To Örebro University

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.

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.

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.

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.

Background

CXLC8 is an important cytokine that attracts immune cells such as neutrophils and macrophages during infections. This cytokine is released from many cell types including epithelial cells such as the urothelial cells found in the urinary tract. CXCL8 release is dramatically increased when urothelial cells meet bacteria such as uropathogenic Escherichia coli, which are the main cause of urinary tract infections. CXCL8 is needed during the course of the infection; nevertheless, high levels have been associated with chronic inflammation and cancer, and are in those cases considered detrimental to health. Lactobacillus is a diverse group of lactic acid bacteria and a major component of our microbiota. Many are also commercially available as probiotics, and even if they have shown effectiveness as preventative supplements for both infections and supporting bowel regularity, much is left to learn about their mode of action. We know that many probiotics are immunomodulators and affect immune cell function, especially in immune cells. However, less is known about their effect on epithelial cell immune function and the possible variation immunomodulation between different Lactobacillus species.   

Results

To test the CXCL8 modulating abilities of Lactobacillus, numerous species were used for screening urothelial cell CXCL8 release. We wanted to assess the role of lactobacilli on resting cells, but also how they affect CXCL8 in cells that are already stimulated with E. coli similar to the case of a urinary tract infection. We found that most lactobacilli had a marginal effect on cytokine release in resting cells not challenged with E. coli. However, E. coli-challenged cells subsequently exposed to lactobacilli showed a significant increase or decrease of CXCL8 levels. Many of the tested species changed cytokine levels severalfold in both directions. Comparing the effects between species using evolutionary trees based on 16S rRNA did not reveal any grouping of effects based on the genetic similarity between the different taxa.

Conclusion

Many lactobacilli have the ability to alter the levels of secreted CXCL8, especially in E. coli-challenged cells. Interestingly, we could not group the effect on CXCL8 based on their evolutionary relationship suggesting that the effects on CXCL8 is analogous in its nature and have evolved independently in many of the tested species. However, as for probiotic activity which is often strain specific, CXCL8 modulating features are also likely to be associated with individual strains. Although we cannot explain the big differences between bacteria, both up- and downregulation of CXCL8 is interesting from a therapeutical perspective. Many pathogens, including uropathogenic E. coli actively inhibit signals that lead to cytokine release and immune migration, and it can in those cases be beneficial to increase CXCL8, whereas decreased cytokine levels might be advantageous in other conditions such as chronic inflammation.  

Epithelial cells are often first responders to microbial invasion, resulting in release of inflammatory mediators such as cytokines and chemokines and subsequent recruitment of immune cells. Several strains from the genus Lactobacillus have been attributed probiotic properties and some of them have been shown to be able to modulate the immune response in vitro and in vivo. L. rhamnosus GR-1, a known probiotic strain was allowed to co-culture with an uroepithelial cell line challenged with heat-killed pathogenic E. coli, resulting in a synergistic up-regulation of the transcription factor NF-κB and increased release of the pro-inflammatory cytokine TNF. Although this L. rhamnosus strain was a poor inducer of uroepithelial NF-κB alone compared to heat-killed E. coli, together with this pathogenic stimulus, it efficiently elicited an epithelial immune response. This effect was greatly reduced if using non-viable lactobacilli suggesting secretion of the active substance. Secreted proteins were isolated and partially mimicked the effect found with viable lactobacilli. Potentiation of the host immune response towards pathogenic E. coli at an early stage using lactobacilli products could facilitate the removal of undesired microbes by activation of the NFκB transcription factor and consequently, epithelial immunity.