Download Review Article 711KB

Review Article
The human microbiome in hematopoiesis and hematologic disorders
Veronica E. Manzo1 and Ami S. Bhatt2
1
School of Medicine, and 2Departments of Medicine and Genetics, Stanford University, Stanford, CA
Humans are now understood to be in
complex symbiosis with a diverse ecosystem of microbial organisms, including
bacteria, viruses, and fungi. Efforts to
characterize the role of these microorganisms, commonly referred as the microbiota, in human health have sought to
answer the fundamental questions of
what organisms are present, how are they
functioning to interact with human cells,
and by what mechanism are these interactions occurring. In this review, we
describe recent efforts to describe the
microbiota in healthy and diseased individuals, summarize the role of various
molecular technologies (ranging from 16S
ribosomal RNA to shotgun metagenomic
sequencing) in enumerating the community structure of the microbiota, and
explore known interactions between the
microbiota and humans, with a focus on
the microbiota’s role in hematopoiesis
and hematologic diseases. (Blood. 2015;
126(3):311-318)
Introduction
The microorganisms living in association with the human body are
collectively called the microbiota, with most microbe-host interactions
being symbiotic as opposed to pathogenic.1,2 There are ;10 to 100
trillion microbes in a human; microbial cells and particles thus outnumber human cells at a ratio of 10:1.3 Microbes reside in and on many
of the body’s surfaces, including the skin, nares, oropharynx, vagina,
and intestine. The majority of human-associated microbes reside within
the colon, and this microbial consortium is established within the first 3
years of life.4-7 Of these lower intestinal microbes, the majority of the
bacteria are in 2 of the 70 phyla: Bacteroidetes and Firmicutes
(Table 1).8
The most comprehensive efforts to catalog the composition and
potential functions of various body sites’ microbiota have been
conducted by the Human Microbiome Project (HMP), a consortiumbased endeavor supported by the National Institutes of Health
Common Fund, and the Metagenomics of the Human Intestinal Tract
project supported by the European commission (definitions of common
terms used in microbiota-related studies are provided in Table 24,9-15).
High-throughput sequencing–based studies have been conducted
to characterize human and microbial genes and genomes, with this
collective combination of genes referred to as the metagenome.16
This information can then be used to help understand the microbial
parts of our genetic and metabolic landscape.4
Host genetics play an important role in establishing and shaping the
microbiota. Single-nucleotide polymorphisms in specific host genomic
loci are believed to impact microbiota composition.17 In contrast,
monozygotic-twin metagenomic studies have revealed notable variability in the composition of microbial communities in healthy
individuals, with twins sharing ,50% of their species-level bacterial
taxa.18 This finding suggests a significant role for environmental and
other factors in shaping microbiota community structures. Viral communities, for example, vary widely among monozygotic twins.19 Complex interplay between host and environmental factors is critical in
shaping the microbiota, and our understanding of these relationships is
evolving rapidly. Many of the mechanisms that define the interface
between microbes and humans have been defined in the context
of the gut microbiota–host relationship (Figure 1); however, our
understanding of the full complexity of host-microbe interactions, even
in this well-studied context, is incomplete. Further research will
likely clarify the mechanistic relationship between the human host,
microbial genes, and the microbiota.
Historically, the microbiota focus has been on bacteria; however,
viruses, bacteriophages, and fungi also contribute to the ecological
diversity in several anatomical niches in humans. The best studied of
these 3 additional components of the microbiota are eukaryotic viruses
(and to a lesser extent, prokaryotic viruses, which are known as
bacteriophages). Eukaryotic viruses are posited to contain a staggering
amount of nucleic acid and amino acid sequence diversity.20 The
identification of known and novel viruses has been potentiated by the
development and use of sequencing-based techniques. Sequencing is
typically followed by computational analysis using programs designed
to identify and phylogenetically characterize viruses.21 Interestingly,
interplay between the bacterial and viral components of the microbiota
has been reported; in a study of the infectivity of poliovirus in a mouse
model, treatment of the mice with antibiotics prior to poliovirus
exposure decreased host susceptibility to disease.22 Abnormalities in
the enteric virome are associated with inflammatory bowel disease,
with an increase in enteric virome richness in Crohn disease and
ulcerative colitis.20 This association suggests a potential relationship between viruses of the microbiota and host inflammation; the
nature of the mechanistic interactions between the virome and the
immune system need to be further elucidated. Although prokaryotic
viruses have been genomically enumerated in the human microbiota,
the understanding of bacteriophage regulation is in its relative infancy.
Submitted April 28, 2015; accepted May 15, 2015. Prepublished online as
Blood First Edition paper, May 26, 2015; DOI 10.1182/blood-2015-04-574392.
© 2015 by The American Society of Hematology
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
Advances in molecular technology
Advances in biological techniques have potentiated great advances in
understanding the human microbiota. Studies of the microbiota began
with microscopy and culturing of organisms. As polymerase chain
reaction and Sanger sequencing became widely used, multilocus
sequence typing (MLST) gained popularity. In this technique, ;470-bp
fragments from several housekeeping genes are amplified and
sequenced to genotypically classify bacterial species.23 This method
311
312
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MANZO and BHATT
Table 1. Major bacterial phyla represented in the human intestine
Phyla
Bacteroidetes
Description
This phylum comprises aerobic and anaerobic Gram-negative bacteria, which commonly function to help break down carbohydrates.
The breakdown products of carbohydrate metabolism have been shown to modify host inflammation. An example of an organism
from this phylum is Bacteriodes fragilis.
Firmicutes
This phylum comprises Gram-positive bacteria with low levels of guanine and cytosine nucleotides. It contains bacteria that are phenotypically
Actinobacteria
This phylum is primarily composed of Gram-positive bacteria with high guanine and cytosine nucleotide levels. These organisms are
Proteobacteria
This phylum is composed of Gram-negative bacteria, many of which are pathogenic to humans. In the gut microbiota of healthy individuals,
diverse and are able to form endospores. An example of an organism from this phylum is Roseburia hominis.
commonly associated with the production of secondary metabolites. An example of an organism from this phylum is Bifidobacterium bifidum.
