Microbiota Changes during Allogeneic Stem Cell Transplantation: Comparison
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Please note this is a comparison between Version 2 by Sirius Huang and Version 1 by Elisabetta Metafuni.

Microbiota changes during allogeneic hematopoietic stem cell transplantation has several known causes: conditioning chemotherapy and radiation, broad-spectrum antibiotic administration, modification in nutrition status and diet, and graft-versus-host disease.

  • fecal microbiota transplantation
  • graft-versus-host disease
  • allogeneic hematopoietic stem cell transplantation

1. Introduction

The intestine is the main site of bacterial, viral, and fungal colonization. All these microorganisms do not merely act as commensal organisms, but actively participate in the digestion of complex carbohydrates and interact with the host immune system in a manner that we still do not fully understand. A healthy bacterial microbiota is involved in heterogeneous activities: development and maturity of the host immune system, digestion of food, synthesis of essential amino acids, short-chain fatty acids (SCFAs), and vitamins, regulation of the immune response, and enhancement of the resistance to pathogenic infection [1]. The vast majority of the bacteria belong to the Bacteroidetes and Firmicutes phyla; these bacteria are in equilibrium with the host’s innate immune system and help to maintain homeostasis, which directly affects host health when altered [2]. The gut microbiota of each individual contains many unique strains not found in others, and inter-individual differences in microbiota composition are much larger than intra-individual variations [3].
It has been known since the 1970s that the microbiota is involved in graft-versus-host disease (GvHD) pathogenesis, as demonstrated by the van Bekkum group in 1974, where mice raised in a germ-free environment did not develop gastrointestinal (GI) GvHD. Just recently, researchers have achieved a partial understanding of the panorama and the intricacies of the microbiota and GvHD [4,5,6][4][5][6]. The microbiota-derived signals can indirectly influence regulatory T lymphocytes (Tregs) by activating innate, gut-resident, antigen-presenting cells (APCs) known as CD103 + CD11b + dendritic cells (DCs) [7]. These DCs subsequently promote adaptive anti-inflammatory responses of Treg cells by producing molecules such as transforming growth factor β (TGF-β) and the vitamin A metabolite, retinoic acid. Tregs are not only responsible for maintaining immunological tolerance in tissues but also actively contribute to tissue repair through the production of amphiregulin [8]. This mechanism is important for homeostasis and modulates the immune response against the gut flora. An impaired mechanism can lead to a pro-inflammatory state that can be common in inflammatory bowel diseases, non-alcoholic steatohepatitis, type 2 diabetes, obesity, and, with some peculiarities, in acute GvHD (aGvHD) [9,10][9][10].
To recognize the complexity of the microbiota, it is essential to acknowledge that any factors disrupting or weakening this system can predispose to infections and autoimmunity. In the setting of allogeneic hematopoietic stem cell transplantation (HSCT), damage to the microbiota is not solely due to the conditioning regimen but also to the preceding chemotherapy and antibiotic treatments, which consistently undermine the microbiota [11,12][11][12].

2. Microbiota Changes during Allogeneic Stem Cell Transplantation

Significant changes were reported in microbiota composition during the HSCT procedure, both in terms of diversity and in terms of taxonomy [13]. These changes might contribute to post-transplant outcomes, such as GvHD incidence, transplant-related mortality (TRM), infectious complications, and overall survival (OS). A reduced intestinal microbial diversity after HSCT was reported to affect survival outcomes [14], while a low microbial diversity at engraftment was significantly associated with a high risk of TRM and severely reduced OS [15,16][15][16]. Predominant genera in samples obtained from patients with reduced microbial diversity were Enterococcus, Streptococcus, Enterobacteriaceae, and Lactobacillus [4,15][4][15]. Patients who died showed an abundance of Proteobacteria including Enterobacteriaceae, while surviving patients showed an abundance of Lachnospiraceae and Actinomycetaceae [15].

