the host colonization by microbiota is a gradual and complex process initiated by the exposure of newborns to the maternal vagina or skin microbiota , according to the mode of delivery , which fluctuates throughout life due to external and internal factors , including dietary habits , antibiotic treatments and host diseases .
the adult human intestine contains approximately 4  10 microorganisms including viruses , fungi and 500 to 1000 bacterial species .
the bacterial density increases along the gastrointestinal tract to reach 3.8  10 bacteria per gram of fecal matter in the colon , and the composition of this microbiota also varies according to spatial localization .
, escherichia coli , whereas the colon is enriched in dietary fiber - consuming bacteria , such as clostridium sp . and bacteroides species .
depending on the origin of their vendor , the small intestine of mice can also be colonized by segmented filamentous bacteria ( sfb ) .
the gut microbiota and microbiota - derived metabolites are essential for healthy host functions , influencing the immune system , the nervous system ( the gut brain axis ) , the endocrine system and the metabolism . despite its key role within the organism ,
the microbiota encompasses millions of proteins and putative antigens to which the host must be tolerant .
physical and chemical protective barriers , such as the mucus layer , production of antimicrobial peptides , the secretion of immunoglobulin a ( iga ) and the epithelium per se , allow microbiota sequestration within the lumen and prevent damage associated with bacterial dissemination . when the gut barrier integrity is impaired , the immune system can respond to antigens of the microbiota , inducing chronic and noxious inflammation , as well as a disequilibrium in bacterial species composition with direct consequences on the different key physiological systems influenced by the microbiota .
the role of the intestinal microbiota in shaping the immune system was clearly demonstrated by the use of axenic mice .
these animals , born and raised under germ - free conditions , display a strong decrease in the expression of most cytokines ( except type 2 cytokines ) , antimicrobial peptides , iga , cd4 t cells and b cells .
the immune system can also influence the equilibrium of the microbiota in favor of certain species .
one well - described example of this reciprocal interaction is the il-23/il-22 network . in response to
microbiota - triggered toll - like receptor ( tlr ) activation , cx3cr1 myeloid cells overexpress the pro - inflammatory cytokines il-23 and il-1 , which in turn induces il-22 expression by cd4 t cells and innate lymphoid cell 3 ( ilc3 ) .
il-22 contributes to the gut barrier integrity and can influence the composition of the microbiota by inducing the expression of antimicrobial peptides within intestinal epithelial cells ( iecs ) , as well as the restoration of mucus production after inflammation or the maintenance of iec fucosylation .
although the role of pro - inflammatory cytokines , including il-23 , il-1 , il-17a and il-6 has largely been described in shaping both the immune system and the intestinal microbiota , renewed investigations suggested that certain anti - inflammatory cytokines , in particular transforming growth factor - beta ( tgf- ) , a highly conserved cytokine , are also key modulators of the microbiota and the host immune cell cross talk . in lower life forms , including in the soil - inhabiting , bacterial - feeding nematode , caenorhabibditis elegans , and in insects such as drosophila melanogaster , two tgf- homologs , namely dbl-1 and dpp , found in these organisms , respectively , increase in response to bacterial entry in the gut and largely contribute to the gut immune response by enhancing antimicrobial peptides production , thus controlling bacterial homeostasis .
the role of tgf- in the interplay between gut bacteria and the immune system is even more evident in mammals with a more complex gut flora and immune system , implying a primacy of this cytokine in the evolution of the relationship between the host organism and the microbiota . in this review ,
we highlight recent findings and provide an insight into the cross talk between the tgf- , the intestinal microbiota and the lymphoid cells of the lamina propria of the mammalian gut .
tgf- is a potent immunosuppressive cytokine involved in the development and functions of numerous immune cells , including t and b cells , but also dendritic cells ( dcs ) and natural killer ( nk ) cells .
tgf-2 and tgf-3 mainly have a role in muscle and bone development , whereas tgf-1 ( tgf- ) expression predominates in immune cells .
all three isoforms signal through a common serine  threonine kinase receptor complex composed of tgf-ri and tgf-rii subunits .
signaling is initiated by binding to tgf-rii , which leads to its auto - phosphorylation , and in turn phosphorylates the tgf-ri subunit ( figure 1 ) .
the latter then phosphorylates the receptor - associated r - smads , smad2 and/or smad3 , which bind to smad4 ( cosmad ) and translocate into the nucleus to regulate gene transcription . among the tgf--activated genes ,
smad7 encodes a protein that binds to the cytoplasmic domain of tgf-ri and negatively regulates tgf- signaling .
