Siddhrth S.Ghosh ,Jing Wng ,Pul J.Ynnie ,Shoh Ghosh ,,*

a Department of Internal Medicine,Virginia Commonwealth University Medical Center,Richmond,VA,USA

b Hunter Homes McGuire Veterans Affairs Medical Center,Richmond,VA,USA

Keywords:

Intestinal barrier

Liver dysfunction

Lipopolysaccharide (LPS)

Inflammation

Gut-liver axis

ABSTRACT

Dietary,environmental or genetic changes contribute to the development of several metabolic and neurological diseases.Extensive research efforts have been directed towards examining how these factors modulate gut bacterial composition.However,the mechanisms by which changes in gut bacterial diversity affect host function are not completely defined.Intestinal barrier dysfunction is being increasingly recognized as an early or initiating step in communicating bacterial diversity-dependent changes to the host.Here,we review the functional composition of the intestinal barrier and describe the consequences of the breach of this barrier.The functional layers of the intestinal barrier provide an initial defense and prevent the infiltration of bacteria or bacterial products from the gut lumen to the underlying lamina propria.Mesenteric lymph nodes subsequently act as the first firewall where dendritic cells from the lamina propria transport the infiltrated bacteria or bacterial products during the early stages of barrier dysfunction.When overwhelmed,the liver functions as the second firewall to detoxify bacterial endotoxins,such as lipopolysaccharide (LPS),and limit systemic involvement.Continuous translocation of LPS from the leaky gut to the liver results in liver damage or dysfunction that leads to the development of type 2 diabetes,atherosclerosis,heart disease,heart failure and also neurological diseases,such as Alzheimer’s and Parkinson’s disease.Thus,targeted restoration of intestinal barrier function represents a novel strategy for the attenuation of multiple diseases.

1.Introduction

Metabolic diseases (e.g.,type 2 diabetes (T2D),fatty liver disease,heart disease and stroke)can be viewed as a group of chronic diseases that share similar features of metabolic disturbances.Dietary overnutrition,a lack of balance between essential dietary components,the downstream effects of this on gut bacterial homeostasis and the integrity of intestinal barrier function underlie the initiation of these metabolic disturbances.1,2Dietary components shape the diversity of gut microbiota and directly or indirectly modulate intestinal barrier function via changes in microbial metabolism.Western-type diets containing high fat and cholesterol are shown to not only deleteriously affect the gut microbiota(dysbiosis),but also lead to gut barrier dysfunction resulting in translocation of the bacterial endotoxin lipopolysaccharide (LPS)into the systemic circulation,a process termed metabolic endotoxemia.3,4This low-grade chronic inflammation was first shown to alter glucose metabolism,5,6but is now known to contribute to the development of almost all metabolic diseases.7In addition,the presence of LPS in the systemic circulation is also identified as a causal or complicating factor in several diseases,such as autism,8Alzheimer’s disease,9Parkinson’s disease,10arthritis,11obesityinduced osteoarthritis,12asthma,13several autoimmune diseases and multiple sclerosis.14,15A chronic inflammatory state,as well as endotoxin tolerance leading to a compensatory hypo-inflammatory state,is thought to underlie immune-escape of cancer cells directly linking circulating LPS to the likely development of multiple cancers.16,17High LPS levels in the portal vein are also linked to the development and progression of hepatocellular carcinoma.18These diverse reports establish the causal relationship between gutderived LPS and the development of diseases and highlight the importance of understanding the consequences of LPS translocation from the gut in relation to disease development.It is noteworthy that recognition of LPS as a likely causative factor has appropriately shifted the emphasis from extensive examination of gut dysbiosis per se to intestinal barrier dysfunction.

The first step in the translocation of bacteria or bacterial products,such as LPS,from the gut lumen is their movement to the subepithelial lamina propria (discussed below in detail) where direct contact with immune cells results in the secretion of proinflammatory mediators that perpetuate local inflammation.Subsequently,LPS is transported to the liver via the portal circulation before reaching the systemic circulation with the potential to affect distant organs.This route of transport underscores the importance of the gut-liver axis in modulating the effects of metabolic endotoxemia.

