microbiote en hématologie -...

Microbiote en hématologie

Florent Malard, MD, PhD Hématologie Clinique et Thérapie Cellulaire

Université Pierre & Marie Curie Hôpital Saint-Antoine

Paris

ü  100 trillion microorganisms: 10 times the number of human cells in our body1

ü Predominantly not yet cultured to date (~70% of non-cultured dominant species)1

ü Central to Food-Microbiota-Host interactions (microbiome and human genome crosstalks in immune,

neural and endocrine functions)2 ü Mutualistic association derived from a long co-evolution2 ü  The microbiome can be modulated (unlike the human

genome)3

The Human Intestinal Microbiota

1.  Savage DC. Annu Rev Microbiol. 1977;31:107-33. 2.  Ratner RE. ADA/JDRF Research Symposium Summary. March 2016. 3.  Ursell LK et al. Gastroenterology. 2014;146(6):1470-6.

Nutritional transition : potentially major impact on our gut bugs

Courtesy of Walter Wahli

1.3 MY

100’000 to 130’000 generations with fiber-rich diet (>60% of energy from fruits, veg, roots, nuts,..)

2 gen. with <10% fiber diet

‘Natural’ History of the Genus Homo

The hygiene hypothesis (Bach JF. N Engl J Med. 2002;347(12):911-20.) The “old friends hypothesis” (Rook GA. Clin Rev Allergy Immunol. 2012;42(1):5-15.)

Missing Microbes (Blaser MJ. 2014)

Altering mother-to-child vertical transfer of the microbiota, via

•  Duration of gestation (preterm births) •  Mode of delivery (cesarean section >30% in Europe; >80% in different

places in the world) •  Hygiene of neonatal environment •  Exposure to antibiotics in mothers, neonates and infants •  Early life food and feeding mode (formula milk; weaning diet) •  Maternal microbiomes as a consequence of environmental drivers

including long term dietary habits Through several generations

Perinatal management and environment: potentially major impact on our gut bugs

‘Natural’ History of the Genus Homo

Determined in Early Childhood, Species Richness Differentiates Human Population-Groups

Yatsunenko T et al. Nature. 2012;486(7402):222-7.

Species richness

Spe

cies

ric

hnes

s

Impact, over generations, of : •  Birth ‘management’? •  Nutritional transition? •  Repeated antibiotherapy? Etc.

… are we altering human biology?

determined in early childhood

Fecal samples from N=531 individuals

OTU, operational taxonomic units.

*P < 0.05, **P < 0.005

Critical Transition in Chronic Immune Diseases (concept)

Sustained alteration of the Gut Microbiota

Sustained low grade to overt inflammation

Dysbiosis

stressor

stressor

Homo sapiens ‘dysbioticus’

Eubiosis of the gut microbiota

Physiological Immune tone symbiosis

Reversible imbalance of the gut microbiota crosstalk

genetic predisposition, infection, diet, lifestyle & environmental

triggers

Transient low grade inflammation

Homo sapiens ‘modernicus’

Homo sapiens ‘symbioticus’

Based on Scheffer M et al. Nature. 2001;413(6856):591-6 and Kéfi S et al. PLoS One. 2014;9(3):e92097.

Microbiota Modulation: from Nutrition to Bacteriotherapy

Developing Strategies to Address Microbiota and HSCT

Probiotics •  Re-introducing endogenous

flora (autologous FMT) •  Re-introducing selected

bacteria with beneficial potential

Postbiotics

•  Identifying and introducing bacterial metabolites that mediate the anti-inflammatory effects

Prebiotics • Encourage “normal” eating • Gastric nutritional supplementation • Flora-targeted nutritional supplements

Antibiotics

•  Selecting antibiotics that spare bacteria with beneficial potential

HSCT, hematopoietic stem cell transplantation.

Chemotherapy and Microbiota Cooperation: Complex Microbiota to Reinforce Immunotherapy

Translocation of Gram+ bacteria into secondary lymphoid

organs

Priming of Th1/Th17 (pTh17)

Late recruitment of CD3+TILs and Th1 into tumor bed

T-bet Rorγ

t

IFNγ

IL-17

2 3

CTX

Homeostasis

Loss of integrity

and dysbiosis

1

Antibiotics

Viaud S et al. Cell Death Differ. 2015;22(2):199-214. Iida N et al. Science. 2013;342(6161):967-70.

CTX, cyclophosphamide, TILs, tumour infiltrating lymphocytes.

Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota (Vétizou M et al. Science. 2015;350(6264):1079-84.)

•  Tumors in antibiotic-treated or germ free mice did not respond to CTLA-4 blockade

•  This defect is overcome by gavage with B. fragilis, by immunization with B. fragilis components, or by adoptive transfer of B. fragilis-specific T cells.

•  CTLA-4 blockade favors the outgrowth of B. fragilis with anticancer properties.

Gut Microbiota Affects Checkpoint Blockade Efficacy

CTLA, Cytotoxic T lymphocyte antigen-4.

Snyder A et al. Science. 2015;350(6264):1031-2.

Commensal Bifidobacterium Promotes Antitumor Immunity and Facilitates Anti PD-L1 Efficacy in a Mouse Model of Melanoma

(Sivan A et al. Science. 2015;350(6264):1084-9.)

•  Tumor growth in mice is dependent of gut microbiota (Bifidobacterium associated with antitumor effects)

•  Combination treatment with PD-L1 blockade and Bifidobacterium abolished tumor growth

•  This effect was mediated by augmented dendritic cell function and CD8+ T cell priming

PD-L1, programmed cell death protein 1 ligand 1.

The composition of intestinal microbiota can be manipulated to improve immune responses against cancer

GVHD and the Microbiota

•  Germ-free mice receiving allo-BMT have less GVHD mortality (Jones JM. et al. Radiat Res. 1971;45(3):577-88. Whangbo J et al. Bone Marrow Transplant. 2017;52(2):183-190.)

•  Mice receiving gut decontamination (neomycin/streptomycin) after allo-BMT have less GVHD mortality (van Bekkum DW et al. J Natl Cancer Inst. 1974;52(2):401-4. Shono Y et al. Sci Transl Med. 2016; 8(339): 339ra71.)

•  Lactobacillus can reduce GVHD mortality (Gerbitz A et al. Blood. 2004;103(11):4365-7)

•  Prospective study: broad antibiotics regimen that also eliminated anaerobes resulted in less acute GVHD; no effect on OS (Beelen DW et al. Blood. 1999;93(10):3267-75.)

•  Today: the “germ-free conditions” model in SCT patients is still highly controversial (some centers are still using gut decontamination) (Shono Y. et al. Int J Hematol. 2015;101(5):428-37.)

Allo-BMT, allogeneic bone marrow transplant; GVHD, graft-versus-host disease; OS, overall survival; SCT, stem cell transplantation.

•  Aplastic anemia patients

•  Laminar airflow, "sterile" food, and treated with oral non-absorbable antibiotics and skin cleaning, with a minimum stay in the rooms for 50 days!

GVHD and the Microbiota: Historical Data

GVHD, graft-versus-host disease .

Storb R et al. N Engl J Med. 1983;308(6):302-7.

N=130 patients with severe aplastic anemia conditioned with cyclophosphamide for transplantation of marrow from HLA -identical siblings

Schwab L et al. Nat Med 2014

Bacterial transloca-on a.er TBI recruit neutrophil and induce GVHD L E T T E R S

NATURE MEDICINE VOLUME 20 | NUMBER 6 | JUNE 2014 649

also performed an experiment comparable to the one described in Figure 1f at an animal facility in the United States, where the mice presumably have a different microflora. We saw improvement in the survival of the US mice when they received the anti-Ly6G antibody after TBI and allo-HCT (Fig. 1j) or when mice (Germany) were condi-tioned with busulphan and cyclophosphamide (BU/CY) chemotherapy (Supplementary Fig. 1a) and when we used a minor histocompat-ibility antigen–mismatched model (BALB/B into C57BL/6, Germany) of allo-HCT (Supplementary Fig. 1b).

