Gut Microbiota and Hematopoietic Stem Cell Transplantation: Where Do We Stand?

REVIEW Gut microbiota and hematopoietic stem cell transplantation: where do we stand?

D Zama1, E Biagi2, R Masetti1, P Gasperini1, A Prete1, M Candela2, P Brigidi2 and A Pession1

Advances in biological techniques have potentiated great progresses in understanding the interaction between human beings and the ∼ 10 to 100 trillion microbes living in their gastrointestinal tract: gut microbiota (GM). In this review, we describe recent emerging data on the role of GM in hematopoietic stem cell transplantation, with a focus on immunomodulatory properties in the immune system recovery and the impact in the development of the main complications, as GvHD and infections.

Bone Marrow Transplantation (2017) 52, 7–14; doi:10.1038/bmt.2016.173; published online 27 June 2016

INTRODUCTION MA, USA). Both these technologies merge the low cost per sequence The microorganisms living in association with the human body with the high-throughput sequencing of short DNA fragments. are collectively called microbiota. Human beings share a close The molecular assessment of the human GM, principally relies symbiotic relationship with ∼ 10 to 100 trillion microbes living in on two approaches of NGS of microbial DNA, the sequencing their gastrointestinal tract. Advances in biological techniques with of the V3–V4 regions of 16S ribosomal DNA gene, which allows the introduction of massively parallel next-generation sequencing reconstructing the phylogenetic structure of the ecosystem. (NGS) provided new insights into the composition and complexity The description of the GM compositional structure by of the gut microbiota (GM) by identifying bacterial taxons that are 16S ribosomal DNA sequencing involves a robust and well- 10 not readily cultivated in vitro.1–3 consolidated pipeline, including DNA extraction from stool, PCR The microbiota plays an important role in human health and amplification of the V3–V4 region of the 16S ribosomal DNA, disease. Particularly, GM is involved in energy harvest and storage, library preparation, DNA sequencing, bioinformatics and 11 and in metabolic functions,4 limits the access of enteropathogens biostatistics analyses. Besides providing the compositional to the gastrointestinal tract5 and influences host immunity, being structure of the GM ecosystem at different phylogenetic levels strategic to the education and preservation of our immune (from phylum up to the genus level), the Quantitative Insights Into homeostasis.6,7 Microbial Ecology (QIIME) pipeline allows to compute the inter- Considering the close cross talk between GM and the immune individual diversity (alpha-diversity) and the intra-individual system, an emerging role has been increasingly attributed to GM diversity (beta-diversity) of the ecosystem. Both parameters are after hematopoietic stem cell transplantation (HSCT). New insights key factors to describe the configuration of the gut microbial have been achieved over the past years on the role of GM on ecosystem. The first is the most relevant factor to define a immune recovery after HSCT and onset of infections and GvHD. dysbiotic GM structure, which is always characterized by a 12 In this review, we provide an update on the biology of reduction of the ecosystem diversity. The second is a funda- immune homeostasis in relation to the microbiota and HSCT. mental factor to describe and quantify the overall compositional Methodologies of characterization of GM are summarized with a differences between groups of subjects, such as in case control comprehensive review of the available literature evaluating the studies exploring GM-associated compositional deviations. impact of GM on HSCT outcome. By shotgun NGS of the microbial DNA extracted from stools, it is possible to characterize the GM metagenome, defined as the overall genome content of GM microbes.13–16 Even if a giant METHODOLOGICAL APPROACH TO GUT MICROBIOTA sequencing effort is necessary, the characterization of the GM CHARACTERIZATION microbiome allows provision of useful information on the functional configuration of the ecosystem, which can be easily In the past years, new culture-dependent approaches for GM 17 characterization have been developed, allowing an increase in the translated into the overall metabolic propensity. cultivable fraction of the ecosystem up to the 50%.8 However, it is a matter of fact that, nowadays, the method elected to conduct a GM study still remains the deep sequencing of the microbial DNA THE RELATIONSHIP BETWEEN GUT MICROBIOTA AND THE extracted from stool samples,9 the only approach allowing HOST DEFENSE disclosure of the whole-biological complexity of the ecosystem. The gastrointestinal barrier is the largest surface of the human Deep sequencing of microbial DNA is principally carried out by body, and, in addition to the absorptive role, it provides several the NGS-based technologies, essentially 454-pyrosequencing (Life different functions such as regulation of microbial colonization Sciences-Roche, Branford, CT, USA), Illumina (San Diego, CA, USA) and mucosal immune responses. GM is controlled by specialized and Ion Torrent NGS platforms (Thermo Fisher Scientific, Waltham, intestinal epithelial cells (IECs), secretory goblet cells and Paneth