2.Gut-liver axis

The bidirectional relationship between the gut (and its microbiota) and liver that results in the coordination and integration of signals produced by diet,environmental factors and genetics is referred to as the gut-liver axis.The portal circulation anatomically connects the gut and liver,forming the functional gut-liver axis whereby in addition to absorbed nutrients,any type of substance that crosses the gut barrier (intact or disrupted) reaches the liver with the potential to affect hepatic metabolic pathways or resident immune cells.Reciprocally,the liver secretes bile acids that not only facilitate digestion and the absorption of nutrients but also affect the composition of microbiota and modulate signaling within enterocytes,mainly via the farnesoid X receptor(FXR).Additionally,bile acids are further metabolized by gut bacteria,reabsorbed and return to the liver via the enterohepatic circulation.In turn,hepatic bile acid synthesis is regulated by an active feedback loop of bile acids returning to the liver.Bile acid-dependent FXR activation in the intestine also stimulates the production of fibroblast growth factor 15(FGF15)(mice)or FGF19(humans),which after binding to FGF receptor 4 in liver cells,represses bile acid synthesis.19Thus,the gut-liver axis connects the liver and intestine via bile acid metabolism.

The homeostasis of gut microbiota and the intestinal barrier,which limit the transfer of bacteria or bacterial products,represent two important components that modulate the gut-liver axis.In the last decade,multiple studies have implicated gut microbiota in the pathogenesis of various diseases,and these causative relationships have been extensively reviewed.20-23However,pathological transport of bacteria or bacterial products(e.g.,LPS)is regulated by the integrity of the intestinal barrier,and its central causative role in the development of gut bacteria-associated diseases is being increasingly recognized.Therefore,this review focuses on the components of the intestinal barrier,how these can be affected,the consequences and the sequence of events associated with disruption of the gut barrier that ultimately leads to the development of metabolic diseases,including liver disease.

3.Gut barrier

The intestinal barrier can be perceived as being composed of four layers,including luminal intestinal alkaline phosphatase(IAP),the mucus layer,the epithelial cell layer and antibacterial proteins produced by Paneth cells,which together,prevent the entry of bacteria or bacterial products into the systemic circulation.24This barrier can also be defined in terms of compositional functionality as follows:extracellular or luminal,cellular,immunological and gut vascular barrier (Fig.1).

Fig.1.Functional components of the intestinal barrier.

3.1.Extracellular or luminal barrier

The components present between intestinal epithelial cells and the lumen technically comprise the extracellular or luminal barrier.IAP secreted into the lumen or anchored onto the epithelial cell membrane,outer and inner mucus layer and luminal gut bacteria that modulate this mucus layer and regulate the local production of antimicrobial peptides and immunoglobulins,are the major components of this extracellular barrier.IAP catalyzes the dephosphorylation of bacterial endotoxin LPS and pathogen-associated molecular patterns(flagellin,CpG DNA),resulting in detoxification and reduced local intestinal inflammation.25In addition,it also dephosphorylates adenosine triphosphate (ATP),thereby facilitating the growth of commensal bacteria and maintaining bacterial homeostasis.26Consistently,oral IAP supplementation inhibits the absorption of LPS,and enteral IAP is thought to represent a novel approach to maintain the integrity of intestinal barrier function in critically ill patients with decreased IAP in ileal fluid samples.27,28

While the inner mucus layer is firmly attached to the underlying epithelial cell layer and does not allow bacteria to penetrate,the outer mucus layer is loose and harbors commensal bacteria that prevent colonization by pathogenic bacteria.An intact and functional mucus layer is crucial for the maintenance of the gut barrier,and alterations in this layer lead to inflammation and are associated with multiple diseases,including inflammatory bowel disease and ulcerative colitis.29-31It is noteworthy that diets low in fiber enhance degradation of the mucus layer resulting in increased inflammation,and dietary fiber supplementation improves the integrity of the mucus layer that results in alleviation of colitis,32,33non-alcoholic steatohepatitis (NASH),34glucose intolerance and atherosclerosis.35

In addition to IAP and the mucus layer,gastric and biliary fluids also cause modifications in the composition of gut bacteria,which affects intestinal homeostasis,often with pathological consequences.36-38