2.1. Antibiotics Affect the Gut Microbiota

A patient candidate for HSCT has already received several courses of broad-spectrum antibiotics. These therapeutic interventions have profoundly influenced the composition of the patient’s gut microbiota [17]. Notably, the most significant impact on the microbiota arises from the antimicrobial regimens administered both prior to and after transplantation. Different antibiotic classes exert distinct effects on the gut microbiota, which largely depend on the activity spectrum of the drug and may promote a different proinflammatory pattern in the gut flora. Particularly, the use of broad-spectrum antibiotics leads to a reduction in microbiota diversity and richness.
It has been demonstrated in mice that treatment with levofloxacin and cefepime had selected a gut flora with high abundances of Clostridia compared with meropenem-treated mice, and this difference was consistent with fecal butyrate levels. Additionally, both levofloxacin- and cefepime-treated mice had significantly lower abundances of Bacteroides thetaiotaomicron, compared with meropenem-treated mice. Notably, meropenem usage has been associated with the proliferation of Bacteroides thetaiotaomicron, a Gram-negative obligate anaerobe, that exhibits the capacity to metabolize dietary polysaccharides and host-derived glycans, including mucin. Therefore, Clostridia regulates intestinal immunity through SCFA production, while Bacteroides thetaiotaomicron is detrimental in determining mucus integrity. This observation provides a plausible explanation for the correlation between the utilization of carbapenems, frequently employed in the context of pre-engraftment febrile neutropenia, and the heightened severity of GI GvHD [18].
Different antibiotics used in the HSCT setting might impact GvHD-related mortality, probably due to the modification induced by antibiotics in microbiota composition [4]. A study conducted by Shono et al. on 857 patients who underwent T-cell-replete HSCT showed that among the twelve most frequently used antibiotics, piperacillin-tazobactam and imipenem-cilastatin were associated with different GvHD-related mortality rates. Additionally, these antibiotics were linked to an increased incidence of grade II–IV aGvHD with a higher occurrence of upper GI GvHD. Conversely, exposure to aztreonam or cefepime correlated with reduced GvHD-related mortality. Microbial composition in subjects and mice treated with imipenem-cilastatin showed a decreased presence of Clostridiales, which is believed to regulate anti-inflammatory processes in the gastrointestinal tract, inducing Tregs via SCFAs metabolites [19]. The use of antibiotic prophylaxes like ciprofloxacin and broad-spectrum systemic antibiotics reduced commensal bacteria, favoring the overgrowth of Enterococci [20]. Antibiotic therapy is not only used for infections but is also employed as a gut decontamination strategy. Weber et al. compared two decontamination schedules: ciprofloxacin 500 mg twice a day and metronidazole 500 mg three times a day, starting 8 days before HSCT until 14 days post-engraftment (n = 200) and rifaximin 200 mg twice a day (n = 194). Results indicated that gut decontamination with rifaximin was associated with similar rates of infectious complications compared with ciprofloxacin/metronidazole but preserved a high intestinal microbiota diversity and mitigated the negative effects of systemic antibiotics on microbial composition [21]. Previously hospitalized patients compared with de novo admitted exhibited a reduced expression of predominant commensal strains together with an increased presence of Enterococci [20]. The selection of antibiotics with a narrower spectrum of activity, particularly targeting anaerobic bacteria, may mitigate intestinal GVHD by minimizing the extent of microbiota alterations [22]. The results are presented Table 1.
Table 1.
Microbiota changes due to conditioning regimen.
References Population Antibiotic Microbiota Changes Effect
Hayase et al. [18] Mice Cefepime and Levofloxacin

vs.

meropenem		 ↑ Clostridiales

↓ Bacteroides thetaiotaomicron

↑ Enterococcus		 ↓ severe GvHD
Shono et al. [19] T-cell-replete HSCT human and mice Piperacillin-tazobactam

Imipenem-cilastatin

Azteronam and Cefepime		 ↓ Actinobacteria

↓ Clostridiales ↑ Erisipelotrichia and Enterococcus

↔ Clostridiales		 ↑ grade II–IV aGvHD

↑ GvHD-related mortality

↓ GvHD-related mortality
Holler et al. [20] Human HSCT Ciprofloxacin and broad-spectrum antibiotics ↑ Enterococci

↓ classical commensal bacteria		 ↑ GI-GvHD
Weber et al. [21] Human HSCT Ciprofloxacin and metronidazole

vs.

rifaximin		 ↔ Enterobacteriaceae ↓ GI-GvHD

↓ TRM

↑ OS
GvHD: graft-versus-host disease; HSCT: hematopoietic stem cell transplantation; GI: gastrointestinal; TRM: transplant-related mortality; OS: overall survival; ↑:increased abundance; ↓: reduced abundance; ↔: unmodified abundance.

