The Potential of Gut Commensals in Reinforcing Intestinal Barrier Function and Alleviating Inflammation

The intestinal microbiota, composed of pro- and anti-inflammatory microbes, has an essential role in maintaining gut homeostasis and functionality. An overly hygienic lifestyle, consumption of processed and fiber-poor foods, or antibiotics are major factors modulating the microbiota and possibly leading to longstanding dysbiosis. Dysbiotic microbiota is characterized to have altered composition, reduced diversity and stability, as well as increased levels of lipopolysaccharide-containing, proinflammatory bacteria. Specific commensal species as novel probiotics, so-called next-generation probiotics, could restore the intestinal health by means of attenuating inflammation and strengthening the epithelial barrier. In this review we summarize the latest findings considering the beneficial effects of the promising commensals across all major intestinal phyla. These include the already well-known bifidobacteria, which use extracellular structures or secreted substances to promote intestinal health. Faecalibacterium prausnitzii, Roseburia intestinalis, and Eubacterium hallii metabolize dietary fibers as major short-chain fatty acid producers providing energy sources for enterocytes and achieving anti-inflammatory effects in the gut. Akkermansia muciniphila exerts beneficial action in metabolic diseases and fortifies the barrier function. The health-promoting effects of Bacteroides species are relatively recently discovered with the findings of excreted immunomodulatory molecules. These promising, unconventional probiotics could be a part of biotherapeutic strategies in the future.Peer reviewe

Development of a Time-Dependent Oral Colon Delivery System of Anaerobic Odoribacter splanchnicus for Bacteriotherapy

Odoribacter (O.) splanchnicus is an anaerobic member of the human intestinal microbiota. Its decrease in abundance has been associated with inflammatory bowel disease (IBD), non-alcoholic fatty liver, and cystic fibrosis. Considering the anti-inflammatory properties of O. splanchnicus and its possible use for IBD, intestinal isolate O. splanchnicus 57 was here formulated for oral colonic release based on a time-dependent strategy. Freeze-drying protocol was determined to ensure O. splanchnicus 57 viability during the process. Disintegrating tablets, containing the freeze-dried O. splanchnicus 57, were manufactured by direct compression and coated by powder-layering technique with hydroxypropyl methylcellulose (Methocel™ E50) in a tangential-spray fluid bed. Eudragit® L was then applied by spray-coating in a top-spray fluid bed. Double-coated tablets were tested for release, showing gastric resistance properties and, as desired, lag phases of reproducible duration prior to release in phosphate buffer pH 6.8. The cell viability and anti-inflammatory activity of the strain were assessed after the main manufacturing steps. While freeze-drying did not affect bacterial viability, the tableting and coating processes were more stressful. Nonetheless, O. splanchnicus 57 cells survived manufacturing and the final formulations had 106-107 CFU/g of viable cells. The strain kept its anti-inflammatory properties after tableting and coating, reducing Escherichia coli lipopolysaccharide-induced interleukin-8 cytokine release from HT-29 cells. Overall, O. splanchnicus 57 strain was formulated successfully for oral colon delivery, opening new ways to formulate pure cultures of single anaerobic strains or mixtures for oral delivery

Colonic Mucosal Microbiota and Association of Bacterial Taxa with the Expression of Host Antimicrobial Peptides in Pediatric Ulcerative Colitis

Inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), are chronic debilitating disorders of unknown etiology. Over 200 genetic risk loci are associated with IBD, highlighting a key role for immunological and epithelial barrier functions. Environmental factors account for the growing incidence of IBD, and microbiota are considered as an important contributor. Microbiota dysbiosis can lead to a loss of tolerogenic immune effects and initiate or exacerbate inflammation. We aimed to study colonic mucosal microbiota and the expression of selected host genes in pediatric UC. We used high-throughput 16S rDNA sequencing to profile microbiota in colonic biopsies of pediatric UC patients (n = 26) and non-IBD controls (n = 27). The expression of 13 genes, including five for antimicrobial peptides, in parallel biopsies was assessed with qRT-PCR. The composition of microbiota between UC and non-IBD differed significantly (PCoA, p = 0.001). UC children had a decrease in Bacteroidetes and an increase in several family-level taxa including Peptostreptococcaceae and Enterobacteriaceae, which correlated negatively with the expression of antimicrobial peptides REG3G and DEFB1, respectively. Enterobacteriaceae correlated positively with the expression siderophore binding protein LCN2 and Betaproteobacteria negatively with DEFB4A expression. The results indicate that reciprocal interaction of epithelial microbiota and defense mechanisms play a role in UC

