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Pathological bacterial translocation in liver cirrhosis

Open AccessPublished:August 29, 2013DOI:https://doi.org/10.1016/j.jhep.2013.07.044

      Abbreviations:

      AMP (antimicrobial peptides), APC (antigen-presenting cell), BDL (bile duct ligation), BT (bacterial translocation), CCl4 (carbon-tetrachloride), DC (dendritic cell), FxR (Farnesoid X receptor), GALT (gut-associated lymphoid tissue), GI (gastrointestinal), GNB (gram-negative bacteria), HE (hepatic encephalopathy), IEL (interepithelial lymphocyte), IFN (Interferon), IgA (Immunoglobulin A), IL (interleukin), LPS (lipopolysaccharide), MCP-1 (monocyte chemotatic protein-1), MDP (Muramyl-Dipeptide), MLCK (myosin light chain kinase), MLN (mesenteric lmyph nodes), NO (nitric oxide), NOD (nucleotide binding oligomerisation domain 2), NFkB (nuclear factor kB), PGN (Peptidoglycans), ROS (reactive oxygen species), SBP (spontaneous bacterial peritonitis), SDD (selective gut decontamination), SIBO (small intestinal bacterial overgrowth), TJ (tight junction), TLR (toll-like-receptor), TNF (tumor necrosis factor), ZO (zonula occludens)

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      • Bacterial translocation in liver cirrhosis: Site and role in fibrogenesis
        Journal of HepatologyVol. 61Issue 3
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          I read with great interest the comprehensive review by Wiest and colleagues [1] on pathological bacterial translocation in liver cirrhosis. They remarkably analyzed the compartments involved and their influencing factors. My concern regards the possible site(s) of bacterial translocation (BT), and the role of BT in the progress of both precirrhotic chronic liver damage, particularly fibrosis, and installed liver cirrhotic lesions themselves.
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      • Reply to: “Bacterial translocation in liver cirrhosis: Site and role in fibrogenesis”
        Journal of HepatologyVol. 61Issue 3
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          We greatly appreciate the comments raised by Dr Matuchansky regarding our recent review on pathological bacterial translocation (BT) in liver cirrhosis [1]. As for the site of BT in cirrhosis we acknowledge the investigation in compensated cirrhotic patients utilizing a multisugar test [2]. The study described by Dr Matuchansky reports in cirrhotic individuals an increased sucralose/erythritol ratio in 5–24 h urine (supposed to indicate colonic permeability) whereas the lactulose/rhamnose ratio in 0–5 h urine (supposed to represent small intestinal permeability) was not altered.  
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      Introduction

      Humans harbor nearly 100 trillion intestinal bacteria, which in terms of numbers, represents around ten times more microbial cells than eukaryotic cells. The gastrointestinal (GI) tract, the largest surface area of the body with an epithelial surface of approximately 400 m2, is in constant exposure to these live microorganisms. Their peaceful coexistence demonstrated by the lack of pro-inflammatory responses against commensal bacteria implicates the presence of clearly defined lines of communication. In fact, bacterial translocation (BT), being defined as translocation of bacteria and/or bacterial products (lipopolysaccharides, peptidoglycans, muramyl-dipeptides, bacterial DNA, etc.) from the gut to mesenteric lymph nodes (MLN) [
      • Berg R.D.
      • Garlington A.W.
      Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model.
      ], is a physiological process in healthy conditions and crucial for host immunity. In contrast, in cirrhosis “pathological” BT develops with a sustained increase in quantity (rate and/or degree) of BT. However, at least in humans, lack of access to MLN and/or upstream compartments towards the mucosal barrier until now hamper establishment of “cut-off” levels for physiological levels of BT in individual patients. Nonetheless, there appears to exist a hierarchy of three barriers against pathological BT, each of which encompasses a distinct set of mechanisms (Fig. 1). First, there are mediators that limit direct contact between the intestinal bacteria and the epithelial cell surface. Secondly, a layer of immune protection involves the rapid detection and killing of bacteria that manage to penetrate. Finally, a set of immune responses minimizes exposure of bacteria to the systemic immune system. In advanced liver cirrhosis, at each of these levels marked alterations have been developed throughout the course of the disease.
      Figure thumbnail gr1
      Fig. 1Compartments and key players involved in mediating pathological BT and the associated host response. Three different routes (1–3) of bacterial translocation can be separated: (1) direct sampling of luminal bacteria (l products) by dendritic cells via processes between epithelial cells, not affecting tight junction function; (2) injured/inflamed epithelium with dysfunctional epithelial barrier and (3) M-cells overlying Peyer Patches as specialized cells providing access of microbial products to antigen-presenting cells. Moreover, three different levels of barriers (I–III) against bacterial translocation are shown: (I) lumen and secretory component (e.g., inner and outer mucus layer, antimicrobial peptides) of gut barrier; (II) mechanical epithelial barrier and the gut-associated lymphatic tissue (GALT) beneath with response elements to BT (e.g., TNF and other pro-inflammatory cytokines) and autonomic nervous system; (III) systemic immune system as third barrier in case of spreading of bacteria(l products) beyond MLN including hematogenous (portal venous) and lymphatic (ductus thoracicus) route of delivery. APC, antigen presenting cell; PRR, pattern recognition receptors; TNF, tumour necrosis factor.

      Background and potential relevance of pathological bacterial translocation

      Pathological BT has been termed the “Achilles heel” in liver disease [
      • Benten D.
      • Wiest R.
      Gut microbiome and intestinal barrier failure–the “Achilles heel” in hepatology?.
      ] playing an important role in the pathogenesis and complications of cirrhosis. The most evidenced clinical expression of pathological BT is spontaneous bacterial peritonitis (SBP). SBP often originates from bacteria in the gut that belong to the normal intestinal microbiota. It has been shown that green fluorescent protein (GFP) labelled Escherichia coli administered orally to cirrhotic rats reveal the presence of these bacteria not only in the intestinal lumen but also in the mesenteric lymph nodes (MLN) and ascites [
      • Teltschik Z.
      • Wiest R.
      • Beisner J.
      • Nuding S.
      • Hofmann C.
      • Schoelmerich J.
      • et al.
      Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.
      ] (Fig. 2). However, not only culture-positive SBP and/or bacteremia impact on the cirrhotic host, but also increased inflow of translocating bacterial products into the hepato-splanchnic as well as systemic circulation. Augmented pro-inflammatory response to gut-derived products and failure to control invading bacteria and -products in concert with host susceptibility determine remote organ injury. This may include acute-on-chronic liver failure, hepato-renal-syndrome and hepatic encephalopathy [
      • Garcia-Tsao G.
      • Wiest R.
      Gut microflora in the pathogenesis of the complications of cirrhosis.
      ]. Therefore, understanding the physiology of gut-bacteria interactions and the pathogenesis of BT can lead to new therapeutic targets in the prevention of infections and other complications of cirrhosis.
      Figure thumbnail gr2
      Fig. 2Visualization of Green fluorescent protein (GFP)-marked E. coli in different compartments after oral gavage in an ascitic rat with cirrhosis. 6 h after oral inoculation of 108 CFU/ml GFP-labeled E. coli, stool along the GI tract, ascites and mesenteric lymph nodes (MLN) were harvested. This clarifies the translocation of those marked bacteria from the gut to MLN as well as into ascites representing the pathophysiological “road” for the development of SBP in advanced cirrhosis. Adapted from Teltschik et al.
      [
      • Teltschik Z.
      • Wiest R.
      • Beisner J.
      • Nuding S.
      • Hofmann C.
      • Schoelmerich J.
      • et al.
      Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.
      ]
      .
      Figure thumbnail gr3
      Fig. 3Compartments and influencing factors promoting pathological BT in cirrhosis. Alterations in the three compartments (= “inner circle”) act in concert and most likely synergistically promote pathological bacterial translocation; each of the compartments per se influences any of the other (symbolized by arrow heads on the circle) and in end-stage disease may lead to a vicious circle. Influencing factors are multiple and can impact on each of the compartments.

      Compartments involved in pathological bacterial translocation

      Gut associated lymphoid tissue (GALT)

      The GALT represents the largest immunological organ in the human body. Despite the vast improvements made in understanding how the microbiota influence host immunity, very little is still known of the intestinal immune system in cirrhosis.
      The innate immune system is considered the “first line of defence” against invading bacteria or their associated products. Invading bacteria are detected by the innate immune system through the recognition of highly conserved bacterial motifs that are present in all bacteria (microbial-associated molecular patterns, MAMPs) by germline-coded pattern-recognition receptors (PRR) on intestinal cells [
      • Akira S.
      • Takeda K.
      • Kaisho T.
      Toll-like receptors: critical proteins linking innate and acquired immunity.
      ]. PRR are located on both the cell surface and within endosomal compartments and these receptors can be further divided into two subgroups: Toll-like receptors (TLRs) and cytoplasmic NLR (nucleotide binding domain, leucine-rich repeats) proteins.
      The mucosal immune system is not ignorant to the commensal bacteria, rather microbial antigens are continuously sampled via various routes (Fig. 1): (1) Dendritic cells (DCs) that underlie the epithelium may open tight junctions (TJ) between epithelial cells, sending processes into the lumen that directly sample microbes [
      • Rescigno M.
      • Urbano M.
      • Valzasina B.
      • Francolini M.
      • Rotta G.
      • Bonasio R.
      • et al.
      Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria.
      ]; lamina propria DCs compromise two different subsets: CD103+CX3CR1 DCs (inducing development of regulatory T cells) and CD103CX3CR1+ DCs (with features of macrophages, promoting TNF-production and development of Th1/Th17 T cells) (2) through interaction with antigenic material in underlying tissue that occurs particularly when epithelial integrity is compromised; or (3) through sampling by specialized M cells within villous epithelium or the follicle-associated epithelium overlying Peyer patches [
      • Hase K.
      • Kawano K.
      • Nochi T.
      • Pontes G.S.
      • Fukuda S.
      • Ebisawa M.
      • et al.
      Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.
      ]. Alterations in these sampling mechanisms in presence of pathological BT in cirrhosis have yet to be delineated. It has been shown that in CCl4-induced cirrhotic ascitic rats translocation of bacterial DNA associates with an increase in total number of intestinal CD103+ DC’s in the lamina propria (as well as MLN) [
      • Munoz L.
      • Jose B.M.
      • Ubeda M.
      • Lario M.
      • Diaz D.
      • Frances R.
      • et al.
      Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.
      ]. However, whether in cirrhosis these CD103+ DC’s or other subsets of DC’s (e.g., CX3CR1+) or mononuclear cells are actually the “transporter” of living bacteria to MLN with pathological BT remains unanswered.
      In response to BT, gut epithelial cells release chemokines that induce the recruitment of DCs to the mucosa. Once activated mature intestinal DCs have the capability to induce and prime mucosal B and T cells ultimately shaping the adaptive mucosal immune system. After maturation, these B and T cells are released into the blood stream and, due to surface expression of the appropriate homing markers, home back to reside within the lamina propria. Microbial antigens presented to B cells induce a commensal-specific IgA response that aids to prevent the commensals from straying beyond the gut mucosa [
      • Hapfelmeier S.
      • Lawson M.A.
      • Slack E.
      • Kirundi J.K.
      • Stoel M.
      • Heikenwalder M.
      • et al.
      Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.
      ]. Interestingly, mice deficient in the TLR-adapter molecule MyD88 on B cells lack commensal-specific immunoglobulin-response with insufficient bacterial killing that leads to lethal dissemination of commensal bacteria during colonic damage [
      • Kirkland D.
      • Benson A.
      • Mirpuri J.
      • Pifer R.
      • Hou B.
      • DeFranco A.L.
      • et al.
      B cell-intrinsic MyD88 signaling prevents the lethal dissemination of commensal bacteria during colonic damage.
      ]. In cirrhotic patients, reductions in memory B cells and hypo-responsiveness to TLR9-stimulation has been reported [
      • Doi H.
      • Iyer T.K.
      • Carpenter E.
      • Li H.
      • Chang K.M.
      • Vonderheide R.H.
      • et al.
      Dysfunctional B-cell activation in cirrhosis resulting from hepatitis C infection associated with disappearance of CD27-positive B-cell population.
      ]. However, to what degree this contributes to pathological BT is currently unknown. Likewise, the role of intestinal T cells is ill defined in liver cirrhosis but deserves more attention. T cells are critical in host defense against the translocation of enteric bacteria [
      • Gautreaux M.D.
      • Deitch E.A.
      • Berg R.D.
      T lymphocytes in host defense against bacterial translocation from the gastrointestinal tract.
      ]. In the absence of T cells, there is spontaneous systemic BT of members of the commensal microbiota, such as E. coli [
      • Owens W.E.
      • Berg R.D.
      Bacterial translocation from the gastrointestinal tract of athymic (nu/nu) mice.
      ]. Moreover, T cell depletion not only causes accumulation of bacteria in MLN in healthy rats but leads to spreading of bacteria to extraintestinal sites in alcohol- and burn-injured rats [
      • Choudhry M.A.
      • Fazal N.
      • Goto M.
      • Gamelli R.L.
      • Sayeed M.M.
      Gut-associated lymphoid T cell suppression enhances bacterial translocation in alcohol and burn injury.
      ].

