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Modulation of cellular innate immune responses by lactobacilli
Örebro University, School of Science and Technology, Örebro University, Sweden.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Lactobacillus is a genus of lactic acid bacteria frequently used as healthpromoting probiotics. Using probiotics to treat or prevent infections is a novel experimental approach with vast impact on future therapy. Lactobacillus rhamnosus GR-1 is a probiotic investigated for its ability to reduce urogenital disease including urinary tract infections caused by pathogenic Escherichia coli. L. rhamnosus GR-1 has been shown to modulate immunity, thought to influence its probiotic effect. In this thesis, the aim was to study immunomodulation by L. rhamnosus GR-1 and other lactobacilli, with emphasis on elicited immune responses such as nuclear factor-kappaB (NF-κB) activation and cytokine release from human urothelial cells.

Viable, heat-killed, and isolated released products from L. rhamnosus GR-1 augmented NF-κB activation in E. coli-challenged urothelial cells. Blocking of lipopolysaccharide binding to toll-like receptor 4 completely quelled this augmentation. Size-fractionation, urothelial cell challenge, and two-dimensional gel electrophoresis of L. rhamnosus GR-1 released products presented several candidate proteins with NF-κB modulatory actions including chaperonin GroEL, elongation factur Tu, and a protein from the NLP/P60 protein family. While tumor necrosis factor was correspondingly augmented by L. rhamnosus GR-1, the release of two other cytokines, interleukin (IL)-6 and CXCL8, was reduced. Similar effects were observed in macrophage-like cells stimulated with L. rhamnosus GR-1.

Many immunomodulatory effects of lactobacilli are believed to be species and strain dependent. Therefore, twelve Lactobacillus strains were used to screen for their effects on CXCL8 release from urothelial cells. A majority of these strains were able to influence CXCL8 release from the cells. Phylogenetic analysis revealed close evolutionary linkage between lactobacilli with similar actions on CXCL8. Increased knowledge on probiotic bacterial products and the mechanism(s) of action could lead to improved future treatments for infections.

Place, publisher, year, edition, pages
Örebro: Örebro universitet , 2012. , 84 p.
Series
Örebro Studies in Life Science, 10
Keyword [en]
cytokines, immunomodulation, lactobacilli, probiotics, urinary tract infections, urothelium
National Category
Biological Sciences
Research subject
Biology
Identifiers
URN: urn:nbn:se:oru:diva-22138ISBN: 978-91-7668-872-4 (print)OAI: oai:DiVA.org:oru-22138DiVA: diva2:510509
Public defence
2012-06-01, Hörsal BIO, Forumhuset, Örebro universitet, Fakultetsgatan 1, Örebro, 13:00 (English)
Opponent
Supervisors
Available from: 2012-03-16 Created: 2012-03-16 Last updated: 2016-08-12Bibliographically approved
List of papers
1. Released substances from lactobacilli influence immune responses in human epithelial cells
Open this publication in new window or tab >>Released substances from lactobacilli influence immune responses in human epithelial cells
2010 (English)In: Abstracts of the 3rd Swedish-Hellenic Life Sciences Research Conference, Athens, March 25-27, 2010 / [ed] Fragiskos Kolisis, Nikolaos Venizelos, 2010, 367-368 p.Conference paper, (Refereed)
National Category
Medical and Health Sciences Biological Sciences
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-12300 (URN)
Conference
The 3rd Swedish-Hellenic Life Sciences Research Conference, Athens, March 25-27, 2010
Note

in vivo 24 (2010).

Available from: 2010-10-22 Created: 2010-10-22 Last updated: 2016-08-10Bibliographically approved
2. Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4
Open this publication in new window or tab >>Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4
2012 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 12, 15- p.Article in journal (Refereed) Published
Abstract [en]

Background: Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation.

Results: Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR 1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR 1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB.

Conclusions: The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR 1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1 modulated the activation of the NF-kappaB through increased levels of TLR4 on the bladder cells and altered subsequent release of cytokines from urothelial cells. By influencing immunological factors such as TLR4, important in the process of fighting pathogens, lactobacilli could facilitate pathogen recognition and infection clearance.