To determine the anatomical locations in which neutrophils accu-mulate upon allo-HCT, we used a recently described bioluminescence

method5 that detects a key neutrophil enzyme, active myeloperoxi-dase. We observed peak myeloperoxidase activity on days 3 and 4 following allo-HCT and used complete Freund’s adjuvant as a posi-tive control (Fig. 2a,b). Nonmyeloablative conditioning (fludarabine and cyclophosphamide) led to less myeloperoxidase signal compared to myeloablative conditioning (busulphan and cyclophosphamide) (Fig. 2b). To more precisely locate the myeloperoxidase signal, we performed ex vivo imaging of the organs from the abdominal region where we detected the signal. On day 3, the main signal came from the terminal ileum (Fig. 2c). Histological analysis of the terminal ileum confirmed increased infiltration of Ly6G+ cells (Fig. 2d) and

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200

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Figure 1 Anti-Ly6G antibody treatment improves survival after MHC-mismatched allo-HCT. (a,b) Microarray-based analysis of RNA expression levels of multiple genes in the terminal ileum of BALB/c mice after TBI (9 Gy), n = 4 for each group. (a) Tile display for the 50 most significantly regulated genes, expressed by Robust Multichip Average (RMA) signal values of 4 individual samples from the following groups: untreated, 24 h after TBI and 48 h after TBI. Red square, Ly6G is the second most significantly regulated gene. (b) Expression levels of Ly6g in the intestines at different time points after TBI. Each data point represents an individual animal. Untreated versus 24 h, P < 0.0001; untreated versus 48 h, P < 0.0001, Benjamini-Hochberg q value 2.675 × 10−5. An unpaired Student’s t-test (two- sided) was applied to calculate P values. All P values were calculated using a Benjamini-Hochberg multiple-testing correction. The experiment was performed once. (c,d) The proportion of CD45+CD11b+Ly6G+ neutrophils in peripheral blood at different time points after allo-HCT from C57BL/6 into BALB/c mice. Relative (c) and absolute (d) numbers of neutrophils are shown from mice injected with 500 g anti-Ly6G antibody or isotype control antibody per mouse on day 1 of allo-HCT. Bar diagrams represent mean s.e.m., n = 6 for each time point, with results pooled from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test (two-sided). (e) The proportion of monocytes and macrophages in peripheral blood at day 2 after allo-HCT from C57BL/6 into BALB/c mice. Bar diagrams represent mean s.e.m., n = 6 for each time point (except for day 3 where n = 7), with results pooled from two independent experiments. NS, not significant, P > 0.05, unpaired Student’s t-test (two-sided). (f) Survival of BALB/c recipients receiving allo-HCT from C57BL/6 donors (n = 12 per group). Data are pooled from two experiments (6 mice per experiment at Freiburg University, Germany). Isotype IgG versus anti-Ly6G, P = 0.007. Differences in animal survival were analyzed by log-rank test. (g) IFN- in the serum of the indicated groups on day 7 (from mice injected with 500 g anti-Ly6G antibody per mouse on day 1 of allo-HCT). Data (mean s.e.m.) are pooled from two independent experiments. *P < 0.05, unpaired Student’s t-test (two-sided). (h) Scored histopathologic changes in the small intestines, large intestines and liver isolated on day 7 after allo-HCT (from mice injected with 250 g anti-Ly6G antibody per mouse on day 1 of allo-HCT). Data (mean s.e.m.) are pooled from two independent experiments. **P < 0.01, ***P < 0.001, unpaired Student’s t-test (two-sided). (i) Percentage survival of syn-HCT recipients (B6D2F1 in B6D2F1 model) injected with 500 g anti-Ly6G antibody or isotype control antibody on days 1 to 5 after syn-HCT. Results are representative of two experiments. (j) Percentage survival of BALB/c recipients receiving allo-HCT from C57BL/6 donors. Data are pooled from the three independent experiments (Shreveport, Louisiana, USA, 1 experiment; Salt Lake City, Utah, USA, 2 experiments). Isotype IgG versus anti-Ly6G, P = 0.0014. Differences in animal survival were analyzed by log-rank test.

L E T T E R S

652 VOLUME 20 | NUMBER 6 | JUNE 2014 NATURE MEDICINE

rectum, and signals from SPIO+ E. coli in a dipole-shaped pattern had appeared in the tissue surrounding the intestinal wall (Supplementary Fig. 2c,d). We detected SPIO in the mesenteric lymph follicles close to the intestinal wall (Supplementary Fig. 2e), saw co-localization of neutrophils and bacteria in the intestinal tissue (Supplementary Fig. 3a–g) and, consistent with intestinal leakage, detected increased serum lipopolysaccharide (LPS) levels after TBI followed by allo-HCT (Supplementary Fig. 3h).

To assess whether the recruitment of neutrophils to the ileum following TBI might be caused by invading bacteria, we compared the frequency of neutrophils in the ilea of mice with normal flora with that in the ilea of mice raised under germ-free conditions. Germ-free mice did not show the increased frequency of neutrophils that we observed in the ilea of mice with normal flora after TBI (Fig. 4a), which is compatible with the finding that intestinal inflammation is regulated by the microbiota16.

The marker Ly6G, used to deplete neutrophils, is also present on other cell types17. Therefore, to validate the role of neutrophils in GVHD, we generated chimeric mice using BM from Mcl1fl/fl; Lyz2tm1(cre) mice, which specifically lack neutrophils18. We confirmed the selective lack of neutrophils (Fig. 4b), and found that Mcl1fl/fl; Lyz2tm1(cre) BM chimeras had improved survival (Fig. 4c) and reduced histological GVHD severity after allo-HCT (Fig. 4d) compared to chimeric Mcl1+/+; Lyz2tm1(cre) controls.

To clarify the link between invading bacteria and neutrophil activa-tion, we depleted endogenous neutrophils via injection of anti-Ly6G antibody and then transferred HoxB8 neutrophil progenitors expressing GFP9 into the mice (Supplementary Fig. 1g and Fig. 4e). To assess the contribution of bacterial activation of neutrophils, we compared WT neutrophils to neutrophils lacking TLR2, TLR3, TLR4, TLR7 and TLR9 (TLR2/3/4/7/9−). As these cells are unresponsive to most bacterial pathogen–associated molecular patterns, we expected them to be less

dMcl1fl/fl; Lyz2tm1(cre) (n = 8)Mcl1+/+; Lyz2tm1(cre) (n = 8)

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his

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3

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4

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ls o

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)

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NSP = 0.025

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4

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Time after allo-HCT (d)


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200

150

100

50

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100

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80

60

40

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Figure 4 Neutrophil recruitment into the intestines is dependent on commensal microflora, and neutrophil-mediated tissue damage requires neutrophil activation via TLRs. (a) The percentage of intestinal neutrophils (CD45+, F4-80−, Gr-1+ and CD11b+) relative to all intestinal leukocytes is displayed for irradiated (3 d after 9-Gy TBI) and untreated mice under normal or germ-free conditions. Data are pooled from three experiments. Normal flora and untreated or normal flora and irradiated, n = 6; germ-free and irradiated, n = 6; germ-free and untreated, n = 8. (b) Frequency of neutrophils and other cells in the peripheral blood on day 28 after syn-HCT in BM chimeras with BM derived from Mcl1fl/fl; Lyz2tm1(cre) or littermate control (Mcl1+/+; Lyz2tm1(cre)) mice. Data are pooled from two experiments. (c,d) BM chimeras with BM derived from Mcl1fl/fl; Lyz2tm1(cre) or Mcl1+/+; Lyz2tm1(cre) mice after undergoing allo-HCT as described for the BALB/c C57BL/6 combination. (c) Survival of chimeric C57BL/6 mice is shown (Mcl1fl/fl; Lyz2tm1(cre) versus Mcl1+/+; Lyz2tm1(cre), P = 0.002). The experiment was performed twice and the results were pooled. (d) Histopathological GVHD severity of chimeric C57BL/6 mice after allo-HCT (BALB/c C57BL/6) is shown (Mcl1fl/fl; Lyz2tm1(cre) versus Mcl1+/+; Lyz2tm1(cre)). The small intestines, large intestines and liver were isolated on day 5 after allo-HCT and scored. The combined score for inflammation and apoptosis is shown. Data are presented as mean s.e.m. (e). Frequency of GFP+ cells analyzed by immunofluorescence microscopy on day 7 in ilea of mice that received allo-HCT as described for the BALB/c C57BL/6 combination and mice that received allo-HCT and recipient-type GFP+ HoxB8 cells overexpressing Bcl-xL (Bcl-xL tg) (P < 0.001). The experiment was performed twice and the results were pooled (n = 14 for each group). (f) Survival of C57BL/6 recipients is shown. Mice received an allo-HCT as described for the BALB/c C57BL/6 combination and recipient type (C57BL/6) HoxB8 cells with a WT genotype, Bcl-xL overexpression or deficiency of TLR2, TLR3, TLR4, TLR7 and TLR9. The experiment was performed twice and the results were pooled. WT versus TLR2/3/4/7/9−, P = 0.003; WT versus Bcl-xL overexpression, P = 0.012. Neutro, neutrophils. (g) The IL-12 and IFN- levels in the serum of the indicated groups on day 5. The groups represent n = 4 (HoxB8 Bcl-xL tg), n = 5 (TLR2/3/4/7/9−) and n = 4 (WT) animals. Data are presented as mean s.e.m. (h) The small intestines, large intestines and liver were isolated on day 5 after allo-HCT and scored. The combined score for inflammation and apoptosis is shown with representative images for the individual groups. Data are presented as mean s.e.m. Histol., histology. (i) Survival of C57BL/6 recipients (BALB/c C57BL/6 combination) is shown for a group that received BM/Tc alone or additional donor-type HoxB8 cells with a WT genotype. BM/Tc alone versus BM/Tc + donor HoxB8 WT cells, P < 0.001. (j) The frequency of myeloperoxidase-positive (MPO+) cells per HPF was correlated with the GVHD severity in patients that had undergone diagnostic colonoscopy after allo-HCT (GVHD grade 0, n = 8; GVHD grade I–II, n = 14; GVHD grade III–IV, n = 15). The patient characteristics are detailed in Supplementary Table 1.