Proteobacteria are classically believed to be less abundant than Bacteroidetes, Firmicutes, and Actinobacteria. However, bacteria in this
phylum frequently expand in dysbiosis or other disease states. An example of an organism from this phylum is Escherichia coli.
has been developed for a handful of pathogenic organisms and
has been helpful in characterizing the relatedness of organisms that
have been isolated and cultured from disease outbreaks. However,
it is inherently limited by its inability to comprehensively identify organisms that lack defined sequences for MLST or are undiscovered. Approximately 60% to 70% of the bacteria in the
human intestinal tract cannot be cultured with currently available
methods.24 These so-called unculturable organisms can often be
identified by 16S ribosomal (r) RNA sequence analysis.25 The
phylogenetic analysis of bacterial 16S rRNA genes (ribosomal
DNAs), amplified directly from complex communities, thus provides an efficient strategy for exploring the biodiversity of a particular
biota.24 Recent data from an analysis of 16S rRNA sequencing
revealed that 62% of the 395 bacterial phylotypes were novel and
;80% of the RNA sequences were from previously uncultivated
species.8 16S rRNA sequencing formed the core of the HMP
microbiota characterization efforts, but it has limitations that include
biases introduced by copy-number variations and challenges in
delineation of closely related prokaryotic species.26 Several different
bacterial species may share identical regions of the16S rRNA
sequence, thus making definitive species-level taxonomic identification impossible. This limitation has recently been overcome by the
application of high-throughput shotgun sequencing, which has
helped to create reference genome databases for newly isolated
and cultured organisms and has facilitated the discovery of near
full genomes of novel, previously uncultured organisms in mixed
samples.27,28 Shotgun sequencing (for whole genome sequencing of
isolates and characterization of metagenomic samples) has become
more accessible as the cost of sequencing has decreased, and
was employed in a subset of samples characterized by the HMP;
additionally, this method has been more broadly applied in more
recent studies. Although this is a relatively accurate method for
taxonomic identification of microbial components of a mixed
population, it is computationally and time intensive (often requiring
“supercomputing” infrastructure) and is often underpowered to
reliably identify rare members of the microbial community.
Table 2. Glossary of terms related to microbiota studies
Term
Gnotobiology
Definition
The scientific investigation of organisms that are either free of microorganisms or are
Reference
9
associated only with specified microorganisms.
Germ-free (axenic) mouse models
Mice free of all microorganisms, including those that are typically found in the gut.
Microbiota
The collection of all the microorganisms living in association with the human body. These
10
4
communities consist of a variety of microorganisms including eukaryotes, archaea,
bacteria and viruses.
Microbiome
Generally accepted to refer to the genomes (genetic material [DNA or RNA]) of the microbiota.
Dysbiosis
A breakdown in the balance between putative species of “protective” vs “harmful” intestinal
11
microorganisms.
Metagenomics
The study of a collection of genetic material (genomes) from a mixed community of organisms
Monocolonization
Introduction of 1 bacterial species in axenic mice; alternatively, a clinical situation in which a
Oligocolonization
Introduction of multiple bacterial species in axenic mice; alternatively, a clinical situation in
(ie, microbial communities).
single organism dominates the microbiota as a result of disease or treatment characteristics.
which a small number (typically ;2-5) of organisms dominate the microbiota as a result of
disease or treatment characteristics.
Fecal microbiota transfer
Introduction of a fecal suspension derived from a healthy donor into the gastrointestinal
12
tract of a diseased individual. Fecal microbiota transfer can be performed via the introduction
of an auto (self) or allo (other) source of stool or purified bacterial stool product via a multitude
of methods, including administration by nasogastric tube, colonoscopy, or capsulized therapy.
Next-generation whole-genome sequencing
DNA is fragmented, amplified, and then sequenced in a massively parallel fashion. This sequence
13
data can be compared to reference databases to infer the taxonomic community structure of
a population of organisms; alternatively, novel organisms/strains can be discovered by de novo
sequence assembly methods.
16S ribosomal RNA/DNA sequencing
16S ribosomal RNA/DNA of prokaryotes is amplified via polymerase chain reaction and sequenced
14
to identify the genus or species of individual organisms.
Viral particle isolation and sequencing
Viral particles are separated from host contaminants using methods such as centrifugation and
filtration. Viral particles can then be treated with DNase I to remove contaminating nucleic acids
on the surface of these particles. A variety of molecular methods can be used to characterize the
genomic sequence of the virions.
15
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MICROBIOME IN HEMATOLOGY
313
Figure 1. The role of the gut microbiota and associated products in shaping the intestinal immune
system. Commensal organisms, such as bacteria in
the phyla Bacteriodes and Firmicutes, colonize the
gastrointestinal tract. SCFAs are produced as a result
of carbohydrate fermentation, which increases the
production of IL-10 and decreases the production of
IL-6 and tumor necrosis factor (TNF)-a. Retinoic acid,
which is produced by intestinal dendritic cells, leads to
an increased differentiation of Tregs and decreased
differentiation of inflammatory Th17 cells. The microfold (M)-cells of Peyer patches uptake antigen from the
lumen and deliver it to the dendritic cells and other
antigen-presenting cells located in the lamina propria.
These cells become activated, and B cells secrete
immunoglobulin (Ig)A into the lumen. These bacteriaspecific IgA molecules thus serve to modulate the
luminal microbiota composition. Paneth cells, an intestinal epithelial cell subtype that is prevalent in the small
intestine and ascending colon, also shape the microbial composition by secreting a-defensins and other
antimicrobial proteins in response to bacterial antigens
binding to toll-like receptors (TLRs). TLRs line the gastrointestinal tract, and microbial products such as
lipopolysaccharide (LPS) and flagellin bind to TLR4
and TLR5, respectively, upregulating the expression of
RegIIIg, a secreted antibacterial lectin that limits infection from Gram-positive bacteria. FDC, follicular
dendritic cell; IL, interleukin; SCFA, short chain fatty
acid; Treg, regulatory T cell; Th, helper T.