Place, publisher, year, edition, pages
BioMed Central, 2012
National Category
Microbiology
Research subject
Microbiology; Immunology
Identifiers
urn:nbn:se:oru:diva-21288 (URN)10.1186/1471-2180-12-15 (DOI)000301484000001 ()22264349 (PubMedID)2-s2.0-84856001528 (Scopus ID)
Available from: 2012-02-17 Created: 2012-01-24 Last updated: 2017-02-17Bibliographically approved
3. Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells
Open this publication in new window or tab >>Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells
Show others...
2012 (English)In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 66, no 2, 147-156 p.Article in journal (Refereed) Published
Abstract [en]

Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli.

Place, publisher, year, edition, pages
Hoboken, USA: Wiley-Blackwell, 2012
Keyword
Immune response, cytokines, lactobacilli, urothelium, uropathogen, secretome
National Category
Immunology Microbiology
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-22594 (URN)10.1111/j.1574-695X.2012.00994.x (DOI)000310277300003 ()22620976 (PubMedID)2-s2.0-84867727218 (Scopus ID)
Note

Funding Agencies:

Knowledge Foundation

Magnus Bergvalls Foundation 

Sparbanksstiftelsen Nya 

Carl Tryggers Foundation, Sweden 

Available from: 2012-04-19 Created: 2012-04-19 Last updated: 2016-12-13Bibliographically approved
4. Probiotic Lactobacillus rhamnosus alters inflammatory responses of bladder epithelial and macrophage-like cells in co-culture
Open this publication in new window or tab >>Probiotic Lactobacillus rhamnosus alters inflammatory responses of bladder epithelial and macrophage-like cells in co-culture
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Immunology Microbiology
Research subject
Biology
Identifiers
urn:nbn:se:oru:diva-22595 (URN)
Available from: 2012-04-19 Created: 2012-04-19 Last updated: 2016-12-14Bibliographically approved
5. Lactobacilli differently regulate expression and secretion of CXCL8 in urothelial cells
Open this publication in new window or tab >>Lactobacilli differently regulate expression and secretion of CXCL8 in urothelial cells
2012 (English)In: Beneficial Microbes, ISSN 1876-2883, E-ISSN 1876-2891, Vol. 3, no 3, 195-203 p.Article in journal (Refereed) Published
Abstract [en]

Modulation of the immune response is an established feature of certain lactobacilli. CXCL8 is an inflammatory chemokine released by the urinary tract mucosa after contact with uropathogenic Escherichia coli during urinary tract infection and is crucial for proper infiltration of immune cells. Nevertheless, persistently high levels of CXCL8 are associated with pathogenicity and malignancy. In this study, we tested twelve Lactobacillus strains for their ability to influence CXCL8 release from urothelial cells. We evaluated how strains from different Lactobacillus species could regulate CXCL8 in human 5637 urothelial cells, either resting cells or cells concomitantly challenged with heat-killed E. coli. A majority of the tested species altered CXCL8 release from the urothelial cells after 24 hours of stimulation. Most species increased CXCL8 release, whereas a few lactobacilli efficiently suppressed CXCL8 secretion from E. coli-challenged cells. While strong CXCL8 modulators such as Lactobacillus reuteri and Lactobacillus delbrueckii were unable to degrade CXCL8 in the extracellular environment, effects on IL8 transcription were evident for selected lactobacilli. Although IL8 transcription was affected by lactobacilli, the influence on mRNA transcript did not correlate to the impact on CXCL8 release. Phylogenetic analysis based on a 16S rRNA dendrogram of the tested lactobacilli and their effect on CXCL8 revealed some linkage to specific Lactobacillus groups. Testing the immunomodulatory nature of lactobacilli can prove important when selecting new probiotic microbes. Moreover, we believe that phylogenetic and phenotypic similarities could be used to analyse the traits governing such modulation.

Place, publisher, year, edition, pages
Wageningen Academic Publishers, 2012
Keyword
Lactobacillus; immunomodulation; probiotics; chemokines; cytokines
National Category
Microbiology Nutrition and Dietetics
Research subject
Immunology; Microbiology; Nutrition
Identifiers
urn:nbn:se:oru:diva-21701 (URN)10.3920/BM2012.0011 (DOI)000308740900004 ()22835703 (PubMedID)2-s2.0-84869221939 (Scopus ID)
Funder
Knowledge Foundation
Note

Funding Agencies:

Magnus Bergvalls Foundation

Helge Ax:son Johnsons Foundation 

Available from: 2012-04-19 Created: 2012-02-17 Last updated: 2017-02-15Bibliographically approved

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