the mammalian gastrointestinal ( gi ) tract harbors a dense and diverse microbial community , which is composed primarily of bacteria but also includes fungi , archaea , and viruses ; collectively , these are referred to as the intestinal microbiota .
these microorganisms establish symbiotic relationships with their hosts , playing crucial roles in the digestion of food and exerting a considerable influence on the physiological , metabolic , nutritional , and immunological state of the host [ 24 ] .
recent studies have demonstrated that alterations in the composition of intestinal microbiota are linked to multiple metabolic and inflammatory diseases in humans , including obesity , inflammatory bowel disease ( ibd ) , colorectal cancer , allergy , type 2 diabetes , liver cirrhosis , rheumatoid arthritis , and neurodevelopmental disorders [ 512 ] .
these associations raise fundamental questions regarding the immunomodulatory mechanisms by which components of the intestinal microbiota and their metabolites influence resistance or susceptibility to a broad range of clinically important diseases .
allogeneic hematopoietic stem cell transplantation ( allo - hsct ) is the only curative therapy for hematologic malignant tumors , bone marrow failure , and congenital metabolic disorders .
however , the development of acute graft - versus - host disease ( agvhd ) limits the success of allo - hsct and is fatal to approximately 15% of transplant recipients [ 13 , 14 ] .
agvhd results from an immunological attack on target recipient organs and tissues ( such as the skin , liver , lung , and gi tract ) by donor allogeneic t cells that are transferred along with the allograft .
the development and severity of agvhd in transplant recipients depend on factors such as recipient age , toxicity of the conditioning regimen , hematopoietic graft source , and agvhd prophylaxis approaches .
steroids are the first line of treatment , but patients with steroid - refractory agvhd have a dismal outcome , with long - term mortality rates that can reach 90% .
recent studies have demonstrated a close relationship between intestinal microbiota composition and the severity of agvhd [ 1719 ] .
for many years , our understanding of the composition of mammalian intestinal microbiota depended on culturing and identifying commensal microorganisms .
however , this approach is insufficient to catalog intestinal microbial species because the majority of intestinal bacteria can not be cultured by currently available methods .
the emergence of new molecular profiling techniques , such as 16s rrna sequence - based microbial identification and high - throughput sequencing analysis , has led to a revolution in the understanding of the intestinal microbiota by allowing culture - independent analysis of microbial community composition .
the human gut harbors approximately one hundred trillion microbes , which is ten times the number of human cells in the body .
their combined genomes contain more than five million genes , outnumbering the human genetic potential by two orders of magnitude .
the most abundant phyla in the human intestine are firmicutes and bacteroidetes , which constitute over 90% of the human intestinal microbes .
the firmicutes consist primarily of bacteria belonging to the clostridia class and include a subset of bacilli ( bacillaceae , enterococcaceae , and lactobacillaceae ) , which are capable of oxidizing organic sugars via fermentation to produce large amounts of lactic acid and carbon dioxide [ 24 , 25 ] .
members of the gut bacteria belonging to the bacteroidetes are represented by several bacteroides species , including b. acidifaciens , b. sartorii , and b. uniformis . the remainder of the commensal bacteria , accounting for less than 10% of the total population , belongs to the phyla proteobacteria , fusobacteria , actinobacteria , verrucomicrobia , cyanobacteria , and tm7 .
these bacteria are capable of successfully competing with members of the firmicutes and bacteroidetes in a strict anaerobic environment , such as the colon [ 26 , 27 ] .
the importance of the intestinal microbiota to host health is highlighted by alterations in community composition in metabolic and inflammatory diseases , such as obesity and ibd [ 5 , 6 ] .
the impact of the gut microflora on agvhd has been shown to be highly significant ( table 1 ) .
earlier studies in mice showed that symptoms of agvhd could be reduced with antibiotics and transplantation in germ - free conditions [ 28 , 29 ] .
one recent study showed how intestinal microbiota influences agvhd progression following allogeneic bone marrow transplantation ( allo - bmt ) .
analysis of the microbiota composition indicated that recipient mice that developed agvhd had a dramatic loss of bacterial diversity and a distinct composition compared with recipient mice that did not develop agvhd . in mouse models of agvhd , there is marked expansion of lactobacillus johnsonii and a decrease in both clostridiales and in other members of the phylum firmicutes in the ileum . to determine the connection between l. johnsonii and agvhd , the recipient mice were treated with antibiotics and then gavaged with l. johnsonii prior to allo - bmt .
antibiotic - treated mice showed a loss of clostridiales and an emergence of enterococcus spp .