1Pediatric Oncology and Haematology Unit `Lalla Seràgnoli', Department of Pediatrics, University of Bologna, Sant'Orsola Malpighi Hospital, Bologna, Italy and 2Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy. Correspondence: Dr D Zama, Paediatric Oncology and Haematology Unit `Lalla Seràgnoli', Department of Pediatrics, University of Bologna, Sant’Orsola Malpighi Hospital, Via Massarenti 11, Bologna 40138, Italy. E-mail: [email protected] Received 3 February 2016; revised 10 May 2016; accepted 13 May 2016; published online 27 June 2016 Gut microbiota and hematopoietic stem cell transplantation D Zama et al 8 cells, through the production of an apical mucous layer, the THE IMMUNOMODULATORY EFFECT OF GUT MICROBIOTA ON secretion of broadly targeted antimicrobial proteins (AMPs) and THE ANTICANCER IMMUNE RESPONSE 18–21 the transcytosis of secretory IgA. The role of microbiota in shaping the systemic immune response The composition and products of GM can be considered an was studied in a cancer model and astonishing data suggested that extension of the self and have unexpected effects on GM composition may significantly influence the anticancer immune the immune system. For example, two supposedly genetically response. Commensal bacteria differentially affect the type of identical organisms, such as same-sex inbred mice, can have inflammatory tone required for the response to traditional widely different metabolic and inflammatory responses, chemotherapeutic agents, as cyclophosphamide,39,40 and to depending on the structure of their microbiota.22 In a condition antibodies blocking immune inhibitory pathways, as cytotoxic of immune homeostasis, mutualistic GM components T-lymphocyte antigen (CTLA)-4 and the axis between programmed communicate with the gut epithelium and underlying lymphoid cell death protein 1 (PD-1) and its ligand 1 (PD-L1).41,42 tissues, favoring a normal function of immune phenotype In particular, cyclophosphamide modifies GM composition in with no expression of disease.23 On the contrary, GM dysbiosis the small intestine and induces the translocation of selected can trigger, or predispose to, several inflammatory disorders, as Gram-positive bacteria into secondary lymphoid organs. These fi inflammatory bowel diseases, asthma, autoimmune arthritis and bacteria stimulate the generation of a speci c immune responses – ‘ ’ diabetes.24 27 mediated by pathogenic Th17 cells and memory Th1. Mice that In the gut lumen, a complex link between the immune system were germ-free or treated with antibiotics to kill Gram-positive and the microbiota is spread over several levels. The immune bacteria had a reduced Th17 responses, and their tumors were system response can be mediated by the recognition of microbial resistant to cyclophosphamide. Adoptive transfer of Th17 cells fi 39 ligands or by a direct interaction between cells of the immune partially restored the antitumor ef cacy of cyclophosphamide. In another experiment with antibiotic-treated or germ-free system and luminal, and components of the microorganisms, or mice, tumor-infiltrating myeloid-derived cells responded poorly to throughout the interaction with their metabolic end products, fi platinum chemotherapy. This results in lower cytokine production such as the short-chain fatty acids (SCFA). The nal balance and tumor necrosis, and deficient production of reactive oxygen among all of these signals may favor the immune tolerance or an species and cytotoxicity after chemotherapy. Thus, optimal fl 28 in ammatory immune response. responses to cancer therapy require an intact commensal One example of this interaction is represented by IECs, which microbiota, mediating its effects by modulating myeloid-derived integrate commensal bacteria-derived signals into antimicrobial cell functions in the tumor microenvironment.40 and immunoregulatory responses by recognition of microbial Also the growth of melanoma was different in mice-harboring ligands by Ag-independent pattern recognition receptors (PRRs). distinct commensal GM compositions. In this