3.2.Intestinal epithelial barrier

A monocellular layer of epithelial cells represents the cellular barrier where the plasma membrane of the epithelial cells restricts transcellular transport.In addition,the microvilli of the apical brush border of epithelial cells are negatively charged because of the presence of polar carbohydrates and charged transmembrane proteins,which causes an electrostatic repulsive force towards negatively charged bacterial cell walls.39Within this monolayer,specialized cells,such as goblet and Paneth cells,provide additional components of the intestinal barrier by producing mucus and antibacterial proteins,respectively.Paracellular transport or transport between epithelial cells is also restricted by intricate spatial arrangements of tight junction proteins,including claudins,zonula occludens(ZO-1 and ZO-2)and occludens.24While an intact and functional intestinal barrier limits the translocation of bacteria or bacterial products and prevents intestinal and systemic inflammation,proinflammatory cytokines,such as tumor necrosis factor alpha (TNFα) or interferon gamma (IFNγ),modulate the complex system of tight junction proteins.40,41Strong evidence exists for the presence of qualitative or quantitative changes in tight junction proteins and loss of intestinal epithelial barrier function in the pathogenesis of multiple diseases,including liver disease.42,43

3.3.Immunological barrier

Production and secretion of proteins into the intestinal lumen that directly interact with bacteria represent the immunological barrier.Specialized epithelial cells or Paneth cells produce antimicrobial peptides,such as defensins,cathelicidins,resistin-like molecules,bactericidal-permeability-inducing proteins and lectins.After secretion,these proteins are retained in the mucus layer where they interact with and destroy any bacteria that have infiltrated the mucus layer and attenuate direct interactions between bacteria and the epithelial cell layer.44Immune cells,such as dendritic or plasma cells,constantly patrol the lamina propria,the layer underlying the intestinal epithelial cell monolayer.Upon the detection of bacteria via pathogen-associated molecular patterns,dendritic cells are responsible for presenting the captured antigens to infiltrating plasma cells that leads to the production of secretory immunoglobulin A (sIgA).sIgA is recognized by the polymeric Ig receptor (pIgR) expressed on the basal membrane of enterocytes.Binding of sIgA to pIgR leads to transcytosis of sIgA to the lumen,where it interacts with the mucus layer via the Fc fragment of the IgA antibody.45sIgA binds to microorganisms and toxins,preventing colonization and toxicity/damage to epithelial cells,a process called immune exclusion.46It also promotes antiinflammatory responses at the mucosal surfaces;sIgA facilitates the uptake of pathogens into IgA-producing Peyer’s patches,47and recognition of sIgA by dendritic cells inhibits the secretion of IL-12 inducing T helper 2 (Th2) and regulatory T (Treg) responses.48Additionally,sIgA can bind to intracellular pathogens during its transcytosis to the lumen.Within the lamina propria,sIgA can also bind to immune complexes attenuating inflammatory processes and systemic responses.Further,dendritic cells also transport the infiltrated bacteria or bacterial antigens to mesenteric lymph nodes(MLNs) allowing priming and maturation of B and T lymphocytes that constitute the adaptive immune response in the gut-associated lymphoid tissue.49

3.4.Gut vascular barrier

Functionally,the gut barrier extends beyond the anatomically identifiable intestinal epithelial cell layer and underlying lamina propria that contains the immune cells.The gut-vascular barrier consists of the vascular endothelium and the enteric glial cells that surround it,which are the major components of the enteric nervous system.The terminal end-feet processes of these glial cells found below the epithelial cells often extend to the epithelial basement membrane and blood vessels.Major subpopulations of enteric glial cells are found in the enteric ganglia in the submucosal and myenteric plexuses of the enteric nervous system (see Fig.1).A significant disruption of enteric glial cells is observed in Crohn’s disease,and a diminished network is associated with modulated response to inflammatory mediators.50Conditional genetic ablation of enteric glial cells induces disruption of intestinal integrity and vascular changes leading to fatal hemorrhagic jejunoileitis.51,52Autoimmune targeting of enteric glial cells regulates the development of fulminant enteritis with increased bacterial translocation and bacteremia.53Conversely,the activation of enteric glia by vagal stimulation prevented burn-induced intestinal barrier dysfunction and attenuated histological gut injury.54Loss of the integrity of the vascular endothelial layer leads to impaired permeability.It is noteworthy that large macromolecules that enter the circulation are found only in the liver and not in the spleen,demonstrating transport via the portal circulation and not through lymphatic vessels.55Furthermore,bacteria that cross the disrupted intestinal epithelial barrier do not automatically translocate to the liver and spleen,demonstrating the importance of the gut vascular barrier.56