      MLN at the centre of BT

      In healthy conditions commensal bacteria transported to MLN by DCs induce a local immune response and are killed without inducing systemic immunity. In contrast, if the MLN were surgically removed, bacterial-laden DC carried commensal bacteria to the spleen and ultimately triggering a microbial-specific systemic immune response [
      • MacPherson A.
      • Gatto D.
      • Sainsbury E.
      • Harriman G.R.
      • Hengartner H.
      • Zinkernagel R.M.
      A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria.
      ,
      • Macpherson A.J.
      • Uhr T.
      Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria.
      ]. In humans (or mice) the presence of immunosuppression permits the translocation of intestinal bacteria systemically, which eventually may lead to sepsis and death [
      • Berg R.D.
      • Wommack E.
      • Deitch E.A.
      Immunosuppression and intestinal bacterial overgrowth synergistically promote bacterial translocation.
      ]. Many mechanisms contribute to the spreading of bacterial products and/or living bacteria beyond the MLN in cirrhosis. These include (but are not limited to) relative deficits within both innate and adaptive immunity that result in a reduced chemotactic, opsonic, phagocytic and killing capacity of mononuclear cells [
      • DeMeo A.N.
      • Andersen B.R.
      Defective chemotaxis associated with a serum inhibitor in cirrhotic patients.
      ,
      • Holdstock G.
      • Leslie B.
      • Hill S.
      • Tanner A.
      • Wright R.
      Monocyte function in cirrhosis.
      ,
      • Fiuza C.
      • Salcedo M.
      • Clemente G.
      • Tellado J.M.
      In vivo neutrophil dysfunction in cirrhotic patients with advanced liver disease.
      ,
      • Hassner A.
      • Kletter Y.
      • Shlag D.
      • Yedvab M.
      • Aronson M.
      • Shibolet S.
      Impaired monocyte function in liver cirrhosis.
      ,
      • Propst-Graham K.L.
      • Preheim L.C.
      • Vander Top E.A.
      • Snitily M.U.
      • Gentry-Nielsen M.J.
      Cirrhosis-induced defects in innate pulmonary defenses against Streptococcus pneumoniae.
      ,
      • Tritto G.
      • Bechlis Z.
      • Stadlbauer V.
      • Davies N.
      • Frances R.
      • Shah N.
      • et al.
      Evidence of neutrophil functional defect despite inflammation in stable cirrhosis.
      ], tuftsin activity [
      • Trevisani F.
      • Castelli E.
      • Foschi F.G.
      • Parazza M.
      • Loggi E.
      • Bertelli M.
      • et al.
      Impaired tuftsin activity in cirrhosis: relationship with splenic function and clinical outcome.
      ], and impaired reticuloendothelial system (RES) activity [
      • Rimola A.
      • Soto R.
      • Bory F.
      • Arroyo V.
      • Piera C.
      • Rodes J.
      Reticuloendothelial system phagocytic activity in cirrhosis and its relation to bacterial infections and prognosis.
      ].

      Intestinal barrier dysfunction: secretory and mechanical components

      Only a single layer of epithelial cells separates the sterile host from trillions of live bacteria. This physical barrier functions to deliver critical secretory compounds to the intestinal lumen, such as IgA, mucus proteins and antimicrobial peptides (AMPs) that help to control bacterial attachment and infiltration into the host.

      Mechanical component

      The mucosal epithelium per se closely interacts with antigen-presenting cells beneath and intraepithelial lymphocytes (IEL) within the lamina propria to maintain intestinal integrity. In human cirrhosis, structural changes of the intestinal mucosa including widening of intercellular spaces, vascular congestion, edema, fibromuscular proliferation, decreased villous to crypt ratio, and thickening of the muscularis mucosae have been described [
      • Astaldi G.
      • Strosselli E.
      Peroral biopsy of the intestinal mucosa in hepatic cirrhosis.
      ,
      • Norman D.A.
      • Atkins J.M.
      • Seelig Jr., L.L.
      • Gomez-Sanchez C.
      • Krejs G.J.
      Water and electrolyte movement and mucosal morphology in the jejunum of patients with portal hypertension.
      ,
      • Misra V.
      • Misra S.
      • Dwivedi M.
      • Gupta S.
      Histomorphometric study of portal hypertensive enteropathy.
      ]. It has also been shown by functional studies utilizing dual sugar absorption tests or other test substances that there is an increase in intestinal permeability due to cirrhosis [
      • Campillo B.
      • Pernet P.
      • Bories P.N.
      • Richardet J.P.
      • Devanlay M.
      • Aussel C.
      Intestinal permeability in liver cirrhosis: relationship with severe septic complications.
      ,
      • Ersoz G.
      • Aydin A.
      • Erdem S.
      • Yuksel D.
      • Akarca U.
      • Kumanlioglu K.
      Intestinal permeability in liver cirrhosis.
      ,
      • Pascual S.
      • Such J.
      • Esteban A.
      • Zapater P.
      • Casellas J.A.
      • Aparicio J.R.
      • et al.
      Intestinal permeability is increased in patients with advanced cirrhosis.
      ,
      • Zuckerman M.J.
      • Menzies I.S.
      • Ho H.
      • Gregory G.G.
      • Casner N.A.
      • Crane R.S.
      • et al.
      Assessment of intestinal permeability and absorption in cirrhotic patients with ascites using combined sugar probes.
      ,
      • Norman K.
      • Pirlich M.
      • Schulzke J.D.
      • Smoliner C.
      • Lochs H.
      • Valentini L.
      • et al.
      Increased intestinal permeability in malnourished patients with liver cirrhosis.
      ,
      • Reiberger T.
      • Ferlitsch A.
      • Payer B.A.
      • Mandorfer M.
      • Heinisch B.B.
      • Hayden H.
      • et al.
      Non-selective betablocker therapy decreases intestinal permeability and serum levels of LBP and IL-6 in patients with cirrhosis.
      ].
      TJs maintain a permeability seal at the apicolateral epithelial surface restricting paracellular movement of even very small (2 kDa) molecules and thus, bacteria and macromolecules such as lipopolysaccharides (LPS). More than 50 different TJ-proteins are known and members in the claudin family and the zonula occludens (ZO) proteins [
      • Marchiando A.M.
      • Graham W.V.
      • Turner J.R.
      Epithelial barriers in homeostasis and disease.
      ] are among those most studied. TJ-function is highly dynamic and controlled by signalling molecules including myosin light chain kinases (MLCK). In short-term BDL-mice, increased MLCK activation with concomitant disruption of TJs (diminished expression of occludin and ZO-1) has been reported in colonic epithelium [
      • Hartmann P.
      • Haimerl M.
      • Mazagova M.
      • Brenner D.A.
      • Schnabl B.
      Toll-like receptor 2-mediated intestinal injury and enteric tumor necrosis factor receptor I contribute to liver fibrosis in mice.
      ]. Also in a descriptive pilot study in human cirrhosis, alterations in TJ-proteins in duodenal biopsies with reduced expression of occludin and claudin-1 that gradually increase from crypt to tip of the villi has been demonstrated [
      • Assimakopoulos S.F.
      • Tsamandas A.C.
      • Tsiaoussis G.I.
      • Karatza E.
      • Triantos C.
      • Vagianos C.E.
      • et al.
      Altered intestinal tight junctions’ expression in patients with liver cirrhosis: a pathogenetic mechanism of intestinal hyperpermeability.
      ]. Therefore, in cirrhotic conditions, loosening of TJs may result in an increased accessibility of bacterial products to areas of “free” passage. However, most critical for the translocation of living whole bacteria is the transcellular route. In fact, e.g., for E. coli C25 transcytosis across CaCo-2 cells has been evidenced to occur even independent of changes in paracellular permeability [
      • Macutkiewicz C.
      • Carlson G.
      • Clark E.
      • Dobrindt U.
      • Roberts I.
      • Warhurst G.
      Characterisation of Escherichia coli strains involved in transcytosis across gut epithelial cells exposed to metabolic and inflammatory stress.
      ]. It is epithelial cells under stress that present with decreased transepithelial resistance and increased translocation of commensal bacteria [
      • Lewis K.
      • Lutgendorff F.
      • Phan V.
      • Soderholm J.D.
      • Sherman P.M.
      • McKay D.M.
      Enhanced translocation of bacteria across metabolically stressed epithelia is reduced by butyrate.
      ]. Corresponding investigations on epithelial transcytosis of commensal bacteria in cirrhosis are lacking.
      Epithelial tolerance normally avoids inflammatory changes in response to physiological levels of BT [
      • Mahida Y.R.
      • Johal S.
      NF-kB may determine whether epithelial cell-microbial interactions in the intestine are hostile or friendly.
      ]. However, as soon as inflammation occurs or mucosal load of bacteria(l products) becomes overwhelming, DCs and other monocytes and neutrophils are recruited, perpetuating the process of BT [
      • Shimizu T.
      • Tani T.
      • Hanasawa K.
      • Endo Y.
      • Kodama M.
      The role of bacterial translocation on neutrophil activation during hemorrhagic shock in rats.
      ,
      • Kalff J.C.
      • Schwarz N.T.
      • Walgenbach K.J.
      • Schraut W.H.
      • Bauer A.J.
      Leukocytes of the intestinal muscularis: their phenotype and isolation.
      ]. Indeed, in experimental cirrhosis BT has been found to be associated with mononuclear cell infiltrate in the lamina propria and concomitant submucosal and mesenteric inflammation [
      • Misra V.
      • Misra S.
      • Dwivedi M.
      • Gupta S.
      Histomorphometric study of portal hypertensive enteropathy.
      ,
      • Garcia-Tsao G.
      • Lee F.Y.
      • Barden G.E.
      • Cartun R.
      • West A.B.
      Bacterial translocation to mesenteric lymph nodes is increased in cirrhotic rats with ascites.
      ,
      • Nagral A.S.
      • Joshi A.S.
      • Bhatia S.J.
      • Abraham P.
      • Mistry F.P.
      • Vora I.M.
      Congestive jejunopathy in portal hypertension.
      ]. This is in line with increased fecal concentrations of polymorphonuclear elastase [
      • Saitoh O.
      • Sugi K.
      • Lojima K.
      • Matsumoto H.
      • Nakagawa K.
      • Kayazawa M.
      • et al.
      Increased prevalence of intestinal inflammation in patients with liver cirrhosis.
      ] and calprotectin [
      • Gundling F.
      • Schmidtler F.
      • Hapfelmeier A.
      • Schulte B.
      • Schmidt T.
      • Pehl C.
      • et al.
      Fecal calprotectin is a useful screening parameter for hepatic encephalopathy and spontaneous bacterial peritonitis in cirrhosis.
      ] being observed in cirrhotic patients. But are those inflammatory changes on a mucosal level normal in relation to the rate and severity of pathological translocation present in advanced cirrhosis? There are no comparative studies but it is tempting to speculate that the intestinal inflammatory mucosal response is reduced and thus, the cirrhotic patient is more tolerant to bacteria reaching the epithelium as compared to the non-cirrhotic healthy individual.
      Therefore, at least a state of relative intestinal tolerance to pathological BT can be proposed in cirrhosis. Correspondingly, in cirrhotic rats with culturable pathological BT to MLN there was no significant activation or change in phagocytic and migratory capacity of lamina propria CD103+ DC’s supporting the yet to be proven hypothesis of relative intestinal tolerance to a vast bacterial challenge [
      • Munoz L.
      • Jose B.M.
      • Ubeda M.
      • Lario M.
      • Diaz D.
      • Frances R.
      • et al.
      Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.
      ]. These data are in accordance with the observed “immune paralysis” in patients with sepsis and acute-on-chronic liver failure, attributed to reductions in HLA-DR-expression on circulating monocytes [
      • Poehlmann H.
      • Schefold J.C.
      • Zuckermann-Becker H.
      • Volk H.D.
      • Meisel C.
      Phenotype changes and impaired function of dendritic cell subsets in patients with sepsis: a prospective observational analysis.
      ,
      • Wasmuth H.E.
      • Kunz D.
      • Yagmur E.
      • Timmer-Stranghoner A.
      • Vidacek D.
      • Siewert E.
      • et al.
      Patients with acute on chronic liver failure display “sepsis-like” immune paralysis.
      ]. It is tempting to speculate whether such immunosuppression, which has been reliably associated with increased rates of bacterial infections is present in advanced human cirrhosis with pathological BT of living bacteria to MLN.