Excellent high-throughput, computationally streamlined methods exist
to estimate the identity and relative abundance of microbial organisms
within a metagenomic sample. For example, metagenomic analysis
software programs such as MetaPhlAn expand on the concept of
MLST by identifying hundreds of conserved or “marker” proteins so
that taxonomic composition within a metagenomic population can be
inferred.29 This and related technologies have been broadly applied
in the research setting and are now expanding to the clinical setting
where the ability to rapidly and accurately classify microbial organisms has demonstrated an impact on clinical outcomes.30
Microbiota in human health and disease
Our understanding of the role of the microbiota in human health and
disease is built on epidemiological, observational, molecular, and
animal-modeling experiments. The microbiota plays an important role
in human health and disease. The microbiota is involved in energy
harvest and storage, as well as in a variety of metabolic functions such
as fermenting and absorbing undigested carbohydrates.31 In particular,
the microbiota shapes global immune cell repertoires, thereby altering
host susceptibility to inflammation and infection at sites of colonization
(Figure 1).1,32 Most studies of the microbiota have been performed in
axenic mice (see Table 2 for definition). Recent twin studies have
demonstrated that the environment is a potent regulator of immune
development, and this may include contributions from the microbiota.33
It is now well accepted that essential developmental functions of
mammals, such as proper training and maturation of the immune
system, require the presence of beneficial microorganisms, which are
known as commensals.34
Dysbiosis, or an imbalance in microbiota composition, is postulated
to be a major factor in a variety of human diseases, such as inflammatory bowel disease, graft-versus-host disease (GVHD), and acute HIV
infection.35 In a now classic study, adult germ-free mice were colonized
by the microbiota of obese mice or lean mice; the mice colonized with
an obese microbiota had a significantly greater percentage increase in
total body fat 2 weeks later.9 As has been clearly demonstrated, the
microbiota is critical for the proper maturation of immune cells and
tissues, and plays a role in protection against infectious pathogens.
Furthermore, certain gut microbiota compositions are associated with
disorders of autoimmunity, including inflammatory bowel diseases,
diabetes, asthma, and allergy.34,36,37
Although associations between the microbiota and products of the
hematopoietic system are well established, a mechanistic understanding of how the microbiota directly or indirectly impact hematopoiesis is
limited. The size of the bone marrow myeloid cell pool has been shown
to correlate strongly with the complexity of the intestinal microbiota.38
The immune system begins to develop in utero, but full maturation
requires both genetic and environmental signals that further shape
immunity after birth.39 Germ-free mice have an immature mucosal
immune system, with reduced secondary lymphoid tissues, lower levels
of secretory immunoglobulin A, and fewer intestinal plasma cells.2 In
addition, germ-free mice have lower levels of serum antibodies and are
more susceptible to intestinal and systemic bacterial infections.40
Certain key intestinal compartments, including lymph nodes, Peyer
patches, and intestinal lymphoid follicles, only fully develop after
birth when they undergo a programmed population change to cope
with the microbiota and maintain homeostasis.41
Effects of microbial metabolites
on hematopoiesis
Specific gut bacteria or bacterial products directly suppress intestinal
inflammation (eg, colitis) in mice through a variety of immune
mechanisms.42-44 Bacterial byproducts in the gut microbiome can
also impact allergic airway disease by way of interaction of bacterial
metabolites with cells of the bone marrow compartment.45
SCFAs serve many important roles, from regulating ion absorption
and gut motility in the intestine, to modulating immune responses.46
314
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MANZO and BHATT
Table 3. Described associations between specific microorganisms, the microbiota, and hematologic disorders
Disease
Aplastic anemia
Implicated microbiota
Aplastic anemia has been reported after infections with human parvovirus B19; hepatitis
Reference
50-52
A, B, C, E, and G; CMV; EBV; echovirus 3; GB virus C; transfusiontransmitted or Torque teno virus; SEN virus; and non–A-E hepatitis viruses.
Megaloblastic anemia
Although changes in the microbiota have not yet been directly associated with this
53
disease, cyanocobalamin (vitamin B12) is synthesized by several genera of intestinal bacteria.
Anemia of chronic inflammation
Hepcidin is a gene that is induced by infection and inflammation. Hepcidin-dependent
54
changes in iron flux can induce anemia in chronic inflammatory states.
Gastric MALT lymphoma
Linked to infection with Helicobacter pylori.
55
Marginal zone lymphomas
HCV might provide the initial antigenic stimulus for B-cell clonal expansion.
56
HTLV-associated acute T-cell leukemia
Etiologically linked to HTLV-1 and with a distinct geographical distribution.
57
Burkitt lymphoma
A subset of the cases of this aggressive lymphoma, especially endemic cases in sub-
58
PTLD
PTLD after solid organ transplant and HCT is associated with EBV, which leads to
Saharan Africa, is associated with EBV infection.
59
uncontrolled B-cell proliferation and tumor formation.
Castleman disease
Human herpesvirus 8 (Kaposi sarcoma–associated herpesvirus) sequences have been
60
described in some cases of multicentric Castleman disease.
ITP
Often secondary to persistent, often inapparent infections (eg, H pylori, CMV, or HCV).
61
Reactive thrombocytosis
Cytokines, such as IL-6, IL-1, and tumor necrosis factor, have been shown to promote in
62
vivo and in vitro megakaryocytopoiesis, or production of platelets. Any inflammatory
process such as bacterial infection, neoplasia, sepsis, multiple trauma, burns, or
pancreatitis that elevates serum interleukin levels (especially IL-6), may increase the
circulating platelet count.
GVHD
Loss of Paneth cells from GVHD results in decreased production of a-defensins.
63
a-Defensins selectively kill noncommensal bacteria while preserving commensal
microbiota. A decrease in the expression of a-defensins may thus result in the loss of
microbial diversity.
Allo-HCT
The gastrointestinal mucosa is damaged, and colonizing bacteria are impacted, leading to
64, 65
an impaired intestinal microbiota with reduced diversity. The diversity of the intestinal
microbiota at engraftment is an independent predictor of mortality in allo-HCT recipients.
Sequencing of free DNA in organ transplant recipients’ plasma revealed an expansion of
Anelloviridae upon immunosuppression. There was a lower anellovirus burden in patients
who suffer from graft rejection. A similar finding would be expected in the HCT population.
