although the etiology of ibd is unknown , many investigators have probed whether a pathogenic cause can be attributed to onset of disease , either in full or in part.5 , 6 , 7 although no pathogen has been shown consistently to be associated with ibd , a subset of cd patients do show increased prevalence of a unique enteropathogenic strain of the b2 phylotype e coli termed aiec.2 , 8 , 9 ileal biopsy specimens from cd and uc patients have shown that aiec are associated primarily with cd ; however , more recent studies have shown an equal prevalence of aiec strains in uc as well as cd , suggesting that this pathobiont may have a greater association with ibd than first thought.10 , 11 , 12 although lf82 is the most referenced aiec , it is only one of many aiec strains that were found in ibd patients.2 , 13 it has been remarkably difficult to identify a specific genetic or molecular marker of aiec , thus in  vitro assays are used to validate aiec .
however , there may be some genetic signatures that differ between the b2 aiec phylotype compared with other aiec phylotypes .
one potential aiec candidate gene is the novel aiec serine protease vat - aiec , which promotes expansion and adherence of aiec to intestinal epithelial cells ( iecs ) .
expression of this gene was approximately 3-fold higher in cd - aiec isolates compared with non - aiec isolates from cd patients or healthy subjects .
it therefore could be considered a selective rather than an exclusive genetic marker for aiec .
aiec are distinct from other strains of e coli because they show nonclassic virulence factors of adherence and invasion ( ie , lack of a type iii secretion system ) .
notably , aiec pathogenesis involves survival and replication in iecs and macrophages in  vivo and in  vitro that further exacerbated barrier dysfunction , this topic is discussed later ( figure  1).9 , 13 martinez - medina and garcia - gil published an excellent comprehensive review on the definition , characteristics , and molecular basis of aiec pathogenicity .
the intestine is lined by a single layer of epithelial cells that are interconnected by transmembrane proteins to create physical connections with one another and to form a selectively permeable barrier .
this leaky barrier serves multiple functions that are essential for intestinal homeostasis : generation of ion solute concentration gradients , absorption of nutrients , antimicrobial peptide secretion ( reviewed by mccole and barrett ) , and protection of the host from toxins and pathogens ( reviewed by peterson and artis ) .
disruption of barrier function can lead to loss of microbiome diversity and promotion of aiec expansion and pathogenesis.1 , 6 iecs express molecular sensors that sample the luminal content for bacterial antigens , and in some cases the bacteria itself , and translate this information to host immune cells .
this process prevents the host immune system from a robust proinflammatory response against commensal bacteria while maintaining the ability to protect against pathogens .
the first identified ibd - associated gene , cytoplasmic nucleotide - binding oligomerization domain 2 ( nod2/card15 ) , a nod - like receptor family member , serves as a sensor to bacterial peptidoglycans ( bps ) ( reviewed by philpott et  al ) .
binding of bps to nod2 results in the activation of nuclear factorb ( nf-b ) and autophagolysis , and the appearance of nod2 at the site of bacterial endocytosis in epithelial cells drives autophagolysis .
mutations in nod2 result in an increased immune response to bacteria and bacterial antigens that is accompanied by impaired autophagolysis of aiec and subsequently survival of aiec in immune cells.23 , 24 conversely , overexpression of nod2 and other genes involved in autophagy ( eg , by activation of the eukaryotic translation initiation factor 2 alpha kinase 4-eukaryotic translation initiation factor 2 alpha - activating transcription factor 4 [ eif2ak4-eif2a - atf4 ] pathway ) , results in an increased autophagy response to aiec and a reduction in aiec survival in macrophages.25 , 26 , 27 thus , functional autophagolysis in both intestinal epithelial cells and macrophages is required for aiec degradation and prevention of aiec - induced barrier dysfunction .
it is clear that proinflammatory cytokine signaling plays a major role in altered intestinal permeability and this may be associated with aiec pathogenesis .
loss of negative regulation of proinflammatory cytokines ( eg , interferon- [ ifn- ] and tumor necrosis factor- [ tnf- ] ) , and , conversely , overstimulation of the proinflammatory response , plays a role in altered intestinal permeability and this may be associated with aiec expansion and pathogenesis.22 , 29 , 30 , 31 a key mediator of signaling by many cytokines , nf-b has been shown to regulate the development and response of the immune system , inflammation , and cancer .
defective nf-b activation can result in a defective immune cell response to bps ( driven by interleukin [ il]8 ) and bacterial lipopolysaccharides , and defective autophagy.33 , 34 , 35 taken together , dysfunctional regulation of proinflammatory cytokines may be associated with an inappropriate response to aiec and may play a role in the promotion of aiec expansion and pathogenesis .