L E T T E R S

652 VOLUME 20 | NUMBER 6 | JUNE 2014 NATURE MEDICINE

rectum, and signals from SPIO+ E. coli in a dipole-shaped pattern had appeared in the tissue surrounding the intestinal wall (Supplementary Fig. 2c,d). We detected SPIO in the mesenteric lymph follicles close to the intestinal wall (Supplementary Fig. 2e), saw co-localization of neutrophils and bacteria in the intestinal tissue (Supplementary Fig. 3a–g) and, consistent with intestinal leakage, detected increased serum lipopolysaccharide (LPS) levels after TBI followed by allo-HCT (Supplementary Fig. 3h).

To assess whether the recruitment of neutrophils to the ileum following TBI might be caused by invading bacteria, we compared the frequency of neutrophils in the ilea of mice with normal flora with that in the ilea of mice raised under germ-free conditions. Germ-free mice did not show the increased frequency of neutrophils that we observed in the ilea of mice with normal flora after TBI (Fig. 4a), which is compatible with the finding that intestinal inflammation is regulated by the microbiota16.

The marker Ly6G, used to deplete neutrophils, is also present on other cell types17. Therefore, to validate the role of neutrophils in GVHD, we generated chimeric mice using BM from Mcl1fl/fl; Lyz2tm1(cre) mice, which specifically lack neutrophils18. We confirmed the selective lack of neutrophils (Fig. 4b), and found that Mcl1fl/fl; Lyz2tm1(cre) BM chimeras had improved survival (Fig. 4c) and reduced histological GVHD severity after allo-HCT (Fig. 4d) compared to chimeric Mcl1+/+; Lyz2tm1(cre) controls.

To clarify the link between invading bacteria and neutrophil activa-tion, we depleted endogenous neutrophils via injection of anti-Ly6G antibody and then transferred HoxB8 neutrophil progenitors expressing GFP9 into the mice (Supplementary Fig. 1g and Fig. 4e). To assess the contribution of bacterial activation of neutrophils, we compared WT neutrophils to neutrophils lacking TLR2, TLR3, TLR4, TLR7 and TLR9 (TLR2/3/4/7/9−). As these cells are unresponsive to most bacterial pathogen–associated molecular patterns, we expected them to be less

dMcl1fl/fl; Lyz2tm1(cre) (n = 8)Mcl1+/+; Lyz2tm1(cre) (n = 8)

GV

HD

his

tolo

gy s

core

P = 0.006

Liver

3

2

1

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HD

his

tolo

gy s

core

P = 0.0002

Largeintestines

4

3

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1

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Smallintestines

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h

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200

150

100

50

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100

50

0

g

Sur

viva

l (%

)0 20 40 60 80 100


100

f BM/Tc/neutro (HoxB8 Bcl-xL tg), n = 10BM/Tc/neutro (HoxB8 WT), n = 10BM/Tc/neutro (HoxB8 TLR2/3/4/7/9–),n = 10

80

60

40

20

00 20 40 60 80


BM/Tc (n = 17)BM/Tc + Donor HoxB8WT (n = 10)

Sur

viva

l (%

) 80

60

40

20

0

100

i

j P = 0.0001

P = 0.016

MP

O+ c

ells

/HP

F

80

60

40

20

00

100

l–ll lll–lVGVHD grade


Bcl-xL tg (n = 4)

TLR2/3/4/7/9– (n = 5)WT (n = 6)

Figure 4 Neutrophil recruitment into the intestines is dependent on commensal microflora, and neutrophil-mediated tissue damage requires neutrophil activation via TLRs. (a) The percentage of intestinal neutrophils (CD45+, F4-80−, Gr-1+ and CD11b+) relative to all intestinal leukocytes is displayed for irradiated (3 d after 9-Gy TBI) and untreated mice under normal or germ-free conditions. Data are pooled from three experiments. Normal flora and untreated or normal flora and irradiated, n = 6; germ-free and irradiated, n = 6; germ-free and untreated, n = 8. (b) Frequency of neutrophils and other cells in the peripheral blood on day 28 after syn-HCT in BM chimeras with BM derived from Mcl1fl/fl; Lyz2tm1(cre) or littermate control (Mcl1+/+; Lyz2tm1(cre)) mice. Data are pooled from two experiments. (c,d) BM chimeras with BM derived from Mcl1fl/fl; Lyz2tm1(cre) or Mcl1+/+; Lyz2tm1(cre) mice after undergoing allo-HCT as described for the BALB/c C57BL/6 combination. (c) Survival of chimeric C57BL/6 mice is shown (Mcl1fl/fl; Lyz2tm1(cre) versus Mcl1+/+; Lyz2tm1(cre), P = 0.002). The experiment was performed twice and the results were pooled. (d) Histopathological GVHD severity of chimeric C57BL/6 mice after allo-HCT (BALB/c C57BL/6) is shown (Mcl1fl/fl; Lyz2tm1(cre) versus Mcl1+/+; Lyz2tm1(cre)). The small intestines, large intestines and liver were isolated on day 5 after allo-HCT and scored. The combined score for inflammation and apoptosis is shown. Data are presented as mean s.e.m. (e). Frequency of GFP+ cells analyzed by immunofluorescence microscopy on day 7 in ilea of mice that received allo-HCT as described for the BALB/c C57BL/6 combination and mice that received allo-HCT and recipient-type GFP+ HoxB8 cells overexpressing Bcl-xL (Bcl-xL tg) (P < 0.001). The experiment was performed twice and the results were pooled (n = 14 for each group). (f) Survival of C57BL/6 recipients is shown. Mice received an allo-HCT as described for the BALB/c C57BL/6 combination and recipient type (C57BL/6) HoxB8 cells with a WT genotype, Bcl-xL overexpression or deficiency of TLR2, TLR3, TLR4, TLR7 and TLR9. The experiment was performed twice and the results were pooled. WT versus TLR2/3/4/7/9−, P = 0.003; WT versus Bcl-xL overexpression, P = 0.012. Neutro, neutrophils. (g) The IL-12 and IFN- levels in the serum of the indicated groups on day 5. The groups represent n = 4 (HoxB8 Bcl-xL tg), n = 5 (TLR2/3/4/7/9−) and n = 4 (WT) animals. Data are presented as mean s.e.m. (h) The small intestines, large intestines and liver were isolated on day 5 after allo-HCT and scored. The combined score for inflammation and apoptosis is shown with representative images for the individual groups. Data are presented as mean s.e.m. Histol., histology. (i) Survival of C57BL/6 recipients (BALB/c C57BL/6 combination) is shown for a group that received BM/Tc alone or additional donor-type HoxB8 cells with a WT genotype. BM/Tc alone versus BM/Tc + donor HoxB8 WT cells, P < 0.001. (j) The frequency of myeloperoxidase-positive (MPO+) cells per HPF was correlated with the GVHD severity in patients that had undergone diagnostic colonoscopy after allo-HCT (GVHD grade 0, n = 8; GVHD grade I–II, n = 14; GVHD grade III–IV, n = 15). The patient characteristics are detailed in Supplementary Table 1.

Schwab L et al. Nat Med 2014

The level of neutrophil infiltra-on correlates with GvHD severity in human GvHD lesions

between models for these 2 outcomes was that disease risk waspredictive of overall survival but not transplant-related mortality. Thefollow-up and outcomes are depicted individually for each subject inFigure 2. Of the 36 deaths, 21 (58%) were transplant related and 15(42%) were due to relapse or progression of disease. Transplant-related deaths were frequently due to GVHD or infection and weremore frequent in patients with lowmicrobiota diversity.We did notobserve any evidence of censoring bias. As inverse Simpson diversitydecreased, the corresponding taxonomic composition of the fecalmicrobiota appeared less complex, with fewer distinct members.In subjects with lower diversity, the microbiota was generally domi-nated by a single bacterial genus. Dominating genera includedEnterococcus, Streptococcus, Enterobacteriaceae (Escherichia andKluyvera), and Lactobacillus. These bacterial taxa were also ob-served in a prior report characterizing the microbiota of this allo-HSCT patient cohort.3

To determine whether the presence or absence of specificbacterial taxa correlated with mortality, we compared the microbiotacomposition of patients who died or survived by the LEfSe method(Table 4). The postengraftment microbiota of subjects who dieddiffered significantly from the microbiota of patients who survived,harboring a greater abundance of g-Proteobacteria, including Enter-obacteriaceae. Conversely, Lachnospiraceae and Actinomycetaceaewere observed in greater relative abundance in subjects who re-mained alive during the follow-up period. We did not observesignificant associations with mortality for any of the remaining taxa.

Discussion

The intestinal microbiota consists of hundreds of distinct bacterialspecies that contribute to host health through a variety of functions

Figure 1. Kaplan-Meier plot of diversity and overall

survival and transplant related mortality.

