In the current issue of HEPATOLOGY, Ewaschuk et al. from the University of Alberta describe the abilities of probiotic bacteria to maintain colonic barrier function and suppress lipopolysaccharide (LPS)-induced hepatic damage in a mouse sepsis model. Cytokine-induced permeability of the gastrointestinal barrier may result from systemic inflammation due to LPS challenge, and the hepatic parenchyma represents a target of Kupffer cell–mediated immunopathology and proinflammatory mediators from the lumen of the gastrointestinal tract (Fig. 1A). Recent evidence indicates that specific probiotic organisms can regulate intestinal barrier function and promote mucosal recovery during pathological conditions such as sepsis (Fig. 1B). Probiotics may affect hepatic and intestinal physiology by different mechanisms, such as the facilitation of the bioavailability of bile acids2 and the provision of substrates for short-chain fatty acid (for example, butyrate) production.3
Sepsis and systemic inflammatory response syndrome have been associated with enhanced intestinal permeability as a potential contributing factor to worse patient outcomes.4 In the study by Ewaschuk et al., Escherichia coli–derived LPS and D-galactosamine were coadministered into the peritoneal activity of mice. A systemic immune response was generated by excessive production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). Presumably, Kupffer cells and other immune cells in the hepatic parenchyma directly contributed to a cytokine surge that was accompanied by up-regulation of interleukin-10 (IL-10). Interestingly, the p35 subunit of IL-12 was not induced in this model. As described in this report, proinflammatory cytokines such as TNF-α and IFN-γ depressed barrier function and elevated intestinal permeability in different models.5, 6 Anti-TNF therapy restored gut barrier function in patients with Crohn's disease and provided evidence that anti-inflammatory strategies may ameliorate intestinal immunopathology by multiple mechanisms.7
The probiotic-mediated restitution of colonic barrier function and the amelioration of hepatic disease may be a primary result of direct effects on intestinal permeability or anti-inflammatory effects. A previous publication documented the protective role of probiotics by demonstrating the ability of an eight-organism combination to promote intestinal barrier function and resistance to enteric pathogen invasion in an IL-10–deficient mouse colitis model.8 Different probiotic agents protected the intestinal epithelium and restored mucosal integrity following acute and chronic intestinal injury.9 Probiotics mediated protective effects on intestinal barrier function by actively secreting soluble mediators8, 10 or by enhancing the production of carbohydrate fermentation byproducts.11 Prebiotics, either alone or in a synbiotic (a combination of prebiotics and probiotics), provide a substrate for the release of barrier-promoting factors in the intestinal lumen. Probiotics may promote barrier function by facilitating tight junction formation and the maintenance of epithelial integrity. A two-organism probiotic combination maintained and enhanced the phosphorylation of tight junction proteins including tight junction protein 1 (TJP1 or ZO-1) and occludin.12 In addition to promoting tight junction formation and maintaining transepithelial resistance, probiotics induced mucin gene expression and the facilitation of mucus layer integrity.13
Probiotics may promote barrier function by delivering anti-inflammatory mediators, resulting in antagonism of proinflammatory cytokine-mediated enhancement of intestinal permeability. Probiotics actively secrete small, anti-inflammatory mediators that may ameliorate acute and chronic inflammation in different animal models.10, 14 Probiotics, including VSL#3, down-regulated specific proinflammatory cytokines, including IFN-γ and TNF-α, via the suppression of key signaling pathways such as nuclear factor kappa B (NF-κB),15 and different probiotics differentially modulated specific cytokines. In the study by Ewaschuk et al., probiotics suppressed the production of colonic TNF-α and IFN-γ, whereas the probiotic-mediated suppression of hepatic cytokines was specific for IL-6. Hepatic TNF-α quantities were not affected by a pretreatment with the eight-organism probiotic combination, VSL#3. A primary effect of the suppression of colonic TNF-α and IFN-γ production may be enhanced restoration of intestinal barrier function and diminished levels of proinflammatory mediators from the intestinal lumen. Conversely, the enhanced production of soluble anti-inflammatory factors by probiotics or soluble factors derived by carbohydrate fermentation may easily permeate an intact intestinal barrier and ultimately account for hepatic recovery. An interesting study showed that lysozyme-modified components of probiotics, when orally administered, protected rats from cecal ligation–induced sepsis. Hepatic macrophages internalized the modified probiotic components and protected animals from severe disease in a cathelicidin-dependent manner.16