Colonic Mucosal Microbiota and Association of Bacterial Taxa with the Expression of Host Antimicrobial Peptides in Pediatric Ulcerative Colitis

Inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), are chronic debilitating disorders of unknown etiology. Over 200 genetic risk loci are associated with IBD, highlighting a key role for immunological and epithelial barrier functions. Environmental factors account for the growing incidence of IBD, and microbiota are considered as an important contributor. Microbiota dysbiosis can lead to a loss of tolerogenic immune effects and initiate or exacerbate inflammation. We aimed to study colonic mucosal microbiota and the expression of selected host genes in pediatric UC. We used high-throughput 16S rDNA sequencing to profile microbiota in colonic biopsies of pediatric UC patients (n = 26) and non-IBD controls (n = 27). The expression of 13 genes, including five for antimicrobial peptides, in parallel biopsies was assessed with qRT-PCR. The composition of microbiota between UC and non-IBD differed significantly (PCoA, p = 0.001). UC children had a decrease in Bacteroidetes and an increase in several family-level taxa including Peptostreptococcaceae and Enterobacteriaceae, which correlated negatively with the expression of antimicrobial peptides REG3G and DEFB1, respectively. Enterobacteriaceae correlated positively with the expression siderophore binding protein LCN2 and Betaproteobacteria negatively with DEFB4A expression. The results indicate that reciprocal interaction of epithelial microbiota and defense mechanisms play a role in UC

Mucosal Prevalence and Interactions with the Epithelium Indicate Commensalism of Sutterella spp.

Sutterella species have been frequently associated with human diseases, such as autism, Down syndrome, and inflammatory bowel disease (IBD), but the impact of these bacteria on health still remains unclear. Especially the interactions of Sutterella spp. with the host are largely unknown, despite of the species being highly prevalent. In this study, we addressed the interaction of three known species of Sutterella with the intestinal epithelium and examined their adhesion properties, the effect on intestinal barrier function and the pro-inflammatory capacity in vitro. We also studied the relative abundance and prevalence of the genus Sutterella and Sutterella wadsworthensis in intestinal biopsies of healthy individuals and patients with celiac disease (CeD) or IBD. Our results show that Sutterella spp. are abundant in the duodenum of healthy adults with a decreasing gradient toward the colon. No difference was detected in the prevalence of Sutterella between the pediatric IBD or CeD patients and the healthy controls. Sutterella parvirubra adhered better than the two other Sutterella spp. to differentiated Caco-2 cells and was capable of decreasing the adherence of S. wadsworthensis, which preferably bound to mucus and human extracellular matrix proteins. Furthermore, only S. wadsworthensis induced an interleukin-8 production in enterocytes, which could be due to different lipopolysaccharide structures between the species. However, its pro-inflammatory activity was modest as compared to non-pathogenic Escherichia coli. Sutterella spp. had no effect on the enterocyte monolayer integrity in vitro. Our findings indicate that the members of genus Sutterella are widely prevalent commensals with mild pro-inflammatory capacity in the human gastrointestinal tract and do not contribute significantly to the disrupted epithelial homeostasis associated with microbiota dysbiosis and increase of Proteobacteria. The ability of Sutterella spp. to adhere to intestinal epithelial cells indicate that they may have an immunomodulatory role.Peer reviewe

Novel strain of Pseudoruminococcus massiliensis possesses traits important in gut adaptation and host-microbe interactions

Fecal microbiota transplantation (FMT) is an efficient treatment for recurrent Clostridioides difficile infection and currently investigated as a treatment for other intestinal and systemic diseases. Better understanding of the species potentially transferred in FMT is needed. We isolated from a healthy fecal donor a novel strain E10-96H of Pseudoruminococcus massiliensis, a recently described strictly anaerobic species currently represented only by the type strain. The whole genome sequence of E10-96H had over 98% similarity with the type strain. E10-96H carries 20 glycoside hydrolase encoding genes, degrades starch in vitro and thus may contribute to fiber degradation, cross-feeding of other species and butyrate production in the intestinal ecosystem. The strain carries pilus-like structures, harbors pilin genes in its genome and adheres to enterocytes in vitro but does not provoke a proinflammatory response. P. massiliensis seems to have commensal behavior with the host epithelium, and its role in intestinal ecology should be studied further.Peer reviewe

