Gut matters: microbe-host interactions in allergic diseases.

The human body can be considered a metaorganism made up of its own eukaryotic cells and trillions of microbes that colonize superficial body sites, such as the skin, airways, and gastrointestinal tract. The coevolution of host and microbes brought about a variety of molecular mechanisms, which ensure a peaceful relationship. The mammalian barrier and immune functions warrant simultaneous protection of the host against deleterious infections, as well as tolerance toward harmless commensals. Because these pivotal host functions evolved under high microbial pressure, they obviously depend on a complex network of microbe-host interactions. The rapid spread of immune-mediated disorders, such as autoimmune diseases, inflammatory bowel diseases, and allergies, in westernized countries is thus thought to be due to environmentally mediated disturbances of this microbe-host interaction network. The aim of the present review is to highlight the importance of the intestinal microbiota in shaping host immune mechanisms, with particular emphasis on allergic diseases and possible intervention strategies.

Molecular crosstalk of probiotic bacteria with the intestinal immune system: clinical relevance in the context of inflammatory bowel disease.

It is current knowledge that the intestinal microbiota plays a major role in the development and maintenance of intestinal health. Intestinal epithelial cells (IEC) constitute the interface between the gut lumen and the innate and adaptive immune system. To maintain intestinal homeostasis, the organized and diffuse compartments of the gut-associated lymphoid tissue have to process the continuously varying information at the interface between the luminal side and the host. Dysregulated intestinal immune responses towards commensal bacteria are an important factor in the pathogenesis of inflammatory bowel diseases (IBD). In contrast to the colitogenic effects of enteric bacteria, clinical and experimental studies showed that specific probiotic strains are protective in the context of chronic intestinal inflammation. Although the molecular understanding of bacteria-host interaction is improving, the anti-inflammatory mechanisms induced by these probiotic bacteria are just starting to be unraveled. The present review is meant to summarize and discuss the clinical relevance of probiotics, but it also seeks to give an overview about currently known probiotic mechanisms in the context of chronic intestinal inflammation with a focus on IEC.

Posttranslational inhibition of proinflammatory chemokine secretion in intestinal epithelial cells: implications for specific IBD indications.

BACKGROUND AND AIM: Inflammatory bowel diseases (IBD) are immune-mediated chronic diseases that are characterized by an overreaction of the intestinal immune system to the intestinal microbiota. VSL#3, a mixture of 8 different lactic acid bacteria, is a clinically relevant probiotic compound in the context of IBD, but the bacterial structures and molecular mechanisms underlying the observed protective effects are largely unknown. The intestinal epithelium plays a very important role in the maintenance of the intestinal homeostasis, as the intestinal epithelial cells (IEC) are capable of sensing, processing, and reacting upon signals from the luminal microbiota and the intestinal immune system. This immune regulatory function of the IEC is lost in IBD owing to dysregulated activation of the IEC. Thus, the aim of this study was to reveal protective mechanisms of VSL#3 on IEC function. RESULTS: In vitro, VSL#3 was found to selectively inhibit activation-induced secretion of the T-cell chemokine interferon-inducible protein (IP)-10 in IEC. Cell wall-associated proteins of VSL#3-derived Lactobacillus casei (L. casei) were identified to be the active anti-inflammatory component of VSL#3. Mechanistically, L. casei did not impair initial IP-10 protein production, but induced posttranslational degradation of IP-10 in IEC. Feeding studies in tumor necrosis factor (TNF)Delta ARE/+ mice, a mouse model for experimental ileitis, revealed that neither VSL#3 nor L. casei is capable of reducing ileal inflammation. Even preweaning feeding of VSL#3 did not prevent the development of severe ileitis in TNF Delta ARE/+ mice. In contrast, VSL#3 feeding studies in IL-10-/- mice, a model for experimental colitis, revealed that VSL#3 has local, intestinal compartment-specific protective effects on the development of inflammation. Reduced histopathologic inflammation in the cecum of IL-10-/- mice after VSL#3 treatment was found to correlate with reduced levels of IP-10 protein in primary cecal epithelial cells. CONCLUSION AND OUTLOOK: These results suggest that the inhibitory effect of VSL#3-derived L. casei on IP-10 secretion in IEC is an important probiotic mechanism that contributes to the anti-inflammatory effects of VSL#3 in specific subsets of patients with IBD. An important future aim is the identification of the active probiotic protein, which could serve as a basis for the development of new efficient therapies in the context of IBD.