      Secretory component

      Much knowledge has been gained recently on this relatively impermeable compartment that may define a confined space, allowing the host to specifically monitor and regulate bacteria that are in close contact with the intestinal surface.
      AMPs include defensins, cathelicidines, resistin-like molecules, bactericidial-permeability-inducing proteins and lectins. Among defensins only α- and β-defensins have been identified in the intestinal tract. All mature defensins have broad range antimicrobial activity by disrupting the structure and function of microbial membranes. α-defensin genes are expressed only in a few cell types, which in humans are predominantly neutrophils and Paneth cells, strategically located at the bottom of each intestinal crypt. The secretion of AMPs by Paneth cells is directly linked to bacteria and LPS exposure [
      • Vaishnava S.
      • Behrendt C.L.
      • Ismail A.S.
      • Eckmann L.
      • Hooper L.V.
      Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface.
      ] and functions to help maintain homeostasis at the intestinal host-microbial interface [
      • Ayabe T.
      • Satchell D.P.
      • Wilson C.L.
      • Parks W.C.
      • Selsted M.E.
      • Ouellette A.J.
      Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria.
      ,
      • Ogle C.K.
      • Noel J.G.
      • Guo X.
      • Wells D.A.
      • Valente J.F.
      • Ogle J.D.
      • et al.
      The ability of endotoxin-stimulated enterocytes to produce bactericidal factors.
      ]. In contrast, β-defensins are expressed constitutively by most epithelial cells in both the small and large intestine [
      • Schroeder B.O.
      • Wu Z.
      • Nuding S.
      • Groscurth S.
      • Marcinowski M.
      • Beisner J.
      • et al.
      Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1.
      ]. CCl4-induced ascitic cirrhotic rats with but not without BT to MLN present with a relative deficiency in Paneth cell defensins particularly in the small intestine [
      • Teltschik Z.
      • Wiest R.
      • Beisner J.
      • Nuding S.
      • Hofmann C.
      • Schoelmerich J.
      • et al.
      Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.
      ]. In contrast, levels of β-defensins are unchanged or elevated in presence of increased BT, demonstrating a normal ß-defensin response in cirrhotic rats. The observed deficit in α-defensins was accompanied by a diminished in vitro antibacterial activity against various Enterobacteriacea. The potential mechanisms mediating the impairment in Paneth cell function in cirrhosis are so far unknown but appear not to relate to the level of portal hypertension since pre-hepatic portal hypertensive rats show no alterations in Paneth-cell products [
      • Teltschik Z.
      • Wiest R.
      • Beisner J.
      • Nuding S.
      • Hofmann C.
      • Schoelmerich J.
      • et al.
      Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense.
      ]. In addition, down-regulation of regenerating islet-derived proteins (RegIIIβ and RegIIIγ) has been demonstrated in the small intestine of mice as well as humans after chronic alcohol intake [
      • Yan A.W.
      • Fouts D.E.
      • Brandl J.
      • Starkel P.
      • Torralba M.
      • Schott E.
      • et al.
      Enteric dysbiosis associated with a mouse model of alcoholic liver disease.
      ]. These lectins are known to bind cell wall peptidoglycans of gram-positive bacteria and function as bactericidal proteins even at low micromolar concentrations [
      • Cash H.L.
      • Whitham C.V.
      • Behrendt C.L.
      • Hooper L.V.
      Symbiotic bacteria direct expression of an intestinal bactericidal lectin.
      ,
      • Mukherjee S.
      • Partch C.L.
      • Lehotzky R.E.
      • Whitham C.V.
      • Chu H.
      • Bevins C.L.
      • et al.
      Regulation of C-type lectin antimicrobial activity by a flexible N-terminal prosegment.
      ]. Therefore, deficiency in various AMPs (α-defensins, RegIII proteins) likely leads to decreased mucosal killing activity resulting in a shift of the bacterial composition facilitating bacterial overgrowth and increases in BT in cirrhosis.

      Mucus

      Mucins create a layer of glycoproteins that prevents direct contact of bacteria with the microvillus [
      • Aranow J.S.
      • Fink M.P.
      Determinants of intestinal barrier failure in critical illness.
      ]. MUC2 is the major secretory mucin being stimulated by a wide array of bioactive factors including microbes/-products, and inflammatory cytokines [
      • Kim Y.S.
      • Ho S.B.
      Intestinal goblet cells and mucins in health and disease: recent insights and progress.
      ]. The “firm” dense inner mucus layer likely traps immune exclusion molecules [
      • Meyer-Hoffert U.
      • Hornef M.W.
      • Henriques-Normark B.
      • Axelsson L.G.
      • Midtvedt T.
      • Putsep K.
      • et al.
      Secreted enteric antimicrobial activity localises to the mucus surface layer.
      ] rendering it sterile [
      • Johansson M.E.
      • Phillipson M.
      • Petersson J.
      • Velcich A.
      • Holm L.
      • Hansson G.C.
      The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria.
      ]. In contrast, the “loose” outer layer is the habitat for commensal bacteria that consume the mucus proteins as a carbon source [
      • Johansson M.E.
      • Larsson J.M.
      • Hansson G.C.
      The two mucus layers of colon are organized by the MUC2 mucin, whereas the outer layer is a legislator of host-microbial interactions.
      ] and provides specific binding sites for bacterial adhesins [
      • Kline K.A.
      • Falker S.
      • Dahlberg S.
      • Normark S.
      • Henriques-Normark B.
      Bacterial adhesins in host-microbe interactions.
      ]. Thus, it is important to differentiate between bacteria that are found within the intestinal lumen and those inhabiting the mucus. In fact, the mucosa-associated microbiome differs from stool flora in cirrhotic patients, particularly in those with hepatic encephalopathy [
      • Bajaj J.S.
      • Hylemon P.B.
      • Ridlon J.M.
      • Heuman D.M.
      • Daita K.
      • White M.B.
      • et al.
      Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation.
      ]. In addition, recent elegant studies in alcoholic patients indicate increased mucus thickness in the duodenum, suggesting changes induced by cirrhosis and/or alcohol [
      • Hartmann P.
      • Chen P.
      • Wang H.J.
      • Wang L.
      • McCole D.F.
      • Brandl K.
      • et al.
      Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice.
      ]. Surprisingly, MUC2 deficient mice are protected from bacterial overgrowth in response to alcohol most likely due to increases in mucosal antimicrobial peptides (RegIIIβ and RegIIIγ) [
      • Hartmann P.
      • Chen P.
      • Wang H.J.
      • Wang L.
      • McCole D.F.
      • Brandl K.
      • et al.
      Deficiency of intestinal mucin-2 ameliorates experimental alcoholic liver disease in mice.
      ] further emphasizing the proposed role of mucus as an active key player in host-microbial interactions [
      • Nishida A.
      • Lau C.W.
      • Zhang M.
      • Andoh A.
      • Shi H.N.
      • Mizoguchi E.
      • et al.
      The membrane-bound mucin Muc1 regulates T helper 17-cell responses and colitis in mice.
      ].
      Bile inhibits small intestinal bacterial overgrowth (SIBO) [
      • Fouts D.E.
      • Torralba M.
      • Nelson K.E.
      • Brenner D.A.
      • Schnabl B.
      Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease.
      ], has a trophic effect on the intestinal mucosa [
      • Levi A.C.
      • Borghi F.
      • Petrino R.
      • Bargoni A.
      • Fronticelli C.M.
      • Gentelini S.
      Modifications of the trophism of intestinal mucosa after intestinal and bilio-pancreatic diversion in the rat.
      ], decreases epithelial internalization of enteric bacteria [
      • Wells C.L.
      • Jechorek R.P.
      • Erlandsen S.L.
      Inhibitory effect of bile on bacterial invasion of enterocytes: possible mechanism for increased translocation associated with obstructive jaundice.
      ], exerts detergent actions with anti-adherence effects and neutralizes endotoxins [
      • Bertok L.
      Physico-chemical defense of vertebrate organisms: the role of bile acids in defense against bacterial endotoxins.
      ,
      • Van Bossuyt H.
      • Desmaretz C.
      • Gaeta G.B.
      • Wisse E.
      The role of bile acids in the development of endotoxemia during obstructive jaundice in the rat.
      ]. However, bile also impacts on intestinal immunity by providing retinoids necessary to imprint intestinal CD103+ DC with the ability to generate gut-tropic T cells [
      • Jaensson-Gyllenbeck E.
      • Kotarsky K.
      • Zapata F.
      • Persson E.K.
      • Gundersen T.E.
      • Blomhoff R.
      • et al.
      Bile retinoids imprint intestinal CD103+ dendritic cells with the ability to generate gut-tropic T cells.
      ]. In cirrhosis, marked decreases in intestinal intraluminal concentrations of bile acids have been ascribed to decreased secretion and increased deconjugation by enteric bacteria. In experimental models the absence of bile in the intestine has been shown to facilitate BT [
      • Clements W.D.
      • Parks R.
      • Erwin P.
      • Halliday M.I.
      • Barr J.
      • Rowlands B.J.
      Role of the gut in the pathophysiology of extrahepatic biliary obstruction.
      ,
      • Parks R.W.
      • Clements W.D.
      • Smye M.G.
      • Pope C.
      • Rowlands B.J.
      • Diamond T.
      Intestinal barrier dysfunction in clinical and experimental obstructive jaundice and its reversal by internal biliary drainage.
      ] and to enhance susceptibility for further translocation in response to endotoxins [
      • Reynolds J.V.
      • Murchan P.
      • Leonard N.
      • Clarke P.
      • Keane F.B.
      • Tanner W.A.
      Gut barrier failure in experimental obstructive jaundice.
      ]. Notably these effects are attenuated after oral administration of bile acids [
      • Lorenzo-Zuniga V.
      • Bartoli R.
      • Planas R.
      • Hofmann A.F.
      • Vinado B.
      • Hagey L.R.
      • et al.
      Oral bile acids reduce bacterial overgrowth, bacterial translocation, and endotoxemia in cirrhotic rats.
      ].
      Conjugated bile acids are natural ligands for several nuclear receptors, of which the transcription factor farnesoid X receptor (FxR) has gained much attention [
      • Gadaleta R.M.
      • van Erpecum K.J.
      • Oldenburg B.
      • Willemsen E.C.
      • Renooij W.
      • Murzilli S.
      • et al.
      Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease.
      ]. Intestinal FxR limits bacterial overgrowth and BT, which has been demonstrated in BDL mice [
      • Inagaki T.
      • Moschetta A.
      • Lee Y.K.
      • Peng L.
      • Zhao G.
      • Downes M.
      • et al.
      Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor.
      ]. A specific FxR agonist (GW4064) repressed bacterial overgrowth, attenuated mucosal injury and reduced bacterial invasion into MLN in wild type but not in mice genetically deficient in FxR [
      • Inagaki T.
      • Moschetta A.
      • Lee Y.K.
      • Peng L.
      • Zhao G.
      • Downes M.
      • et al.
      Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor.
      ]. Activation of FxR by GW4064 led to the identification of several novel FxR target genes, including those that promote antimicrobial defense. How these FxR target genes function to maintain intestinal homeostasis will surely be an active area of future investigations.