experiment, the PRRs are receptors, which specifically recognize and bind presence of commensal Bifidobacterium modulates T-cell distinctive microbial molecular ligands, such as lipopolysaccharide, infiltration of the tumor, resulting in favorable outcomes and flagellin, peptidoglycans and formylated peptides. The most enhanced response to immunotherapy with PD-L1 antibodies.41 studied PRRs are toll-like receptors (TLRs). In addition to The immunostimulatory role of GM composition was also IECs, TLRs are also expressed cells of the innate immune studied in a mouse model of melanoma treated with monoclonal system (macrophages and dendritic cells (DCs)) and cells of the Ab directed against CTLA-4, a different negative regulator of adaptive immune system (T- and B cells). Functional consequence T-cell activation. The GM composition, particularly the presence of TLR engagement depends on the responding cell type of Bacteroidales, affects Th1 immune responses, facilitating tumor of the cell where the ligand is encountered. Unlike pathogens control in mice and patients, although sparing intestinal integrity. that use TLR pathways to trigger inflammation, commensal Tumors in antibiotic-treated or germ-free mice did not respond to bacteria exploit the TLR pathway to maintain intestinal CTLA blockade. This defect was overcome by gavage with homeostasis.29,30 B. fragilis by immunization with B. fragilis polysaccharides, or by fi 42 Another example is represented by microfold cells, specialized adoptive transfer of B. fragilis-speci c T cells. All these IECs concentrated in the follicle-associated epithelium,31 which observations support the idea that the composition of gut continuously sample luminal Ags for presentation to the microbes could be manipulated for stimulate commensals underlying dendritic cells.32–34 The activation of these cells, associated with antitumor immunity. by commensal bacteria, such as Lactobacillus reuteri and L. casei, modulate immune system by the differentiation of naive T-helper GUT MICROBIOTA AND HEMATOPOIETIC STEM CELL (Th) cells into Treg cells.35 TRANSPLANTATION Immune response is also coupled with information coming from Evolution of GM composition during HSCT the local gut throughout the capability of DCs of monitoring 36 GM structure before the transplantation well approximates the availability of ATP derived from commensal bacteria. commonly reported healthy-like profile, in terms of richness and DC-associated sensors of cellular energy resources are regulated – diversity of the ecosystem1 both in adults and children.43 45 It is by AMP-activated protein kinases, as mammalian target of also efficient in terms of SCFA production, the most reliable rapamycin. This pathway affects both the innate and adaptive biomarker of microbiota–host mutualistic configuration.6 arms of the immune system. Indeed, in response to ATP This observation demonstrates the high dynamicity of the GM, stimulation, the secretion of interleukin (IL)-6 and IL-23 by 37 which recovers its layout before the HSCT, in spite of the activated DCs induces Th17 cell differentiation, and inhibits treatments received by these patients in terms of chemotherapy Treg development and memory T-cell formation, promotes the and antibiotic exposure (Figure 1). differentiation of Th1, Th2 and Th17 cells, and regulates HSCT and all the related standard procedures, such as 36,38 CD8+ T-cell trafficking. conditioning regimen, antibiotic exposure, diet and anti-acid All these mechanisms are only a part of the complex interaction prophylaxis, represent an upset event with a profound regulating local and systemic inflammatory response, arising from modification of the GM structure and disruption of its mutualistic the relationship between immune system and GM, and asset.43,44 In particular, during the neutropenic period, the species underscore how the GM exerts a key role in the defense system richness of the gut ecosystem (alpha-diversity) decreased of ~ 30% of the host. compared with the pre-HSCT. The GM structure is characterized by