Allo-HCT, allogeneic HCT; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HCT, hematopoietic cell transplantation; HCV, hepatitis C virus; HTLV, human Tcell lymphotropic virus; ITP, idiopathic thrombocytopenic purpura; MALT, mucosa-associated lymphatic tissue; PTLD, posttransplantation lymphoproliferative
disorder.
SCFAs achieve these widespread functions by activating several G
protein–coupled cell surface receptors, such as GPR43, which is
expressed by granulocytes and some myeloid cells, and GPR109a,
a receptor for niacin and C4 expressed by gut epithelial cells,
adipocytes, macrophages, and dendritic cells. SCFAs suppress nuclear factor kB and the production of inflammatory cytokines such as
IL-6 and tumor necrosis factor a, except for IL-10, which increases.47
These molecules increase generation of Th1, Th17, and IL-101 cells
but decrease the proliferation of T cells and B cells in the mouth.46,48
Butyrate, a type of SCFA, enhances T-cell apoptosis and decreases
accumulation of T cells in inflamed colonic mucosa by upregulating
Fas.49 Retinoic acid, a metabolite of dietary vitamin A, can promote
induced Treg generation and inhibit Th17 and type 1 regulatory T–like
cell responses.35 Polysaccharide A, a carbohydrate produced by the
human commensal bacterium Bacteroides fragilis, is sufficient to
ameliorate T cell–driven colitis in an IL-10–dependent manner.32
Given the evidence supporting a role for the microbiota in normal
hematopoiesis, it is unsurprising that alterations in the microbiota, as
well as in specific microbes, are associated with hematologic disorders.
In some cases, pathogens are believed to trigger hematologic disorders;
in others, disruption of homeostasis in the microbiota is associated with
clinically significant outcomes in patients with hematologic disorders.
Microbial organisms have been demonstrated, in some way, to impact
every compartment of the hematopoietic system. Here, we outline major
hematologic disease and describe associations with infections and/or
alterations of the microbiota (Table 350-65).
Microbiota in hematological diseases
Anemias
Many anemias, such as aplastic anemia and anemia of chronic inflammation, are associated with infections and inflammatory processes,
suggesting there may be an important relationship between red blood
cells and the microbiota. Aplastic anemia, characterized by pancytopenia and hypoplastic bone marrow, can follow hepatitis A, B, C, E, and
G infection and is associated with parvovirus B19, CMV, and EBV.50
In anemia of chronic inflammation, inflammatory cytokines induce
hepcidin expression and alter iron homeostasis. Furthermore, infections
with various bacterial species have been shown to induce hepcidin.66
Although not yet reported, a direct link between the gut microbiota and
hepcidin in anemia of chronic inflammation would be predicted to exist
and be of potential clinical significance.
Iron homeostasis, critical in red blood cell metabolism, is also
important in regulating bacterial infections. Disorders of iron excess,
such as hemochromatosis and chronic hemolysis, render patients susceptible to certain bacterial infections. A randomized controlled study
in African children assessed the effects of increased amounts of iron in
the colon and discovered significant decreases in lactobacilli, a type of
beneficial bacterium, and increased levels of enterobacteria, which
includes Escherichia coli and Salmonella species.49 Although existing
studies support the role of iron in mediating gut microbiota
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MICROBIOME IN HEMATOLOGY
315
homeostasis, additional research is necessary to more fully elucidate
this relationship.
that an intact microbiota is required for optimal response to these
therapies.
Lymphomas
Gastrointestinal dysbiosis in HCT
Many lymphomas are associated with the presence of specific
microorganisms. Relatively strong evidence exists supporting the role
of several pathogenic organisms in lymphomagenesis; these organisms
include EBV, HCV, Helicobacter pylori, and HIV. Lymphoma of
gastric MALT type has been linked to infection with H pylori.
Regression of MALT lymphoma has been demonstrated in up to 70%
of patients treated with antibiotics that eradicate H pylori infection,
suggesting a role for this common gastric microbiota resident in disease
persistence.67 In marginal zone lymphomas, HCV is hypothesized to
provide the initial antigenic stimulus for B-cell clonal expansion as part
of the multistep progression toward lymphomagenesis.56 Other
infection-lymphoma relationships have been described between
the human T-cell lymphotropic virus 1 and adult T-cell leukemia/
lymphoma, between EBV and endemic Burkitt lymphoma and
posttransplantation lymphoproliferative disorder, and between human herpesvirus 8 and multicentric Castleman disease.57-60
Immunocompromised patients with inherited, acquired, or iatrogenic
disorders are exquisitely sensitive to developing dysbiosis. The type of
dysbiosis that can occur varies depending on body site and underlying
disorder but, interestingly, is strongly associated with clinical outcomes
in certain patient populations. Normally, the human gastrointestinal
tract contains a diverse population of microbes, with several types of
obligate anaerobic bacteria. During allo-HCT, the gastrointestinal
mucosa is damaged and colonizing bacteria are impacted, leading to
marked decreases in overall bacterial diversity. In fact, decreased
diversity of the intestinal microbiota at engraftment was shown to be an
independent predictor of mortality (after multivariate adjustment for
other clinical predictors) in allo-HCT recipients.64 The association
between low microbiota diversity and mortality was highly significant
in a single-institution study; additional investigations in multiple
centers will help to clarify the generalizability of this provocative
finding. Efforts to understand the potential mechanistic link between
loss of microbiota diversity and poor outcomes will also be informative.
Although some allo-HCT patients were able to maintain intestinal
bacterial diversity throughout the course of transplantation, others had
a gut microbiota in which a single bacterial taxon predominated and
replaced the previously complex microbial composition.64 One hypothesis linking dysbiosis and mortality is that these dramatic alterations of the intestinal microbiota, in conjunction with neutropenia and
mucosal barrier injury, can lead to complications such as septicemia.70
Alternatively, given the strong link between microbiota diversity and
the generation of immunologic diversity in the setting of development,
another hypothesis is that low microbiota diversity may impact the
robustness and speed of immune reconstitution. Another example of
the extreme loss of diversity in a subset of post-HCT patients was
described in another single-institution study of a cohort of patients with
an antibiotic-responsive colitis syndrome. There, next-generation sequencing of colon biopsy samples revealed that the majority of bacterial
DNA sequences recovered from the sample were from a novel bacterium,
Bradyrhizobium enterica.27 Again, this was a single-institution study;
given the significant differences in antibiotic usage (this center used an
unusual gut decontamination protocol of neomycin and polymixin), it is
difficult to ascertain the generalizability of this organism-disease association to other transplantation centers. However, this finding points out
the impressive oligo- or monoclonality of the tissue-invasive or adherent
gut microbiota in a subset of post-HCT patients.