      IgA antibodies

      On a daily basis 2–5 g of sIgA is secreted into the gut lumen accounting for more than 70% of total body immunoglobulin production. IgA antibodies effectively bind and aggregate bacteria preventing mucosal adherence and colonization (immune exclusion) [
      • Spaeth G.
      • Gottwald T.
      • Specian R.D.
      • Mainous M.R.
      • Berg R.D.
      • Deitch E.A.
      Secretory immunoglobulin A, intestinal mucin, and mucosal permeability in nutritionally induced bacterial translocation in rats.
      ]. Despite increased BT in IgA-deficient mice [
      • MacPherson A.
      • Gatto D.
      • Sainsbury E.
      • Harriman G.R.
      • Hengartner H.
      • Zinkernagel R.M.
      A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria.
      ], commensal-related sepsis is not observed in IgA-deficient animals or humans, which may be due to the overcompensatory function of IgM in the absence of mucosal IgA [
      • Harriman G.R.
      • Bogue M.
      • Rogers P.
      • Finegold M.
      • Pacheco S.
      • Bradley A.
      • et al.
      Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes.
      ]. In cirrhotic patients, decreased fecal IgA concentrations as well as decreased secretion of mucosal IgA into the jejunum have been reported [
      • Saitoh O.
      • Sugi K.
      • Lojima K.
      • Matsumoto H.
      • Nakagawa K.
      • Kayazawa M.
      • et al.
      Increased prevalence of intestinal inflammation in patients with liver cirrhosis.
      ], suggesting a potential relationship between IgA and BT, and the development of infections in cirrhosis, although this hypothesis has yet to be proven.

      Intestinal microbiota; qualitative and quantitative changes

      The intestinal microflora consists of a dynamic mixture of microbes with considerable quantitative and qualitative differences among individuals and particularly among species. Additionally, only a small proportion of the enteric bacteria can currently be examined by conventional culture techniques [
      • Gill S.R.
      • Pop M.
      • Deboy R.T.
      • Eckburg P.B.
      • Turnbaugh P.J.
      • Samuel B.S.
      • et al.
      Metagenomic analysis of the human distal gut microbiome.
      ] limiting diagnostic measures. The proximal small intestine (duodenum, jejunum) is sparsely populated with bacteria; however, from the ileum on there is a sharp increase in microbial density, from 105 colony forming units (CFU)/ml in the jejunum to 108 in distal ileum and cecum, up to 1012 in the colon [
      • Marteau P.
      • Pochart P.
      • Dore J.
      • Bera-Maillet C.
      • Bernalier A.
      • Corthier G.
      Comparative study of bacterial groups within the human cecal and fecal microbiota.
      ].

      Quantitative changes

      Small intestinal bacterial overgrowth (SIBO) has arbitrarily been defined as >105 CFU/ml and/or the presence of colonic bacteria in upper jejunal aspirate [
      • Corazza G.R.
      • Menozzi M.G.
      • Strocchi A.
      • Rasciti L.
      • Vaira D.
      • Lecchini R.
      • et al.
      The diagnosis of small bowel bacterial overgrowth. Reliability of jejunal culture and inadequacy of breath hydrogen testing.
      ]. Using this gold standard, the prevalence of SIBO in cirrhotic patients ranges from 48% to 73% [
      • Chesta J.
      • Silva M.
      • Thompson L.
      • del Canto E.
      • Defilippi C.
      Bacterial overgrowth in small intestine in patients with liver cirrhosis.
      ,
      • Bauer T.M.
      • Steinbruckner B.
      • Brinkmann F.E.
      • Ditzen A.K.
      • Schwacha H.
      • Aponte J.J.
      • et al.
      Small intestinal bacterial overgrowth in patients with cirrhosis: prevalence and relation with spontaneous bacterial peritonitis.
      ,
      • Bauer T.M.
      • Schwacha H.
      • Steinbrückner B.
      • Brinkmann F.E.
      • Ditzen A.K.
      • Aponte J.J.
      • et al.
      Small intestinal bacterial overgrowth in human cirrhosis is associated with systemic endotoxemia.
      ,
      • Bode J.C.
      • Bode C.
      • Heidelbach R.
      • Durr H.K.
      • Martini G.A.
      Jejunal microflora in patients with chronic alcohol abuse.
      ,
      • Pardo A.
      • Bartoli R.
      • Lorenzo-Zuniga V.
      • Planas R.
      • Vinado B.
      • Riba J.
      • et al.
      Effect of cisapride on intestinal bacterial overgrowth and bacterial translocation in cirrhosis.
      ]. SIBO has been shown to be particularly frequent in those with more severe liver disease [
      • Morencos F.C.
      • Las Heras C.G.
      • Martin R.L.
      • Lopez Arias M.J.
      • Ledesma F.
      • Pons R.F.
      Small bowel bacterial overgrowth in patients with alcoholic cirrhosis.
      ,
      • Yang C.Y.
      • Chang C.S.
      • Chen G.H.
      Small-intestinal bacterial overgrowth in patients with liver cirrhosis, diagnosed with glucose H2 or CH4 breath tests.
      ] and in those with a prior history of SBP and/or hepatic encephalopathy [
      • Chang C.S.
      • Yang S.S.
      • Kao C.H.
      • Yeh H.Z.
      • Chen G.H.
      Small intestinal bacterial overgrowth versus antimicrobial capacity in patients with spontaneous bacterial peritonitis.
      ,
      • Jun D.W.
      • Kim K.T.
      • Lee O.Y.
      • Chae J.D.
      • Son B.K.
      • Kim S.H.
      • et al.
      Association between small intestinal bacterial overgrowth and peripheral bacterial DNA in cirrhotic patients.
      ]. In advanced liver cirrhosis it has been linked to the development of BT, SBP and endotoxemia [
      • Bauer T.M.
      • Schwacha H.
      • Steinbrückner B.
      • Brinkmann F.E.
      • Ditzen A.K.
      • Aponte J.J.
      • et al.
      Small intestinal bacterial overgrowth in human cirrhosis is associated with systemic endotoxemia.
      ,
      • Pardo A.
      • Bartoli R.
      • Lorenzo-Zuniga V.
      • Planas R.
      • Vinado B.
      • Riba J.
      • et al.
      Effect of cisapride on intestinal bacterial overgrowth and bacterial translocation in cirrhosis.
      ,
      • Guarner C.
      • Runyon B.A.
      • Young S.
      • Heck M.
      • Sheikh M.Y.
      Intestinal bacterial overgrowth and bacterial translocation in cirrhotic rats with ascites.
      ]. In fact, in cirrhosis SIBO is one of the main factors that promotes BT and the occurrence of BT to MLN in experimental models routinely associates with SIBO [
      • Guarner C.
      • Runyon B.A.
      • Young S.
      • Heck M.
      • Sheikh M.Y.
      Intestinal bacterial overgrowth and bacterial translocation in cirrhotic rats with ascites.
      ,
      • Perez-Paramo M.
      • Munoz J.
      • Albillos A.
      • Freile I.
      • Portero F.
      • Santos M.
      • et al.
      Effect of propranolol on the factors promoting bacterial translocation in cirrhotic rats with ascites.
      ]. A direct relationship between the density and composition of bacteria populating a segment of the intestine and numbers of viable bacteria of this strain present in MLN has been demonstrated in mouse models [
      • Steffen E.K.
      • Berg R.D.
      Relationship between cecal population levels of indigenous bacteria and translocation to the mesenteric lymph nodes.
      ]. Importantly, in the absence of SIBO in experimental cirrhosis BT occurs rarely (0–11%). However, since BT does not occur in up to half of the cirrhotic animals with SIBO, it appears that SIBO is supportive but not sufficient per se for BT to occur. Therefore, other factors, most likely a decrease in local immunity, play the most important role in inducing BT. For instance, in experimental ethanol-induced liver injury, increases in BT do occur prior to changes in intestinal flora [
      • Yan A.W.
      • Fouts D.E.
      • Brandl J.
      • Starkel P.
      • Torralba M.
      • Schott E.
      • et al.
      Enteric dysbiosis associated with a mouse model of alcoholic liver disease.
      ]. SIBO in cirrhosis has traditionally been attributed, at least partly, to a decrease in small-bowel motility and intestinal transit time [
      • Bauer T.M.
      • Steinbruckner B.
      • Brinkmann F.E.
      • Ditzen A.K.
      • Schwacha H.
      • Aponte J.J.
      • et al.
      Small intestinal bacterial overgrowth in patients with cirrhosis: prevalence and relation with spontaneous bacterial peritonitis.
      ,
      • Chang C.S.
      • Chen G.H.
      • Lien H.C.
      • Yeh H.Z.
      Small intestine dysmotility and bacterial overgrowth in cirrhotic patients with spontaneous bacterial peritonitis.
      ,
      • Chesta J.
      • Defilippi C.
      • Defilippi C.
      Abnormalities in proximal small bowel motility in patients with cirrhosis.
      ,
      • Sadik R.
      • Abrahamsson H.
      • Björnsson E.
      • Gunnarsdottir A.
      • Stotzer P.O.
      Etiology of portal hypertension may influence gastrointestinal transit time.
      ,
      • Stewart J.J.
      • Battarbee H.D.
      • Farrar G.E.
      • Betzing K.W.
      Intestinal myoelectrical activity and transit time in chronic portal hypertension.
      ]. The proposed contribution of proton pump inhibitors for the development of SIBO [
      • Bauer T.M.
      • Schwacha H.
      • Steinbrückner B.
      • Brinkmann F.E.
      • Ditzen A.K.
      • Aponte J.J.
      • et al.
      Small intestinal bacterial overgrowth in human cirrhosis is associated with systemic endotoxemia.
      ,
      • Fried M.
      • Siegrist H.
      • Frei R.
      • Froehlich F.
      • Duroux P.
      • Thorens J.
      • et al.
      Duodenal bacterial overgrowth during treatment in outpatients with omeprazole.
      ] and SBP [
      • Bajaj J.S.
      • Zadvornova Y.
      • Heuman D.M.
      • Hafeezullah M.
      • Hoffmann R.G.
      • Sanyal A.J.
      • et al.
      Association of proton pump inhibitor therapy with spontaneous bacterial peritonitis in cirrhotic patients with ascites.
      ,
      • Bajaj J.S.
      • Ratliff S.M.
      • Heuman D.M.
      • Lapane K.L.
      Proton pump inhibitors are associated with a high rate of serious infections in veterans with decompensated cirrhosis.
      ] has recently been questioned in a large cohort of cirrhotic patients [
      • Terg R.
      • Casciato P.
      • Garbe C.
      • Cartier M.
      • Stieben T.E.
      • Mendizabal M.
      • et al.
      Proton pump inhibitor therapy does not increase the incidence of bacterial infection in decompensated cirrhotic patients. A nationwide multicenter, prospective study.
      ]. Nonetheless, hypo- and achlorhydria have been observed in cirrhotics even without acid suppressive medication, resulting in higher pH in the small intestine and under these circumstances have been associated with SIBO [
      • Shindo K.
      • Machida M.
      • Miyakawa K.
      • Fukumura M.
      A syndrome of cirrhosis, achlorhydria, small intestinal bacterial overgrowth, and fat malabsorption.
      ].