with respect to the clinical intersection between altered expression of genes in ibd and genes involved in aiec expansion , patients who are at risk of developing cd  also  overexpress carcinoembryonic antigen  related cell - adhesion molecule 6 ( ceacam6 ) , placing them at a higher risk for aiec adherence and invasion via interaction of bacterial type 1 pili and long polar fimbriae ( lpf ) to host ceacam6 .
ceacam6 , normally expressed on the apical membrane of human epithelial cells from the large intestine , is regulated by the proinflammatory cytokines ifn- and tnf-.36 , 38 studies using isolated human peyer s patches or mouse ileal biopsy specimens containing peyer s patches showed that bacterial pili specifically targeted peyer s patch m - cells ( discussed later ) expressing ceacam6 ; however , lpf - independent aiec invasion and adherence also has been shown in other types of intestinal epithelial cells.36 , 39 nevertheless , aiec can up - regulate ceacam6 indirectly via activation of proinflammatory cytokines , thus leading to increased expression of ceacam6 and further binding and internalization of aiec .
carcinoembryonic antigen human bacterial artifical chromosome 10 ( ceabac10 ) transgenic mice that overexpress human ceacams show no clinical evidence of colitis ; however , these same mice infected with aiec show a barrier defect .
it would be expected , therefore , that patients with nod2 mutations and/or increased expression of ileal ceacam6 are more at risk to develop ibd . in support of this statement , ceacam6 and
the pore - forming tj protein claudin-2 , isolated from ileal biopsy specimens of cd patients , are not only co - localized , but expression levels are both increased compared with control subjects , thereby promoting a favorable environment for aiec adherence and invasion and further exacerbation of barrier dysfunction , ultimately leading to a vicious cycle of proinflammatory cytokine release and increased barrier permeability .
interestingly , although ceacam6 is overexpressed in ileal mucosa of ibd patients , the protein is not normally expressed in ileal mucosa .
the mechanism of aiec internalization involves macropinocytosis and vacuolization of the bacteria into iecs and macrophages.9 , 42 , 43 , 44 aiec have been found in late endosomes of intestinal epithelial cells , as evident by co - localization with the lysosomal marker lysosomal - associated membrane protein-1 ( lamp1).24 , 44 similarly , mycobacterium avium subspecies paratuberculosis has been localized in endosomes of intestinal epithelial cells .
the process of internalization into endosomes also occurs during reorganization of tjs and is discussed later .
aiec - containing endosomes mature into phagolysosomes , which then can exit iecs , as discussed later .
alternatively , aiec replication is permitted in autophagolysis - deficient , aiec - containing phagosome / endosome / lysosomes , leading to loss of organelle membrane integrity , release of the bacteria into the cytoplasm , and basolateral exit of the bacteria from the iec ( figure  1).46 , 47 aiec translocation through the epithelial barrier is dependent on type 1 pili and binding of lpf to glycoprotein 2 ( gp2 ) to aiec disruption of epithelial junctions microfold cells ( m - cells).39 , 43 , 48 m - cells are required for the development of host immunity , are involved in bacterial sampling,50 , 51 , 52 , 53 and can serve as an alternative gateway for aiec39 , 54 into and through the epithelium ( reviewed by pravda ) .
the aiec - containing endosome can mature into a phagosome and exit basolaterally from the iec , where it can be processed by macrophages ( figure  1).42 , 44 after basolateral exit from epithelial cells , aiec exposure to lamina propria immune cells induces a proinflammatory response and a tnf-dependent increase in m - cell development , thereby further permitting the adherence and invasion of aiec and exacerbation of barrier permeability .
aiec invasion elicits a proinflammatory response by triggering secretion of proinflammatory cytokines from iecs , resulting in recruitment of immune cells,13 , 57 and , subsequently , nf-b signaling , which allows survival of aiec in macrophages . in summary
, cytokines play a key role in the pathogenesis of aiec and aiec - induced epithelial barrier dysfunction .
in addition to the unknown etiology of ibd , it is uncertain whether inflammation precedes epithelial barrier dysfunction or if barrier dysfunction results in chronic inflammation such as that observed in ibd . however , some evidence points to an underlying barrier defect that renders individuals susceptible to disease .
first - degree relatives of cd patients show asymptomatic barrier dysfunction associated with subclinical immune activation , indicating that a barrier defect may precede or is at least an early event in the disease.59 , 60 in addition , altered permeability across the intestinal epithelium is a predictor of relapse in human cd ; whereas increased permeability precedes inflammation in rodent models , canine models , and human disease,59 , 64 , 65 , 66 and has been shown to be the underlying mechanism of ibd in these patients .
pathologic stimuli , such as aiec , in part influence epithelial barrier integrity by altering the network of proteins that regulate barrier permeability known as the intercellular apical junctional complex ( ajc ) .
the ajc , comprised of several different proteins with distinct responsibilities , directs the paracellular movement of ions and solutes and thus is responsible to maintain appropriate epithelial barrier permeability .