Platelet disorders
As a rapidly turning-over component of the blood, platelet concentrations are described to be uniquely sensitive to the presence of
microbial organisms. Infections have been associated with ITP. For
example, there is strong evidence for an association between infection
with H pylori and ITP, where platelets can be activated by H pylori
antibodies, by FcgIIA, or through an interaction between H pylori–
bound von Willebrand factor and platelet glycoprotein IB.68 Other
infections that are associated with thrombocytopenia include CMV and
varicella zoster virus. CMV can infect megakaryocytes and supporting
stroma to produce an acute ITP-like syndrome that occurs in
immunocompromised individuals.68 CMV can also cause severe
congenital thrombocytopenia and delayed platelet recovery after
bone marrow transplantation. HCV-infected patients have a higher
incidence of ITP than expected, but the pathophysiology is complex
and poorly understood.61
In addition to infection-induced thrombocytopenia, reactive
thrombocytosis is a commonly associated condition in the setting of
systemic infections. Any inflammatory process, such as bacterial
infection or sepsis, that elevates serum interleukin levels (especially
IL-6) may increase the circulating platelet count.62 It is important to
distinguish this presumably benign reactive condition from malignant thrombocythemic conditions.
GVHD
Impact of the microbiota on treatment
outcome
The composition of the gastrointestinal microbiota, as has been discussed, impacts normal hematopoiesis and can be involved in disease
pathogenesis. It is then, perhaps, no surprise that there is evidence
supporting the role of the microbiota in mediating treatment response
and outcomes in response to chemotherapy, immunotherapy, and
stem cell transplantation. Indeed, 2 recent reports demonstrated
that the microbiota is a modifier of response to platinum-based
chemotherapy, cyclophosphamide, and cytosine guanine dinucleotide oligonucleotide immunotherapy.67,69 Disruption of the microbiota was associated with impaired tumor regression, suggesting
Another common HCT complication involving the gastrointestinal
tract is GVHD. There are many mechanistic models that suggest a role
for components of the microbiota in the development of GVHD.71 Low
gut microbiota diversity post–allo-HCT is associated with the use of
systemic antibiotics, and, in a recent study, this association was more
pronounced in gastrointestinal GVHD.72
Several immunologic cell types play a critical role in modulating
and responding to the gut microbiota. Antigens from colonic
commensal microbiota are important in educating Tregs.73 Tregs and
IL-10–producing type 1 regulatory T–like cells compose the major
regulatory populations in the intestine.35 Paneth cells, which are
located next to intestinal stem cells within the crypts of the intestinal
lumen, secrete antimicrobial peptides and a-defensins. a-Defensins
are active against many Gram-negative and Gram-positive bacteria,
fungi, and enveloped viruses but are typically selectively active against
316
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MANZO and BHATT
noncommensals. These molecules are hypothesized to function largely
by forming pores in bacterial cell walls or by disrupting the cell
membrane.74 In GVHD, for example, Paneth cells are lost, resulting in
decreased expression of a-defensins, which can lead to an expansion
of noncommensal bacteria and decreased microbial diversity.63
In addition to Tregs and Th1 and Th17 cells, the innate immune
system also plays a critical role in recognizing pathogens and targeting
them for destruction through specialized molecular structures. Pattern
recognition receptors are expressed by cells of the innate immune
system and are responsible for recognizing pathogen-associated molecular patterns, the evolutionarily conserved components of pathogens. Upon pathogen-associated molecular pattern engagement, pattern
recognition receptors trigger intracellular signaling cascades, ultimately culminating in the expression of a variety of proinflammatory
molecules, which together orchestrate the early host response to
infection. Pathogen-associated molecular pattern engagement and
downstream signaling is a prerequisite for the subsequent activation
and shaping of adaptive immunity.75 It is hypothesized that these
innate immune cells play a critical role in shaping the microbiota
through selective targeting of certain microbial organisms. As
molecular tools for the dissection of immune cell subsets and
microorganisms evolve, it is anticipated that additional mechanisms
whereby host-microbe homeostasis is maintained will be identified.
Tools and approaches to modify the microbiota
As more is learned about the microbiota, tools and approaches to
modify the microbiota are also being developed in the hope of improving health outcomes. Interventions range from biological therapies
such as fecal microbiota transfer (FMT), to modification of the diet, to
personalized and targeted antibiotic therapies designed to tailor the
exact composition of the microbiota. FMT is the introduction of a fecal
suspension derived from a healthy donor into the gastrointestinal tract
of a diseased individual. FMT can be performed via the introduction
of stool or a purified bacterial stool product via nasogastric tube,
colonoscopy, or capsulized therapy. This technique has been used to
successfully treat recurrent Clostridium difficile–associated disease.76
FMT results in a change in bacterial composition that is accompanied
by resolution of patient symptoms.76 In a recent investigation of
capsulized therapy, whereby pills derived from an “optimized human
stool source” are administered in 2 successive days, .70% efficacy was
achieved with 1 round of therapy; this rate rose to .90% with a second
round of therapy. Although FMT has gained traction in specific clinical
centers and has been demonstrated to be safe and effective for
immunocompetent patients, the application of this therapy has not yet
been widely applied to patients with hematologic diseases. Recently, 2
cases of successful FMT have been reported in post–allo-HCT patients;
given the very high incidence of C difficile–associated disease in this
patient population, FMT provides a potentially attractive treatment
approach.77,78 Concerns about infectious organisms transmitted
through FMT will need to be addressed prior to broad application of
this therapy.
Although pharmacologic and biological approaches to altering the
microbiota predominate, another way to influence these microbial
communities is through the diet. It has been known for some time that
microbial community structures can change drastically in response to
long-term differences in dietary intake. Recent studies have shown that
short-term consumption of diets composed entirely of animal or plant
products can also alter microbial community structure.79 In these studies,
alteration of the diet overwhelmed the interindividual differences in
microbial gene expression.