      Qualitative changes

      The full microbial richness in the human population reaches up to 40,000 species and the bacterial metagenome may exceed the human genome by 100 fold [
      • Human Microbiome Project Consortium
      Structure, function and diversity of the healthy human microbiome.
      ]. However, only 30–40 species amount to about 98–99% of the microbiota, and Firmicutes and Bacteroidetes are the predominant intestinal phyla across all vertebrates [
      • Eckburg P.B.
      • Bik E.M.
      • Bernstein C.N.
      • Purdom E.
      • Dethlefsen L.
      • Sargent M.
      • et al.
      Diversity of the human intestinal microbial flora.
      ]. Using culture-independent techniques such as pyro-sequencing, analyses of fecal contents could demonstrate reductions in microbial diversity and distinct dysbiosis in both animal models as well as human cirrhosis [
      • Fouts D.E.
      • Torralba M.
      • Nelson K.E.
      • Brenner D.A.
      • Schnabl B.
      Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease.
      ,
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      ]. The microbiota of cirrhosis has been associated with the depletion of the beneficial phyla Lachnospiraceae (particularly clostridiae) [
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      ,
      • Gomez-Hurtado I.
      • Santacruz A.
      • Peiro G.
      • Zapater P.
      • Gutierrez A.
      • Perez-Mateo M.
      • et al.
      Gut microbiota dysbiosis is associated with inflammation and bacterial translocation in mice with CCl4-induced fibrosis.
      ] and bacteroidetes (mainly family of Bacteroidaceae) [
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      ] and enrichment in the phyla Proteobacteria (mainly class of Gammaproteobacteria and among those particularly Enterobacteriaceae) [
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      ,
      • Gomez-Hurtado I.
      • Santacruz A.
      • Peiro G.
      • Zapater P.
      • Gutierrez A.
      • Perez-Mateo M.
      • et al.
      Gut microbiota dysbiosis is associated with inflammation and bacterial translocation in mice with CCl4-induced fibrosis.
      ]. Interestingly, particularly the depletion of clostridiae resulted in a pronounced pro-inflammatory profile [
      • Gomez-Hurtado I.
      • Santacruz A.
      • Peiro G.
      • Zapater P.
      • Gutierrez A.
      • Perez-Mateo M.
      • et al.
      Gut microbiota dysbiosis is associated with inflammation and bacterial translocation in mice with CCl4-induced fibrosis.
      ] and correlated negatively with Child-Pugh score [
      • Chen Y.
      • Yang F.
      • Lu H.
      • Wang B.
      • Chen Y.
      • Lei D.
      • et al.
      Characterization of fecal microbial communities in patients with liver cirrhosis.
      ]. Moreover, the particular relevance of alterations in the mucosa-associated microbiome has been evidenced by distinct differences between cirrhotic patients with and without hepatic encephalopathy being associated with increased levels of inflammation [
      • Bajaj J.S.
      • Hylemon P.B.
      • Ridlon J.M.
      • Heuman D.M.
      • Daita K.
      • White M.B.
      • et al.
      Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation.
      ]. Finally, similar dysbiosis is observed in inflammatory bowel disease (reviewed in Danese [
      • Fava F.
      • Danese S.
      Intestinal microbiota in inflammatory bowel disease: friend of foe?.
      ]). In conjunction with recent findings that mucosal inflammation per se modifies microbial composition inducing the expansion of microorganisms with genotoxic capabilities (such as E. coli) [
      • Arthur J.C.
      • Perez-Chanona E.
      • Muhlbauer M.
      • Tomkovich S.
      • Uronis J.M.
      • Fan T.J.
      • et al.
      Intestinal inflammation targets cancer-inducing activity of the microbiota.
      ] it remains to be seen whether inflammation is the cause or the consequence of changes in microbial composition in those entities.
      Anaerobic bacteria do not readily translocate whereas aerobic gram negative bacteria translocate easily and even across a histologically intact intestinal epithelium [
      • Wells C.L.
      Colonization and translocation of intestinal bacterial flora.
      ,
      • Steffen E.K.
      • Berg R.D.
      • Deitch E.A.
      Comparison of translocation rates of various indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node.
      ]. Moreover, anaerobes outnumber aerobes by 100:1 and limit the colonization and overgrowth of other potentially invasive microbes, thereby confining potentially pathogenic bacteria. In fact, selective elimination of anaerobic bacteria facilitates SIBO and translocation of facultative bacteria [
      • Wells C.L.
      • Maddaus M.A.
      • Reynolds C.M.
      • Jechorek R.P.
      • Simmons R.L.
      Role of anaerobic flora in the translocation of aerobic and facultatively anaerobic intestinal bacteria.
      ]. Bacteria that translocate most readily are facultative intracellular pathogens (e.g., Salmonella, Listeria), able to resist phagocytic killing. In contrast, commensal bacteria are easily killed after phagocytosis, surviving only when host defenses are impaired. Gram-negative bacteria (GNB) (specifically E. coli, K. pneumoniae, P. aeruginosa and other Enterobacteriaceae), enterococci and other streptococci, have been found to be the most adept at translocating to MLN [
      • Steffen E.K.
      • Berg R.D.
      • Deitch E.A.
      Comparison of translocation rates of various indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node.
      ]. Interestingly, these species and among those particularly E. coli are those that most frequently cause spontaneous bacterial infections in cirrhotic patients [
      • Wiest R.
      • Rath H.C.
      Bacterial translocation in the gut.
      ,
      • Bert F.
      • Johnson J.R.
      • Ouattara B.
      • Leflon-Guibout V.
      • Johnston B.
      • Marcon E.
      • et al.
      Genetic diversity and virulence profiles of Escherichia coli isolates causing spontaneous bacterial peritonitis and bacteremia in patients with cirrhosis.
      ,
      • Garcia-Tsao G.
      Spontaneous bacterial peritonitis.
      ,
      • Navasa M.
      • Follo A.
      • Llovet J.M.
      • Clemente G.
      • Vargas V.
      • Rimola A.
      • et al.
      Randomized, comparative study of oral ofloxacin versus intravenous cefotaxime in spontaneous bacterial peritonitis.
      ,
      • Fleig W.E.
      • Grothe W.
      • Lotterer E.
      • Behl S.
      Spontaneous bacterial peritonitis (SBP). Retrospective and prospective data from a multicenter study on prevalence, diagnosis and therapy in Germany.
      ]. As described for other disease patterns which are accompanied by BT, for example intestinal obstruction, burn injury or starvation, the translocation of almost exclusively coliform bacteria underlines the pronounced preference of these Gram-negative strains to translocate [
      • Katouli M.
      • Nettebladt C.G.
      • Muratov V.
      • Ljungqvist O.
      • Bark T.
      • Svenberg T.
      • et al.
      Selective translocation of coliform bacteria adhering to caecal epithelium of rats during catabolic stress.
      ,
      • Wells C.L.
      Relationship between intestinal microecology and the translocation of intestinal bacteria.
      ]. Certain E. coli strains (e.g., biochemical phenotype C1–C4 or C25) have been reported to translocate more efficiently than others across intestinal mucosa when it is exposed to metabolic and inflammatory stress [
      • Macutkiewicz C.
      • Carlson G.
      • Clark E.
      • Dobrindt U.
      • Roberts I.
      • Warhurst G.
      Characterisation of Escherichia coli strains involved in transcytosis across gut epithelial cells exposed to metabolic and inflammatory stress.
      ,
      • Katouli M.
      • Nettebladt C.G.
      • Muratov V.
      • Ljungqvist O.
      • Bark T.
      • Svenberg T.
      • et al.
      Selective translocation of coliform bacteria adhering to caecal epithelium of rats during catabolic stress.
      ,
      • Ljungdahl M.
      • Lundholm M.
      • Katouli M.
      • Rasmussen I.
      • Engstrand L.
      • Haglund U.
      Bacterial translocation in experimental shock is dependent on the strains in the intestinal flora.
      ]. However, in cirrhosis E. coli isolates from SBP cases are genetically diverse [
      • Bert F.
      • Johnson J.R.
      • Ouattara B.
      • Leflon-Guibout V.
      • Johnston B.
      • Marcon E.
      • et al.
      Genetic diversity and virulence profiles of Escherichia coli isolates causing spontaneous bacterial peritonitis and bacteremia in patients with cirrhosis.
      ].

      Influencing factors on compartments promoting bacterial translocation (Fig. 3)