Conditioning regimen Loss of GM diversity associated with TRM antibiotics, diet GvHD Specific dysbioses: - Higher loss of diversity, - Drop of health-promoting faecalibacterium, - Increase of enterococcus.

Intestinal domination: - Enterococcus Vancomycin-resistant bacteremia - Proteobacteria GRAM negative bacteremia GM biodiversity

Healthy-like GM profile Disruption of mutualistic asset Ecosystem recovery (normal SCFA production) (invasion of new species (>90%))

Day 0 Engraftment Day 60 Figure 1. Hematopoietic stem cell transplantation (HSCT) temporarily disrupts diversity, individual signature and short-chain fatty acid production of the gut microbiota. HSCT resulted in a profound disturbance of the gut ecosystem that can be measured in the loss of ~ 30% in average of the pre-HSCT alpha-diversity. A full color version of this figure is available at the Bone Marrow Transplantation journal online. a massive ‘invasion’ of new species and o10% of the pre-existing proved to be a reliable predictor of the overall mortality in species being conserved. These alterations bring about a loss of transplanted patients. Indeed, in a cohort of 80 transplanted the individual layout.43 Extreme shifts in the intestinal microbiota adults, low-, intermediate- and high-diversity intestinal diversity of were observed in two-third of patients, documenting either the GM at the engraftment defined three groups with different Enterococcal or streptococcal domination.46 This deep modifica- overall survival at 3 years, 36, 60, and 67%, respectively.45 tion of GM is also accompanied by a loss in the abundance of The increase of the mortality rate in this study was primarily health-promoting species, for example Faecalibacterium and due to transplant-related causes, suggesting a link between that Ruminococcus.43 lack of microbial diversity and infection and/or GvHD.45 Specific factors were identified to be associated with a specific Also the level of urinary excretion of 3-indoxyl sulfate, an GM layout following HSCT. Increase in Enterococci and simulta- indirect marker of GM health, was found to predict poor, early and neous loss of commensal bacteria was found in patients receiving long-term outcomes of transplanted patients.53 3-Indoxyl sulfate is both oral ciprofloxacin and systemic broad-spectrum antibiotics. A a product of L-tryptophan metabolism by commensal bacteria possible explanation of this phenomenon is that the commensal expressing tryptophanase and acts as intercellular signal in bacteria are important inducers of AMPs, such as α-defensins microbial communities, modulating mucosal barrier function and produced by Paneth cells, selectively active against non- expression of pro- and anti-inflammatory genes in the IECs. Low commensal Gram-negative and Gram-positive bacteria,47 target- 3-indoxyl sulfate levels were the result of a disrupted GM ing highly conserved and essential features of bacterial biology. signature, and were associated with significantly higher After prolonged use of these antibiotics, the presence of transplant-related mortality and worse overall survival 1 year commensal bacteria in GM declines and, consequently, also AMPs, after HSCT.53 (Clinical studies evaluating GM in HSCT are such as C-type lectin regenerating islet-derived protein III α (REG3α), summarized in Table 1). which is the major peptide preventing Enterococcal overgrowth.48 Also in patients with gastrointestinal GvHD a marked reduction in GvHD the secretion α-defensins due to the allogeneic T-cell response has GvHD is associated with major shifts in the composition of the GM been described, resulting in profound damage to Paneth cells. As both in murine and human recipients of HSCT.54 In mouse models, mentioned above, these peptides regulate the pathogen flora in specific dysbiosis associated with GvHD are characterized by loss the gut, although preserving commensals. So the final result is the of the overall diversity, increase of members of loss of diversity of endogenous mutualists, paving the way to the the bacterial orders Enterobacteriales (Escherichia, Klebsiella, proliferation of alloctonous opportunistic bacteria.49 and Enterobacter species) and Lactobacillales (Lactobacillus, Interestingly, a small percentage of the bacterial species resists Enterococcus and Streptococcus species), and reduction of during HSCT and is conserved throughout the whole longitudinal obligate anaerobic bacteria from the order of the survey, as a consequence of the ecosystem being upset, and Clostridiales (Faecalibacterium, Roseburia, Ruminococcus and ‘reappear’ later on. These persisting species could act as 'founders' Blautia).49,54,55 to reconstruct a healthy-like ecosystem once conditions allow.43 An experimental mouse model demonstrated that microbiota, Indeed, Bacteroides species, which are the most represented in turn, could modulate the severity of intestinal inflammation. among the persisting ones, are known to act as a bacterial Indeed, GvHD was aggravated eliminating Lactobacillales from the reservoir to maintain long-term colonization or repopulate the gut – flora of the mice before HSCT, whereas the reintroduction of after the ecosystem disruption.50 52 On the contrary, the great predominant species of Lactobacillus mediated significant majority of the newly acquired phylotypes, invading temporarily 54 protection against GvHD. Another elegant experimental mouse the ecosystem, is replaced later on during the GM recovery. In fact, model compared either L.rhamnosus GG or ciprofloxacin alone after a period of ~ 60 to 100 days after HSCT, the ecosystem from day 7 before transplantation and throughout the recovers the initial amount of richness, underlying once again the post-transplantation period, or ciprofloxacin for 7 days, followed high dynamicity of the GM. by L. rhamnosus GG for the post-transplantation period showing that oral administration of L. rhamnosus GG before and after The role of GM in the HSCT transplantation results in improved survival and reduced acute The relevant impact of GM in the HSCT was recently demon- GvHD. Furthermore, culturing of mesenteric lymph node tissue, as strated. Particularly, the diversity of the gut microbial community a sign of an affected mucosal barrier function due to the

