Epithelial interactions of Gram-negative commensals in human gastrointestinal tract

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Title: Epithelial interactions of Gram-negative commensals in human gastrointestinal tract
Author: Hiippala, Kaisa
Contributor: University of Helsinki, Faculty of Agriculture and Forestry
Doctoral Programme in Microbiology and Biotechnology
Human Microbiome Research Program/Faculty of Medicine
Publisher: Helsingin yliopisto
Date: 2020-11-06
Language: en
Belongs to series: Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, Biologicae - URN:ISSN:2342-5431
URI: http://urn.fi/URN:ISBN:978-951-51-6671-5
Thesis level: Doctoral dissertation (article-based)
Abstract: 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.
Subject: mikrobiologia
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