Colonic Mucosal Microbiota and Association of Bacterial Taxa with the Expression of Host Antimicrobial Peptides in Pediatric Ulcerative Colitis

Inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), are chronic debilitating disorders of unknown etiology. Over 200 genetic risk loci are associated with IBD, highlighting a key role for immunological and epithelial barrier functions. Environmental factors account for the growing incidence of IBD, and microbiota are considered as an important contributor. Microbiota dysbiosis can lead to a loss of tolerogenic immune effects and initiate or exacerbate inflammation. We aimed to study colonic mucosal microbiota and the expression of selected host genes in pediatric UC. We used high-throughput 16S rDNA sequencing to profile microbiota in colonic biopsies of pediatric UC patients (n= 26) and non-IBD controls (n= 27). The expression of 13 genes, including five for antimicrobial peptides, in parallel biopsies was assessed with qRT-PCR. The composition of microbiota between UC and non-IBD differed significantly (PCoA,p= 0.001). UC children had a decrease in Bacteroidetes and an increase in several family-level taxa including Peptostreptococcaceae and Enterobacteriaceae, which correlated negatively with the expression of antimicrobial peptides REG3G and DEFB1, respectively. Enterobacteriaceae correlated positively with the expression siderophore binding protein LCN2 and Betaproteobacteria negatively with DEFB4A expression. The results indicate that reciprocal interaction of epithelial microbiota and defense mechanisms play a role in UC

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics

Isolation of Anti-Inflammatory and Epithelium Reinforcing Bacteroides and Parabacteroides Spp. from A Healthy Fecal Donor

Altered intestinal microbiota is associated with systemic and intestinal diseases, such as inflammatory bowel disease (IBD). Dysbiotic microbiota with enhanced proinflammatory capacity is characterized by depletion of anaerobic commensals, increased proportion of facultatively anaerobic bacteria, as well as reduced diversity and stability. In this study, we developed a high-throughput in vitro screening assay to isolate intestinal commensal bacteria with anti-inflammatory capacity from a healthy fecal microbiota transplantation donor. Freshly isolated gut bacteria were screened for their capacity to attenuate Escherichia coli lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) release from HT-29 cells. The screen yielded a number of Bacteroides and Parabacteroides isolates, which were identified as P. distasonis, B. caccae, B. intestinalis, B. uniformis, B. fragilis, B. vulgatus and B. ovatus using whole genome sequencing. We observed that a cell-cell contact with the epithelium was not necessary to alleviate in vitro inflammation as spent culture media from the isolates were also effective and the anti-inflammatory action did not correlate with the enterocyte adherence capacity of the isolates. The anti-inflammatory isolates also exerted enterocyte monolayer reinforcing action and lacked essential genes to synthetize hexa-acylated, proinflammatory lipid A, part of LPS. Yet, the anti-inflammatory effector molecules remain to be identified. The Bacteroides strains isolated and characterized in this study have potential to be used as so-called next-generation probiotics

Epithelial interactions of Gram-negative commensals in human gastrointestinal tract