Increased Pancreatic Protease Activity in Response to Antibiotics Impairs Gut Barrier and Triggers Colitis.

Background & Aims: Antibiotic (ABx) therapy is associated with increased risk for Crohn's disease but underlying mechanisms are unknown. We observed high fecal serine protease activity (PA) to be a frequent side effect of ABx therapy. The aim of the present study was to unravel whether this rise in large intestinal PA may promote colitis development via detrimental effects on the large intestinal barrier. Methods: Transwell experiments were used to assess the impact of high PA in ABx-treated patients or vancomycin/metronidazole-treated mice on the epithelial barrier. Serine protease profiling was performed using liquid chromatography-mass spectrometry/mass spectrometry analysis. The impact of high large intestinal PA on the intestinal barrier in wild-type and interleukin (IL)10-/- mice and on colitis development in IL10-/- mice was investigated using vancomycin/metronidazole with or without oral serine protease inhibitor (AEBSF) treatment. Results: The ABx-induced, high large intestinal PA was caused by significantly increased levels of pancreatic proteases and impaired epithelial barrier integrity. In wild-type mice, the rise in PA caused a transient increase in intestinal permeability but did not affect susceptibility to chemically induced acute colitis. In IL10-/- mice, increased PA caused a consistent impairment of the intestinal barrier associated with inflammatory activation in the large intestinal tissue. In the long term, the vancomycin/metronidazole-induced lasting increase in PA aggravated colitis development in IL10-/- mice. Conclusions: High large intestinal PA is a frequent adverse effect of ABx therapy, which is detrimental to the large intestinal barrier and may contribute to the development of chronic intestinal inflammation in susceptible individuals.

The gut microbiota promotes hepatic fatty acid desaturation and elongation in mice.

Interactions between the gut microbial ecosystem and host lipid homeostasis are highly relevant to host physiology and metabolic diseases. We present a comprehensive multi-omics view of the effect of intestinal microbial colonization on hepatic lipid metabolism, integrating transcriptomic, proteomic, phosphoproteomic, and lipidomic analyses of liver and plasma samples from germfree and specific pathogen-free mice. Microbes induce monounsaturated fatty acid generation by stearoyl-CoA desaturase 1 and polyunsaturated fatty acid elongation by fatty acid elongase 5, leading to significant alterations in glycerophospholipid acyl-chain profiles. A composite classification score calculated from the observed alterations in fatty acid profiles in germfree mice clearly differentiates antibiotic-treated mice from untreated controls with high sensitivity. Mechanistic investigations reveal that acetate originating from gut microbial degradation of dietary fiber serves as precursor for hepatic synthesis of C16 and C18 fatty acids and their related glycerophospholipid species that are also released into the circulation.

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

BACKGROUND & AIMS: The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). METHOD: Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. RESULTS: Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. CONCLUSION: Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS.

Physiological relevance of food grade microcapsules: Impact of milk protein based microcapsules on inflammation in mouse models for inflammatory bowel diseases.

In order to increase beneficial effects of bioactive compounds in functional food and dietary supplements, enormous efforts are put in the technological development of microcapsules. Although these products are often tailor-made for disease susceptible consumer, the physiological impact of microcapsule uptake on the respective target consumer has never been addressed. The present study aimed to assess the relevance of this aspect by analyzing the impact of milk protein based microcapsules on experimental inflammatory bowel disease. Long-term feeding of sodium caseinate or rennet gel microcapsules resulted in significant alterations in the intestinal microbiota of healthy mice. In TNFΔARE/wt mice, a model for chronic ileal inflammation, rennet gel microcapsules resulted in further increased splenomegaly, whereas ileal inflammation was unchanged. In IL10(-/-) mice, a model for chronic colitis, both types of microcapsules induced a local increase of the intestinal inflammation. The present study is the first to demonstrate that, independent of their cargo, microcapsules have the potential to affect the intestinal microbiota and to exert unprecedented detrimental effects on disease-susceptible individuals. In conclusion, the impact of microcapsule uptake on the respective target consumer groups should be thoroughly investigated in advance to their commercial use in functional food or dietary supplements.

Fetal exposure to maternal inflammation does not affect postnatal development of genetically-driven ileitis and colitis.