BLOOD, 14 AUGUST 2014 x VOLUME 124, NUMBER 7 INTESTINAL BACTERIAL DIVERSITY IN ALLO-HSCT 1177

For personal use only.on February 3, 2015. by guest www.bloodjournal.orgFrom

between models for these 2 outcomes was that disease risk waspredictive of overall survival but not transplant-related mortality. Thefollow-up and outcomes are depicted individually for each subject inFigure 2. Of the 36 deaths, 21 (58%) were transplant related and 15(42%) were due to relapse or progression of disease. Transplant-related deaths were frequently due to GVHD or infection and weremore frequent in patients with lowmicrobiota diversity.We did notobserve any evidence of censoring bias. As inverse Simpson diversitydecreased, the corresponding taxonomic composition of the fecalmicrobiota appeared less complex, with fewer distinct members.In subjects with lower diversity, the microbiota was generally domi-nated by a single bacterial genus. Dominating genera includedEnterococcus, Streptococcus, Enterobacteriaceae (Escherichia andKluyvera), and Lactobacillus. These bacterial taxa were also ob-served in a prior report characterizing the microbiota of this allo-HSCT patient cohort.3

To determine whether the presence or absence of specificbacterial taxa correlated with mortality, we compared the microbiotacomposition of patients who died or survived by the LEfSe method(Table 4). The postengraftment microbiota of subjects who dieddiffered significantly from the microbiota of patients who survived,harboring a greater abundance of g-Proteobacteria, including Enter-obacteriaceae. Conversely, Lachnospiraceae and Actinomycetaceaewere observed in greater relative abundance in subjects who re-mained alive during the follow-up period. We did not observesignificant associations with mortality for any of the remaining taxa.

Discussion

The intestinal microbiota consists of hundreds of distinct bacterialspecies that contribute to host health through a variety of functions

Figure 1. Kaplan-Meier plot of diversity and overall

survival and transplant related mortality.

BLOOD, 14 AUGUST 2014 x VOLUME 124, NUMBER 7 INTESTINAL BACTERIAL DIVERSITY IN ALLO-HSCT 1177

For personal use only.on February 3, 2015. by guest www.bloodjournal.orgFrom

Taur Y et al. Blood 2014

Low bacterial diversity at engra.ment is associated with reduced overall survival and increased NRM

n=80

Increased bacterial diversity at D+12 is associated with reduced GVHD-‐related mortality

of patients and then confirmed in a second cohort. Overall survival proba-bilities were estimated using Kaplan-Meier methodology and comparedusing the log rank test. Incidence and overall survival data are presentedrelative to the time of BMT, but patient cohorts were analyzed relative to the

landmark of day 12, the median time of stool collection. We note that nopatients in the study died or developed GVHD before day 16. Comparison ofthe risk of acute GVHD and GVHD-related mortality after adjusting forclinical factors was performed using Cox regression. A receiver operating

Table 1Clinical Characteristics of Allo-BMT Patients Transplanted at Memorial Sloan Kettering Cancer Center with Stool Samples Collected Day 12 after BMT Included inan Identification and Validation Cohort

Cohort 1 Cohort 2

Dates of transplant September 2009 to October 2012 August 2011 to August 2013Age, yr 25-70, median 53 26-75, median 50Gender Female 38%, male 62% Female 32%, male 69%Primary malignancy NHL 38%, AML 38%, ALL 9.4%, Hodgkin disease 6.3%,

CLL 6.3%, MDS 3.1%NHL 35%, AML 37%, ALL 12%, Hodgkin disease 3.9%,CLL 7.8%, MDS 3.9%

Disease risk High 45%, intermediate 36%, low 19% High 35%, intermediate 27%, low 37%Graft source Peripheral blood 55%, cord blood 42%, bone marrow 3.1% Peripheral blood 57%, cord blood 39%, bone marrow 3.9%Donor relationship and HLA Sibling identical (29%), unrelated identical (22%),

unrelated nonidentical (48%)Sibling identical (24%), unrelated identical (29%),unrelated nonidentical (47%)

Conditioning intensity Standard intensity myeloablative 23%, reduced-intensitymyeloablative 44%, nonmyeloablative 33%

Standard intensity myeloablative 14%, reduced-intensitymyeloablative 57%, nonmyeloablative 29%

Stool sample collection day þ8 to þ16, median þ12 þ8 to þ16, median þ12

NHL indicates non-Hodgkin lymphoma; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; CLL, chronic lymphoblastic leukemia; MDS,myelodysplastic syndrome.

A B

C

P = .05

P = .005

P = .04P = .04

Figure 1. Changes in the intestinal flora are associated with differences in GVHD-related mortality. (A) Bacterial diversity was quantified using the inverse Simpsonindex after composition analysis of stool samples from cohort 1 performed by 16S gene sequencing. Patients were stratified by the median diversity index value andanalyzed for cumulative incidence of GVHD-related mortality. (B) Associations of bacterial genera with GVHD-related mortality outcomes were quantified by lineardiscriminant analysis effect size analysis. Position along the vertical axis indicates statistical significance. (C) Patients from cohorts 1 and 2 were stratified by medianBlautia abundance (.05% in both) and analyzed for incidence of GVHD-related mortality.

R.R. Jenq et al. / Biol Blood Marrow Transplant 21 (2015) 1373e1383 1375

Jenq RR et al. BBMT 2015

n=64

Abundance of Bacteria from the Genus Blautia Near Day12 Is Associated with Reduced Acute GVHD RequiringSystemic Immune Suppression

To identify bacterial subsets associated with GVHD-related mortality, we focused on bacterial phylogeny at thegenus level, which is generally the most specific level atwhich 16S deep sequencing still provides reliable classifica-tion. Our approach involved first identifying bacterial generapotentially associated with GVHD-related mortality using ataxonomic discovery analysis, then evaluating candidategenera for their association with the incidence of GVHD-related mortality, and finally evaluating for reproducibilityof observed associations in an additional independent cohortof patients from the same institution. Beginning with taxo-nomic discovery analysis of bacterial genera, we found thatbacteria belonging to the genus Blautia were most signifi-cantly associated with reduced GVHD-related mortality(Figure 1B, P ¼ .01). The Blautia genus notably includesanaerobic intestinal commensal organisms within the bac-terial class Clostridia [38,39]. The genus Veillonella, inturn, was associated with increased GVHD-related mortality(P ¼ .047).

We then evaluated the association of Blautia and Veillo-nella abundance with GVHD-related mortality, stratifyingpatients by the median abundance (.05% and .04%, respec-tively). We found that patients with higher Blautia abun-dance had reduced GVHD-related mortality (Figure 1C,

Figure 3. Association of Blautia abundance with clinical acute GVHD. (A) Patients were stratified by Blautia abundance below or above .05% and evaluated fordevelopment of the indicated severity grades of acute GVHD as well as acute GVHD that required systemic therapy with corticosteroids. (B) Patients were evaluatedfor development of acute GVHD in typical target organs.

Table 3Subgroup Analysis of Patients from the Cohorts Combined Examining theRelationship Between Blautia Abundance and GVHD-Related Mortality

Subgroup No. of Cases(Blautia Low)

No. of Cases(Blautia High)

GVHD-RelatedMortalityLog Rank P

Myeloablativeconditioning

13 9 .1

Reduced-intensityconditioning

27 30 .3

Nonmyeloablativeconditioning

18 18 .02

PBSC graft 31 33 .006Cord blood graft 24 23 .2

PBSC indicates peripheral blood stem cell.P < .05 are depicted in bold.

R.R. Jenq et al. / Biol Blood Marrow Transplant 21 (2015) 1373e1383 1377

Blau-a abundance at D+12 is associated with reduced incidence of acute GVHD

Jenq RR et al. BBMT 2015

E

A

B

Gra

des

2– 4

GV

HD

(%)

C

F Aztreonam or cefepimePiperacillin-tazobactam

0 20 40 600

5

10

15

20

25

0 20 40 600

5

10

15

20

25*P = 0.02 **P = 0.007

0 20 40 600

5

10

15

20

25

Months after HSCT

Aztreonam

No ABX (n = 793)ABX (n = 64)

P = 0.78

0 20 40 600

5

10

15

20

25

Months after HSCT

Cefepime

No ABX (n = 705)ABX (n = 152)

P = 0.98

D

Imipenem-cilastatin

Imipenem-cilastatin

0 1 2 3 40

10

20

30

40

50

Months after HSCT

Upp

er G

I GV

HD

( g

rade

1) (

%)

Imipenem-cilastatin

NoABX (n = 303)ABX (n = 144)

*P = 0.045

0 1 2 3 40

10

20

30

40

50

Months after HSCT

Piperacillin-tazobactam

No ABX (n = 166)ABX (n = 281)

**P = 0.002

0 1 2 3 40

10

20

30

Months after HSCT

Low

er G

I GV

HD

(g

rade

s 1–

4) (

%)

Imipenem-cilastatin

No ABX (n = 303)ABX (n = 144)

*P = 0.036

0 1 2 3 40

10

20

30

Months after HSCT


No ABX (n = 166) ABX (n = 281)