The present doctoral thesis examines the epithelial host-microbe interactions of different Gram-negative commensal bacteria in the human gastrointestinal microbiota, which consists of bacteria, viruses and eukaryotic organisms. Under steady-state conditions, commensal gut bacteria are harmless symbionts co-existing with the host as part of the normal, balanced microbiota and supporting intestinal homeostasis. On the contrary, dysbiotic microbiota with reduced species richness and altered composition is associated with many intestinal and systemic diseases. Constant communication between the microbiota and the host is enabled either by direct contact or secreted effector molecules. During the last decades, the beneficial effects of commensal bacteria on intestinal mucosa, as well as the related molecular mechanisms, have been increasingly investigated. Currently, the research focus is shifting from traditional probiotics to so-called “next-generation probiotics” and their potentially health-promoting molecules, “post-biotics”. In addition to the traditional treatment strategies for intestinal diseases, novel bacteriotherapy alternatives could be utilized to increase the presence or activity of commensal species with immunoregulatory capacity and the ability to enhance the barrier function. First, the colonic mucosal microbiota of pediatric ulcerative colitis (UC) patients was compared to non-inflammatory bowel disease (IBD) controls using colonic biopsies and pyrosequencing. Microbiota richness and diversity did not differ significantly between the UC and control subjects. Compositional microbiota changes were observed in the mucosa of UC patients with increased abundance of Firmicutes and Proteobacteria, especially the family Sutterellaceae, and decreased proportion of Bacteroidetes. Furthermore, the expression of selected host genes related to the barrier function was studied in the UC subjects. Most notably, the expressions of inflammatory cytokine interleukin-8 (IL-8), inflammation marker lipocalin-2 and calcium binding proteins, forming the IBD biomarker calprotectin, were elevated supporting previous findings in the literature. Next, the abundance and prevalence of Sutterella spp., belonging to the family Sutterellaceae that displayed increased abundance in the mucosa of UC patients, were studied using biopsies from IBD, celiac disease (CeD) and non-disease controls. In addition, epithelial interactions of the genus Sutterella were assessed in vitro. A decreasing gradient from the duodenum to the rectum was observed in the abundance of Sutterella spp. in non-disease adult subjects. No difference was detected in the prevalence of Sutterella between pediatric CeD or IBD patients and controls. Sutterella wadsworthensis was able to adhere to mucus, while Sutterella parvirubra had a higher adhesion capacity to enterocytes and showed competitiveness in adhesion against S. wadsworthensis. Sutterella spp. harbor a penta-acylated, less toxic lipopolysaccharide (LPS), which caused only a mild release of proinflammatory IL-8 from the HT-29 enterocyte cell line compared to hexa-acylated Escherichia coli. S. wadsworthensis and its LPS induced a higher IL-8 response in the HT-29 enterocytes compared to the other two species, indicating differences in their proinflammatory capacity. Overall, these findings implicated Sutterella spp. as a highly prevalent, benign gut commensal with mild proinflammatory mucosal interactions. A high-throughput screening method was developed to isolate anti-inflammatory strains from a healthy volunteer who had acted as a donor for fecal microbiota transplantation. In the screening, isolates capable of attenuating inflammation in vitro, i.e. decreasing E. coli LPS-induced IL-8 levels in enterocytes as compared to the LPS control, were considered as potentially anti-inflammatory, and identified using 16S rRNA gene sequencing and finally whole genome sequencing. The majority of the isolates eliciting anti-inflammatory activity belonged to the order Bacteroidales. In vitro epithelial interaction assays studying the Bacteroidales strains revealed no correlation between attenuation capacity and adhesion, indicating that the effect was independent of cell-cell-contact. Furthermore, the culture supernatants of attenuating isolates were also effective in decreasing the LPS induced IL-8 response in enterocytes, which supported our hypothesis concerning the presence of effector molecules. Lastly, one of the Bacteroidales strains isolated from the fecal donor was Odoribacter splanchnicus, which is known as an abundant, short-chain fatty acid producing gut commensal. Bacterial-epithelial interactions of this less studied commensal were assessed in vitro. O. splanchnicus did not adhere to enterocytes or enhance epithelial monolayer integrity, yet the bacterium and its cell-free culture supernatant displayed in vitro inflammation