked shift in their intestinal microbiota. In particular, commensal Enterococcusspecies are suggested to dominate the fecal microbiota of patients following allo-HSCT, while levels of Clostridiumare speculated to deplete. 1The butyrate-forming functionality of commensal clostridia provides protection against graft- versus-host disease (GvHD). 2Disruption of normal gut microbiota, caused by broad-spectrum antibiotics, has been associated with increased incidence of lethal GvHD and poor overall survival (OS) in humans and mice. 3Pre-clinical mouse studies have uncovered that Enterococcusspecies are involved in the initiation of antigen-presenting cell (APC) and CD4 +/RORγ +T-cell infiltration that results in the establishment of colitis. 4

A study by Stein-Thoeringer, Memorial Sloan Kettering Cancer Center (MSKCC), New York, NY, US, and colleagues, investigated the role of enterococci in the establishment of acute GvHD (aGvHD) in both allo-HSCT recipients and pre-clinical allo-HSCT mouse models. The study also explores the theory that Enterococcusvitality is reliant on lactose metabolism and looks at the effect of dietary lactose and lactose-malabsorption genotypes on the severity of GvHD in humans and mice. 5

MULTICENTER PATIENT STUDY

Study design

  • Patients across four transplant centers underwent allo-HSCT following ablative, reduced intensity or nonmyeloablative conditioning ( Table 1)
  • Patients were divided into two main observation cohorts: 
    1. MSKCC cohort
    2. Multicenter validation cohort: Duke University (US), Hokkaido University (Japan) and University Hospital Regensburg (Germany)
  • Patient fecal microbiota was profiled using 16S rRNA gene sequencing over the course of allo-HSCT (day -30 to +24 relative to HSCT; 7-day sliding windows)
  • Enterococcusdomination was defined as relative genus abundance of ≥30% in any fecal sample
  • Pre- and post-transplant fecal samples were collected from patients receiving allo-HSCT for acute myeloid leukemia (AML) in the MSKCC cohort to investigate the microbial metabolic pathways that characterize domination
  • Identification of patients carrying a lactose malabsorption genotype was carried out using the single nucleotide polymorphism (SNP), rs4988235(-13910*T) in 602 patients from the MSKCC cohort with available pretransplant germline DNA samples

Results

  • Patient characteristics
    • In total 1,325 adult allo-HSCT recipients were recruited across the four centers:
      • 1,101 patients in MSKCC cohort
      • 224 patients in the multicenter validation cohort

Table 1. Clinical characteristics of the patient cohort

Overall Cohort

N=1,325

Institution (%)

 

MSKCC

1,101 (83.1)

Regensburg

79 (6.0)

Duke

79 (6.0)

Hokkaido

66 (5.0)

Age at HSCT, year (mean (sd))

52.9 (12.8)

Sex (male, %)

801 (60.5)

Disease (%)

AML

 

485 (36.6)

MDS/MPN

244 (18.4)

NHL

223 (16.8)

ALL

124 (9.4)

Myeloma

113 (8.5)

CLL

33 (2.5)

CML

29 (2.2)

Hodgkins

30 (2.3)

AA

9 (0.7)

Other

35 (2.6)

Graft type (%)

 

BM/PBSC unmodified     

660 (49.8)

Cord

207 (15.6)

PBSC T-cell depleted

458 (34.6)

Conditioning intensity (%)

 

Ablative              

744 (56.2)

Reduced intensity

466 (35.2)

Nonmyeloablative             

115 (8.7)

AA, aplastic anemia; ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; BM, bone marrow; CLL, chronic lymphocytic leukemia; CML, chronic myeloid leukemia; HSCT, hematopoietic stem cell transplantation; MDS/MPN, myelodysplastic syndromes/myeloproliferative neoplasms; MSKCC, Memorial Sloan Kettering Cancer Centre; NLH, Non-Hodgkin Lymphomas; PBSC, peripheral blood stem cells; sd, standard deviation.

  • Microbiota domination
    • Enterococcusdomination was observed in 65% of patients after allo-HSCT across centers
    • E. faeciumwas the dominant enterococcal species across both cohorts, with 40.1% and 46.0% of patients in the MSKCC and multicenter-validation cohorts respectively, reaching domination between days -20 and +80 of allo-HSCT
    • Enterococcusfecal domination in the early allo-HSCT period (day 0 to +21 post allo-HSCT) was associated with significantly reduced OS in both the MSKCC (HR 1.97; p< 0.001) and combined multicenter-validation (HR 1.95; p= 0.03) cohorts
    • Enterococcusfecal domination in the early allo-HSCT period was associated with increased GvHD-related mortality in both the MSKCC (HR 2.04; p= 0.01) and combined multicenter-validation (HR 5.8; p= 0.002) cohorts
  • Lactose and Enterococcus:
    • Metabolic pathways involved in lactose and galactose degradation were enriched in E. faecium-dominated, post-transplant microbiota but were not common in pre-transplant samples
    • Enterococcal expansion following allo-HSCT was associated with a reduction of Clostridiumand fecal butyrate
    • Enterococcal domination was significantly prolonged in patients with lactose malabsorption following termination of broad-spectrum antibiotics when compared to those patients that had lactose absorber genotypes

