Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5 '-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1 beta and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-gamma and TNF-alpha responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products

Bacterial Regulation of Peripheral Immunity : Mechanistic insights from lactobacilli and Staphylococcus aureus

There is a constant cross-talk between our immune system and the colonizing microbiota. The gut resident bacteria produce a broad range of molecules with regulatory activities in both local and distal tissues. Staphylococcus (S.) aureus is a commensal bacterium with high pathogenic potential due to production of several potent virulence factors including staphylococcal enterotoxins (SEs). These SEs are known to induce overwhelming T cell responses, which can result in a serious condition known as toxic shock syndrome. In contrast, several species of bacteria from the genus Lactobacillus exhibit probiotic features and promote beneficial physiological and immunological effects in its host. The underlying mechanisms behind bacterial activation and regulation of peripheral lymphocytes remain elusive. In this thesis, we explored how secreted factors present in the cell free supernatants (CFS) of cultured S. aureus and lactobacilli mechanistically impact the activation of different types of T cells and NK cells. In paper I, we investigated the influence of S. aureus-CFS and SEA on regulatory T cells and found that despite de novo induction of FOXP3 expression, TREG cells also produced pro-inflammatory cytokines, which associated with CD161-expression. In paper II, we could show that S. aureus-CFS and SEA induce proliferation, cytotoxicity and cytokine production in conventional and unconventional T- and NK cells. Moreover, we also showed that the lactobacilli-CFS were able to dampen immune cell activation, which was partly linked to lactobacilli-derived lactate. In paper III, we continued to investigate the mechanism behind Lactobacillus-mediated dampening of induced lymphocyte responses and identified extracellular membrane vesicles to be one of the main components involved in Lactobacillus-mediated regulation of cytokine responses. Other observations made in paper II brought about several questions regarding the ability of SEs to activate unconventional T- and NK cells, which lacks certain receptors known to be required for SE-mediated activation of conventional T cells. In paper IV, we therefore investigated the mechanism behind SE-mediated activation of γδ T-, MAIT- and NK cells and found that SEs indirectly activated γδ T- and NK cells, which required the presence of conventional αβ T cells. In summary, this thesis presents novel insights into how soluble components from bacteria modulate immune cell responses and extends the general understanding of bacterial influence on peripheral immunity. At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 4: Manuscript.</p

Probiotic Lactobacilli Modulate Staphylococcus aureus-Induced Activation of Conventional and Unconventional T cells and NK Cells

Lactobacilli are probiotic commensal bacteria and potent modulators of immunity. When present in the gut or supplemented as probiotics, they beneficially modulate ex vivo immune responsiveness. Further, factors derived from several lactobacilli strains act immune regulatory in vitro. In contrast, Staphylococcus aureus (S. aureus) is known to induce excessive T cell activation. In this study, we aimed to investigate S. aureus-induced activation of human mucosal-associated invariant T cells (MAIT cells), gamma delta T cells, NK cells, as well as of conventional CD4(+) and CD8(+) T cells in vitro. Further, we investigated if lactobacilli-derived factors could modulate their activation. PBMC were cultured with S. aureus 161: 2 cell-free supernatants (CFS), staphylococcal enterotoxin A or CD3/CD28-beads alone, or in combination with Lactobacillus rhamnosus GG-CFS or Lactobacillus reuteri DSM 17938-CFS and activation of T and NK cells was evaluated. S. aureus-CFS induced IFN-gamma and CD107a expression as well as proliferation. Costimulation with lactobacilli-CFS dampened lymphocyte-activation in all cell types analyzed. Preincubation with lactobacilli-CFS was enough to reduce subsequent activation, and the absence of APC or APC-derived IL-10 did not prevent lactobacilli-mediated dampening. Finally, lactate selectively dampened activation of unconventional T cells and NK cells. In summary, we show that molecules present in the lactobacilli-CFS are able to directly dampen in vitro activation of conventional and unconventional T cells and of NK cells. This study provides novel insights on the immune-modulatory nature of probiotic lactobacilli and suggests a role for lactobacilli in the modulation of induced T and NK cell activation

Extracellular membrane vesicles from Limosilactobacillus reuteri strengthen the intestinal epithelial integrity, modulate cytokine responses and antagonize activation of TRPV1