Table 1. Here we report the studies, which evaluate the role of gut microbiota using next-generation sequencing platforms in human patients receiving allogeneic hematopoietic stem cell transplantation

Study Patients Methods of GM analysis Aim Main results

Taur 94 Fecal samples from conditioning regimen to To analyze the risk of bacteremia • Bloodstream infection was detected in 22 (23.4%) et al.54 adults day +35 days after HSCT. during HSCT. patients: 9 were due to VRE, 10 to Gram-negative bacteria. V1–V3 region of the 16 S rRNA gene was ampliﬁed by means of PCR. Puriﬁed PCR • Enterococcal domination (40.4% of pts) increased products were sequenced on a 454 GS FLX the risk of VRE bacteremia. Risk factors were Titanium platform (Roche Diagnostics, leukemia diagnosis and metronidazole Indianapolis, IN, USA). administration.

• Proteobacterial domination (12.8% pts) increased the risk of Gam-negative bacteremia. Proteobacteria domination was reduced 10-fold by fluoroquinolone administration. Taur 80 Fecal samples collected within 7 days To analyze the impact of GM • The diversity of the intestinal microbiota at et al.53 adults following stem cell engraftment. diversity on mortality following engraftment is an independent predictor of the HSCT. mortality. PCR followed by sequencing for microbiota analysis. • On the basis of the overall bacterial diversity, pts were classified in high-, intermediate-, low-diversity groups, which, respectively, correspond to survival at 3 years 36, 60 and 67%. Holler 31 Fecal samples collected at a min. of three time To monitor microbiome shifts in • A major loss of bacterial diversity contributed to et al.52 adults points: before HSCT, during aplastic period, and the course of allogeneic HSCT. the intestinal inflammation. on days 21–28. • Increase in the proportion of Enterococci and NGS of the V3 region of the 16s rRNA gene, PCR decrease in the commensal bacteria was prominent of Enterococci and analysis of urinary indoxyl in patients who developed GI GvHD or suffered sulphate. from active GI GvHD. Jenq 115 Fecal samples collected within 4 days of day 12 To evaluate the relationship • Increased bacterial diversity was associated with et al. 66 adults after T-cell-replete HSCT. between GvHD and the fecal reduced GVHD-related mortality. bacterial composition after 1st 64-patient cohort: 454 GS FLX Titanium HSCT. • Blautia abundance was associated with reduced platfor. GvHD lethality and improved the overall survival. 2nd 51-patient cohort: Illumina MiSeq platform Loss of Blautia was associated with treatment to sequence the V4–V5 region of the 16S rRNA with antibiotics that inhibit anaerobic bacteria and gene. receiving total parenteral nutrition for longer durations. Weber 131 Urinary 3-IS was performed by reversed-phase To evaluate the correlation of • Low urinary 3-IS levels between days 0 and et al.61 adults liquid chromatography–electrospray urinary 3-IS levels as predictors 10 after HSCT were associated with poor, early ionization–tandem MS at the time of HSCT and of outcome of HSCT. and long-term outcome because of an increased early thereafter. frequency of lethal complications mostly as a consequence of GI GvHD. Biagi 10 Fecal samples collected pre-HSCT and at least To analyze the connection • GvHD and non-GvHD subjects had a different et al.51 children three times after HSCT. between the process of GM temporal trajectory of GM ecosystem recovery after reconstruction GvHD in pediatric HSCT. V4 region of the 16S rRNA gene was amplified patients. and subsequently NGS was performed. GC–MS • Specific dysbiosis associated with GvHD: drop of used to determine SCFA production. Faecalibacterium and increase of Enterococcus.

• In pre-HSCT samples, non-GvHD pts had higher abundances of mutualists propionate-producing Bacteroidetes, which persist during the HSCT-induced ecosystem disruption. Haris 66 Fecal samples from conditioning regimen to day To investigate whether changes • 112 PCs occurred in 66 subjects. et al.82 adults +35 days after HSCT. in GM are associated with PCs after HSCT. • High co-morbidity index, fluoroquinolones, low- V1–V3 region of the 16 S rRNA gene was GM baseline diversity and Gammaproteobacteria amplified by means of PCR. Purified PCR domination of fecal microbiota, which included products were sequenced on a 454 GS FLX common respiratory pathogens, predicted PCs. Titanium platform. • In separate analyses, low-baseline diversity was associated with PCs occurring pre-engraftment, although Gammaproteobacteria domination predicted PCs post engraftment and the overall mortality. Abbreviations: 3-IS = 3-indoxyl sulphate; GC–MS = gas chromatography–mass spectrometry; GI = gastrointestinal; GM = gut Microbiota; HSCT = hematopoietic stem cell transplantation; min. = minimum; NGS = next-generation sequencing; pts = patients; PCs = pulmonary complications; rRNA = ribosomal RNA; SCFA = short-chain fatty acids; VRE = vancomycin-resistant Enterococcus.