*P = 0.019

0 1 2 3 40

20

40

60

Months after HSCT

No ABX (n = 303)ABX (n = 144)

*P = 0.0165

0 1 2 3 40

20

40

60

Months after HSCT


NoABX (n = 166)ABX (n = 281)

*P = 0.0167

0 20 40 600

10

20

30

40

Months after HSCT

Treatment-related mortality(non-GVHD)

Received imipenem-cilastatin or piperacillin-tazobactam first-line (n = 306)

Received aztreonam or cefepime first-line (n = 77)

Not treated for neutropenic fever (n = 474)

P = 0.48

0 20 40 600

5

10

15

20

25

Months after HSCT

GVHD-related mortality

*P = 0.029

0 20 40 600

25

50

75

100

Months after HSCT

Per

cent

age

Overall survival

P = 0.87

0 20 40 600

20

40

60

80

Months after HSCT

Relapse-related mortality

P = 0.12

Received imipenem-cilastatin orpiperarr cillin-tazobactam fiff rsrr t-line (n = 306)

Received aztreonrr am orcefeff pime fiff rsrr t-line (n = 77)

Not trerr ated foff r neutroperr nic feff ver (n = 474)

GV

HD

-rela

ted

mor

tality

(%)

Lacto

bacil

lus

Clostrid

ia

Bacter

oidete

s

Erysipe

lotric

hia

Actino

bacte

ria

Entero

cocc

us

Akker

mansia

0.000010.0001

0.0010.01

0.11

10100

100010,000

Fold

cha

nge

**

No ABX (n = 709)ABX (n = 148)

No ABX (n = 557)ABX (n = 300)

Months after HSCT Months after HSCT


Fig. 1. The clinical use of imipenem-cilastatin and piperacillin-tazobactam is as-sociated with increased GVHD-related mortality. (A to D) A retrospective cohortof 857 adult patients was identified as recipients of non–T cell–depleted allo-HSCT atour center from 1992 to 2015. (A) GVHD-related mortality in patients exposed to az-treonam, cefepime, imipenem-cilastatin, or piperacillin-tazobactam. ABX, antibiotics.(B) Analyses of overall grades 2 to 4 GVHD are shown. (C) Analyses of upper GI grade1 GVHD are shown. (D) Analyses of lower GI grades 1 to 4 GVHD are shown. (E) A subsetof patients who had been treated for neutropenic fever was stratified according towhether they received first-line treatment with imipenem-cilastatin or piperacillin-tazobactam, versus aztreonam or cefepime. Outcomes indicated were depicted byKaplan-Meier plots and curves compared by the log-rank test. *P < 0.05, **P < 0.01in (A) to (E). (F) Intestinal microbiota composition analysis using 16S rRNA sequen-cing before and after beginning treatment with the indicated antibiotics in allo-HSCT recipients. *P < 0.05 after multiple comparisons with Holm-Sidak correction.

R E S EARCH ART I C L E

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E

A

B

Gra

des

2– 4

GV

HD

(%)

C


0 20 40 600

5

10

15

20

25

0 20 40 600

5

10

15

20

25*P = 0.02 **P = 0.007

0 20 40 600

5

10

15

20

25

Months after HSCT

Aztreonam

No ABX (n = 793)ABX (n = 64)

P = 0.78

0 20 40 600

5

10

15

20

25

Months after HSCT

Cefepime

No ABX (n = 705)ABX (n = 152)

P = 0.98

D

Imipenem-cilastatin

Imipenem-cilastatin

0 1 2 3 40

10

20

30

40

50

Months after HSCT

Upp

er G

I GV

HD

( g

rade

1) (

%)

Imipenem-cilastatin

NoABX (n = 303)ABX (n = 144)

*P = 0.045

0 1 2 3 40

10

20

30

40

50

Months after HSCT


No ABX (n = 166)ABX (n = 281)

**P = 0.002

0 1 2 3 40

10

20

30

Months after HSCT

Low

er G

I GV

HD

(g

rade

s 1–

4) (

%)

Imipenem-cilastatin

No ABX (n = 303)ABX (n = 144)

*P = 0.036

0 1 2 3 40

10

20

30

Months after HSCT


No ABX (n = 166) ABX (n = 281)

*P = 0.019

0 1 2 3 40

20

40

60

Months after HSCT

No ABX (n = 303)ABX (n = 144)

*P = 0.0165

0 1 2 3 40

20

40

60

Months after HSCT


NoABX (n = 166)ABX (n = 281)

*P = 0.0167

0 20 40 600

10

20

30

40

Months after HSCT





P = 0.48

0 20 40 600

5

10

15

20

25

Months after HSCT


*P = 0.029

0 20 40 600

25

50

75

100

Months after HSCT

Per

cent

age

Overall survival

P = 0.87

0 20 40 600

20

40

60

80

Months after HSCT


P = 0.12




GV

HD

-rela

ted

mor

tality

(%)

Lacto

bacil

lus

Clostrid

ia

Bacter

oidete

s

Erysipe

lotric

hia

Actino

bacte

ria

Entero

cocc

us

Akker

mansia

0.000010.0001

0.0010.01

0.11

10100

100010,000

Fold

cha

nge

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No ABX (n = 709)ABX (n = 148)

No ABX (n = 557)ABX (n = 300)







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The clinical use of imipenem-‐cilasta-n and piperacillin-‐tazobactam is associated with increased GVHD-‐related mortality

Shono Y et al. STM 2016

E

A

B

Gra

des

2– 4

GV

HD

(%

)

C


0 20 40 600

5

10

15

20

25

0 20 40 600

5

10

15

20

25*P = 0.02 **P = 0.007

0 20 40 600

5

10

15

20

25

Months after HSCT

Aztreonam

No ABX (n = 793)ABX (n = 64)

P = 0.78

0 20 40 600

5

10

15

20

25

Months after HSCT

Cefepime

No ABX (n = 705)ABX (n = 152)

P = 0.98

D

Imipenem-cilastatin

Imipenem-cilastatin

0 1 2 3 40

10

20

30

40

50

Months after HSCT

Upp

er G

I GV

HD

( g

rade

1) (

%)

Imipenem-cilastatin

NoABX (n = 303)ABX (n = 144)

*P = 0.045

0 1 2 3 40

10

20

30

40

50

Months after HSCT


No ABX (n = 166)ABX (n = 281)

**P = 0.002

0 1 2 3 40

10

20

30

Months after HSCT

Low

er G

I GV

HD

(g

rade

s 1–

4) (

%)

Imipenem-cilastatin

No ABX (n = 303)ABX (n = 144)

*P = 0.036

0 1 2 3 40

10

20

30

Months after HSCT


No ABX (n = 166) ABX (n = 281)

*P = 0.019

0 1 2 3 40

20

40

60

Months after HSCT

No ABX (n = 303)ABX (n = 144)

*P = 0.0165

0 1 2 3 40

20

40

60

Months after HSCT


NoABX (n = 166)ABX (n = 281)

*P = 0.0167

0 20 40 600

10

20

30

40

Months after HSCT





P = 0.48

0 20 40 600

5

10

15

20

25

Months after HSCT


*P = 0.029

0 20 40 600

25

50

75

100

Months after HSCT

Per

cent

age

Overall survival

P = 0.87

0 20 40 600

20

40

60

80

Months after HSCT


P = 0.12




GV

HD

-rela

ted

mor

tality

(%)

Lacto

bacil

lus

Clostrid

ia

Bacter

oidete

s

Erysipe

lotric

hia

Actino

bacte

ria

Entero

cocc

us

Akker

mansia

0.000010.0001

0.0010.01

0.11

10100

100010,000

Fol

d ch

ange

**

No ABX (n = 709)ABX (n = 148)

No ABX (n = 557)ABX (n = 300)







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maintaining colonic homeostasis and health (25, 26). Despite large dif-ferences in the abundance of Clostridiales, we observed no significantchanges in the amounts of SCFAs in the colon comparing specimensfrom recipients treated with aztreonam or imipenem-cilastatin (fig. S6).

To acquire greater resolution of the bacterial composition betweenaztreonam- and imipenem-cilastatin–treated murine specimens, weperformed metagenomic shotgun sequencing with stool collected onday 21 after allo-HSCT. We found that, concordant with the 16S se-quencing results, imipenem-cilastatin but not aztreonam treatmentresulted in an increased abundance of A. muciniphila (Fig. 4F). How-ever, as the largest percentage of reads from the analysis were determinedto be unclassified, it is possible that additional significant differences inbacterial species composition exist between the two antibiotic treat-ment types. Metagenomic shotgun sequencing analysis also revealeddifferences in gene content between microbiota specimens from micetreated with aztreonam and imipenem-cilastatin, depicted by prin-cipal component analysis of gene orthologs (Fig. 4G). LEfSe analysisof gene pathways indicated that the microbiota genes in mice treatedwith imipenem-cilastatin were enriched for processes including lipo-polysaccharide synthesis, and relatively depleted in several processesincluding D-alanine metabolism (fig. S7). Lipopolysaccharide is wellknown for inducing a proinflammatory cascade in many disease pro-cesses including GVHD (27), whereas reductions in D-alanine contentof lipoteichoic acid can enhance the anti-inflammatory properties of lactobacilli(28, 29).