3.5.Factors affecting the intestinal barrier

The effects of dietary or environmental factors on the intestinal barrier can be either direct (by affecting the different layers of the barrier and intracellular signaling mechanisms) or indirect (by affecting intestinal microbiota).However,for any particular nutrient,the detailed mechanisms are not well understood.Flavonoids,which are abundant in most vegetables,fruits,green and black tea,red wine,chocolate and coffee,maintain the integrity of the intestinal barrier by strengthening tight junctions.57However,Western-type diets that are rich in fat and sugars not only cause dysbiosis but also reduce the thickness of the mucus layer and the number of goblet cells,thereby increasing intestinal permeability by multiple mechanisms.58Similarly,disruption of the intestinal barrier by a deficiency of vitamins and trace minerals,as well as alcohol consumption,involve a combination of multiple events,including tight junction,mucus layer and microbial alterations.59-62Environmental factors,such as medication or stress,also induce intestinal barrier dysfunction via multiple mechanisms.Non-steroidal anti-inflammatory drugs are well known for their damaging effects on the gut.Specifically,they affect cytoskeleton and tight junction homeostasis and modulate the intestinal bacterial composition.63,64Stress is a lifestyle factor that has been linked to deterioration of the intestinal barrier via gut-brain interactions where mucosal disturbances are caused by the activation of a corticotropic-releasing factor-mast cell axis.65However,exercise-induced stress is thought to induce barrier dysfunction that involves the gut microbiota-brain axis.66It is therefore apparent that while the effects of external factors on barrier dysfunction are widely known,the details of the contributing mechanisms are not fully understood.

4.Sequential consequences of gut barrier dysfunction

Translocation of bacteria is a physiological process in which small amounts of microorganisms and their constituents pass from the intestinal lumen to the lamina propria.Dendritic cells,which are relatively ineffective in killing bacteria compared with macrophages,67transport the ingested bacteria to the MLNs.Notably,orally administered commensal bacteria could be recovered from MLNs but not from splenocytes,while intraperitoneal delivery led to clearance by the spleen.68Thus,MLNs confine the commensal bacteria(within the dendritic cells)to the mucosal immune system and avoid systemic priming by these organisms.53This process is also crucial for the modulation of the immune system and the development of immune tolerance.69,70

Disruption of the extracellular/luminal and epithelial cell barrier results in increased bacterial translocation,including pathogenic bacteria into the lamina propria(Fig.2).An efficient immunological barrier consisting of dendritic cells limits this process by translocating the bacteria or bacterial products to MLNs and orchestrating a local immune response.29,30Thus,MLNs are viewed as the primary or first firewall,and the combined functions of the immunological barrier and MLNs limit systemic involvement.However,when this primary firewall is overwhelmed or fails,bacteria or bacterial products reach the liver via the portal circulation,causing portal bacteremia and endotoxemia.Despite the fact that the liver is usually devoid of bacteria,the liver is exposed to trace amounts of bacterial products,such as endotoxins,even in healthy individuals.71The liver acts as the second firewall when the intestinal barrier becomes dysfunctional and MLNs are unable to contain the released bacteria or bacterial products.72-74Variable amounts of bacteria or bacterial products are then delivered to the liver through the mesenteric and portal circulation.75Resident macrophages of the liver or Kupffer cells effectively phagocytose and kill the bacteria limiting transfer to the systemic circulation.In addition,LPS binding protein (LBP),an acute phase protein synthesized by the liver,binds bacterial endotoxin LPS and facilitates binding to Toll-like receptor 4(TLR4)resulting in enhanced uptake by Kupffer cells.76-78Consistently,clearance of Escherichia coli K-12 is delayed in Kupffer cell-depleted mice.79However,the ability of the liver to neutralize the constant flux of bacteria or bacterial products is also limited,and if disruption of the intestinal barrier persists,liver damage and subsequent serious systemic perturbations can occur.