, which was associated with exacerbated agvhd . in contrast , mice reintroduced with l. johnsonii showed no expansion of enterococcus spp . and did not experience increased agvhd lethality or pathology .
these results suggest that l. johnsonii may reduce agvhd severity by preventing the expansion of enterococcus spp .
a clinical research study elucidated variations in the intestinal microbiota of 31 patients receiving allogeneic hsct .
the result of metagenomic analysis showed that patients had a predominance of commensal bacteria at the time of admission .
after transplantation , a relative shift toward enterococci was observed , and this shift was particularly prominent in patients that developed subsequently active gastrointestinal agvhd .
another clinical study revealed that the diversity of intestinal microbiota at engraftment is an independent predictor of mortality in allo - hsct recipients .
first , the graft infused into the patient must contain immunological cells , such as mature t cells .
second , the recipient must be immunocompromised and can not reject cells from the donor .
it is accepted that the development of agvhd can be conceptualized as a three - stage process [ 15 , 36 ] .
underlying diseases , previously administered therapies and the hsct conditioning regimen can damage host tissues , resulting in the production of  danger signals ,  such as proinflammatory cytokines , chemokines , mhc antigens , and costimulatory molecules on host apcs .
both cellular mediators , such as cytotoxic lymphocytes ( ctls ) and nk cells , and inflammatory factors , such as tumor necrosis factor alpha ( tnf- ) , interferon gamma ( ifn- ) , and interleukin-1 ( il-1 ) , can cause the destruction of the target tissue , typically the skin , liver , and gut .
it has been proposed that the gi tract may be particularly critical at the initial stage . both total body irradiation ( tbi ) and high - intensive chemotherapy as part of the conditioning regimen
however , such treatments may also stimulate host tissues to secrete inflammatory cytokines , such as tnf- and il-1 , and their direct influence on epithelial cells of the gi tract allows for the translocation of intestinal microbes and their by - products , including lipopolysaccharide ( lps ) and peptidoglycan , into the systemic circulation ( figure 1 ) .
the symbiotic nature of the intestinal host - microbial relationship is dependent on limiting bacterial penetration of host tissues .
controlling bacterial interactions with the intestinal surface is an important strategy for minimizing bacterial translocation .
the intestinal epithelial surface is the primary interface between the gut bacteria and deeper tissues .
given the enormous numbers of commensal microbes and the persistent invasion of pathogens , it is important that the host immune system monitors and regulates microbial interactions with the intestinal surface .
the intestinal surface maintains an intact barrier through formation of complex protein - protein networks that firmly join together via tight junctions . in hsct
, intestinal bacteria may translocate into deeper tissues from the damaged gi tract and cause infection and septicemia . in a mouse model of agvhd ,
serious impairment of intestinal barrier function in the jejunum was detected , with increased permeability and morphological changes owing to both decreased protein expression and altered localization of the tight junction protein occluding .
there is a significant relationship between gut microbiota and intestinal radiosensitivity . in a mouse model of tbi - bmt ,
several commensal bacteria or their components have the ability to protect intestinal mucosal tissue from irradiation damage .
bacteria - derived flagellin pretreatment protected mice from radiation - induced intestinal mucosal injury and apoptosis via a toll - like receptor 5 ( tlr5)-dependent mechanism . in a human trial , patients taking the probiotic mixture vsl#3 ( a mixture of eight probiotic strains ) experienced radiation - induced diarrhea less frequently than patients taking a placebo in a double - blinded study .