      Any increase in translocation of bacteria(l) products to the GALT triggers a concert of pro-/anti-inflammatory cytokine release. Among those TNF has gained most attention because it increases TJ-permeability in the intestine via decreases in expression of TJ-proteins but also activation of MLCK [
      • Taylor C.T.
      • Dzus A.L.
      • Colgan S.P.
      Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha.
      ]. In BDL mice, increased numbers of infiltrating monocytes in the lamina propria expressing TNF have been demonstrated to disrupt epithelial TJs resulting in pathological BT [
      • Hartmann P.
      • Haimerl M.
      • Mazagova M.
      • Brenner D.A.
      • Schnabl B.
      Toll-like receptor 2-mediated intestinal injury and enteric tumor necrosis factor receptor I contribute to liver fibrosis in mice.
      ]. Most importantly, anti-TNF monoclonal antibody as well as pentoxifylline treatment significantly decreases incidences of BT in experimental cirrhosis [
      • Frances R.
      • Chiva M.
      • Sanchez E.
      • Gonzalez-Navajas J.M.
      • Llovet T.
      • Zapater P.
      • et al.
      Bacterial translocation is downregulated by anti-TNF-alpha monoclonal antibody administration in rats with cirrhosis and ascites.
      ,
      • Corradi F.
      • Brusasco C.
      • Fernandez J.
      • Vila J.
      • Ramirez M.J.
      • Seva-Pereira T.
      • et al.
      Effects of pentoxifylline on intestinal bacterial overgrowth, bacterial translocation and spontaneous bacterial peritonitis in cirrhotic rats with ascites.
      ]. Interestingly, functional polymorphism of the MCP-1 gene, known to confer increased MCP-1 expression and thus increased chemotaxis of monocytes – the major source of TNF – has been shown to be a risk factor for the development of SBP in patients with alcoholic cirrhosis [
      • Gabele E.
      • Muhlbauer M.
      • Paulo H.
      • Johann M.
      • Meltzer C.
      • Leidl F.
      • et al.
      Analysis of monocyte chemotactic protein-1 gene polymorphism in patients with spontaneous bacterial peritonitis.
      ]. TNF-secretion is likewise increased in MLN and serum in experimental and human cirrhosis with ascites [
      • Genesca J.
      • Marti R.
      • Rojo F.
      • Campos F.
      • Peribanez V.
      • Gonzalez A.
      • et al.
      Increased tumour necrosis factor alpha production in mesenteric lymph nodes of cirrhotic patients with ascites.
      ,
      • Munoz L.
      • Albillos A.
      • Nieto M.
      • Reyes E.
      • Lledo L.
      • Monserrat J.
      • et al.
      Mesenteric Th1 polarization and monocyte TNF-alpha production: first steps to systemic inflammation in rats with cirrhosis.
      ,
      • Albillos A.
      • de la Hera A.
      • Reyes E.
      • Monserrat J.
      • Munoz L.
      • Nieto M.
      • et al.
      Tumor necrosis factor-alpha expression by activated monocytes and altered T cell homeostasis in ascitic alcoholic cirrhosis: amelioration with norfloxacin.
      ] and was found to be predictive for bacterial infections post-transplantation [
      • Genesca J.
      • Marti R.
      • Rojo F.
      • Campos F.
      • Peribanez V.
      • Gonzalez A.
      • et al.
      Increased tumour necrosis factor alpha production in mesenteric lymph nodes of cirrhotic patients with ascites.
      ]. Therefore, enhanced TNF levels in the GALT appears to play a central role in promoting pathological BT in cirrhosis. Also IL6 and IFN-gamma have been shown to increase intestinal epithelial permeability [
      • Suzuki T.
      • Yoshinaga N.
      • Tanabe S.
      Interleukin-6 (IL-6] regulates claudin-2 expression and tight junction permeability in intestinal epithelium.
      ] and induce transcytotic translocation of commensal E. coli across epithelial cells [
      • Clark E.
      • Hoare C.
      • Tanianis-Hughes J.
      • Carlson G.L.
      • Warhurst G.
      Interferon gamma induces translocation of commensal Escherichia coli across gut epithelial cells via a lipid raft-mediated process.
      ], respectively. Although increased serum levels of IL6 and IFN-gamma are present in advanced cirrhotic patients [
      • Lee F.Y.
      • Lu R.H.
      • Tsai Y.T.
      • Lin H.C.
      • Hou M.C.
      • Li C.P.
      • et al.
      Plasma interleukin-6 levels in patients with cirrhosis. Relationship to endotoxemia, tumor necrosis factor-alpha, and hyperdynamic circulation.
      ,
      • Sandler N.G.
      • Koh C.
      • Roque A.
      • Eccleston J.L.
      • Siegel R.B.
      • Demino M.
      • et al.
      Host response to translocated microbial products predicts outcomes of patients with HBV or HCV infection.
      ,
      • Wiest R.
      • Weigert J.
      • Wanninger J.
      • Neumeier M.
      • Bauer S.
      • Schmidhofer S.
      • et al.
      Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis.
      ] no data on their role in promoting BT in portal hypertension are available. Important to note is that production of TNF and IL6 stimulated by LPS and/or bacterial DNA is excessively augmented in cirrhosis as compared to healthy controls [
      • Munoz L.
      • Jose B.M.
      • Ubeda M.
      • Lario M.
      • Diaz D.
      • Frances R.
      • et al.
      Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.
      ,
      • Deviere J.
      • Content J.
      • Denys C.
      • Vandenbussche P.
      • Schandene L.
      • Wybran J.
      • et al.
      Excessive in vitro bacterial lipopolysaccharide-induced production on monokines in cirrhosis.
      ,
      • Frances R.
      • Munoz C.
      • Zapater P.
      • Uceda F.
      • Gascon I.
      • Pascual S.
      • et al.
      Bacterial DNA activates cell mediated immune response and nitric oxide overproduction in peritoneal macrophages from patients with cirrhosis and ascites.
      ,
      • Frances R.
      • Rodriguez E.
      • Munoz C.
      • Zapater P.
      • De la M.L.
      • Ndongo M.
      • et al.
      Intracellular cytokine expression in peritoneal monocyte/macrophages obtained from patients with cirrhosis and presence of bacterial DNA.
      ,
      • Tazi K.A.
      • Quioc J.J.
      • Saada V.
      • Bezeaud A.
      • Lebrec D.
      • Moreau R.
      Upregulation of TNF-alpha production signaling pathways in monocytes from patients with advanced cirrhosis: possible role of Akt and IRAK-M.
      ] setting the stage for a vicious circle to perpetuate pathological BT (see hypothesis).
      Reactive oxygen species (ROS) impact on epithelial cells increasing the in vitro internalization rate of E. coli [
      • Schoultz I.
      • McKay C.M.
      • Graepel R.
      • Phan V.C.
      • Wang A.
      • Soderholm J.
      • et al.
      Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C.
      ], modulating responses to bacterial stimuli [
      • Ivison S.M.
      • Wang C.
      • Himmel M.E.
      • Sheridan J.
      • Delano J.
      • Mayer M.L.
      • et al.
      Oxidative stress enhances IL-8 and inhibits CCL20 production from intestinal epithelial cells in response to bacterial flagellin.
      ] and changing brush border glycosylation increasing bacterial adherence [
      • Prabhu R.
      • Balasubramanian K.A.
      Altered glycosylation of surfactant and brush border membrane of the small intestine in response to surgical manipulation.
      ]. In experimental cirrhosis, intestinal mucosal oxidative damage has been evidenced by increased lipid peroxidation and altered enterocyte mitochondrial function [
      • Ramachandran A.
      • Prabhu R.
      • Thomas S.
      • Reddy J.B.
      • Pulimood A.
      • Balasubramanian K.A.
      Intestinal mucosal alterations in experimental cirrhosis in the rat: role of oxygen free radicals.
      ,
      • Chiva M.
      • Guarner C.
      • Peralta C.
      • Llovet T.
      • Gomez G.
      • Soriano G.
      • et al.
      Intestinal mucosal oxidative damage and bacterial translocation in cirrhotic rats.
      ]. Besides ROS, nitric oxide (NO) is important in modulating macrophage function, cytokine release and bactericidal killing capacity [
      • Obermeier F.
      • Gross V.
      • Scholmerich J.
      • Falk W.
      Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide.
      ,
      • Wallace J.L.
      • Miller M.J.
      Nitric oxide in mucosal defense: a little goes a long way.
      ], as well as maintaining gut barrier function [
      • Alican I.
      • Kubes P.
      A critical role for nitric oxide in intestinal barrier function and dysfunction.
      ]. Overproduction of NO, known to be particularly present in the splanchnic circulation [
      • Wiest R.
      • Groszmann R.J.
      Nitric oxide and portal hypertension: its role in the regulation of intrahepatic and splanchnic vascular resistance.
      ], has been shown to be deleterious to the integrity of the intestinal epithelium. In fact, NO at high concentrations induces gastric mucosal damage, decreases the viability of rat colonic epithelial cells [
      • Lopez-Belmonte J.
      • Whittle B.J.
      • Moncada S.
      The actions of nitric oxide donors in the prevention or induction of injury to the rat gastric mucosa.
      ,
      • Tepperman B.L.
      • Brown J.F.
      • Whittle B.J.
      Nitric oxide synthase induction and intestinal epithelial cell viability in rats.
      ], directly dilates TJs in intestinal epithelial monolayers, inhibits ATP-formation and hence, increases intestinal permeability [
      • Forsythe R.M.
      • Xu D.Z.
      • Lu Q.
      • Deitch E.A.
      Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion.
      ,
      • Salzman A.L.
      • Menconi M.J.
      • Unno N.
      • Ezzell R.M.
      • Casey D.M.
      • Gonzalez P.K.
      • et al.
      Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers.
      ]. The importance of iNOS-derived NO production in promoting BT has been evidenced experimentally after insults such as endotoxemia, hemorrhagic shock, or thermal injury but also recently in human cirrhosis [
      • Unno N.
      • Wang H.
      • Menconi M.J.
      • Tytgat S.H.
      • Larkin V.
      • Smith M.
      • et al.
      Inhibition of inducible nitric oxide synthase ameliorates endotoxin-induced gut mucosal barrier dysfunction in rats.
      ,
      • Chen L.W.
      • Hsu C.M.
      • Wang J.S.
      • Chen J.S.
      • Chen S.C.
      Specific inhibition of iNOS decreases the intestinal mucosal peroxynitrite level and improves the barrier function after thermal injury.
      ,
      • Du Plessis J.
      • Vanheel H.
      • Janssen C.E.
      • Roos L.
      • Slavik T.
      • et al.
      Activated intestinal macrophages in patients with cirrhosis release NO and IL-6 that may disrupt intestinal barrier function.
      ]. This has been confirmed in iNOS knockout mice exposed to LPS that exhibit a reduced mortality and absent BT [
      • Mishima S.
      • Xu D.
      • Lu Q.
      • Deitch E.A.
      Bacterial translocation is inhibited in inducible nitric oxide synthase knockout mice after endotoxin challenge but not in a model of bacterial overgrowth.
      ].
      The gut is one of the most intensely innervated organs and the autonomic nervous system has recently been realized to influence mucosal barrier function [
      • Keita A.V.
      • Soderholm J.D.
      The intestinal barrier and its regulation by neuroimmune factors.
      ]. In cirrhotic ascitic rats splanchnic specific sympathectomy has been shown to prevent translocation and spreading of E. coli, being associated with increased chemotaxis and phagocytic capacity of mononuclear cells [
      • Worlicek M.
      • Knebel K.
      • Linde H.J.
      • Moleda L.
      • Scholmerich J.
      • Straub R.H.
      • et al.
      Splanchnic sympathectomy prevents translocation and spreading of E. coli but not S aureus in liver cirrhosis.
      ]. Additional proposed beneficial effects of sympathectomy are accelerated intestinal transit time [
      • Perez-Paramo M.
      • Munoz J.
      • Albillos A.
      • Freile I.
      • Portero F.