It is now evident that humans coexist in an intricate and carefully
orchestrated community with the microbiota, in the gut, skin, and
beyond. Decades of research have demonstrated that the microbiota
composition is closely associated with alterations in human health, and
that in some cases, the composition of the microbiota can directly
impact health outcomes that range from obesity to immunologic
diseases such as allergy and asthma. The development of increasingly
refined tools to define the taxonomy, function, and synthetic capacity of
the microbiota has potentiated the identification of strong associations
between the microbiota and health outcomes. The microbiota can
impact human organ systems directly, as well as remotely, even as far as
the bone marrow compartment. Although our understanding of the role
of the microbiota in normal hematopoiesis and hematologic diseases is
at its relative infancy, we anticipate that the tools now available to define
the microbiota will lead to the appreciation of the microbiota as
a biomarker of disease, with both diagnostic and prognostic utility. In
the future, antibiotics may be used to remove or suppress undesirable
components of the human microbiome, and probiotics or FMT may be
used to introduce or help increase microbial components with known
beneficial functions for the human host.80 Given the critical role of the
hematologic system in monitoring and adapting to the external environment, we anticipate that investigation of the disease-microbiota
relationship will generate new diagnostic, prognostic, and therapeutic
approaches for hematologic disorders.
Acknowledgments
This work was supported by National Institutes of Health National
Cancer Institute grant K08 CA184420, an American Society of
Hematology Scholar Award, the Amy Strelzer Manasevit Award
from the National Marrow Donor Program, and an unrestricted gift
to the Stanford Microbiome Research Fund from the Samarth
Foundation.
Authorship
Contribution: V.E.M. and A.S.B. performed a review of the literature,
wrote the paper, and developed the figure.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Ami S. Bhatt, Stanford University, 269 Campus
Dr, Stanford, CA 94305; e-mail: [email protected].
References
1. Kamada N, Kim Y-G, Sham HP, et al. Regulated
virulence controls the ability of a pathogen to
compete with the gut microbiota. Science. 2012;
336(6086):1325-1329.
2. Khosravi A, Yáñez A, Price JG, et al. Gut microbiota
promote hematopoiesis to control bacterial infection.
Cell Host Microbe. 2014;15(3):374-381.
3. Savage DC. Microbial ecology of the
gastrointestinal tract. Annu Rev Microbiol.
1977;31:107-133.
4. Turnbaugh PJ, Ley RE, Hamady M, FraserLiggett CM, Knight R, Gordon JI. The human
microbiome project. Nature. 2007;449(7164):
804-810.
5. Koenig JE, Spor A, Scalfone N, et al. Succession
of microbial consortia in the developing infant gut
microbiome. Proc Natl Acad Sci USA. 2011;
108(suppl 1):4578-4585.
6. Palmer C, Bik EM, DiGiulio DB, Relman DA,
Brown PO. Development of the human infant
intestinal microbiota. PLoS Biol. 2007;5(7):e177.
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MICROBIOME IN HEMATOLOGY
7. Yatsunenko T, Rey FE, Manary MJ, et al.
Human gut microbiome viewed across age and
geography. Nature. 2012;486(7402):222-227.
28. Conlan S, Kong HH, Segre JA. Species-level
analysis of DNA sequence data from the NIH Human
Microbiome Project. PLoS ONE. 2012;7(10):e47075.
8. Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of
the human intestinal microbial flora. Science. 2005;
308(5728):1635-1638.
29. Segata N, Waldron L, Ballarini A, Narasimhan V,
Jousson O, Huttenhower C. Metagenomic microbial
community profiling using unique clade-specific
marker genes. Nat Methods. 2012;9(8):811-814.
9. Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight
R, Gordon JI. The effect of diet on the human gut
microbiome: a metagenomic analysis in humanized
gnotobiotic mice. Sci Transl Med. 2009;1(6):6ra14.
10. Giraud A. Axenic mice model. Methods Mol Biol.
2008;415:321-336.
11. Tamboli CP, Neut C, Desreumaux P, Colombel
JF. Dysbiosis in inflammatory bowel disease. Gut.
2004;53(1):1-4.
12. Borody TJ, Paramsothy S, Agrawal G. Fecal
microbiota transplantation: indications, methods,
evidence, and future directions. Curr
Gastroenterol Rep. 2013;15(8):337.
13. Loman NJ, Constantinidou C, Chan JZM, et al.
High-throughput bacterial genome sequencing: an
embarrassment of choice, a world of opportunity.
Nat Rev Microbiol. 2012;10(9):599-606.
14. Weisburg WG, Barns SM, Pelletier DA, Lane DJ.
16S ribosomal DNA amplification for phylogenetic
study. J Bacteriol. 1991;173(2):697-703.
15. Djikeng A, Halpin R, Kuzmickas R, et al. Viral
genome sequencing by random priming methods.
BMC Genomics. 2008;9:5.
16. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V,
Mardis ER, Gordon JI. An obesity-associated gut
microbiome with increased capacity for energy
harvest. Nature. 2006;444(7122):1027-1031.
17. Benson AK, Kelly SA, Legge R, et al. Individuality
in gut microbiota composition is a complex
polygenic trait shaped by multiple environmental
and host genetic factors. Proc Natl Acad Sci USA.
2010;107(44):18933-18938.
18. Turnbaugh PJ, Quince C, Faith JJ, et al.
Organismal, genetic, and transcriptional variation
in the deeply sequenced gut microbiomes of
identical twins. Proc Natl Acad Sci USA. 2010;
107(16):7503-7508.
19. Reyes A, Haynes M, Hanson N, et al. Viruses in
the faecal microbiota of monozygotic twins and
their mothers. Nature. 2010;466(7304):334-338.
20. Virgin HW. The virome in mammalian physiology
and disease. Cell. 2014;157(1):142-150.
21. Zhao G, Krishnamurthy S, Cai Z, et al.
Identification of novel viruses using
VirusHunter—an automated data analysis
pipeline. PLoS ONE. 2013;8(10):e78470.