Bone Marrow Transplantation (2017) 7 – 14 © 2017 Macmillan Publishers Limited, part of Springer Nature. Gut microbiota and hematopoietic stem cell transplantation D Zama et al 11 intestinal inflammation, revealed a reduced translocation of commensal microbes to invade underlying tissues and the enteric bacteria.56 This observation indicates that antibiotic bloodstream.71 exposure before HSCT modifies the risk of intestinal GvHD and As a result, systemic bacterial infections are frequent during the that targeted flora reintroduction may potentially reduce the early transplant period.72 Vancomycin-resistant Enterococcus (VRE), severity of GvHD. viridians group Streptococcus and aerobic Gram-negative bacteria Data found in humans, both adults and children are in line with are the most common causes of bloodstream infection following those found in mouse models. In particular, GvHD onset was HSCT.73,74 The complex commensal microbial populations associated with a drop in the abundance of the known colonizing the human intestine provide resistance to infection, health-promoting Faecalibacterium of the order of Clostridiales whereas the increase of pathogens in the gut favors the and high percentages of Enterococcus.43,44 These latter development of resistant bacteria.75 Recently, Taur et al.46 microorganisms are potential contributors to increased intestinal characterized longitudinally the fecal microbiota of 94 patients inflammation in both mice and humans, by induction of IL-1 undergoing HSCT and observed extreme shifts in the intestinal and IL-6 production, resulting in subsequent Th1 and Th17 microbiota in two-thirds of patients with either Enterococcal or activation.44 Thus, the predominance of Enterococci together with Streptococcal domination, with a cutoff definition of 30% for the reduction of commensals Faecalibacterium and Clostridiales, intestinal domination. These perturbations correlated with considered to be important for maintaining tolerance against subsequent development of a corresponding bloodstream the intestinal microbiome,57,58 can favor the consolidation of infection with either VRE or Gram-negative bacteria. intestinal inflammation. On the other hand, intestinal flora Interestingly, viridians group Streptococci and VRE were diversity and a high percentage of the bacterial genus of the historically the most frequently encountered bloodstream Blautia of the order of Clostridiales in the GM in the early period infections in HSCT patients during the pre-engraftment 72,73,76 after HSCT were associated with reduced GvHD lethality.59 period. Enterococcal domination was associated with an Finally, we have successfully demonstrated different temporal increased risk of VRE bacteremia, whereas intestinal domination trajectory of GM ecosystem recovery after HSCT between GvHD by Proteobacteria increased the risk of bacteremia with aerobic and non-GvHD subjects.43 Interestingly, GvHD and non-GvHD Gram-negative bacilli. In almost 50% of the patients with subjects differ also in the pre-HSCT GM signature, which bacteremia, a preceding intestinal domination by a corresponding is projected in the distinctive trajectory of post-HSCT GM organism has been demonstrated and the median time between 46 reconstruction. Non-GvHD subjects have a higher abundance of domination and bacteremia in these patients was 7 days. members of the Bacteroidetes phylum, known to produce The role of antibiotics administered during HSCT on GM SCFA from dietary fiber,55 influencing both the magnitude diversity was extensively evaluated. Metronidazole was associated 77 and the quality of the immune response.60 SCFA are end products with increased risk of Enterococcal domination, supporting the idea that anaerobic bacteria contribute to colonization of microbial fermentation of complex plant polysaccharides 78 that cannot be digested by humans alone, because our genomes resistance. Fluoroquinolones protected against proteobacterial do not encode an adequate repertoire of glycoside hydrolases.61 domination, reducing the risk of Gram-negative bloodstream infections. This finding supports prior studies of fluoroquinolone SCFA are one of the clearest examples of how diet and 79 nutrient processing by the microbiota combine to shape prophylaxis in neutropenic patients and sheds light on the immune responses. mechanism of protection. The complex polysaccharides fermenters members of the Clostridia species are associated with the increased production Pulmonary complications of SCFA (acetate, propionate and butyrate), and consequent Several studies have shown gut dysbiosis can trigger inflammation expansion and activation of colonic regulatory T cells (Tregs) by in asthma and allergic airways diseases,26 cystic fibrosis80 and 57,62 IECs production of transforming growth factor-β. In particular, infection-mediated lung inflammation81 also in the absence of butyrate modify the cytokine production profile inducing potent lung colonization or infection. Intestinal permeability may be anti-inflammatory cytokine IL-10, induce colonic Tregs through implicated in the translocation of bacteria and/or microparticles, 61 63 upregulation of gut-homing molecules and Foxp3. It also such as lipopolysaccharide, on the pathway to acute lung injury 64 promotes intestinal epithelial barrier integrity, limiting exposure and sepsis-associated acute respiratory distress syndrome. of the mucosal immune system to luminal microbes, preventing Harris et al.82 recently investigated the role of GM in gut–lung aberrant the inflammatory responses and enhancement of cross talk after HSCT aiming to evaluate the role of GM in the hematopoiesis of DC precursors with a low ability to activate the onset of pulmonary complications (PCs). They found 112 PCs allergy-related Th type 2 cells.26 (defined by radiographic parenchymal abnormalities in the setting Interestingly, dysbiosis of patients with GvHD, share similar of respiratory signs/symptoms and associated mortality) in 66 allo- features to those found in patients with inflammatory bowel transplanted patients. High co-morbidity index, fluoroquinolones, diseases. Indeed, reduced microbial richness, impaired abundance low-baseline diversity and interestingly Gammaproteobacteria of butyrate producers and relative abundance of Gram-positive domination of fecal microbiota predicted PCs. Gammaproteobac- bacteria (especially Clostridium clusters IV and XIVa) were teria is a class of bacteria that includes respiratory pathogens such associated with intensity of intestinal inflammation in both.65 as Klebsiella pneumoniae and K. oxytoca. These findings may The levels of Enterobacteriaceae at the diagnosis were negatively suggest direct translocation of bacteria to the lungs during early correlated with follow-up clinical outcomes and increased transplant or indirect lung injury caused by microbiota stimulation abundance was associated with the grade of inflammation.66 Like of a systemic or local inflammatory response as possible in GvHD also in Crhon’s disease, lower levels of Firmicutes, such as mechanisms; alternatively, associations might reflect overall Faecalibacterium prausnitizi a major producer of the SCFA butyrate, microbiota status or antimicrobial use. were found.67,68 Graft-versus-tumor Infection Considering that intestinal bacteria are modulators of systemic Pretransplant conditioning transiently ablates circulating immune responses Peled et al.83 also hypothesized that granulocytes and monocytes, and markedly increases suscept- components of the intestinal flora could be associated with ibility to disseminated infections.69,70 In addition, radiation and graft-versus-tumor activity, modifying relapse rate after HSCT. For chemotherapy-mediated destruction of gut epithelial cells enables the first time, an association between relapse after HSCT and GM