attenuation capacity. Furthermore, the spent medium of the O. splanchnicus strain induced a higher anti-inflammatory cytokine interleukin-10 release in relation to tumor necrosis factor alpha in peripheral blood mononuclear cells compared to O. splanchnicus cells or the E. coli control. Outer membrane vesicles (OMVs) were isolated from O. splanchnicus culture medium. The treatment of enterocyte monolayer with O. splanchnicus OMVs prior to LPS stimulation caused a significant decrease in IL-8 levels. The anti-inflammatory effect was more consistent with OMVs than bacterial cells. Taken together, O. splanchnicus seems to primarily exert beneficial interaction with the host. Commensal bacteria and intestinal gut epithelium are engaged in constant cross-talk mediated by direct cell-cell contact and/or secreted bacterial effector molecules. The delicate balance of mucosal microbiota enhancing the barrier function and keeping the intestinal immune cells alerted at an appropriate level is susceptible to disturbances potentially leading to dysbiosis. In this context, the identification of gut homeostasis promoting bacteria and their metabolites, as undertaken in this study, is a vital part of novel, personalized bacteriotherapy using a defined bacterial cocktail to assist in restoring intestinal equilibrium.Tässä väitöskirjassa tutkittiin tiettyjen Gram-negatiivisten kommensaalibakteerien vuorovaikutusta ihmisen suolistoepiteelin kanssa. Ihmisen suolistomikrobisto koostuu bakteereista, viruksista ja eukaryooteista. Normaaliolosuhteissa kommensaalibakteerit ovat osa toimivaa, tasapainoista suolistomikrobistoa eläen symbioosissa isännän kanssa. Epätasapainoinen mikrobisto on liitetty moniin suolistosairauksiin, jossa bakteerien lajikirjo on vähentynyt ja mikrobiston koostumus muuttunut. Suoliston bakteerit kommunikoivat jatkuvasti isännän kanssa muun muassa tuottamalla efektorimolekyylejä, joiden mahdolliset suolistoterveyttä edistävät ominaisuudet ovat herättäneet kiinnostusta viimeisen vuosikymmenen aikana. Perinteisten probioottien lisäksi tutkimuskohteena ovatkin nykyään kommensaalibakteerit eli niin sanotut uuden sukupolven probiootit sekä niiden tuottamat molekyylit. Ensimmäisessä osatyössä tutkittiin haavaisesta paksusuolentulehduksesta (UC) kärsivien lasten epiteelimikrobistoa ja paksusuolen epiteelisolujen geeniekspressiota, joita verrattiin terveisiin verrokkeihin. Mikrobiston lajirikkaudessa tai monipuolisuudessa ei ollut merkittäviä eroja UC-potilaiden ja terveiden välillä. Koostumukseltaan mikrobistot olivat erilaisia, sillä UC potilailla havaittiin Firmikuuttien ja Proteobakteerien, etenkin Sutterellaceae-heimon, lisääntyneet osuudet mikrobistossa. UC-potilaiden epiteelisoluissa interleukin-8 –sytokiinin, inflammatiomarkkerin lipokalin-2 sekä kalprotektiinin ekspressiot olivat suurentuneet verrattuna terveisiin kontrolleihin. Seuraavassa osatyössä tutkittiin Sutterella-bakteerin suhteellista osuutta ja esiintyvyyttä tulehduksellisissa suolistosairauksissa (IBD), keliakiassa ja terveissä verrokeissa sekä bakteerin vuorovaikutusta suolistoepiteelin kanssa in vitro-kokeilla. Terveillä verrokeilla Sutterella-bakteerin suhteellinen osuus koko mikrobistosta väheni tasaisesti biopsioissa pohjukaissuolesta peräsuoleen. Sutterella-suvun esiintyvyydessä ei ollut eroja keliakia- tai IBD-potilailla verrattuna kontrolleihin. Sutterella wadsworthensis sitoutui in vitro mukukseen, kun taas Sutterella parvirubra sitoutui paremmin epiteelisoluihin. Sutterella-bakteeri tuottaa ulkopinnalleen penta-asyloitua, vähemmän toksista lipopolysakkaridia (LPS), joka aiheutti vain lievän IL-8-tuoton HT-29-epiteelisolulinjassa verrattuna Escherichia coli-bakteeriin. Sutterella-suvun bakteerit vaikuttavat olevan harmittomia, laajalti esiintyviä suolistokommensaaleja, joiden vuorovaikutus epiteelin kanssa on vain lievästi tulehdusta aiheuttavaa. Kahdessa viimeisessä osatyössä tutkittiin anti-inflammatorisia Bacteroidales-isolaatteja, jotka eristettiin terveeltä ulosteensiirtoluovuttajalta. Sadoista bakteeri-isolaateista seulottiin mahdollisesti anti-inflammatorisia kantoja testaamalla bakteerien kykyä vähentää IL-8-tuottoa LPS-indusoiduissa epiteelisoluissa. Suurin osa anti-inflammatorisista isolaateista kuului Bacteroidales-lahkoon. Bakteerien kyky vähentää IL-8-tuottoa ei korreloinut adheesiokyvyn kanssa, viitaten ettei solu-solu –kontakti ollut tarpeellinen. Lisäksi Bacteroidales-isolaattien kasvuliemet, joista bakteerit olivat siivilöity pois, alensivat tulehdusta epiteelisoluissa. Mahdollisten efektorimolekyylien osuutta attenuaatiossa tutkittiin Odoribacter splanchnicus-isolaatilla, jonka tuottamat ulkomembraanivesikkelit vähensivät tasaisesti IL-8-tuottoa LPS-indusoiduissa epiteelisoluissa. O. splanchnicus-kasvuliemi aiheutti myös suuremman anti-inflammatorisen IL-10-sytokiinin tuoton ihmisen mononukleaarisissa leukosyyteissä E. coliin verrattuna