interestingly , smad2/3-dependent and smad4-independent signaling pathways have been described , including the smad2/3 interaction with trim33 ( tif-1 ) and ikks , which can act either in combination or in competition with smad4 during the differentiation steps of a given cell .
moreover , tgf-ri can also activate other pathways , such as the mapk / mek , jnk / p38 and akt / pi3k , which could phosphorylate the variable linker regions of smad23 .
in the gut , iecs as well as selective immune cells , including dcs are an important source of bioactive tgf-. tgf- is a polypeptide secreted as an inactive form bound to the latency - associated peptide , which masks its binding site to tgf-rii ( figure 1 ) . to be activated , the latent complex needs to undergo conformational changes or protein cleavage .
several molecules can mediate this activation , including furin , metalloproteinases and the integrins ( itg ) v6 and v8 .
the tgf-r complex is widely expressed by immune cells , as well as other cells .
the importance of tgf- signaling in gut immune cell homeostasis has been highlighted by studies that used either genetically altered tgf- signaling pathways , the deletion of factors important for tgf- activation or administration of anti - tgf-. in mice , germ - line ablation of smad3 is associated with gut inflammation and abscesses , which are totally absent when animals are crossed on a rag - ko background . dc - specific deletion of itg 8 chain causes inflammatory bowel diseases ( ibd ) associated with unchecked t - lymphocyte activation .
interestingly , under inflammatory conditions , cd4 foxp3 regulatory t cells ( tregs ) constitute a unique v8-expressing t - cell subset , and are thus capable of counter - regulating gut inflammation through the provision of bioactive tgf-. tgf-1-deficient mice spontaneously develop multi - organ autoimmune lesions by the age of 3 weeks .
t lymphocytes are key effectors of this autoimmunity , as mice specifically lacking tgf-r in t cells develop a similar inflammation to that observed in tgf-1-deficient animals .
it is worth noting that the intestine is not severely affected in these models , probably due to the early onset of the autoimmune disease .
indeed , an incomplete deprivation of tgf- signaling in t cells , due to the expression of a dominant negative receptor of tgf-r , is associated with a slower disease progression with intestinal inflammation . in patients with crohn 's disease and ulcerative colitis , the two major forms of ibd known to be associated with an excessive immune response directed against components of commensal bacteria , an impairment of tgf-
signaling has largely been reported and seems likely associated with an increase in smad7 expression in t cells .
in addition to t cells , gut pathologies were also associated with impairment of tgf- signaling in b cells , dcs and nk cells .
the smad3/4 pathway enhances iga class switching and secretion of iga within the lumen , which contribute to maintaining gut barrier integrity against microbiota , and the deletion of tgf-rii in cd11c cells , affecting both dcs and nk cells , leads to spontaneous colitis .
compared with specific pathogen - free animals , the levels of tgf- expression are largely impaired in the gut of germ - free mice .
iecs are most likely an important source of bioactive tgf- when considering the huge number of cells they represent in the gut . epithelial cell injury and gut inflammation
have both been demonstrated to enhance tgf- production by iecs , and there is growing evidence to suggest that iec - derived tgf- is also regulated by the microbiota .
several clostridium species were shown to produce short - chain fatty acids , such as butyrate , acetate and propionate , capable of exacerbating tgf- production by colonic ecs .
though the exact mechanisms by which clostridium - derived short - chain fatty acids increase tgf- in iecs remain to be unraveled , it is noteworthy that not only do the clostridium species increase tgf- secretion by iecs in the colon , but also the expression of metalloproteinases at the surface of iecs , providing a large source of bioactive tgf- within the colon .
microbiota - derived products were also reported to influence the tgf- production by lamina propria dcs , including atp ( adenosine 5-triphosphate ) , which increases the expression of tgf- in a cd70 subset of dcs of the small intestine .
similarly to the effects of short - chain fatty acids on iecs , microbiota - derived atp can both directly increase iec tgf- production and indirectly increase active tgf- levels by promoting integrin v8 expression at the surface of dcs .
after dextran sodium sulfate treatment , clostridium butyricum was recently reported to enhance tgf- production by colonic lamina propria dcs mainly in a tlr2 , ap1erk pathway - dependent manner .
furthermore , microbiota - mediated tlr signaling in intestinal dcs also promotes the expression of the integrin v8 essential for tgf- activation .
the long co - evolution of commensal bacteria and the mammalian gut has resulted in multiple mechanisms to increase tgf- levels , which include microbiota - induced tgf- production as well as enhancement of active and hence functional tgf- levels . although direct and indirect mechanisms may be used by iecs and dcs in response to different commensal bacteria and bacteria - derived products
, future investigations should address the specific contributions of each mechanism during homeostasis , healing , inflammation initiation and inflammation resolution more precisely .