The roles of systemic IL-6 and peroxisome proliferator-activated receptor gamma (PPAR-γ) represent potentially key contributions of the study by Ewaschuk et al. The cytokine IL-6 is regulated partly by NF-κB activation and has multiple effects. Reports have indicated that IL-6 may be a critical proinflammatory mediator in sepsis, and multiple studies have documented serum IL-6 as a potential biomarker of sepsis in human patients.17 Interestingly, IL-6 may enhance intestinal barrier function by up-regulating keratin gene expression in intestinal epithelial cells18 and protecting enterocytes from stress-induced apoptosis.19 The authors of the current study did not report effects on colonic IL-6 quantities, and perhaps the maintenance or increased production of IL-6 in the intestinal mucosa may serve as a protective feature of probiotics and commensal bacteria. PPAR-γ may serve as a key mediator of probiotic-mediated anti-inflammatory effects, and relationships have been uncovered between PPAR-γ and IL-6. A PPARG allele (encoding variant PPAR-γ) was recently associated with reduced serum IL-6 levels in human individuals evaluated for obesity-related genetic polymorphisms.20 Probiotics up-regulated PPAR-γ as a strategy to regulate NF-κB signaling and inflammatory responses.21 Stress-induced alterations of colonic permeability were abrogated by the induction of PPAR-γ production via agonists such as rosiglitazone.22 In the current study, the induction of PPAR-γ by orally administered probiotics may account for systemic anti-inflammatory effects and associated enhancement of intestinal barrier function. A related issue is the role of the regulator, PPAR-γ, and its role in the regulation of NF-κB signaling and proinflammatory cytokine production. If PPAR-γ is a negative regulator of NF-κB signaling, why does IL-6 seem to be differentially regulated in comparison with other NF-κB–regulated cytokines such as TNF-α and IL-12? The results suggest that PPAR-γ is a more potent regulator of IL-6 production and that IL-6 may be a key cytokine with respect to hepatic inflammation.
Clinical studies have highlighted the potential contributions of probiotics and probiotic-derived molecules in the amelioration of liver disease. One previous report in this journal documented that synbiotics significantly reduced blood ammonia levels and reversed minimal hepatic encephalopathy in patients with cirrhosis.23 Detrimental effects on hepatobiliary physiology such as biliary obstruction may be associated with enhanced intestinal permeability, and probiotics including the VSL#3 component, Lactobacillus plantarum, as a single agent may restore barrier function.24 A recent clinical study25 reported that critically ill patients in the intensive care unit setting demonstrated improved intestinal barrier function following the oral administration of probiotics. However, a recent review of eight randomized studies including 999 adult intensive care unit patients concluded that probiotics and synbiotics lacked beneficial effects with respect to established outcome criteria.26 Finally, documented reports of infection due to probiotics in vulnerable patient groups emphasize the point that specific probiotic strains must be evaluated for patient safety27 and that efficacy is dependent on the strain-specific properties of individual probiotic formulations.
In summary, probiotics may attenuate liver disease associated with systemic inflammation by promoting intestinal barrier function or direct immunoregulatory effects. Physiological translocation across the intestinal barrier is a known phenomenon that may be associated with occult bacteremia in seemingly healthy children and adults.28 Pathological bacterial translocation may result from the penetration of excessive numbers of intestinal bacteria, including subgroups of organisms and bacterially derived mediators with proinflammatory features, resulting in hepatic injury and inflammation. The oral administration of probiotics may remodel intestinal microbial communities and facilitate restoration of barrier function and immunomodulation. The article by Ewaschuk et al. points to the stimulation of PPAR-γ as a potentially useful feature of probiotic strains to be considered for trials in patients with liver injury. Not all probiotics are created equal. The selection of specific probiotic strains that are screened for particular features may result in safe and effective agents for patients predisposed to sepsis and liver disease. Future preventative and therapeutic strategies may be enhanced by probiotic-based regimens that address both defects in intestinal barrier function and the regulation of systemic inflammatory mediators.