As mentioned above, we detected anincrease in A. muciniphila in the flora ofimipenem-cilastatin–treated mice using16S rRNA deep sequencing (Fig. 4E). Thisbacterium has the ability to degrade mucusas a carbohydrate source (30, 31). Usingour metagenomic shotgun sequencingresults, we determined whether genes pre-dicted to encode secretory mucolytic en-zymes were differentially present in micetreated with each antibiotic. The identifica-tion and characterization of bacterial mu-colytic enzymes are still a young field, but arecent study examined the whole genomicsequence of A. muciniphila (ATCC BAA-835), isolated from human feces (31). Theauthors identified two strong candidatesfor mucus degradation: Amuc_0953 (a sul-fatase) and Amuc_2164 (a glycosyl hydro-lase), which both contained predictedsecretory signal peptide cleavage sites aswell as predicted mucin-binding domains.We quantified the presence of sequenceswith homology to these two genes andfound that both were markedly enrichedin specimens from imipenem-cilastatin–treated mice (Fig. 4H). We then soughtto characterize the mucus layer of the co-lon in antibiotic-treated transplanted mice.Using periodic acid–Schiff staining, we ob-served a marked reduction in the thick-ness of the mucus layer in recipients

treated with imipenem-cilastatin on day 21 compared to aztreonam-treated recipients (Fig. 5, A and B). No differences in the numbers ofmucus-producing goblet cells between aztreonam- and imipenem-cilastatin–treated recipients were seen, suggesting that mucus production was notimpaired (Fig. 5C). Moreover, by using a general bacterial 16S rRNAprobe (EUB338) (32) coupled with MUC2 staining, we directly visualizedthe inner mucus layer in the colon of antibiotic-treated recipients andconfirmed a marked thinning of the mucus layer of mice treated withimipenem-cilastatin. Markedly, we also observed dissemination of bac-teria past the colonic epithelial barrier in histopathology specimens fromimipenem-cilastatin–treated mice (Fig. 5D), whereas this was not seenin aztreonam-treated mice. We further assessed epithelial barrier func-tion in recipients by performing oral administration of fluorescein isothio-cyanate (FITC)–dextran, a poorly absorbed carbohydrate that can enterthe bloodstream if the epithelial barrier has been compromised.Consistent with the pathological findings mentioned above, we detecteda greater amount FITC in the serum of imipenem-cilastatin–treated miceas early as 5 days after initiation of antibiotics (Fig. 5E), indicatinggreater loss of gut integrity in these recipients. Together, these resultsdemonstrate that imipenem-cilastatin treatment can exacerbate GVHDthrough a combination of factors including compromised barrier func-tion with thinning of the protective mucus layer and reduced numbersof colonic B cells, increased infiltration of granulocytes, elevated

0

5

10

15µm

AztreonamImipenem

**

Goblet cells

0

0.5

1.0

1.5

Fold

cha

nge

P = 0.279

B CMucus thickness

D

Aztreonam Imipenem

EUBMUC2DAPI

10 µm

0

500

1000

1500

2000

FITC

-dex

tran

(ng/

ml)

AztreonamImipenem

*E

A

ImipenemAztreonam50 µM

Fig. 5. Mice treated with imipenem-cilastatin in the setting of GVHD result in the loss of the colonicmucus layer and impaired intestinal barrier function. (A to C) Colon tissues from mouse recipientswere fixed by water-free methanol-Carnoy’s fixative on day 21, stained with periodic acid–Schiff stain,and visualized by light microscopy. Orange triangles in (A) indicate the location of the inner mucuslayer. 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 means ± SEM (n = 10). **P < 0.01by Mann-Whitney U test. (D) Immunostaining of colon sections for MUC2 (green) with general bacterial16S rRNA gene in situ hybridization (FISH) probe EUB338 (EUB) (red) counterstained with Hoechst(blue). Data are representative of two independent experiments (n = 10). Orange arrowheads indicateinner mucus layer; red arrowheads indicate bacteria penetrating beyond the mucus layer and colonicepithelium. (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 after transplant. The graph showsplasma FITC-dextran concentrations. Data are representative of two independent experiments. Values rep-resent means ± SEM (n = 6 to 8). *P < 0.05 by Mann-Whitney U test.




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E

A

B

Gra

des

2– 4

GV

HD

(%)

C


0 20 40 600

5

10

15

20

25

0 20 40 600

5

10

15

20

25*P = 0.02 **P = 0.007

0 20 40 600

5

10

15

20

25

Months after HSCT

Aztreonam

No ABX (n = 793)ABX (n = 64)

P = 0.78

0 20 40 600

5

10

15

20

25

Months after HSCT

Cefepime

No ABX (n = 705)ABX (n = 152)

P = 0.98

D

Imipenem-cilastatin

Imipenem-cilastatin

0 1 2 3 40

10

20

30

40

50

Months after HSCT

Upp

er G

I GV

HD

( g

rade

1) (

%)

Imipenem-cilastatin

NoABX (n = 303)ABX (n = 144)

*P = 0.045

0 1 2 3 40

10

20

30

40

50

Months after HSCT


No ABX (n = 166)ABX (n = 281)

**P = 0.002

0 1 2 3 40

10

20

30

Months after HSCT

Low

er G

I GV

HD

(g

rade

s 1–

4) (

%)

Imipenem-cilastatin

No ABX (n = 303)ABX (n = 144)

*P = 0.036

0 1 2 3 40

10

20

30

Months after HSCT


No ABX (n = 166) ABX (n = 281)

*P = 0.019

0 1 2 3 40

20

40

60

Months after HSCT

No ABX (n = 303)ABX (n = 144)

*P = 0.0165

0 1 2 3 40

20

40

60

Months after HSCT


NoABX (n = 166)ABX (n = 281)

*P = 0.0167

0 20 40 600

10

20

30

40

Months after HSCT





P = 0.48

0 20 40 600

5

10

15

20

25

Months after HSCT


*P = 0.029

0 20 40 600

25

50

75

100

Months after HSCT

Per

cent

age

Overall survival

P = 0.87

0 20 40 600

20

40

60

80

Months after HSCT


P = 0.12




GV

HD

-rela

ted

mor

tality

(%)

Lacto

bacil

lus

Clostrid

ia

Bacter

oidete

s

Erysipe

lotric

hia

Actino

bacte

ria

Entero

cocc

us

Akker

mansia

0.000010.0001

0.0010.01

0.11

10100

100010,000

Fold

cha

nge

**

No ABX (n = 709)ABX (n = 148)

No ABX (n = 557)ABX (n = 300)







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Akkermansia muciniphila is increased a.er imipenem treatment and is associated with mucus degrada-on

Shono Y et al. STM 2016

D

E

F

0

25

50

75

Abu

ndan

ce (%

)

***

***

0

25

50

75

Abu

ndan

ce (%

)

***

Aztreonam Imipenem

Aztreonam Imipenem

A B

−1

0

1

2

−2 −1 0 1 2PC1 (60.4%)

PC

2 (3

0.1%

)

Aztreonam

Imipenem

–6 –4 –2 0 2 4 6

10–3

10–2

10–1

LDA score (log10)

Order

P = 0.05

Favors imipenemtreatment

Favors aztreonamtreatment

Verrucomicrobiales

Enterobacteriales

Clostridiales

P value

G

Aztreonam Imipenem

0.00

0.25

0.50

0.75

1.00

0.00

0.25

0.50

0.75

1.00

2 4 6 2 4 6Sample

Rel

ativ

e sp

ecie

s ab

unda

nce Species (unclassified removed)

Akkermansia muciniphila Anaerostipes sp. 3_2_56FAA Clostridiales bacterium VE202−09 Enterobacter cloacae Enterococcus faecalis Enterococcus gallinarum Klebsiella oxytoca Lachnospiraceae bacterium A2 Lachnospiraceae bacterium COE1 Lactobacillus johnsoniiOther

−0.01

0.00

0.01

0.02

−0.04 0.00 0.04

PC

2 (1

3.4%

)

PC1 (69.2%)

Imipenem

Aztreonam

–6 –4 –2 0 2 4 6

10–3

10–2

10–1

LDA score (log10)

Genus

P = 0.05

Favors imipenemtreatment

Favors aztreonamtreatment

AkkermansiaAnaerotruncus

Klebsiella

P value

Order

Genus

Weighted UniFrac distance

KEGG orthology euclidean distance

Aztreonam Imipenem

0.00

0.25

0.50

0.75

1.00

0.00

0.25

0.50

0.75

1.00

2 4 6 2 4 6

Species Akkermansia muciniphila

Lactobacillus johnsoniiUnclassifiedOther

Sample

AztreonamImipenem

H

0.0000

0.0002

0.0004

0.0006

% o

f map

ped

read

s

Amuc_0953**

0.0000

0.0001

0.0002

0.0003

0.0004

0.0005Amuc_2164

**

C

Clostrid

iales

Entero

bacte

riales

Lacto

bacil

lales

Bacter

oidale

s

Lacto

bacil

lus

Entero

cocc

us

Akker

mansia

Verru

comicr

obial

es

Rel

ativ

e sp

ecie

s ab

unda

nce

Lachnospiraceae bacterium A2

ImipenemAztreonam

V V

Actinobacteria (c)