2.2. Conditioning Affects the Gut Microbiota

By the time a patient arrives at the time of transplantation, he/she has already received varying amounts of chemotherapy and has been exposed to several antibiotics in most cases [23]. Therefore, the patient’s intestinal microbiota has been injured, displays a reduced α-diversity, and is going to receive a conditioning regimen with or without radiotherapy. This means that on a weakened flora susceptible to damage, further depletion will occur. Although we know that chemotherapy impairs intestinal microbiota, the mechanisms by which this occurs are still not completely clear [23,24][23][24].
Data about changes in the microbiota composition after HSCT are sometimes conflicting. Holler et al. compared pre- and post-HSCT fecal microbial composition finding an increase in Enterococci [20,25][20][25] and a reduction in Firmicutes and other phyla for all patients [20,26][20][26]. Recently, Kouidhi et al. reported a comparison between HSCT patients and healthy controls. At the phylum level, Actinobacteria were more represented in the control group compared with Proteobacteria and Verrucomicrobia in the HSCT group. At the genus level, patients in the HSCT group showed a lower abundance of Faecalibacterium, Alistipes, and Prevotella 9 and a higher abundance of Bacteroides, Escherichia/Shigella, Klebsiella, and Akkermansia [27]. It is well established that a conditioning regimen involving radioactive sources can lead to dysbiosis of the microbiota, and that radiation-induced enteritis is exacerbated by this dysbiosis. This understanding is derived not only from direct observations of microbiota damage in total body irradiation (TBI) regimens but also from extensive experience in oncology and the widespread use of radiotherapy [28].
In radiation enteritis, as in chemotherapy regimens, an increased abundance of bacteria belonging to the Actinobacteria and Proteobacteria phyla is reported. These bacteria are conditional pathogens such as Escherichia coli. Conversely, microorganisms from the Firmicutes and Bacteroides phyla are reduced by radiations [29]. In a recent paper, Gu and colleagues reported microbiota changes during myeloablative transplantation, including rabbit thymoglobulin administration. Microbiota diversity began to decline from the start of the conditioning regimen and continued to diminish over the course of HSCT until day 12 after HSCT, where diversity reached the lowest value. After that, diversity gradually increased over time. Intestinal domination varied from beneficial genus Bacteroides before conditioning to pathogenic genera such as Enterococcus, Klebsiella, and Escherichia during the engraftment phase [16].
The reduction in microbiota diversity described early after HSCT is a byproduct of conditioning regimens and restoration of pre-HSCT levels is possible over time in the absence of GvHD or other modifying events [27,30][27][30]. A reduced microbial diversity after HSCT was associated with high GvHD lethality [14], while the persistence of high microbiota diversity after HSCT was associated with a lower risk of death and TRM, without an increased relapse rate [4]. Results are resumed in Table 2.
Table 2.
Microbiota changes due to conditioning regimen.
References Population Microbiota Changes Effect
Stein-Thoeringer et al. [25] Human and mice HSCT ↑ Enterococcus genus and Enterococcus faecium species ↓ OS

↑ GvHD-related mortality
Chiusolo et al. [26] Human HSCT ↑ Bacteroidetes

↓ Firmicutes		 -

-
Kouidhi [27] HSCT

vs.

Healthy subjects		 ↑ Proteobacteria and Verrucomicrobia phyla

↓ Faecalibacterium, Alistipes, and Prevotella 9 genera

↑Bacteroides, Escherichia/Shigella,

↑ Klebsiella, Veionella and Akkermansia genera

↑ Actinobacteria phylum

↑ Faecalibacterium, Alistipes, and Prevotella 9 genera		 -

-

↑ GvHD

↓ GvHD
Jian et al. [28] Radiotherapy ↓ Lactobacillus and Bifidobacterium

↑ Staphylococcus and Escherichia coli		 ↑ radiation enteritis

↓ intestinal barrier function

↑ inflammation
Gu et al. [16] Myeloablative conditioning plus ATG ↓ Bacteroides genus and

↑ Enterococcus, Klebsiella, and Escherichia genera		 ↓ OS for low microbial diversity
HSCT: hematopoietic stem cell transplantation; GvHD: graft-versus-host disease; ATG: anty-thymocyte globulins; OS: overall survival; ↑:increased abundance; ↓: reduced abundance.

2.3. Diet Affects Microbiota

The role of diet and nutrition in HSCT patients is often underappreciated, but growing evidence suggests a link between the diet, the microbiota, and clinical outcomes. Although we do not yet know exactly how this element can be used to deliberately select a tolerogenic microbiota, some evidence suggests its potential role in the HSCT setting. It has been demonstrated that enteral nutrition, compared to parenteral nutrition, is protective against the development of GvHD and reduces TRM while increasing OS [31,32][31][32].
In this regard, it is even more intriguing evidence provided by Khuat et al., who demonstrated in preclinical models and clinical trials that obesity has a negative effect on HSCT outcomes in both mice and humans. Obesity is specifically associated with an increased risk of aGVHD with GI involvement. This effect appeared restricted to the gut and relies on increased production of pro-inflammatory cytokines by donor CD4+ T-cells. In the murine model, the pre-transplant diet and consequently the selected microbiota can influence TRM, often related to GI aGvHD. Indeed, it has been observed that mice with diet-induced obesity (DIO) exhibited increased gut permeability and translocation of endotoxins across the gut barrier, along with reduced diversity in their gut microbiota. After HSCT, these changes in DIO mice promoted aGvHD and led to a more severe clinical presentation [33].

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