PRE-CLINICAL MOUSE STUDY

Study Design

  • Three mouse models of allo-HSCT were utilized to investigate GvHD related mortality:
    1. Major histocompatibility complex (MHC)-matched, minor-antigen-mismatched allo-HSCT (C57BL/6-to-129S1/Sv transplant [C57BL/6→129S1/Sv])
    2. MHC-disparate model after irradiation conditioning (C57BL/6→ BALB/c)
    3. MHC-matched, minor antigen-mismatched after busulfan and cyclophosphamide conditioning (LP/J→ C57BL/6)
  • Mice received bone marrow (BM) or T cell-replete bone marrow (BM+T [2 × 10 6T cells])
  • 16S rRNA gene sequencing was carried out on fecal samples
  • Microbiota domination and GvHD:
    • LP/J→ C57BL/6 mice were colonized with a community of six bacterial strains ( Akkermansia muciniphila, Lactobacillus johnsonii, Blautia producta, Bacteroides sartorii, Clostridium bolteae, and Parabacteroides diastonis) on day -21 of allo-HSCT (LP/J→ gnotobiotic C57BL/6)
      • One group of mice then underwent E. faecalis OG1RFcocolonization on day -21 of allo-HSCT
      • One group of mice then underwent E. faecalis OG1RFcocolonization post-transplant
  • Lactose, Enterococcusand GvHD:
    • Lactose-free chow was fed to C57BL/6→BALB/c and LP/J→ C57BL/6 mice
    • Mice were subjected to chemotherapy conditioning at days-7 to -3 relative of HSCT
    • Flow cytometric analysis of donor T-cells was carried out on day +14 of allo-HSCT

Results

  • Microbiota domination and GvHD:
    • Across the gnotobiotic mouse models, Enterococcusdominated the gut microbiota at day +8 of HSCT of all mice that developed aGvHD
    • E. faecalisexpansion was independent of antibiotic administration and dependent on GvHD development, as it was not seen in control mice receiving T cell–depleted allografts and therefore did not develop GvHD
    • Mice receiving BM+T allo-HSCT elicited significantly reduced OS in mouse models 1 (p= 0.013), 2 (p< 0.001) and 3 (p< 0.01), compared to those receiving BM allo-HSCT
    • E. faecaliscolonization resulted in significantly elevated interferon-γ serum concentrations
    • E. faecaliscolonization resulted in increased numbers of donor T-cells, activated CD4 +T-cells and CD4 +RORg +T helper 17 (T H17) cells in colon lamina propria
    • Post-transplant administration of  E. faecalis OG1RFsignificantly reduced OS (p< 0.001) and aggravated GvHD in mice receiving BM+T allo-HSCT
  • Lactose, Enterococcusand GvHD:
    • Metabolic pathways involved in lactose and galactose degradation were enriched in E. faecalis–dominated, post-transplant microbiota of mice with GvHD
    • Enterococcal expansion was associated with a reduction in Clostridiumand fecal butyrate in mice with GvHD
    • A lactose-free diet significantly reduced the abundance of post-transplant Enterococcusand incidence of experimental GvHD
    • A lactose-free diet resulted in a reduction of activated and proliferating CD4 +T-cells and CD4 +Tbet +(T H1) T-cells
    • A lactose-free diet significantly increased post-transplant survival (%) in mice that had undergone allo-HSCT, both with (p<0.01) or without (p<0.05) prior chemotherapeutic conditioning

Conclusions

  • Fecal Enterococcusspecies domination following allo-HSCT is a risk factor for aGvHD development and increased GvHD-associated mortalities
  • Lactose fuels the expansion of enterococci and could be targeted to prevent intestinal and systemic T-cell activation with subsequent development of diseases such as GvHD
  • Maintenance of enterococcal domination following administration of broad-spectrum antibiotics may be reduced by restricting the intake of lactose
  • Observations from the study provide rationale for a lactose-free diet, to improve patient outcome following allo-HSCT
  1. Taur Y. et al., Intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation. Clin Infect Dis. 2012 Oct; 55(7): 905–14. DOI: 10.1093/cid/cis580
  2. Mathewson N.D. et al., Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease. Nat Immunol. 2016 May; 17(5): 505–513. DOI: 10.1038/ni.3400
  3. Shono Y. et al., Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice. Sci Transl Med. 2016 May 18; 8(339): 339ra71. DOI: 10.1126/scitranslmed.aaf2311
  4. Geva-Zatorsky N. et al., Mining the Human Gut Microbiota for Immunomodulatory Organisms. Cell. 2017 Feb 23; 168(5): 928–943. DOI: 10.1016/j.cell.2017.01.022
  5. Stein-Thoeringer C.K. et al., Lactose drives Enterococcus expansion to promote graft-versus-host disease. Science. 2019 Nov 29; 366(6469): 1143–1149. DOI: 10.1126/science.aax3760