Mehanizmi aktivacije naravne imunosti celic črevesnega epitelija z izbranimi probiotičnimi sevi bakterij

Paveljšek, Diana (author), Rogelj, Irena (mentor), Jerala, Roman (co-mentor)

Manipulacija črevesne mikrobiote s koristnimi mikroorganizmi predstavlja obetavno alternativo in podporo za zdravljenje črevesnega vnetja in motenj v delovanju črevesne epitelne bariere. Probiotiki lahko namreč stimulirajo tako pridobljeni kot tudi naravni imunski odziv. Namen raziskave je bil zato pojasniti signalne poti, ki jih v črevesnih epitelnih celicah sprožijo probiotični bakterijski sevi Lactobacillus gasseri K7 (K7), Lactobacillus fermentum L930BB (L930BB), Bifidobacterium animalis subsp. animalis IM386 (IM386) in Lactobacillus plantarum WCFS1 (WCFS1). Izhodišče raziskave je bila in vivo študija, v kateri so na miših s povzročenim kolitisom proučevali zaščitni vpliv sevov L930BB in IM386. Rezultati analize transkriptoma mišjega kolona so pokazali spremembe v izražanju genov, ki so povezani z aktivacijo proti-apoptostskih poti preko fosfatidil inozitol 3-kinaze (PI3K)/Akt in aktivacijo poti, ki vodijo v regulacijo aktinskega citoskeleta in tesnih stikov preko protein-kinaze C (PKC). Tako reorganizacija aktinskega citoskeleta, kot tudi zmanjšana apoptoza, prispevata k obnavljanju monosloja črevesnih epitelnih celic. V in vitro poskusih smo dokazali, da so aktivirane signalne poti posledica signalizacije preko receptorja naravne imunosti, Toll-u podobnega receptorja 2 (TLR2). Z uporabo pretočne citometrije smo potrdili, da so probiotični sevi in ligand za TLR2 sposobni zmanjšati s citokini povzročeno celično smrt. S pregledovanjem črevesne epitelne celične linije Caco-2 pod konfokalnim mikroskopom pa smo opazili, da probiotični sevi, ob simulaciji vnetnih razmer s H2O2, zmanjšajo internalizacijo proteina tesnih stikov ZO-1 in tako stabilizirajo medcelične povezave. V stanju vnetja se je ob dodatku probiotičnih sevov, zmanjšala tudi permeabilnost celičnega monosloja, kar dodatno dokazuje zaščitni vpliv izbranih sevov in njihovo zmožnost podpore vzdrževanja črevesne epitelne bariere.

• Language: Slovenian
• Keywords: probiotiki, Lactobacillus, Bifidobacterium, transkriptom, signalne poti, črevesna epitelna bariera, imunski odziv, Toll-u podobni receptor, protein-kinaza C, fosfatidil inozitol 3-kinaza, tesni stiki, aktinski citoskelet, apoptoza
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [D. Paveljšek]
• Year: 2019
• PID: 20.500.12556/RUL-107836
• UDC: 579.61:579.86:616.34:577.27.085
• COBISS.SI-ID: 5058936
• Publication date in RUL: 30.05.2019

Secondary language

• Language: English
• Title: Mechanisms of innate immunity activation in intestinal epithelial cells with selected probiotic bacterial strains

Abstract

Manipulation of the intestinal microbiota with beneficial microorganisms represents a promising alternative and support to the treatment of intestinal inflammation and disorders in intestinal epithelial barrier function. Probiotics can stimulate both the adaptive and innate immune responses. The aim of the study was therefore to clarify the signalling pathways in intestinal epithelial cells triggered by probiotic bacterial strains Lactobacillus gasseri K7 (K7), Lactobacillus fermentum L930BB (L930BB), Bifidobacterium animalis subsp. animalis IM386 (IM386) and Lactobacillus plantarum WCFS1 (WCFS1). The starting point of the research was an in vivo study in which the protective effect of strains L930BB and IM386 was examined in a mouse model with induced colitis. Results of mice colon transcriptome analysis revealed changes in gene expression, involved in activation of anti-apoptotic pathways through phosphatidylinositol 3-kinase (PI3K)/Akt and activation of pathways that lead to regulation of actin cytoskeleton and tight junctions through protein kinase C (PKC). Reorganisation of actin cytoskeleton and decreased apoptosis are both helpful in intestinal epithelial cell monolayer reconstitution. We proved in in vitro experiments that activated signalling pathways are a consequence of signalization through the innate immune receptor Toll-like receptor 2 (TLR2). With the use of flow cytometry we confirmed that probiotic strains and TLR2 ligand are able to reduce cytokine induced cell death. By examining the intestinal epithelial cell line Caco-2 under the confocal microscope, we observed that probiotic strains, in addition to the simulation of inflammatory conditions with H2O2, reduce the internalization of the tight junction protein ZO-1 and thus stabilize intercellular connections. In inflammatory conditions, the addition of probiotic strains also reduced the permeability of the cell monolayer, further proving the protective effect of selected strains and their ability to support the maintenance of the intestinal epithelial barrier.

• Keywords: probiotics, Lactobacillus, Bifidobacterium, transcriptome, signalling pathways, intestinal epithelial barrier, immune response, Toll-like receptor, protein kinase C, phosphatidylinositol 3-kinase, actin cytoskeleton, apoptosis