BACKGROUND: Chronic inflammatory disorders have been increasing in incidence over the past decades following geographical patterns of industrialization. Fetal exposure to maternal inflammation may alter organ functions and the offspring's disease risk. We studied the development of genetically-driven ileitis and colitis in response to maternal inflammation using mouse models. METHODS: Disease susceptible (TnfΔARE/+ and IL10-/-) and disease-free (Tnf+/+ and IL10-/+) offspring were raised in inflamed and non-inflamed dams. Ileal, caecal and colonic pathology was evaluated in the offspring at 8 or 12 weeks of age. Ly6G-positive cells in inflamed sections from the distal ileum and distal colon were analysed by immunofluorescence microscopy. Gene expression of pro-inflammatory cytokines was measured in whole tissue specimens by quantitative PCR. Microarray analyses were performed on laser microdissected intestinal epithelium. Caecal bacterial communities were assessed by Illumina sequencing of 16S rRNA amplicons. RESULTS: Disease severity, the number of infiltrated neutrophils as well as Tnf and Il12p40 mRNA expression were independent of maternal inflammation in the offspring of mouse models for ileitis (TnfΔARE/+) and colitis (IL10-/-). Although TNF-driven maternal inflammation regulated 2,174 (wild type) and 3,345 (TnfΔARE/+) genes in the fetal epithelium, prenatal gene expression patterns were completely overwritten after birth. In addition, co-housing experiments revealed no change in phylogenetic diversity of the offspring's caecal microbiota in response to maternal inflammation. This is independent of the offspring's genotype before and after the onset of tissue pathology. CONCLUSIONS: Disease risk and activity in mouse models of chronic ileitis and colitis was independent of the fetal exposure to maternal inflammation. Likewise, maternal inflammation did not alter the diversity and composition of offspring's caecal microbiota, clearly demonstrating that changes of the gene expression program in the fetal gut epithelium were not relevant for the development of chronic inflammatory disorders in the gut.

Lactocepin as a protective microbial structure in the context of IBD.

Probiotics have been shown to exert beneficial effects in the context of different diseases including inflammatory bowel diseases (IBD). However, clinical use of probiotics is hampered by lack of understanding of the protective mechanisms and by safety concerns regarding the application of high numbers of live bacteria in patients. The identification of protective microbial structure-function relationships might enable to overcome these restraints and might lead to innovative therapies using the isolated active microbial structures. In our study, we aimed to characterize the protective mechanisms of VSL#3, a clinically relevant probiotic mixture in IBD. We found Lactobacillus casei/paracasei-produced lactocepin to selectively degrade pro-inflammatory chemokines, resulting in reduced immune cell infiltration and reduced inflammation in experimental IBD models. As immune cell recruitment is a major proinflammatory mechanism our findings suggest that lactocepin might be of broad therapeutic relevance in an array of inflammatory diseases like IBD, allergic skin inflammation and psoriasis.

Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines.

The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L. casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, T cell infiltration and inflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD.

Safety assessment of probiotics for human use.

The safety of probiotics is tied to their intended use, which includes consideration of potential vulnerability of the consumer or patient, dose and duration of consumption, and both the manner and frequency of administration. Unique to probiotics is that they are alive when administered, and unlike other food or drug ingredients, possess the potential for infectivity or in situ toxin production. Since numerous types of microbes are used as probiotics, safety is also intricately tied to the nature of the specific microbe being used. The presence of transferable antibiotic resistance genes, which comprises a theoretical risk of transfer to a less innocuous member of the gut microbial community, must also be considered. Genetic stability of the probiotic over time, deleterious metabolic activities, and the potential for pathogenicity or toxicogenicity must be assessed depending on the characteristics of the genus and species of the microbe being used. Immunological effects must be considered, especially in certain vulnerable populations, including infants with undeveloped immune function. A few reports about negative probiotic effects have surfaced, the significance of which would be better understood with more complete understanding of the mechanisms of probiotic interaction with the host and colonizing microbes. Use of readily available and low cost genomic sequencing technologies to assure the absence of genes of concern is advisable for candidate probiotic strains. The field of probiotic safety is characterized by the scarcity of studies specifically designed to assess safety contrasted with the long history of safe use of many of these microbes in foods.

Post-translational inhibition of IP-10 secretion in IEC by probiotic bacteria: impact on chronic inflammation.

BACKGROUND: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC). METHODOLOGY/PRINCIPAL FINDINGS: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF(DeltaARE) and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells. CONCLUSIONS/SIGNIFICANCE: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.