tregs are characterized by constitutively high levels of the forkhead family transcription factor foxp3 , which is deemed to provide them with their regulatory activity .
although tregs constitute approximately 10% of the cd4 t cells in most organs , they are more abundant within the lamina propria , often exceeding values of 20% and 30% in the small intestine and in the colon , respectively . in the gut , tregs can regulate the different branches of the mucosal immune response through several mechanisms .
they inhibit t - cell responses to maintain immune tolerance to dietary and microbiota - derived antigens .
tregs express high levels of ctla-4 , icos , il-10 , il-35 and to some extent tgf-. however , foxp3-cre ; tgf1 mice , with tregs unable to produce tgf- , remain healthy without any signs of ibd , implying that production of tgf- by tregs is not essential for their regulatory functions in the gut .
however , a cellular source of bioactive tgf- , such as dcs , is essential to sustain the high levels of foxp3 in tregs and their regulatory functions .
tregs differentiate either in the thymus ( ttreg ) or in the periphery of naive t cells ( ptreg ) .
the ttreg cells and ptreg cells appear to have non - redundant immunoregulatory functions in the gut .
the deprivation of tgf- signaling in thymocytes was associated with a delay in the ttreg cell development potentially due to their apoptosis . within the foxp3 locus ,
conserved noncoding dna sequences ( cns1 - 3 ) promote stability , size and composition of the foxp3 treg populations . in the gut , the binding of smad2/3/4 to the cns1 induces foxp3 expression in peripheral naive t cells .
cns1-deficient mice display unaffected ttreg cell populations , but a defective induction of foxp3 in the gut - associated lymphoid tissue . unlike foxp3-deficient mice
the relative proportion of ttreg cells and ptreg cells in the intestine remains a matter of debate .
although dietary antigens induce the development of ptreg cells in the small intestine , independently of microbiota , commensal bacteria and their derived products have an instrumental role in the differentiation of ptregs within the colon . 

clostridium butyricum enhances tgf- production by lamina propria dcs of colon , which was associated with ptreg differentiation . among bacterial products ,
butyrate , a clostridium - derived short - chain fatty acid , not only induces tgf- expression by iecs and potentially helps enforce ptreg - cell differentiation , but also acts directly on ptreg - cell differentiation by inhibiting histone deacetylase , resulting in histone 3 lysine 27 acetylation ( h3k27ac ) on cns1 ( and cns2 at high concentrations of butyrate ) , opening the chromatin and leading to the transcription of foxp3 .
it is noteworthy that in the colon of patients with ulcerative colitis , the levels of butyrate and the capacity of treg cells to suppress effector t cells are reduced , whereas the levels of tgf- are largely reported to be increased .
this could either be due to the increased expression of smad7 in t cells ( including tregs ) from patients with ibd or to different but complementary roles of tgf- and microbiota - products in ptreg - cell differentiation .
retinoic acid ( ra ) , a vitamin a - derived metabolite largely produced in the gut by cd103 dcs , and iecs not only contributes to mucosal homing but also has a role in tolerance and inflammation .
cd103 dcs exhibit an exacerbated ability to induce ptreg - cell differentiation in vitro , which is dependent on their ra production and tgf- signaling in t cells . on stimulation , in the presence of tgf-
tgf--induced high expression levels of il-6r at the surface of t cells , which constrains treg differentiation , can be reversed by the addition of ra into the culture medium . of note ,
vitamin a - deficient mice were reported to have a comparable proportion and number of total intestinal tregs implying that ttregs can replenish the intestinal treg compartment in the absence of ra - induced ptreg .
interestingly , the gut microbiota was recently reported to repress ra synthesis . though this repressive role was observed in the context of colitis - associated cancer and the contribution of specific bacteria or bacterial - derived products repressing ra
remains unclear , these recent works suggest that microbiota could finely tune the differentiation of colonic ptreg cells by either promoting tgf- or repressing ra synthesis . within the gut ,
these rort foxp3 cells were shown to be very stable and to strongly express immunosuppressive molecules , such as ctla-4 , lag-3 , tim3 and icos .
mice lacking rort expression , specifically in foxp3-expressing cells , do not develop spontaneous colitis but are more susceptible to chemically induced colitis and t - cell - driven colitis .
the development of rort - expressing tregs is also dependent on the wide - range of commensal bacterial species , including the butyrate - expressing clostridia species , but also sfb and b. thetaiotaomicron , as well as on the levels of vitamin a , il-6 and il-23 .