Bacterial extracellular membrane vesicles (MV) are potent mediators of microbe-host signals, and they are not only important in host-pathogen interactions but also for the interactions between mutualistic bacteria and their hosts. Studies of MV derived from probiotics could enhance the understanding of these universal signal entities, and here we have studied MV derived from Limosilactobacillus reuteri DSM 17938 and BG-R46. The production of MV increased with cultivation time and after oxygen stress. Mass spectrometry-based proteomics analyses revealed that the MV carried a large number of bacterial cell surface proteins, several predicted to be involved in host-bacteria interactions. A 5 '-nucleotidase, which catalyze the conversion of AMP into the signal molecule adenosine, was one of these and analysis of enzymatic activity showed that L. reuteri BG-R46 derived MV exhibited the highest activity. We also detected the TLR2 activator lipoteichoic acid on the MV. In models for host interactions, we first observed that L. reuteri MV were internalized by Caco-2/HT29-MTX epithelial cells, and in a dose-dependent manner decreased the leakage caused by enterotoxigenic Escherichia coli by up to 65%. Furthermore, the MV upregulated IL-1 beta and IL-6 from peripheral blood mononuclear cells (PBMC), but also dampened IFN-gamma and TNF-alpha responses in PBMC challenged with Staphylococcus aureus. Finally, we showed that MV from the L. reuteri strains have an antagonistic effect on the pain receptor transient receptor potential vanilloid 1 in a model with primary dorsal root ganglion cells from rats. In summary, we have shown that these mobile nanometer scale MV reproduce several biological effects of L. reuteri cells and that the production parameters and selection of strain have an impact on the activity of the MV. This could potentially provide key information for development of innovative and more efficient probiotic products

Activation of human γδ T cells and NK cells by Staphylococcal enterotoxins requires both monocytes and conventional T cells

Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-, γδ T-, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST-1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST-1. SE-induced IFN-γ expression in MAIT-, γδ T-, and NK cells was clearly reduced by neutralization of IL-12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore, co-cultures of isolated cell populations revealed that the ability of SEA to activate γδ T- and NK cells was fully dependent on the presence of both monocytes and αβ T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections

Restriction Endonucleases from Invasive <i>Neisseria gonorrhoeae</i> Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

<div><p>The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. <i>Neisseria gonorrhoeae</i> infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.</p></div

Bacterial infection induces micronuclei formation in VK2/E6E7 cells.

<p>VK2/E6E7 cells were infected with <i>N. gonorrhoeae</i> for 24 h. Cytokinesis was blocked with cytochalasin B for 36 h and BNC from infected and control cells were analyzed for micronuclei formation. A. Average numbers of observed micronuclei/1000 BNC ± standard deviation from 3 independent experiments are shown (*<i>p</i><0.05). B. DIC image showing a representative Giemsa-stained BNC (blue), containing one micronucleus (arrow) and several intracellular bacteria (arrowheads), captured using a 63× objective. Scale bar represents 10 µm. C. Frequencies of micronuclei formation and DI observed in 3 independent experiments where 1000 BNC were scored in each experiment are shown.</p

Lysates of <i>N. gonorrhoeae</i> fragments pECFP-N1 and damage DNA from VK2/E6E7 cells.

<p>A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).</p

Microinjection of bacterial lysates in the cytoplasm of VK2/E6E7 cells causes DSBs.

<p>A. Interphase VK2/E6E7 cells were subjected to cytoplasmic microinjection of bacterial MS11 P+ lysate, MS11 P+ HI lysate, or PBS. Cells were incubated for 20–24 h and then stained for DSBs with 53BP1 antibodies. The graph shows the average number of 53BP1 positive cells counted in two independent experiments under each condition. Control cells are non-injected cells. B. Images showing DIC and fluorescent images of representative cells microinjected with MS11 P+ lysate (left) or PBS (right). FITC-dextran (green) was co-injected into the cytoplasm to identify microinjected cells. Scale bar represents 10 µm.</p

Gonococcal infection causes durations of mitosis, nuclear swelling, and targets regulatory mitotic genes and proteins.

<p>A. VK2/E6E7 cells were synchronized and bacteria were allowed to adhere and invade cells for 24 h. Cells were then fixed and stained with DAPI. The nuclear area of 300 infected and 300 uninfected cells were measured. Data were analyzed using a paired 2-tailed Student's <i>t</i>-test. The average nuclear areas observed in 3 independent experiments are shown (*<i>p</i><0.05). Error bars represent S.E.M. B. Cells were infected with <i>N. gonorrhoeae</i> and observed for 24 h by live-cell microscopy. DIC images were captured every 15 min at randomly selected positions, and times spent transitioning from prophase to cytokinesis were measured for 150 mitotic cells. The graph shows mean ± standard deviation values for the time required for progression from prophase to cytokinesis, measured in 3 independent experiments. C. VK2/E6E7 cells were infected with <i>N. gonorrhoeae</i> for 24 h. RNA from control cells and infected cells was isolated and qPCR was performed. Average mRNA levels of <i>MAD1L1</i> and <i>MAD2L1</i> from 3 independent experiments are shown (*<i>p</i><0.05). Error bars represent S.E.M. Values were normalized against <i>TUBA1A</i> expression and analyzed using a paired 2-tailed Student's <i>t</i>-test. D. Protein expression in infected cells and control cells was analyzed in western blots using antibodies against of MAD1 and MAD2. GAPDH or α-tubulin was used as loading controls. E. Western blot band intensities were quantified and analyzed with a paired 2-tailed Student's <i>t</i>-test, using GAPDH or α-tubulin expression as controls. Mean expression levels of each protein observed in 3 independent western blots are shown (*<i>p</i><0.05). Error bars represent S.E.M.</p