another study showed that a lactobacillus rhamnosus gg- ( lgg- ) derived soluble protein , p40 , ameliorates intestinal injury and colitis , reduces apoptosis , and preserves barrier function by transactivation of the egf receptor ( egfr ) in intestinal epithelial cells .
the paneth cell is an intestinal epithelial cell that plays a key role in limiting bacterial penetration into host tissues .
these cells have a much longer half - life than other cells found in the small intestine .
paneth cells can secrete a number of microbicidal proteins , including -defensins , which selectively kill pathogens , while preserving commensals .
when paneth cells sense bacterial signals , they react by discharging their microbicidal granule contents into the gut lumen . therefore , paneth cells are critical to the immune response to pathogens and the maintenance of a noninflammatory commensal flora in the small intestine .
current studies show that paneth cells are targeted by agvhd , resulting in a substantial loss of paneth cells in the intestine and marked reduction in the expression of -defensins in recipients with agvhd .
restriction fragment length polymorphism ( rflp ) of intestinal microbial communities showed loss of physiologic diversity among the microbiota and the overwhelming expansion of a rare bacterium .
16s rrna gene sequencing demonstrated that this peak was almost certainly due to the presence of escherichia coli in the intestine of recipients suffering from agvhd .
another clinical study showed that enumeration of duodenal paneth cells is a readily available index of disease severity that provides important information regarding agvhd prognosis .
septicemia is the most life - threatening infection following allogeneic hsct , and gram - negative bacteria are the most dominant pathogens of septicemia .
agvhd is regarded as one of the major predisposing factors for the development of septicemia .
lps can enter the circulation through the impaired mucosal barrier after the conditioning regimen . in experimental studies ,
probiotic microorganisms have been shown to alter the composition of the intestinal microflora and thereby mediate anti - inflammatory effects . modifying the intestinal microbiota by oral administration of lgg before and after transplantation resulted in improved survival and reduced agvhd .
connections between pathogen - associated molecular patterns ( pamps ) and pathogen recognition receptors ( prrs ) control adaptive immune responses in inflammatory disorders , including agvhd . intestinal bacteria and their components
the stimulation of prrs leads to transcription of inflammatory genes and upregulation of proinflammatory cytokines and class ii and costimulatory molecules , resulting in local tissue inflammation and migration of leukocytes .
intestinal mucosal surfaces are rich in resident innate immune cells , such as macrophages and dendritic cells ( dcs ) . signaling through prrs
regulates the activity of dcs , leading to phagocytosis , chemokine receptor expression , cytokine secretion and migration from peripheral tissue to draining lymph nodes , and antigen presentation ( figure 1 ) .
several tlrs have been described to recognize different pamps , including gram - positive bacteria - derived lipoproteins ( tlr2 ) , gram - negative bacteria - derived lps ( tlr4 ) , bacterial flagellin ( tlr5 ) , rna ( tlr3 , tlr7 , and tlr8 ) , cytosine - phosphorothioate - guanine ( cpg ) dna ( tlr9 ) , profilin ( tlr11 , tlr12 ) , and bacterial 23s ribosomal rna ( tlr13 ) [ 50 , 51 ] .
tlr downstream signaling activates a complex signaling cascade , eventually leading to host resistance against pathogens by increased production of cytokines , chemokines , adhesion molecules , and antimicrobial peptides , as well as to enhanced antigen presentation by apcs .
pamps are recognized not only by tlrs but also by nucleotide - binding oligomerization domain- ( nod- ) like receptors ( nlrs ) .
nlrs include proteins such as nalps , nod1 , and nod2 , which are involved in the secretion of inflammatory cytokines , such as il-1 and il-18 .
as recognized molecular patterns of invading and resident microbes , tlrs are fundamental to controlling intestinal tissue homeostasis in studies that involve intestinal epithelial cell damage . in experimental agvhd ,
tlrs appear to have a significant role in disease outcomes . in models of hsct ,
however , the role of tlr4 in agvhd is still unclear , with conflicting findings in different studies [ 54 , 55 ] .