5.Gut barrier/liver dysfunction and disease development

A sustained disruption of the intestinal barrier,overwhelming of the MLN firewall and Kupffer cell-dependent inflammatory responses to bacteria or bacterial products that reach the liver result in local hepatic inflammation.This leads to further recruitment of systemic inflammatory cells,such as neutrophils,T-cells and monocytes,perpetuating liver inflammation.80Unresolved hepatic inflammation underlies the resulting hepatocyte cell death by apoptosis and necrosis.81Another consequence of sustained hepatic inflammation and hepatocyte cell death is the activation and proliferation of hepatic stellate cells that leads to the development of hepatic fibrosis.82,83

Recent studies also indicate that in addition to the direct effects of alcohol on hepatic parenchymal cells,alcohol-mediated dysbiosis,intestinal barrier dysfunction and activation of hepatic immune cells,play a significant role in the development of alcoholic liver disease.84-86Consistently,targeted improvement in gut barrier function is being explored as a novel therapeutic strategy for modulation of alcoholic liver disease,87and restoration of gut barrier function underlies the beneficial effects of several dietary or herbal medicine components.88-92Similarly,intestinal barrier dysfunction and hepatic inflammation resulting from subsequent translocation of LPS and other pathogen-associated molecular patterns (PAMPs) to the liver are being increasingly recognized as causative factors in the development of non-alcoholic fatty liver disease(NAFLD)and NASH.93-95Consistently,Nier et al.96reported altered levels of intestinal permeability markers in the early stages of NAFLD in children,and Volynets et al.97found increased intestinal permeability in subjects with NAFLD ranging from simple steatosis to steatohepatitis.

Fig.2.Stages in the disruption of the intestinal barrier function and associated consequences.

Liver dysfunction resulting from dysbiosis and subsequent increased intestinal permeability also underlies the development of several other metabolic and neurological diseases.Impaired clearance of LPS by the liver overwhelmed with continuous translocation from the leaky gut leads to an increase in the hepatic secretion of proinflammatory cytokines.This persistent immune activation alters the expression of hepcidin,leading to iron deficiency and a redox imbalance in the liver.98Collectively,these hepatic changes alter various aspects of hepatic metabolism leading to the development of multiple diseases,such as T2D,99-102atherosclerosis,heart disease and heart failure.103-107Lu et al.108identified liver dysfunction as a major contributor to neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).Diminished clearance of amyloid-beta peptide (Aβ) by dysfunctional livers has recently been identified as a novel mechanism underlying the progression of Alzheimer’s disease.109

Consistent with the role of intestinal barrier and liver dysfunction in disease development,targeted improvement by supplementation with Akkermansia muciniphila in overweight or obese subjects led to improved liver function and inflammation,improved insulin sensitivity and a reduction in plasma cholesterol,without an overall change in gut microbiota,providing the proofof-concept and establishing the therapeutic potential of improving intestinal barrier and liver dysfunction.110Earlier studies from our laboratory have also demonstrated attenuation of glucose intolerance and atherosclerosis by oral supplementation with curcumin that improves intestinal barrier function,111,112providing a novel mechanism of action of largely non-absorbable curcumin with numerous health beneficial effects.24Collectively,these studies highlight the importance of targeted modulation of intestinal barrier function as a novel therapeutic strategy and suggest changes in intestinal barrier function as the major mechanism underlying the beneficial effects of several phytochemicals.

6.Conclusions

Although gut dysbiosis is extensively researched and thought to contribute to the development of multiple diseases,the critical role of intestinal barrier dysfunction is gaining considerable importance as the central mechanism that effectively translates luminal bacterial changes to modulate host metabolism.Intestinal barrier dysfunction occurs in response to dietary,environmental or genetic factors that can affect the various components of the intestinal barrier.The major consequence of the disruption of a homeostatic intestinal barrier is the translocation of bacteria or bacterial products (e.g.,LPS),first to the lamina propria,then to MLNs and ultimately to the liver where continuous exposure to LPS results in liver dysfunction.The development of several metabolic and neurological diseases is directly related to liver dysfunction,and targeted improvement of intestinal barrier function leading to improved liver function results in the attenuation of these diseases.As reviewed here,extending our current knowledge of this gutliver axis in disease development and the future development of targeted strategies to improve function of the multiple layers of the intestinal barrier will provide novel avenues for the attenuation of these diseases.

Authors’contributions

J.Wang and P.J.Yannie performed all the experiments in the laboratory reported in the publications,including this review.S.S.Ghosh collected the information.S.S.Ghosh and S.Ghosh organized and wrote the review.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was partially supported by Basic Innovative Science Award(1-16-IBS-105)from the American Diabetes Association to S.Ghosh.