although not shown in these studies , we could hypothesize that tgf- , described to induce the co - expression of foxp3 and rort in cd4 t cells is also one of the key cytokines involved in the development of rort - expressing tregs . in conclusion
, bacteria and bacteria - derived products from the gut microbiota can influence tgf- levels both under healthy and pathological conditions .
whether the imbalance in gut bacteria composition ( dysbiosis ) , observed in patients with ibd , similarly affects tgf- production and its corresponding alterations on the immune cell tolerance remains to be addressed .
the type 17 helper ( th17 ) cells constitute a subset of cd4 t cells that express the transcription factor rort and produce cytokines , such as il-17a / f , il-22 , often in association with other cytokines , including il-10 , gm - csf and ifn , depending on local inflammatory conditions .
th17 cells are commonly associated with the development of chronic inflammatory diseases , particularly autoimmunity and tumorigenesis .
however , tissue - protective roles have also been attributed to th17 cells in the gut .
neutralization of il-17a with antibodies increases tissue damage in patients with crohn 's disease , and il-17a injection into mice maintains the tight junctions in the epithelium and therefore the intestinal barrier integrity .
th17-cell differentiation from naive cd4 t cells requires antigen stimulation and was largely reported to be influenced by tgf-. th17 cells generated with tgf- and il-6 produce il-17a but fail to be pathogenic without further exposure to il-23 .
moreover , th17 cells can be generated in the absence of tgf- signaling but in the presence of il-23 , il-6 and il-1. these tgf--independently generated th17 cells were described as strongly pathogenic , reinforcing the importance of tgf- in the control of pathogenic features of th17 cells . in cd4 t cells , in the presence of il-6 , tgf- signaling promotes the expression of both rorc and ror encoding for rort and ror , respectively , two transcriptional factors essential for th17 differentiation .
interestingly , the induction of rorc and ror by tgf- is independent of smad4 , and an opposite role for smad2 and smad3 in the regulation of rort transcriptional activity was described . using mice with specific ablation of tgf- in cd4 t cells
, it has been proposed that the autocrine production of tgf- by cd4 t cells is essential for th17-cell differentiation .
however , a recent study revealed that effector cd4 t cells are unable to convert tgf- into its bioactive form suggesting that some other cells , able to activate the tgf- , are mandatory for th17-cell differentiation .
thus , within the gut , it is likely that iecs and dcs , which express large amounts of tgf- and can lead to its activation , contribute to th17-cell differentiation . alongside its ability to enhance ptreg - cell differentiation ,
ra was shown to inhibit il-6r and il-23r upregulation induced by tgf- and il-6 , respectively .
however , ra was also proposed to work in concert with microbial - driven signals through tlr-5 on dcs of the lamina propria to enhance t - cell differentiation into th17 cells .
these observations suggest that depending on the accessibility of microbial - derived products for immune cell actors and thus on the gut barrier integrity of the host , ra can differently influence th17-cell differentiation .
specific bacteria , such as atp - producing bacteria , or iec - adhering bacteria like sfb , were described to induce intestinal th17 cells .
although most intestinal th17 cells bear a tcr specific for sfb antigens , sfb antigens alone do not drive th17 differentiation and tlr signaling is not required for th17 cells , as myd88-deficient mice display a normal number of intestinal th17 cells compared with control littermates .
the binding of sfb to iecs directly induces the expression of the serum amyloid a1 and 2 ( saa1/2 ) and reactive oxygen species , which in turn promote th17-cell differentiation .
whether sfb promote the expression of tgf- by iecs and/or dcs has so far not been addressed . however , a source of bioactive tgf- is required to induce th17-cell differentiation in response to the commensal bacteria .
thus , within the gut , both microbiota and tgf- signaling in cd4 t cells promote the differentiation of th17 cells .
the contribution of these two key actors is largely influenced by the inflammatory conditions , which in turn influence the profile of cytokines secreted by th17 cells and their functions .
ilcs are the most recently described immune cells to be associated with the regulation of gut homeostasis .
ilcs share common features with cd4 t cells but lack t - cell lineage markers and have been categorized into at least three subsets ilc1 , ilc2 and ilc3 , based on differential transcription factor expression patterns .
ilc1 express the transcription factor t - bet , the surface marker cd127 , nkp44 ( or nkp46 in mice ) and produce cytokines , such as tnf- and ifn-. in the human gut , ilc1s have a strong tropism for the epithelium where they produce ifn- in response to il-12 and il-15 .
a recent study , targeting tgfbr2 in ilc1 , showed that tgf- , in a smad4-independent manner , is required for ilc1 development in the salivary gland .
so far , the role of tgf- signaling on intestinal ilc1 development and function has not been tested and needs further investigations .