evaluation of the expression of tlrs on t lymphocytes and monocytes in 34 patients showed that levels of tlr5 on monocytes and t lymphocytes are positively correlated with agvhd , whereas levels of tlr1 and tlr9 are negative predictors .
the pathophysiology of agvhd is a multistep process that eventually results in t helper 1-driven ( th1-driven ) tissue damage .
recently , increasing evidence indicates the involvement of t helper 17 ( th17 ) and regulatory t cells ( tregs ) in agvhd pathogenesis .
one clinical study showed that in situ quantification of the th17/treg ratio was a specific marker of human agvhd .
murine experimental studies provide inconsistent results on the role of th17 in agvhd [ 5961 ] .
in contrast , treg contributes to tolerance acquisition to donor antigen in solid organ transplantation and protects the development of fatal agvhd in murine model .
the homeostasis of steady - state mucosal t cell subsets is controlled by signals from various components of the intestinal microbiota [ 63 , 64 ] .
treg and th17 cells are the most abundant lamina propria cd4 t cell subsets at steady state .
tregs are crucial in inhibiting excessive inflammatory responses toward intestinal bacteria [ 65 , 66 ] .
th17 cells are characterized by the production of il-17 and other effector cytokines , such as il-17f and il-22 .
th17 cell - derived cytokines function as important activators of innate immune mechanisms , such as recruitment of neutrophils and induction of antimicrobial peptide production from epithelial cells .
treg and th17 cell numbers in the gut are controlled by signals from different species of the commensal microbiota [ 68 , 69 ] .
colonic treg cells are induced by bacteria belonging to group iv and xiva clostridia , and small intestinal th17 cells are induced by segmented filamentous bacteria ( sfb ) [ 7072 ] .
patients undergoing allo - hsct have a substantially increased risk of bacterial , fungal , and viral infection . multiple approaches to decrease the risk of infections after hsct
have been explored , including laminar airflow housing , the use of antibiotics , and prophylactic antiviral and antifungal therapies .
antibiotic resistance in pathogenic bacteria has been an increasing threat to human health during the last decade , and it is widely accepted that the antibiotic resistance development and spread in microbes can be largely attributed to the abuse and misuse of antibiotics . in addition , breakdown of the normal microbial community by antibiotic use increases the risk of pathogen infection and the overgrowth of harmful disease .
clostridium difficile infection ( cdi ) is the most common cause of severe diarrhea and occurs with increased frequency after broad - spectrum antibiotic treatment [ 75 , 76 ] .
results from a retrospective study showed that cdi is strongly associated with agvhd and increased nonrelapse mortality in allo - hsct patients . in recent years
, fecal microbiota transplantation ( fmt ) has emerged as an efficacious method for the treatment of cdi [ 78 , 79 ] .
fmt refers to infusion of a fecal suspension from a healthy individual into the gi tract of a patient to restore healthy intestinal microbiota and cure disease .
an alternative solution to this problem is to screen some beneficial bacteria from stool and only infuse these bacteria as a cocktail into the gi tract [ 80 , 81 ] .
probiotics are live microorganisms , which when administered in adequate amounts confer a health benefit to the host .
various animal and human studies have demonstrated that some probiotic strains can successfully modify the mucosal immune response due to species and strain specificity of the probiotics [ 82 , 83 ] .
the most commonly used probiotics are bifidobacterium , bacillus , and the lactic acid bacteria ( lab ) , including genus lactobacillus and streptococcus .
lactobacillus rhamnosus atcc 53103 , isolated from a healthy human intestinal commensal , is one of the most widely used and well - documented probiotic strains [ 17 , 45 , 8587 ]
. some of the beneficial impacts of probiotics have been scientifically documented , and three main categories of probiotics contributing to host health have been described [ 83 , 88 , 89 ] . first , certain probiotics can exclude or inhibit pathogens .
second , probiotics can enhance the function of the intestinal epithelial barrier by modulating various signaling pathways , inducing mucus and antimicrobial peptide production , enhancing tight junction functioning , and preventing apoptosis .
third , probiotics can modulate host immune responses , resulting in both local and systemic effects .