22. Kuss SK, Best GT, Etheredge CA, et al. Intestinal
microbiota promote enteric virus replication and
systemic pathogenesis. Science. 2011;
334(6053):249-252.
23. Maiden MCJ, Bygraves JA, Feil E, et al.
Multilocus sequence typing: a portable approach
to the identification of clones within populations of
pathogenic microorganisms. Proc Natl Acad Sci
USA. 1998;95(6):3140-3145.
24. Suau A, Bonnet R, Sutren M, et al. Direct analysis
of genes encoding 16S rRNA from complex
communities reveals many novel molecular
species within the human gut. Appl Environ
Microbiol. 1999;65(11):4799-4807.
25. Amann R, Springer N, Ludwig W, Görtz HD,
Schleifer KH. Identification in situ and phylogeny
of uncultured bacterial endosymbionts. Nature.
1991;351(6322):161-164.
26. Klappenbach JA, Saxman PR, Cole JR, Schmidt TM.
rrndb: the Ribosomal RNA Operon Copy Number
Database. Nucleic Acids Res. 2001;29(1):181-184.
27. Bhatt AS, Freeman SS, Herrera AF, et al. Sequencebased discovery of Bradyrhizobium enterica in cord
colitis syndrome. N Engl J Med. 2013;369(6):
517-528.
30. Wilson MR, Naccache SN, Samayoa E, et al.
Actionable diagnosis of neuroleptospirosis by
next-generation sequencing. N Engl J Med. 2014;
370(25):2408-2417.
31. Gill SR, Pop M, Deboy RT, et al. Metagenomic
analysis of the human distal gut microbiome.
Science. 2006;312(5778):1355-1359.
32. Mazmanian SK, Round JL, Kasper DL.
A microbial symbiosis factor prevents intestinal
inflammatory disease. Nature. 2008;453(7195):
620-625.
33. Brodin P, Jojic V, Gao T, et al. Variation in the
human immune system is largely driven by nonheritable influences. Cell. 2015;160(1-2):37-47.
34. Chow J, Lee SM, Shen Y, Khosravi A, Mazmanian
SK. Host-bacterial symbiosis in health and
disease. Adv Immunol. 2010;107:243-274.
35. Barnes MJ, Powrie F. Regulatory T cells reinforce
intestinal homeostasis. Immunity. 2009;31(3):
401-411.
36. Karlsson FH, Tremaroli V, Nookaew I, et al. Gut
metagenome in European women with normal,
impaired and diabetic glucose control. Nature.
2013;498(7452):99-103.
37. Qin J, Li Y, Cai Z, et al. A metagenome-wide
association study of gut microbiota in type 2
diabetes. Nature. 2012;490(7418):55-60.
38. Balmer ML, Schürch CM, Saito Y, et al.
Microbiota-derived compounds drive steady-state
granulopoiesis via MyD88/TICAM signaling.
J Immunol. 2014;193(10):5273-5283.
39. Weissman IL. Developmental switches in the
immune system. Cell. 1994;76(2):207-218.
40. Smith K, McCoy KD, Macpherson AJ. Use of
axenic animals in studying the adaptation of
mammals to their commensal intestinal
microbiota. Semin Immunol. 2007;19(2):59-69.
41. Sawa S, Cherrier M, Lochner M, et al. Lineage
relationship analysis of RORgammat1 innate
lymphoid cells. Science. 2010;330(6004):
665-669.
42. Atarashi K, Tanoue T, Oshima K, et al. Treg
induction by a rationally selected mixture of
Clostridia strains from the human microbiota.
Nature. 2013;500(7461):232-236.
43. Round JL, Mazmanian SK. The gut microbiota
shapes intestinal immune responses during
health and disease. Nat Rev Immunol. 2009;9(5):
313-323.
44. Smith PM, Howitt MR, Panikov N, et al. The
microbial metabolites, short-chain fatty acids,
regulate colonic Treg cell homeostasis. Science.
2013;341(6145):569-573.
45. Trompette A, Gollwitzer ES, Yadava K, et al. Gut
microbiota metabolism of dietary fiber influences
allergic airway disease and hematopoiesis. Nat
Med. 2014;20(2):159-166.
46. Kim CH, Park J, Kim M. Gut microbiota-derived
short-chain Fatty acids, T cells, and inflammation.
Immune Netw. 2014;14(6):277-288.
47. Singh N, Thangaraju M, Prasad PD, et al.
Blockade of dendritic cell development by
bacterial fermentation products butyrate and
propionate through a transporter (Slc5a8)dependent inhibition of histone deacetylases.
J Biol Chem. 2010;285(36):27601-27608.
48. Kurita-Ochiai T, Fukushima K, Ochiai K. Volatile
fatty acids, metabolic by-products of
periodontopathic bacteria, inhibit lymphocyte
317
proliferation and cytokine production. J Dent Res.
1995;74(7):1367-1373.
49. Zimmerman MA, Singh N, Martin PM, et al.
Butyrate suppresses colonic inflammation through
HDAC1-dependent Fas upregulation and Fasmediated apoptosis of T cells. Am J Physiol
Gastrointest Liver Physiol. 2012;302(12):
G1405-G1415.
50. Rauff B, Idrees M, Shah SA, et al. Hepatitis
associated aplastic anemia: a review. Virol J.
2011;8:87.
51. Ishimura M, Ohga S, Ichiyama M, et al. Hepatitisassociated aplastic anemia during a primary
infection of genotype 1a torque teno virus. Eur J
Pediatr. 2010;169(7):899-902.
52. Mishra B, Malhotra P, Ratho RK, Singh MP,
Varma S, Varma N. Human parvovirus B19 in
patients with aplastic anemia. Am J Hematol.
2005;79(2):166-167.
53. Albert MJ, Mathan VI, Baker SJ. Vitamin B12
synthesis by human small intestinal bacteria.
Nature. 1980;283(5749):781-782.
54. Pigeon C, Ilyin G, Courselaud B, et al. A new
mouse liver-specific gene, encoding a protein
homologous to human antimicrobial peptide
hepcidin, is overexpressed during iron overload.
J Biol Chem. 2001;276(11):7811-7819.