      • Santos M.
      • et al.
      Effect of propranolol on the factors promoting bacterial translocation in cirrhotic rats with ascites.
      ], prevention of gram-negative bacterial overgrowth [
      • Freestone P.P.
      • Williams P.H.
      • Haigh R.D.
      • Maggs A.F.
      • Neal C.P.
      • Lyte M.
      Growth stimulation of intestinal commensal Escherichia coli by catecholamines: a possible contributory factor in trauma-induced sepsis.
      ] and improvement in gastrointestinal permeability [
      • Reiberger T.
      • Ferlitsch A.
      • Payer B.A.
      • Mandorfer M.
      • Heinisch B.B.
      • Hayden H.
      • et al.
      Non-selective betablocker therapy decreases intestinal permeability and serum levels of LBP and IL-6 in patients with cirrhosis.
      ]. Propranolol has likewise been used and found to lower rate of BT in experimental cirrhosis [
      • Perez-Paramo M.
      • Munoz J.
      • Albillos A.
      • Freile I.
      • Portero F.
      • Santos M.
      • et al.
      Effect of propranolol on the factors promoting bacterial translocation in cirrhotic rats with ascites.
      ] as well as incidence of infectious complications in cirrhotic patients [
      • Senzolo M.
      • Cholongitas E.
      • Burra P.
      • Leandro G.
      • Thalheimer U.
      • Patch D.
      • et al.
      Beta-Blockers protect against spontaneous bacterial peritonitis in cirrhotic patients: a meta-analysis.
      ]. In contrast, parasympathetic input and effects on BT have not been addressed in portal hypertension. However, vagal nerve stimulation attenuates inflammatory response to endotoxin [
      • Borovikova L.V.
      • Ivanova S.
      • Zhang M.
      • Yang H.
      • Botchkina G.I.
      • Watkins L.R.
      • et al.
      Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin.
      ] and intestinal inflammation [
      • de Jonge W.J.
      • van der Zanden E.P.
      • The F.O.
      • Bijlsma M.F.
      • van Westerloo D.J.
      • Bennink R.J.
      • et al.
      Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway.
      ], protecting against burn-induced intestinal injury [
      • Costantini T.W.
      • Bansal V.
      • Krzyzaniak M.
      • Putnam J.G.
      • Peterson C.Y.
      • Loomis W.H.
      • et al.
      Vagal nerve stimulation protects against burn-induced intestinal injury through activation of enteric glia cells.
      ]. Finally, neural stimulation of mast cells modulates intestinal barrier function and mast cell stabilization with ketotifen has been reported to reduce splanchnic inflammatory response in portal hypertensive rats [
      • Sanchez-Patan F.
      • Anchuelo R.
      • Vara E.
      • Garcia C.
      • Saavedra Y.
      • Vergara P.
      • et al.
      Prophylaxis with ketotifen in rats with portal hypertension: involvement of mast cell and eicosanoids.
      ].
      Diet and nutrition are key for host-microbiome interactions. Starvation has deleterious effects on gut mucosal integrity, epithelial cell proliferation and synthesis of mucins and antimicrobial peptides [
      • Hodin C.M.
      • Lenaerts K.
      • Grootjans J.
      • de Haan J.J.
      • Hadfoune M.
      • Verheyen F.K.
      • et al.
      Starvation compromises Paneth cells.
      ]. Moreover, mucosal epithelial cells under metabolic stress perceive commensal bacteria as threat responding with increased endocytotic activity and resulting in increased inflammatory response [
      • Nazli A.
      • Yang P.C.
      • Jury J.
      • Howe K.
      • Watson J.L.
      • Soderholm J.D.
      • et al.
      Epithelia under metabolic stress perceive commensal bacteria as a threat.
      ]. Liver cirrhosis in advanced stages is frequently associated with malnutrition [
      • Cheung K.
      • Lee S.S.
      • Raman M.
      Prevalence and mechanisms of malnutrition in patients with advanced liver disease, and nutrition management strategies.
      ], which has been reported to contribute to enhanced BT [
      • Casafont F.
      • Sanchez E.
      • Martin L.
      • Aguero J.
      • Romero F.P.
      Influence of malnutrition on the prevalence of bacterial translocation and spontaneous bacterial peritonitis in experimental cirrhosis in rats.
      ] and increased permeability [
      • Norman K.
      • Pirlich M.
      • Schulzke J.D.
      • Smoliner C.
      • Lochs H.
      • Valentini L.
      • et al.
      Increased intestinal permeability in malnourished patients with liver cirrhosis.
      ].
      Susceptibility genes for pathological BT have recently been reported mainly influencing innate host defense mechanisms. NOD2 is highly expressed in monocytes and Paneth cells [
      • Lala S.
      • Ogura Y.
      • Osborne C.
      • Hor S.Y.
      • Bromfield A.
      • Davies S.
      • et al.
      Crohn’s disease and the NOD2 gene: a role for paneth cells.
      ] and recognizes muramyl dipeptide (MDP), a component of the peptidoglycan present in the bacterial wall of gram-positive and -negative bacteria. After ligand recognition, NOD2 switches on the NFkB- and MAPK cascade culminating in the induction of pro-inflammatory cytokines and chemokines. Mutant NOD2 has been implicated in the pathogenesis of mucosal inflammation in Crohn’s disease [
      • Hugot J.P.
      • Chamaillard M.
      • Zouali H.
      • Lesage S.
      • Cezard J.P.
      • Belaiche J.
      Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn‘s disease.
      ,
      • Ogura Y.
      • Bonen D.K.
      • Inohara N.
      • Nicolae D.L.
      • Chen F.F.
      A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease.
      ] and in gastrointestinal graft-versus-host disease [
      • Holler E.
      • Rogler G.
      • Herfarth H.
      • Brenmoehl J.
      • Wild P.J.
      • Hahn J.
      • et al.
      Both donor and recipient NOD2/CARD15 mutations associate with transplant-related mortality and GvHD following allogeneic stem cell transplantation.
      ], conditions also known to associate with increased BT. Three common single nucleotide polymorphisms in NOD2 (the frame shift mutation 1007fs (3020insC, SNP 13) the two missense mutations R702W (2104C >T, SNP 8) and G908R [2722G >C, SNP 12] have been most thoroughly investigated. The presence of any of those mutated NOD2 alleles has been reported to be an independent risk factor for SBP [
      • Bruns T.
      • Peter J.
      • Hagel S.
      • Pfeifer R.
      • Prinz P.
      • Stallmach A.
      Homozygous carrier of the NOD2 1007fs frame-shift mutation presenting with refractory community-acquired spontaneous bacterial peritonitis and developing fatal pulmonary mucormycosis: a case report.
      ,
      • Bruns T.
      • Peter J.
      • Reuken P.A.
      • Grabe D.H.
      • Schuldes S.R.
      • Brenmoehl J.
      • et al.
      NOD2 gene variants are a risk factor for culture-positive spontaneous bacterial peritonitis and monomicrobial bacterascites in cirrhosis.
      ,
      • Appenrodt B.
      • Grunhage F.
      • Gentemann M.G.
      • Thyssen L.
      • Sauerbruch T.
      • Lammert F.
      Nucleotide-binding oligomerization domain containing 2 (NOD2) variants are genetic risk factors for death and spontaneous bacterial peritonitis in liver cirrhosis.
      ]. Mechanisms for increased BT by deficient NOD2 function include (i) promotion of bacterial overgrowth via impaired Paneth cell function and diminished production of subgroups of AMPs [
      • Kobayashi K.S.
      • Chamaillard M.
      • Ogura Y.
      • Henegariu O.
      • Inohara N.
      • Nunez G.
      • et al.
      Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract.
      ,
      • Bevins C.L.
      • Stange E.F.
      • Wehkamp J.
      Decreased Paneth cell defensin expression in ileal Crohn’s disease is independent of inflammation, but linked to the NOD2 1007fs genotype.
      ]. Indeed, MDP induces bacterial killing in vitro in ileal crypts and intestinal crypts lacking NOD2 are unable to kill bacteria efficiently [
      • Petnicki-Ocwieja T.
      • Hrncir T.
      • Liu Y.J.
      • Biswas A.
      • Hudcovic T.
      • Tlaskalova-Hogenova H.
      • et al.
      Nod2 is required for the regulation of commensal microbiota in the intestine.
      ]. (ii) Impaired intracellular bacterial killing after engulfment into mononuclear cells of the GALT via, e.g., failure to recruit autophagy protein ATG16L1 and thus impaired wrapping of invading bacteria by autophagosomes [
      • Travassos L.H.
      • Carneiro L.A.
      • Ramjeet M.
      • Hussey S.
      • Kim Y.G.
      • Magalhaes J.G.
      • et al.
      Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry.
      ].
      TLR2 is expressed on the cell surface of macrophages and other immune competent cells and recognizes PAMPs of gram-positive organisms. Among the multiple polymorphisms existing in the TLR2 gene, increased risk for SBP has been reported for cirrhotic patients with the TLR2–16934 TT genotype and carriers with both TLR2 GT tandem repeat alleles present in frequencies greater than 20 in both alleles [
      • Nischalke H.D.
      • Berger C.
      • Aldenhoff K.
      • Thyssen L.
      • Gentemann M.
      • Grunhage F.
      • et al.
      Toll-like receptor (TLR) 2 promotor and intron 2 polymorphisms are associated with increased risk for spontaneous bacterial peritonitis in liver cirrhosis.
      ]. Although not reported for every TLR2 variant the TLR2–16934 TT genotype has been found to associate with increased TLR2 function [
      • Bielinski S.J.
      • Hall J.L.
      • Pankow J.S.
      • Boerwinkle E.
      • Matijevic-Aleksic N.
      • He M.
      • et al.
      Genetic variants in TLR2 and TLR4 are associated with markers of monocyte activation: the Atherosclerosis Risk in Communities MRI Study.
      ]. This underlines the promotive role of TLR2 in mediating pathological BT in BDL mice since, TLR2 deficient mice present with markedly attenuated BT to MLN and endotoxinemia [
      • Hartmann P.
      • Haimerl M.
      • Mazagova M.
      • Brenner D.A.
      • Schnabl B.
      Toll-like receptor 2-mediated intestinal injury and enteric tumor necrosis factor receptor I contribute to liver fibrosis in mice.
      ]. In cirrhotic patients TLR2 and NOD2 variants seem to represent supplementary risk factors since simultaneous presence of both unfavourable polymorphisms markedly increases the risk for SBP in cirrhotic patients [
      • Nischalke H.D.
      • Berger C.
      • Aldenhoff K.
      • Thyssen L.
      • Gentemann M.
      • Grunhage F.
      • et al.
      Toll-like receptor (TLR) 2 promotor and intron 2 polymorphisms are associated with increased risk for spontaneous bacterial peritonitis in liver cirrhosis.
      ]. This underlines the known interaction of NOD2 and TLRs in particular the modulation of TLR2-dependent cytokine responses by NOD2 [
      • Netea M.G.
      • Ferwerda G.
      • de Jong D.J.
      • Jansen T.
      • Jacobs L.
      • Kramer M.
      • et al.
      Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release.
      ]. The latter is particularly relevant for bacterial killing, which appears to be dose dependent [
      • Girardin S.E.
      • Philpott D.J.
      • Lemaitre B.
      Sensing microbes by diverse hosts. Workshop on pattern recognition proteins and receptors.
      ,
      • Borm M.E.
      • van Bodegraven A.A.
      • Mulder C.J.
      • Kraal G.
      • Bouma G.
      The effect of NOD2 activation on TLR2-mediated cytokine responses is dependent on activation dose and NOD2 genotype.
      ]. Most interestingly to note, benefit of probiotica-pulsed DC treatment in experimental colitis depends on intact functional NOD2- and TLR2-signalling [
      • Foligne B.
      • Zoumpopoulou G.
      • Dewulf J.
      • Ben Younes A.
      • Chareyre F.
      • Sirard J.C.
      • et al.
      A key role of dendritic cells in probiotic functionality.
      ]. Therefore, alterations in NOD2- and TLR2-function at various cellular sites appear to play a key role for the susceptibility of pathological BT.