the rationale for using probiotics to treat gastrointestinal disorders is supported by several clinical studies . in one study ,
ingestion of bifidobacterium infantis 35624 was shown to alleviate symptoms of irritable bowel syndrome ( ibs ) associated with normalization of the ratio of anti - inflammatory to proinflammatory cytokines . to assess the effects of probiotics in diarrhea , a meta - analysis that included 63 studies ( n = 8014 )
was carried out ; the results showed that probiotic strains appear to be safe and have clear beneficial effects in shortening the duration and reducing stool frequency in acute infectious diarrhea .
animal models indicate the close connection between gut microbiota and ibd , and several kinds of probiotics were evaluated in human clinical trials [ 92 , 93 ] .
escherichia coli nissle 1917 shows efficacy and safety in maintaining a remission equivalent in patients with ulcerative colitis .
the once daily high dose probiotic vsl#3 is effective in maintaining antibiotic - introduced remission for at least one year in patients with recurrent or refractory pouchitis .
there are several bottlenecks that need to be overcome before probiotics can be applied in clinical treatment .
one important limitation is the delivery of viable cells to the lower gi tract without a significant loss of cell viability and metabolic features through the harsh conditions of the stomach . in addition , compared to animal models , a major challenge for understanding the interactions between intestinal microbiota and human hosts is the heterogeneity of microbial composition that can colonize the intestine
. different components of microbiota can have very different effects on the host , and the composition of microbial communities can be influenced by a variety of factors , including diet , antibiotic therapy , and environmental exposure to microbes [ 97 , 98 ] .
an increasing number of studies have identified an important interaction between intestinal microbiota and mammalian host health and disease .
recent evidence has demonstrated that specific species of intestinal bacteria appear to be specialized in their ability to induce particular immune cell subsets .
probiotic bacteria can modulate immune responses to promote health by altering the composition of intestinal microbiota .
manipulation of the intestinal microbiota through probiotics or their components holds great promise for the treatment of inflammatory diseases , including agvhd . despite some notable successes from probiotics trials ,
there are numerous obstacles to overcome before probiotic strains can be utilized as agvhd therapeutics .
thus , they should be tested in vitro and then evaluated for their suitability and efficacy in vivo .
hence , there remains a pressing need to screen for probiotic strains that display more remarkable therapeutic responses to agvhd .
second , although changes in microbiota associated with agvhd progression are apparent , it is unknown which exact components of the microbiota are responsible for aggravating or ameliorating agvhd .
therefore , monitoring intestinal microbiota changes of patients before and after agvhd is of vital significance .
however , causality of agvhd in response to probiotic - induced microbiota changes still has not been demonstrated .
along with research on screening for safe and effective probiotic strains moving towards increasing profundity , more attention should be focused on the immune mechanisms governing probiotic effects in agvhd .
several creative therapeutic methods might originate from a better understanding of the relationship among probiotic strains , intestinal microbiota , and immune regulation during agvhd .
 the mammalian intestinal microbiota is a complex ecosystem that plays an important role in host immune responses . 
 recent studies have demonstrated that alterations in intestinal microbiota composition are linked to multiple inflammatory diseases in humans , including acute graft - versus - host disease ( agvhd ) . 
 agvhd is one of the major obstacles in allogeneic hematopoietic stem cell transplantation ( allo - hsct ) , characterized by tissue damage in the gastrointestinal ( gi ) tract , liver , lung , and skin . here , we review the current understanding of the role of intestinal microbiota in the control of immune responses during agvhd . 
 additionally , the possibility of using probiotic strains for potential treatment or prevention of agvhd will be discussed . 
1. Introduction
2. Altered Intestinal Microbiota Composition and Diversity Associated with aGVHD
3. Intestinal Barrier Damage and LPS-Induced Septicemia during aGVHD 
4. Connections among Intestinal Microbiota, Innate Immune Receptors, and T Cell Differentiation during aGVHD
5. Probiotics and Their Potential Application in the Treatment of aGVHD
6. Conclusions and Future Perspectives 