the structural units of the ajc responsible for regulating barrier integrity and permeability are adherens junctions ( aj ) and apical tjs .
ajs and tjs are associated with scaffolding proteins that cooperate with the cytoskeleton of the cell providing the ajc with a dynamic means of regulation .
dysregulation of tj proteins is observed in ibd patients ( table  1).68 , 69 , 70 , 71 that the ajc is required for barrier integrity has led to the hypothesis that defects in the ajc may play a role in ibd pathogenesis .
alteration of the ajc can occur in response to exposure to aiec and other enteropathogenic e coli.71 , 72 , 73 , 74 aiec have been shown to decrease transepithelial electrical resistance of iecs owing to disruption of the ajc.47 , 67 the molecular interaction of aiec with iecs induces an inflammatory response leading to the overproduction of proinflammatory cytokines , with ifn- and tnf- as the major effectors , and this is a key step in the pathogenesis of ibd . ifn- and tnf- both
have been shown to increase gut permeability by acting on tj proteins and tj strand complexity,76 , 77 which then further exacerbates barrier dysfunction .
aiec - dependent , cytokine - induced alterations in aj and tj proteins is discussed later .
as previously discussed , an increase in expression of both ceacam6 and claudin-2 is associated with increased barrier permeability .
interestingly , targeting of adherens junction proteins , occludin , and claudin 3/4 and other tight junction proteins by helicobacter pylori , vibrio cholera , and enteropathogenic e coli , respectively , has been shown previously,78 , 79 supporting the possibility that aiec directly bind to tj proteins .
it has been shown that aiec results in increased claudin-2 expression in mice and human beings , and because aiec pathogenesis is not dependent solely on interaction of ceacam6 with aiec , it is possible that claudin-2 could serve as a gateway for aiec entry into iecs while co - internalization of tj proteins and aiec may promote aiec invasion and increased barrier permeability .
whether aiec are able to bind directly to tj proteins has yet to be shown experimentally .
distinct from the functions of adherens junctions , tjs serve as a paracellular gateway for passive fluid movement and solute flux and limit the passive movement of proteins and lipids , thereby generating a concentration gradient as well as playing a role in polarization of epithelia .
the conglomeration of proteins that make up the tjs include the claudin family of transmembrane proteins , junctional adhesion molecule - a ( jam - a ) , occludin , and zonula occludens members 13 ( others not included in this article are reviewed elsewhere ) ( figure  1 ) .
claudin-2 is a prominent member of the claudin family because it forms a cation - selective pore that permits paracellular sodium and water flux.80 , 81 overexpression of claudin-2 results in an increase in epithelial barrier permeability both in  vitro and in  vivo.82 , 83 , 84 , 85 il13 and tnf- induce a barrier defect in uc by up - regulating claudin-2 to facilitate the pore pathway , although il6 also has been shown to increase claudin-2 expression in intestinal epithelial cells.86 , 87 that epithelial barrier leakiness is related inversely to tj strand complexity supports the association of increased levels of claudin-2 with increased barrier permeability in ibd.70 , 88 conversely , a decrease in claudin-2 expression results in a tighter epithelial barrier .
adherens junctions are primarily responsible for cell  cell recognition as well as initiating and maintaining cell  cell contact and polarization .
the major aj protein is e - cadherin , a transmembrane protein associated with the catenin family of cytoplasmic proteins ( figure  1 ) .
e - cadherin plays a critical role in adhesion of cells and is required for the formation of tjs .
e - cadherin single - nucleotide polymorphisms and dysregulation of e - cadherin are associated with ibd ( reviewed by mccole ) , supporting a role for aj defects in ibd - associated barrier dysfunction . furthermore ,
aiec , in addition to other intestinal pathogens , are able to displace e - cadherin in  vitro,47 , 93 potentially providing another mechanism of invasion and barrier disruption .
jam - a is another psd95-dlga - zonula occludens-1 homology domain ( pdz)-domain  containing integral protein localized to tjs .
jam - a shows pdz - domain  dependent interactions with the scaffolding protein zonula occludens 1 ( zo-1 ) and cell polarity partitioning defective protein-3 .
expression of jam - a was shown to be decreased together with zo-1 in ibd , and loss of jam - a results in increased epithelial barrier permeability.101 , 102 it is unknown whether aiec directly results in the down - regulation of jam - a or if it is a consequence of down - regulation of zo-1 ; however , aiec may regulate jam - a indirectly via the release of inflammatory cytokines from infected macrophages . nevertheless , it is possible that aiec , in addition to altering zo-1 , may alter jam - a and partitioning defective protein-3dependent targeting of tj proteins to the membrane , resulting in disrupted tj complex integrity and altered cell polarity , further promoting ibd pathogenesis .