      Stage of disease, route and site of pathological bacterial translocation

      Severity of liver disease

      Rate and degree of pathological BT increases with severity of liver disease (Fig. 4). Pathological translocation of vital bacteria to MLN is a phenomenon of the decompensated stage. In experimental cirrhosis, this occurs only in animals with ascites but not in rats without ascites [
      • Garcia-Tsao G.
      • Lee F.Y.
      • Barden G.E.
      • Cartun R.
      • West A.B.
      Bacterial translocation to mesenteric lymph nodes is increased in cirrhotic rats with ascites.
      ,
      • Wiest R.
      • Das S.
      • Cadelina G.
      • Garcia-Tsao G.
      • Milstien S.
      • Groszmann R.J.
      Bacterial translocation in cirrhotic rats stimulates eNOS-derived NO production and impairs mesenteric vascular contractility.
      ]. These data are in accordance with studies in cirrhotic patients demonstrating significant increases in lipopolysaccharide-binding-protein (long-term marker of gram-negative pathological BT) [
      • Albillos A.
      • DelaHera A.
      • Gonzalez M.
      • Moya J.
      • Calleja J.
      • Monserrat J.
      • et al.
      Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement.
      ] and intestinal permeability [
      • Zuckerman M.J.
      • Menzies I.S.
      • Ho H.
      • Gregory G.G.
      • Casner N.A.
      • Crane R.S.
      • et al.
      Assessment of intestinal permeability and absorption in cirrhotic patients with ascites using combined sugar probes.
      ,
      • Kalaitzakis E.
      • Johansson J.E.
      • Bjarnason I.
      • Bjornsson E.
      Intestinal permeability in cirrhotic patients with and without ascites.
      ] in ascitic cirrhotics but not in patients without ascites as compared to healthy controls. Correspondingly, modulators of pathological BT such as SNS and malnutrition are typically features of the decompensated stadium [
      • Henriksen J.H.
      • Moller S.
      • Ring-Larsen H.
      • Christensen N.J.
      The sympathetic nervous system in liver disease.
      ,
      • Sam J.
      • Nguyen G.C.
      Protein-calorie malnutrition as a prognostic indicator of mortality among patients hospitalized with cirrhosis and portal hypertension.
      ]. In principle however, level of portal hypertension [
      • Reiberger T.
      • Ferlitsch A.
      • Payer B.A.
      • Mandorfer M.
      • Heinisch B.B.
      • Hayden H.
      • et al.
      Non-selective betablocker therapy decreases intestinal permeability and serum levels of LBP and IL-6 in patients with cirrhosis.
      ] and liver insufficiency [
      • Andreu M.
      • Sola R.
      • Sitges-Serra A.
      • Alia C.
      • Gallen M.
      • Vila M.C.
      • et al.
      Risk factors for spontaneous bacterial peritonitis in cirrhotic patients with ascites.
      ,
      • Guarner C.
      • Sola R.
      • Soriano G.
      • Andreu M.
      • Novella M.T.
      • Vila M.C.
      • et al.
      Risk of a first community-acquired spontaneous bacterial peritonitis in cirrhotics with low ascitic fluid protein levels.
      ,
      • Tandon P.
      • Garcia-Tsao G.
      Bacterial infections, sepsis, and multiorgan failure in cirrhosis.
      ] are the driving forces for BT. The latter appears to be the culprit since, chronic pre-hepatic portal hypertension without liver insufficiency does not lead to pathological BT [
      • Garcia-Tsao G.
      • Albillos A.
      • Barden G.E.
      • West A.B.
      Bacterial translocation in acute and chronic portal hypertension.
      ], whereas in galactosamine-induced liver failure, a model that does not develop portal hypertension, BT is observed in all liver failure animals compared to only 0–16% in controls [
      • Kasravi F.B.
      • Wang L.
      • Wang X.D.
      • Molin G.
      • Bengmark S.
      • Jeppsson B.
      Bacterial translocation in acute liver injury induced by D-galactosamine.
      ]. Also SIBO is observed in increasing frequency with worsening of severity of liver diseases [
      • Pande C.
      • Kumar A.
      • Sarin S.K.
      Small-intestinal bacterial overgrowth in cirrhosis is related to the severity of liver disease.
      ], reaching incidences above 80% in advanced cirrhotic patients with ascites [
      • Jun D.W.
      • Kim K.T.
      • Lee O.Y.
      • Chae J.D.
      • Son B.K.
      • Kim S.H.
      • et al.
      Association between small intestinal bacterial overgrowth and peripheral bacterial DNA in cirrhotic patients.
      ]. However, today no data on the exact determinant of liver insufficiency mediating the risk of BT and/or utilization of quantitative liver function tests (indocyanine green clearance or methacetin breath test) for prediction of BT are available. Nonetheless, surrogate markers of pathological BT such as systemic endotoxin levels incrementally increase in relation with severity of liver cirrhosis graded by Child-classification [
      • Chan C.C.
      • Hwang S.J.
      • Lee F.Y.
      • Wang S.S.
      • Chang F.Y.
      • Li C.P.
      • et al.
      Prognostic value of plasma endotoxin levels in patients with cirrhosis.
      ,
      • Lin R.S.
      • Lee F.Y.
      • Lee S.D.
      • Tsai Y.T.
      • Lin H.C.
      • Lu R.H.
      • et al.
      Endotoxemia in patients with chronic liver diseases: relationship to severity of liver diseases, presence of esophageal varices, and hyperdynamic circulation.
      ]. Moreover, direct data on culturable BT to MLN revealed a significantly higher rate in Child C cirrhotic patients (30%) as compared to Child B or A (8% and 3%, respectively) patients and Child-Pugh score was the only independent predictor for pathological BT [
      • Cirera I.
      • Bauer T.M.
      • Navasa M.
      • Vila J.
      • Grande L.
      • Taura P.
      • et al.
      Bacterial translocation of enteric organisms in patients with cirrhosis.
      ]. In contrast, presence of bacterial DNA or LPS in MLN has been evidenced to occur already in pre-ascitic animals [
      • Ubeda M.
      • Munoz L.
      • Borrero M.J.
      • Diaz D.
      • Frances R.
      • Monserrat J.
      • et al.
      Critical role of the liver in the induction of systemic inflammation in rats with preascitic cirrhosis.
      ,
      • Bigatello L.M.
      • Broitman S.A.
      • Fattori L.
      • DiPaoli M.
      • Pontello M.
      • Bevilacqua G.
      • et al.
      Endotoxemia, encephalopathy, and mortality in cirrhotic patients.
      ] and the detection of bacterial DNA in the systemic circulation was not associated with differences in severity of liver insufficiency [
      • Such J.
      • Frances R.
      • Munoz C.
      • Zapater P.
      • Casellas J.A.
      • Cifuentes A.
      • et al.
      Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites.
      ]. This hints to different mechanisms responsible for translocation of bacterial DNA as compared to viable bacteria in liver cirrhosis. Finally, gastrointestinal hemorrhage has been shown to increase BT in healthy animals [
      • Baker J.W.
      • Deitch E.A.
      • Li M.
      • Berg R.D.
      • Specian R.D.
      Hemorrhagic shock induces bacterial translocation from the gut.
      ] and portal hypertensive rats are particularly susceptible for shock-induced BT to MLN and blood [
      • Sorell W.T.
      • Quigley E.M.
      • Jin G.
      • Johnson T.J.
      • Rikkers L.F.
      Bacterial translocation in the portal-hypertensive rat: studies in basal conditions and on exposure to hemorrhagic shock.
      ]. Pre-existing increases in intestinal permeability in portal hypertension most likely is the underlying mechanism since (i) prior exposure to bacterial DNA has been shown to strongly aggravate systemic inflammation and gut barrier loss in experimental hemorrhagic shock [
      • Luyer M.D.
      • Buurman W.A.
      • Hadfoune M.
      • Wolfs T.
      • Van’t Veer C.
      • Jacobs J.A.
      • et al.
      Exposure to bacterial DNA before hemorrhagic shock strongly aggravates systemic inflammation and gut barrier loss via an IFN-gamma-dependent route.
      ] and (ii) higher intestinal permeability in decompensated cirrhotic patients with active GI hemorrhage is an independent predictor for development of bacterial infections [
      • Kim B.I.
      • Kim H.J.
      • Park J.H.
      • Park D.I.
      • Cho Y.K.
      • Sohn C.I.
      • et al.
      Increased intestinal permeability as a predictor of bacterial infections in patients with decompensated liver cirrhosis and hemorrhage.
      ].
      Figure thumbnail gr4
      Fig. 4Stages of liver disease and hypothesis on development of pathological BT. Left: normal healthy conditions with “normal” exclusively low levels of translocation of bacteria(l products); Middle: increases in paracellular translocation of bacterial products stimulate an augmented pro-inflammatory cytokine response and release of ROS and NOx within the GALT; these mediators impact on the mechanical and secretory barrier as well as most likely on the flora; Right: in ascitic cirrhotic conditions in presence of IBO and a proposed state of epithelial tolerance, enhanced transcytosis of viable bacteria develops ultimately leading to immune paralysis in the GALT (which could lead to a vicious circle perpetuating BT by a relative lack of bacterial killing).

      BT-route

      Despite obvious differences in size, chemical structure and receptor-ligand interactions between all various types of bacterial products as well as viable bacteria, the question how this impacts on the route of translocation namely para- vs. transcellular and lymphatic vs. hematogenous is unanswered. This could be of clinical relevance, since the lymphatic route connects the gut with the lung and mesenteric lymph duct ligation has been shown to protect from hemorrhagic shock induced pulmonary injury in rats [
      • Sambol J.T.
      • Xu D.Z.
      • Adams C.A.
      • Magnotti L.J.
      • Deitch E.A.
      Mesenteric lymph duct ligation provides long term protection against hemorrhagic shock-induced lung injury.
      ]. In respect to vital culturable bacteria, experimental models of severe inflammatory insults reveal their appearance in the portal circulation earlier and to an excessively higher degree than in the lymphatic system [
      • Mainous M.R.
      • Tso P.
      • Berg R.D.
      • Deitch E.A.
      Studies of the route, magnitude, and time course of bacterial translocation in a model of systemic inflammation.
      ]. In experimental cirrhosis positive portal culture has likewise been reported in the majority of cases with BT to MLN [
      • Llovet J.M.
      • Bartoli R.
      • Planas R.
      • Cabre E.
      • Jimenez M.
      • Urban A.
      • et al.
      Bacterial translocation in cirrhotic rats. Its role in the development of spontaneous bacterial peritonitis.
      ]. This also points towards the importance of hematogenous spreading of viable bacteria after crossing the epithelial barrier in cirrhosis. Nonetheless, comparative and kinetic studies assessing the lymphatic and portalvenous route in parallel are not available in liver cirrhosis.

      Site of bacterial translocation

      The colon is used to harbour a vast number of bacteria, and normally is more efficient at eliminating translocating bacteria and presents with higher transepithelial resistance than the small bowel [
      • Powell D.W.
      Barrier function of epithelia.
      ]. Experimental studies have shown that after inoculation of equal concentrations of E. coli into small or large bowel, BT occurs at higher a rate after small bowel inoculation [
      • Koh I.H.
      • Guatelli R.
      • Montero E.F.
      • Keller R.
      • Silva M.H.
      • Goldenberg S.
      • et al.
      Where is the site of bacterial translocation–small or large bowel?.
      ]. In addition, proximal gut colonization has been associated with increased BT and septic morbidity in surgical intensive care patients [
      • MacFie J.
      • O’Boyle C.
      • Mitchell C.J.
      • Buckley P.M.
      • Johnstone D.
      • Sudworth P.
      Gut origin of sepsis: a prospective study investigating associations between bacterial translocation, gastric microflora, and septic morbidity.
      ,
      • Marshall J.C.
      • Christou N.V.
      • Horn R.
      • Meakins J.L.
      The microbiology of multiple organ failure. The proximal gastrointestinal tract as an occult reservoir of pathogens.
      ], indicating that small intestinal bacterial overgrowth has the greatest potential for promoting BT. As for liver cirrhosis, this is supported by a study that showed that lower BT rates in cisapride-treated cirrhotic animals were associated to lower jejunal but not cecal bacterial counts [
      • Pardo A.
      • Bartoli R.
      • Lorenzo-Zuniga V.
      • Planas R.
      • Vinado B.
      • Riba J.
      • et al.
      Effect of cisapride on intestinal bacterial overgrowth and bacterial translocation in cirrhosis.
      ]. Histological changes however, have been shown to be most marked in the cecum in experimental cirrhosis [
      • Misra V.
      • Misra S.
      • Dwivedi M.
      • Gupta S.
      Histomorphometric study of portal hypertensive enteropathy.
      ,
      • Garcia-Tsao G.
      • Lee F.Y.
      • Barden G.E.
      • Cartun R.
      • West A.B.
      Bacterial translocation to mesenteric lymph nodes is increased in cirrhotic rats with ascites.
      ,
      • Nagral A.S.
      • Joshi A.S.
      • Bhatia S.J.
      • Abraham P.
      • Mistry F.P.
      • Vora I.M.
      Congestive jejunopathy in portal hypertension.
      ]. Moreover, elegant recent loop-experiments, assessing intestinal permeability by local injection of FITC-marked dextran or GFP-marked E. coli at different sites in BDL mice, revealed that the cecum and colon are the sites with the largest rate of BT and increase in intestinal permeability [
      • Hartmann P.
      • Haimerl M.
      • Mazagova M.
      • Brenner D.A.
      • Schnabl B.
      Toll-like receptor 2-mediated intestinal injury and enteric tumor necrosis factor receptor I contribute to liver fibrosis in mice.
      ,
      • Fouts D.E.
      • Torralba M.
      • Nelson K.E.
      • Brenner D.A.
      • Schnabl B.
      Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease.
      ]. Most interestingly, these changes did precede alterations of the microbiome re-inforcing the primary role of permeability and host response to BT. However, these experiments were performed 1 day after BDL and thus, in non-cirrhotic conditions and it remains to be seen whether this applies also to other models and cirrhotic stages of liver disease.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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