(p), phylum(c), class(o), order(f), family(g), genus

(p), phylum(c), class(o), order(f), family(g), genus

(g)(o)

(o)

(o)

(g)

(g)(f)

(f)Firmicu

tes (p

)

Clostri

dia (c

)

Gammaproteobacteria (c)Proteobacte

ria (p

)

errucomicrobiae (c)

errucomicrobia (p)

Fig. 4. Mice treated withimipenem-cilastatin in the set-ting of GVHD show increasedabundance of Akkermansia.(A) Stool specimens obtained frommice treated with imipenem-cilastatin or aztreonamwere col-lected on day 21 and analyzedby 16S rRNA gene sequencing(as in Fig. 2), followed by prin-cipal components (PC) analysisof weighted and normalizedUniFrac distances. Proportion ofvariance accounted for by eachprincipal component is indicated.(B and C) Differential taxonomicabundance between aztreonam-and imipenem-cilastatin–treatedrecipients was analyzed by lineardiscriminant analysis (LDA) cou-pled with effect size measure-ments (LEfSe) (B) and by LEfSeprojected as a cladogram (C).Data are representative of morethan five independent experi-ments in (A) to (C). (D and E)Comparisons of bacterial abun-dance at the phylogenetic lev-els of order (D) and genus (E).Data are combined from six in-dependent experiments (n =32 to 36). ***P < 0.001 bymulti-ple comparisons, corrected byHolm-Sidak test. (F to H) Stoolspecimens collected from micewith GVHD treated with antibio-tics were collected on day 21and evaluated by metagenomicshotgun sequence analysis. (F)Comparison of bacterial speciesabundance determined by taxo-nomy. Numbers 1 through 6along the x axis represent theindividual subjects. (G) Princi-pal component analysis ofquantification of sequencereads from KEGG (Kyoto Ency-clopedia of Genes andGenomes)gene orthologs comparingspecimens from mice treatedwith aztreonam and imipenem-cilastatin. (H) Quantification ofgene sequences by homologywas performed on stool spe-cimens collected on day 21.Amuc_0953 (a sulfatase) andAmuc_2164 (a glycosyl hydro-lase) are two predicted secretedmucolytic genes found in thegenome of A. muciniphila ATCCBAA-835, isolated from humanfeces. **P<0.01byMann-WhitneyU test.




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•  Occurs commonly due to the effects of radiation/ chemotherapy: incidence correlates with intensity of conditioning1

•  Often occurs independent of GVHD1

•  GVHD onset is associated with increases in Lactobacilus (mice and humans)2

•  Reduced caloric intake leads to loss of Blautia and Clostridiales3

•  Blautia abundance early post-transplant is associated with better OS and lower GVHD-related mortality3

•  Post-transplant low microbial diversity is associated with increased NRM3

GVHD and the Microbiota: Impact of Reduced Oral Intake and Antibiotics

GVHD, graft-versus-host disease; NRM, non-relapse mortality; OS, overall survival.

1.  Shono Y. et al. Int J Hematol. 2015 May;101(5):428-37. 2.  Jenq RR et al. J Exp Med. 2012 May 7;209(5):903-11. 3.  Jenq RR et al. Biol Blood Marrow Transplant. 2015;21(8):1373-83.

Interactions Between Nutrition, GVHD and the Microbiota: Which Model?

Can the flora modulation impact on GVHD?

BMT, bone marrow transplant; GVHD, graft-versus-host disease.

Fecal microbiota transplanta-on is a promising treatment of for steroid-‐resistant acute GVHD of the gut

Kakihana et al. Blood 2016

40

Beclomethasone

FK

mPSLPSL 20 15

TAZ/PIPC CFPM+VCM

Diarrhea

Fecal incontinent

FMT

PrebioticsMeals

Case 1

20

FMT

BeclomethasoneFK

Octreotide

FentanylPrebiotics

MealsLVFX CFPM+VCM

mPSL PSL 20

DiarrheaHematochezia

Case 2

30

CAZ LVFX

Abdominal pain

PSL30

BeclomethasoneFK

FMT

CFPM+VCM CAZ

Diarrhea

Skin eruption

Case 3

PrebioticsMeals

100 8050 30

mPSL

Case 4

i) ii)

iii) iv)

ST

Feces Graft Feces Graft

Donor

Patient*1

PreNext day 1w 2w 4w

Corynebacterium

Clostridium

Lactobacillus

Bacteroides


Donor

Patient

PreNext day 1w

Next day 1w 2w 4w

Lactobacillus

Bacteroides

Streptococcus

Prevotella

Bifidobacterium

Feces Graft

Donor

Patient

PreNext day 1w 2w 4w

Lactobacillus

Bacteroides

Bifidobacterium

Faecalibacterium

Staphylococcus


Donor

Patient

PreNext day 1w 2w

Next day

Bifidobacterium

Bacteroides

Lactobacillus

Escherichia

Escherichia

day0 day7 day14 day21 day28 day35 day0 day7 day14 day21 day28 day35 day42

day0 day7 day14 day21 day28

FMT

FK

Octreotide

PrebioticsLVFX CFPM+TEIC

mPSL

Rituximab

Diarrhea

Hematochezia

200 16080100

Dexamethasone32mg

MEPM+TEIC TAZ/PIPC(+TEIC)

day0 day7 day14 day21 day28

Antibiotics Antibiotics

AntibioticsAntibiotics

5020

Beclomethasone

A

BacteroidesStreptococcusEnterococcusFusobacteriumGranulicatellaPhascolarctobacteriumMegasphaeraSubdoligranulumTannerellaBarnesiellaCoprobacillusPseudoflavonifractorGemellaAkkermansiaAllisonellaTM7AnaerotruncusParvimonasDelftiaPseudomonas

BifidobacteriumClostridiumCorynebacteriumAnaerostipesAlistipesParasutterellaAcidam inococcusArthrobacterTuricibacterLachnospiraDesulfovibrioDyellaShuttleworthiaLactococcusRhodanobacterAcinetobacterFinegoldiaPropionibacteriumDesulfotomaculumRobinsoniella

PrevotellaRuminococcusVeillonellaCatenibacteriumKlebsiellaDialisterFlavonifractorOlsenellaAtopobiumShigellaAcidovoraxScardoviaRalstoniaStenotrophomonasAdlercreutziaAnaerococcusPantoeaDechloromonasPeptostreptococcus

FaecalibacteriumParabacteroidesEubacteriumActinomycesCoriobacteriumDoreaRothiaSutterellaEggerthellaCoprococcusOscillibacterLachnobacteriumCampylobacterLachnoanaerobaculumSphingomonasOxalobacterOribacteriumButyricimonasMicrobacteriumUndefined

LactobacillusEscherichiaStaphylococcusBlautiaCollinsellaRoseburiaMitsuokellaSlackiaAlloscardoviaOdoribacterTerrahaemophilusParaprevotellaBilophilaBrevundimonasSolobacteriumBradyrhizobiumNocardiaActinobaculumJanthinobacteriumSynergistes

Figure 1.

2086 KAKIHANA et al BLOOD, 20 OCTOBER 2016 x VOLUME 128, NUMBER 16

For personal use only.on July 18, 2017. by guest www.bloodjournal.orgFrom

PATIENT ADMISSION

Feces collec-on and analysis

Metagenomics Bacteriology (MDRB, pathogens, C. difficile)

Biochemistry

Pa-ent inclusion

Informa:on and consent

Detec:on of resistant bacteria or pathogenic microorganisms => exclusion of

the study

Diagnos-c of AML

D0

Feces collec-on and analysis

Control of microbiota recovery

Metagenomics Bacteriology (MDRB, C.

difficile) Biochemistry

Feces collec-on and analysis Determina-on of dysbiosis

Metagenomics Biochemistry

Bacteriology (MDRB, C. difficile)

AFMT ABer aplasia comple:on

≈D29 2 days before discharge

D30-‐D31 (D25 to D36)

1 day before discharge

≈D40 At hospital entry

Hospitaliza-on for Induc-on chemotherapy (≈30 days)

Hospitaliza-on for consolida-on chemotherapy (≈30 days)

Pa:ent at home (≈10 days)

Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6

Chemotherapy Aplasia An-bio-cs

≈D70 At hospital discharge

≈M6 ≈M12

Pa-ent follow-‐up Collec:on of clinical informa:ons

Hospitaliza:ons in ICU, new medica:ons (D70, M6) Status (alive/death, remission/relapse), AE, SAE (D70,

M6, M12)

Main participation period ≈ 70 days Follow-up (1 year)

Autologous Fecal Transplantation in Newly Diagnosed AML Patients

AE, adverse event; AFMT, autologous fecal microbiota transplantation; AML, acute myeloid leukemia; ICU, intensive care unit; MDRB, multidrug resistant bacteria; SAE, serious adverse event .