© 2017 Macmillan Publishers Limited, part of Springer Nature. Bone Marrow Transplantation (2017) 7 – 14 Gut microbiota and hematopoietic stem cell transplantation D Zama et al 12 composition was found. In a cohort of 160 allografted adults of GM should pave the way to targeting the microbiota in a affected by oncological disease, the taxons most significantly rational way in experimental and clinical studies, necessary to associated with freedom from relapse were members of the explore its beneficial effects in HSCT. human oral flora, including Streptococcus anginosus. Patients with higher abundance of this bacterium had less relapse after transplantation, whereas Enterococcus faecium was associated CONFLICT OF INTEREST with the increased risk of relapse.83 The authors declare no conflict of interest.

FUTURE PERSPECTIVES ACKNOWLEDGEMENTS The increasing knowledge of the relationship between GM and This work was supported by a grant from Fondazione Umberto Veronesi (Milan). immune system opens countless diagnostic and therapeutic opportunities in transplanted patients. Several diagnostic and REFERENCES therapeutic strategies can be hypothesized to transfer from the bench to the bedside of this knowledge. 1 Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M et al. fi Diversity of the human intestinal microbial flora. Science 2005; 308: Identi cation of high-risk patients with an increased probability – of transplant-related mortality could help to prevent or treat early 1635 1638. 2 Dethlefsen L, Huse S, Sogin ML, Relman DA. The pervasive effects of an antibiotic life-threatening complications linked to dysbiosis of the GM. 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