55. Bayerdörffer E, Neubauer A, Rudolph B, et al;
MALT Lymphoma Study Group. Regression of
primary gastric lymphoma of mucosa-associated
lymphoid tissue type after cure of Helicobacter
pylori infection. Lancet. 1995;345(8965):
1591-1594.
56. Franco V, Florena AM, Iannitto E. Splenic
marginal zone lymphoma. Blood. 2003;101(7):
2464-2472.
57. Matutes E. Adult T-cell leukaemia/lymphoma.
J Clin Pathol. 2007;60(12):1373-1377.
58. Tao Q, Robertson KD, Manns A, Hildesheim A,
Ambinder RF. Epstein-Barr virus (EBV) in
endemic Burkitt’s lymphoma: molecular analysis
of primary tumor tissue. Blood. 1998;91(4):
1373-1381.
59. Taylor AL, Marcus R, Bradley JA. Post-transplant
lymphoproliferative disorders (PTLD) after solid
organ transplantation. Crit Rev Oncol Hematol.
2005;56(1):155-167.
60. Soulier J, Grollet L, Oksenhendler E, et al.
Kaposi’s sarcoma-associated herpesvirus-like
DNA sequences in multicentric Castleman’s
disease. Blood. 1995;86(4):1276-1280.
61. Pockros PJ, Duchini A, McMillan R,
Nyberg LM, McHutchison J, Viernes E. Immune
thrombocytopenic purpura in patients with chronic
hepatitis C virus infection. Am J Gastroenterol.
2002;97(8):2040-2045.
62. Kaser A, Brandacher G, Steurer W, et al.
Interleukin-6 stimulates thrombopoiesis
through thrombopoietin: role in inflammatory
thrombocytosis. Blood. 2001;98(9):2720-2725.
63. Eriguchi Y, Takashima S, Oka H, et al. Graftversus-host disease disrupts intestinal microbial
ecology by inhibiting Paneth cell production of
a-defensins. Blood. 2012;120(1):223-231.
64. Taur Y, Jenq RR, Perales MA, et al. The effects
of intestinal tract bacterial diversity on mortality
following allogeneic hematopoietic stem cell
transplantation. Blood. 2014;124(7):1174-1182.
65. De Vlaminck I, Khush KK, Strehl C, et al.
Temporal response of the human virome to
immunosuppression and antiviral therapy.
Cell. 2013;155(5):1178-1187.
66. Peyssonnaux C, Zinkernagel AS, Datta V, Lauth
X, Johnson RS, Nizet V. TLR4-dependent
hepcidin expression by myeloid cells in response
to bacterial pathogens. Blood. 2006;107(9):
3727-3732.
318
BLOOD, 16 JULY 2015 x VOLUME 126, NUMBER 3
MANZO and BHATT
67. Iida N, Dzutsev A, Stewart CA, et al. Commensal
bacteria control cancer response to therapy by
modulating the tumor microenvironment. Science.
2013;342(6161):967-970.
68. Cines DB, Liebman H, Stasi R. Pathobiology of
secondary immune thrombocytopenia. Semin
Hematol. 2009;46(1 Suppl 2):S2-S14.
72. Holler E, Butzhammer P, Schmid K, et al.
Metagenomic analysis of the stool microbiome
in patients receiving allogeneic stem cell
transplantation: loss of diversity is associated
with use of systemic antibiotics and more
pronounced in gastrointestinal graft-versus-host
disease. Biol Blood Marrow Transplant. 2014;
20(5):640-645.
69. Viaud S, Saccheri F, Mignot G, et al. The
intestinal microbiota modulates the anticancer
immune effects of cyclophosphamide. Science.
2013;342(6161):971-976.
73. Lathrop SK, Bloom SM, Rao SM, et al. Peripheral
education of the immune system by colonic
commensal microbiota. Nature. 2011;478(7368):
250-254.
70. Taur Y, Xavier JB, Lipuma L, et al. Intestinal
domination and the risk of bacteremia in patients
undergoing allogeneic hematopoietic stem cell
transplantation. Clin Infect Dis. 2012;55(7):
905-914.
74. Mathewson N, Reddy P. The microbiome and
graft versus host disease. Current Stem Cell
Reports. 2015;1(1):39-47.
71. Shono Y, Docampo MD, Peled JU, Perobelli SM,
Jenq RR. Intestinal microbiota-related effects on
graft-versus-host disease. Int J Hematol. 2015;
101(5):428-437.
75. Mogensen TH. Pathogen recognition and
inflammatory signaling in innate immune
defenses. Clin Microbiol Rev. 2009;22(2):
240-273.
76. Khoruts A, Dicksved J, Jansson JK, Sadowsky
MJ. Changes in the composition of the human
fecal microbiome after bacteriotherapy for
recurrent Clostridium difficile-associated diarrhea.
J Clin Gastroenterol. 2010;44(5):354-360.
77. de Castro CG Jr, Ganc AJ, Ganc RL, Petrolli MS,
Hamerschlack N. Fecal microbiota transplant after
hematopoietic SCT: report of a successful case.
Bone Marrow Transplant. 2015;50(1):145.
78. Neemann K, Eichele DD, Smith PW, Bociek R,
Akhtari M, Freifeld A. Fecal microbiota
transplantation for fulminant Clostridium difficile
infection in an allogeneic stem cell transplant
patient. Transpl Infect Dis. 2012;14(6):
E161-E165.
79. David LA, Maurice CF, Carmody RN, et al. Diet
rapidly and reproducibly alters the human gut
microbiome. Nature. 2014;505(7484):559-563.
80. Preidis GA, Versalovic J. Targeting the human
microbiome with antibiotics, probiotics, and
prebiotics: gastroenterology enters the
metagenomics era. Gastroenterology. 2009;
136(6):2015-2031.
2015 126: 311-318
doi:10.1182/blood-2015-04-574392 originally published
online May 26, 2015
The human microbiome in hematopoiesis and hematologic disorders
Veronica E. Manzo and Ami S. Bhatt
Updated information and services can be found at:
http://www.bloodjournal.org/content/126/3/311.full.html
Articles on similar topics can be found in the following Blood collections
Review Articles (589 articles)
Transplantation (2092 articles)
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.