ilc2s are identified as lineage negative gata-3 st2 crth2 . in response to the il-33 , an iec - produced cytokine , gut ilc2s produce amphiregulin ( areg ) , involved in tissue repair , and areg - deficient mice are more susceptible to dextran sodium sulfate , a microbiota - dependent colitis model . 
 in vitro
, tgf- was shown to be a potent inducer of areg in human lung adenocarcinoma .
whether tgf- , a microbiota - derived cytokines expressed by iecs , could be involved in areg production by intestinal ilc2s remains to be elucidated .
ilc3s express the transcription factor rort and secrete cytokines , such as il-17a , and il-22 , in association with ifn- , gm - csf or alone .
ilc3s have been divided into three subsets : lti ( lymphoid tissue inducer ) , nkp46 ilc3 and nkp46 ilc3 . in the adult colon ,
the role of the microbiota on both the development and functions of ilc3s remains controversial . through their production of il-22 , both nkp46 and
nkp46 ilc3s have a redundant function with an important role in microbiota segregation , pathogen clearance , maintenance of the intestinal barrier integrity , mucosal healing .
interestingly , tgf- signaling was recently proposed to control the balance between nkp46 and nkp46 ilc3 ; however , the source of tgf- and the actors of the tgf- signaling pathway involved remain unknown .
thus several recent studies have established a link between the ilc biology , tgf- and microbiota . regarding the role of ilcs in gut homeostasis , and the protective role of tgf- in the gut
, future investigations should address whether tgf- induced by the microbiota could allow ilcs to regulate the intestinal barrier integrity and in turn protect against bacterial and parasitic entry in the deeper layers of the gut .
immunoglobulin a ( iga ) can uniquely pass through the epithelium and additionally modulate luminal bacterial composition . in the gut , iga secreted from b cells can bind to polymeric ig receptors on the surface of iecs and subsequently translocate into the lumen .
iga can be categorized into two subsets : high - affinity igas , which neutralize pathogens , and low - affinity igas , which prevent bacterial adhesion by aggregating them together .
it has been proposed that specific commensal bacteria have a crucial role in iga production .
sfb , but not escherichia coli , are capable of inducing iga secretion in peyer 's patches and in isolated lymphoid follicles .
so far , the mechanisms linking sfb and iga production remain unclear and require further investigations .
tgf- is a master regulator of iga production among all of the other iga - modulating immune cell factors and cytokines , with b - cell - specific deletion of tgfr resulting in the loss of iga - producing b cells .
tgf--induced iga production is dependent on canonical pathways , as evidenced by the fact that smad2 , smad3 and smad4 deficiency results in the decrease in iga levels .
interestingly , tgf- can directly and indirectly control iga production . indeed , in addition to its effect on b cells , tgf- also regulates t - follicular - helper - cell development , a cd4 t - cell subset involved in the control of isotype - switched antibody production . 

alcaligenes species , gut - associated lymphoid tissue - resident bacteria , as well as lactobacillus gasseri sbt2055 , a probiotic bacterium , were shown to induce tgf- production by small intestinal dcs in a tlr2-dependent manner and in turn induce iga production by b cells .
thus by influencing tgf- production in the gut , the microbiota regulates iga secretion and thus the strength of the intestinal barrier .
largely overlooked until the last decade , the interplay between the microbiota and the mammalian host organism is now widely regarded as essential for host homeostasis and health . in this review
, we have highlighted the complexity of this interplay within the gut and the central role of tgf- in regulating these interactions .
tgf- levels in the gut are directly and indirectly modulated by the gut microbiota , which impacts the development and functions of immune cell subsets , which in turn regulates microbiota sequestration within the mammalian lumen ( table 1 and figure 2 ) .
. this area of research should contribute to fully understanding the effects of tgf- on microbiota homeostasis and the risks of utilizing either neutralizing tgf- antibodies or tgf- analogs in human patients .
 the relationship between host organisms and their microbiota has co - evolved towards an inter - dependent network of mutualistic interactions . 
 this interplay is particularly well studied in the gastrointestinal tract , where microbiota and host immune cells can modulate each other directly , as well as indirectly , through the production and release of chemical molecules and signals . in this review , 
 we define the functional impact of transforming growth factor - beta ( tgf- ) on this complex interplay , especially through its modulation of the activity of local regulatory t cells ( tregs ) , type 17 helper ( th17 ) cells , innate lymphoid cells ( ilcs ) and b cells . 
Introduction
TGF- signaling pathways and gut immunity
Microbiota-induced TGF-
TGF- signaling, microbiota and Tregs
TGF- signaling, microbiota and type 17 helper cells
TGF- signaling, microbiota and ILCs
IgA
Concluding remarks