ClinicalTrials.gov Identifier: NCT02928523.

[FMT-SR-GVHD] Fecal Microbiota Transplantation for Steroid Refractory Gastro-Intestinal Acute GVHD after Allo-HSCT

Diagnosis(of(((GI(aGVHD(

Donor(screening((metagenomics,(

bacteriology,(virology,(biochemistry)!

Cor<costeroids(

Feces/blood(collec<on((metagenomics,(bacteriology,(

virology,(biochemistry,(immunomonitoring)!

Donor(feces(collec<on(

HFMT(1!

Pa<ent(screening((preFinclusion)!

History(of(an<biotherapy((metagenomics,(

bacteriology,(virology,(biochemistry)!

!

Pa$ent!follow,up:!post,transplant!clinical!tolerance,!collec$on!of!adverse!effects!

!

3,5!days! 21days!

Cor<costeroids!

7!days!

Evalua<on(of(GI(aGVHD(response((

((

Donor(feces(collec<on(

Diagnosis(of(SR(GI(aGVHD(

7,10!d!

HFMT(2!

≈M3 ≈M6 D28

Pa<ent(inclusion(consent!

D0 D7

Feces/blood(collec<on(

(metagenomics,(bacteriology,(virology,(

biochemistry,(immunomonitoring)!

Feces/blood(collec<on((metagenomics,(bacteriology,(

virology,(biochemistry,(immunomonitoring)!

Clinical(status(

Allo-HSCT, allogeneic hematopoietic stem cell transplantation; GVHD, graft-versus-host disease; HFMT, Home Fecal Microbiota Transplantation; GI, gastro-intestinal. ClinicalTrials.gov Identifier: NCT02928523.

Cases of FMT Performed at Saint-Antoine Hospital (Paris, France)

•  Male, 64 years old •  AML in PIF •  Re-induction course by gemtuzumab in association to

chemotherapy, complicated by a sepsis and skin infection related to multiresistant Pseudomonas aeruginosa

•  Presence of a multiresistant Pseudomonas aeruginosa considered as a contraindication to allo-HSCT

•  FMT before undergoing HSCT •  Allo-HSCT form a MUD •  No episodes of infection related to multiR Pseudomonas

aeruginosa (negative after FMT)

AML, acute myeloid leukemia; allo-HSCT, allogeneic hematopoietic stem cell transplantation; FMT, Fecal Microbiota Transplantation; MUD, matched unrelated donor, PIF, primary induction failure.

16 years old girl, AML RIC haploiden-cal allo-‐HSCT with PT-‐Cy

Β-‐lactams/Carbapenem

Vancomycine

Metronidazole/Fidaxomicine

Immunosuppressive therapy

1 A

1st FMT 2nd FMT

VRE -‐VRE + VRE +

36

37

38

39

40

0 30 60 90 120 150 180 210 240 270 300 330 360

VRE -‐VRE -‐

Body te

mpe

rature

(°C)

Days after HSCT

1st CDI 2nd CDI

K. Pneumoniae ESBL sepsis

20 years old woman, Ph-‐ ALL sequen-al MUD allo-‐HSCT

Β-‐lactams/Carbapenemes

Vancomycin

Teicoplanin

Immunosuppressive therapy

Body te

mpe

rature

(°C)

Days after HSCT

36

37

38

39

40

0 30 60 90 120 150 180 210 240 270 300

1st FMT 2nd FMT

VRE + VRE -‐

1 B

Take Home Messages

•  Humans share a core microbiome and yet differ by genes, species, enterotypes (ecology) and gene count (microbiota diversity)

•  Microbiome gene count / microbiota diversity is a key stratifier with diagnostic and prognostic value in several immune disorders including major diseases of modern world increasing in incidence

•  Dysbiosis is an altered state of microbe-host symbiosis with auto-aggravating signals from both sides

•  Dietary modulation of microbiome gene count is possible i.e. with low glycemic index carbohydrates – high/diverse fiber diet

•  Microbiota modulation (e.g. fecal transplation) could be considered as a reinforcement strategy in current therapy including HSCT

HSCT, hematopoietic stem cell transplantation.

The Future of Hematopoietic Stem Cell Transplantation

•  INSERM U938, team Mohty •  Mohamad Mohty •  Beatrice Gaugler •  Florent Malard •  Eolia Brissot •  Agathe Farge •  Vincent Jachiet •  Myriam Labopin •  Arsène Mekinian •  Nicolas Stocker •  Yishan Ye

•  Bone Marrow Transplant Group -‐ Hematology department -‐ Saint-‐Antoine Hospital: •  Mohamad Mohty •  Georgia Babpaglia •  Ramdane Belhocine •  Eolia Brissot •  Remy Dulery •  Florent Malard •  Clemence Mediavilla

and reduced levels of Firmicutes including Blautia species afterchemotherapy.52 However, the causal relationship between thechemotherapy and microbiota is hard to delineate because manyof these patients received prophylactic antibiotics concurrentlywith the chemotherapy.

The intestinal microbiota and GVHD

A normal and healthy intestinal microbiota is diverse and dominatedby obligate anaerobic bacteria.16,19 From the time of admission todischarge from the hospital, many HCT patients lose this diversity,and the changes that occur are influenced by antibiotic treatmentas well as development of GVHD.53-55 An impact of the microbiotaon GVHD development was first proposed in the 1970s when it wasdemonstrated that mice kept under germ-free conditions developedless gutGVHD.56,57 These resultswere followed up by clinical studiesthat initially showed promising results for intestinal decontamina-tion in HCT patients,58,59 though later studies have not reproducedthese benefits,60-62 possibly due to varying success of the decontami-nating regimens. In addition, a large prospective study targetinganaerobic bacteria in HCT patients showed decreased GVHDdevelopment, indicating that selective decontamination may havebeneficial effects.63

The loss of intestinal diversity seen in HCT patients is generallyassociated with loss of Clostridia species known to produce SCFAsfrom dietary fiber.64 Butyrate is preferred as an energy source ofintestinal epithelial cells and a recent article suggests that reducedamounts of butyrate are found in intestinal epithelial cells after allo-

HCT in mice, while the addition of these SCFAs reduced gutdamage and GVHD.65 Interestingly, this effect was also mimickedby the administration of a mix of bacteria, known to be highproducers of butyrate, belonging to the Clostridia class. Althoughthis is so far the only experimental evidence of efficacy of SCFAs ina GVHD model, a clinical study from our group also showedthat high intestinal abundance of Blautia, a genus belonging tothe Clostridia class, correlates with reduced risk of death fromGVHD.66 Antibiotic treatment is probably the main factor thatinfluences the microbiota changes observed during the course oftransplant andwe recently showed that the choice of antibiotic regimenused to treat neutropenic fever influences the incidence of GVHD.Administration of the broad-spectrum antibiotics imipenem-cilastatinand piperacillin-tazobactam was associated with increased GVHD-relatedmortality at 5 years.67 This retrospective study did not findan association between metronidazole and reduced GVHD asreported by Beelen et al,63 which could be due to multiple factorsincluding the use of different antibiotic combinations in theretrospective study, aswell as geographic and cultural differences in theintestinal flora. Fecal microbiota transplant (FMT) can be used torestore a disrupted intestinalflora and a recent pilot studywith the aim toevaluate the safety of FMT in HCT patients with steroid-refractory orsteroid-dependent GVHD showed promising results; the treatment waswell received and 3 of 4 patients showed resolution of signs andsymptoms of GVHD 28 days after FMT.68 However, this very smallstudy did not address long-term efficacy and larger studies with thecapacity to control for confounding factors will be necessary todetermine the efficacy of FMT as a novel treatment of GVHD.Together, these results indicate that the microbiota present in the gutmay not only mirror GVHD host pathology but that the reverse may

Healthy Gut GvHDSmall Intestine Small IntestineLarge Intestine Large Intestine

Lumen

Mucus

Epithelium

Th17

Th1

Lamina Propria

Enterococcus

Proteobacteria

Akkermansia

Blautia

Th1

Th17

Treg

defensins

DC

IgA

Goblet Cell

Treg

Th2

Macrophage

Paneth Cells

Lgr5+ Stem Cells

Figure 1. Intestinal damage and dysbiosis linked to GVHD. Upon HCT, multiple factors such as conditioning toxicity, antibiotic treatment, and immune activation mediateGVHD. GVHD progression associates with injury to stem cell compartments along with Paneth cells (small intestine) and goblet cells. This leads to increased intestinal

permeability, inflammation, and reduction of the mucus layer and antimicrobial products (eg, defensins). Antibiotic treatment and a limited amount of nutrients in the gut alsopromote gut dysbiosis, furthering gastrointestinal damage and disease. DC, dendritic cell; IgA, immunoglobulin A.

BLOOD, 23 FEBRUARY 2017 x VOLUME 129, NUMBER 8 GVHD AND INTESTINAL MICROBIOTA 929

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Intes-nal microbiota and acute GVHD

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