occludin , a 65-kilodalton integral transmembrane protein , is involved in the stability and regulation of tjs .
although down - regulation of occludin is observed in cd and uc patients , the uc - associated proinflammatory cytokine il13 alone was not able to abolish occludin protein levels in a colorectal cancer cell line ( ht-29 ) . this discrepancy was attributed to a dilution of the number of cells isolated from the inflamed tissue of uc patients .
nevertheless , it has been shown from in  vitro work that occludin plays a key role in promoting barrier function and is redistributed away from tight junctions after enterohemorrhagic e coli infection of human colonoid monolayers.17 , 74 further work to elucidate the in  vivo role of occludin is needed because mice lacking occludin do not show defects in tj morphology despite histologic abnormalities in some tissues .
proinflammatory cytokine - dependent phosphorylation of myosin light chain ( mlc ) by myosin light chain kinase ( mlck ) or rho - associated kinase results in rapid reorganization of the ajc.67 , 83 , 105 although it remains to be confirmed , aiec could affect occludin distribution indirectly through bacteria - dependent induction of proinflammatory cytokines from immune cells , activation of mlck or rho - associated kinase , and subsequent redistribution of occludin .
zo is a pdz homology domain containing plaque protein involved in scaffolding of tj proteins to the cytoskeleton .
zo-1 , -2 , and -3 contain pdz domains that facilitate anchorage of tj proteins such as claudins , occludin , and jam - a to the cytoplasm and thereby play a key role in tj formation and regulation .
tnf- induces an increase in tj permeability via nf-b  dependent down - regulation of zo-1 protein and mlck - dependent95 , 96 , 109 , 110 , 111 redistribution of tj proteins in an apoptosis - independent manner ( reviewed by bruewer et  al ) . similarly , aiec disrupt tj composition by altering zo-1 , resulting in increased appearance of gaps between cells.47 , 93 conversely , probiotic and commensal bacteria promote up - regulation of zo proteins and strengthening of the epithelial barrier ( reviewed by ulluwishewa et  al ) , potentially supporting the notion that when probiotic bacteria outcompete pathogens , this not only will return the balance of the microbiota , but also enable restitution of the tj complex . in the absence of bacteria , regulation of the ajc
is mediated by the interaction of the cytoskeleton with the proteins of the ajc or by cytokines .
ifn- induces macropinocytosis of occludin , jam - a , and claudin-1 via myosin 2dependent vacuolarization of the apical membrane into early endosomes , resulting in an increase in barrier permeability .
that aiec also are internalized by macropinocytosis illustrates a direct disruption of the ajc by the bacteria and establishes an additional virulence factor for the invasion of aiec .
data to date clearly support the notion that ibd pathogenesis is dependent on dysregulation of multiple factors including disruption of the intestinal barrier and immune dysregulation , and is associated with profound alterations in gut microbial communities . altered intestinal homeostasis drives expansion of aiec and results in compromised barrier function .
aiec are able to invade , survive , and replicate within host cells , leading to exacerbation of barrier dysfunction by disruption of tj proteins , and ultimately contributing to ibd pathogenesis .
if aiec does indeed play an important role in ibd pathogenesis it will be vital to better understand unresolved questions regarding its appearance in disease . specifically : ( 1 ) what are the host genetic and environmental factors that promote aiec expansion in some patients but not all ; ( 2 ) how is regional variation in aiec expansion determined ; and ( 3 ) can therapeutic interventions ( probiotics , prebiotics , small molecules ) specifically neutralize or modify aiec pathogenic behavior ?
aiec are capable of compromising intestinal epithelial barrier properties both directly and indirectly to enhance their pathogenic impact .
therefore , in addition to the earlier - described approaches , it will be of key interest to determine if strategies designed to enhance the epithelial barrier can minimize the pathogenic impact of aiec and its putative contribution to ibd .
 pathobiont expansion , such as that of adherent - invasive escherichia coli ( aiec ) , is an emerging factor associated with inflammatory bowel disease . 
 the intestinal epithelial barrier is the first line of defense against these pathogens . 
 inflammation plays a critical role in altering the epithelial barrier and is a major factor involved in promoting the expansion and pathogenesis of aiec . 
 aiec in turn can exacerbate intestinal epithelial barrier dysfunction by targeting multiple elements of the barrier . 
 one critical element of the epithelial barrier is the tight junction . 
 increasing evidence suggests that aiec may selectively target protein components of tight junctions , leading to increased barrier permeability . 
 this may represent one mechanism by which aiec could contribute to the development of inflammatory bowel disease . 
 this review article discusses potential mechanisms by which aiec can disrupt epithelial tight junction function and intestinal barrier function . 
AIEC
Interaction of AIEC With IECs
AIEC-Dependent Disruption of the Apical Junctional Complex
Conclusions