Tomoyuki Shimazu

Immunobiotic Lactobacillus jensenii Elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the toll-like receptor signaling pathway

ABSTRACT The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

Immunobiotic Lactobacillus jensenii Modulates the Toll-Like Receptor 4-Induced Inflammatory Response via Negative Regulation in Porcine Antigen-Presenting Cells

ABSTRACT Previously, we demonstrated that Lactobacillus jensenii TL2937 attenuates the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial cells. In view of the critical importance of antigen-presenting cell (APC) polarization in immunoregulation, the objective of the present study was to examine the effect of strain TL2937 on the activation patterns of APCs from swine Peyers patches (PPs). We demonstrated that direct exposure of porcine APCs to L. jensenii in the absence of inflammatory signals increased expression of interleukin-10 (IL-10) and transforming growth factor β in CD172a+ APCs and caused them to display tolerogenic properties. In addition, pretreatment of CD172a+ APCs with L. jensenii resulted in differential modulation of the production of pro- and anti-inflammatory cytokines in response to TLR4 activation. The immunomodulatory effect of strain TL2937 was not related to a downregulation of TLR4 but was related to an upregulation of the expression of three negative regulators of TLRs: single immunoglobulin IL-1-related receptor (SIGIRR), A20, and interleukin-1 receptor-associated kinase M (IRAK-M). Our results also indicated that TLR2 has an important role in the anti-inflammatory activity of L. jensenii TL2937, since anti-TLR2 antibodies blocked the upregulation of SIGIRR and IRAK-M in CD172a+ APCs and the production of IL-10 in response to TLR4 activation. We performed, for the first time, a precise functional characterization of porcine APCs from PPs, and we demonstrated that CD172a+ cells were tolerogenic. Our findings demonstrate that adherent cells and isolated CD172a+ cells harvested from swine PPs were useful for in vitro study of the inflammatory responses in the porcine gut and the immunomodulatory effects of immunobiotic microorganisms.

Immunoregulatory Effect of Bifidobacteria Strains in Porcine Intestinal Epithelial Cells through Modulation of Ubiquitin-Editing Enzyme A20 Expression

Background We previously showed that evaluation of anti-inflammatory activities of lactic acid bacteria in porcine intestinal epithelial (PIE) cells is useful for selecting potentially immunobiotic strains. Objective The aims of the present study were: i) to select potentially immunomodulatory bifidobacteria that beneficially modulate the Toll-like receptor (TLR)-4-triggered inflammatory response in PIE cells and; ii) to gain insight into the molecular mechanisms involved in the anti-inflammatory effect of immunobiotics by evaluating the role of TLR2 and TLR negative regulators in the modulation of proinflammatory cytokine production and activation of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways in PIE cells. Results Bifidobacteria longum BB536 and B. breve M-16V strains significantly downregulated levels of interleukin (IL)-8, monocyte chemotactic protein (MCP)-1 and IL-6 in PIE cells challenged with heat-killed enterotoxigenic Escherichia coli. Moreover, BB536 and M-16V strains attenuated the proinflammatory response by modulating the NF-κB and MAPK pathways. In addition, our findings provide evidence for a key role for the ubiquitin-editing enzyme A20 in the anti-inflammatory effect of immunobiotic bifidobacteria in PIE cells. Conclusions We show new data regarding the mechanism involved in the anti-inflammatory effect of immunobiotics. Several strains with immunoregulatory capabilities used a common mechanism to induce tolerance in PIE cells. Immunoregulatory strains interacted with TLR2, upregulated the expression of A20 in PIE cells, and beneficially modulated the subsequent TLR4 activation by reducing the activation of MAPK and NF-κB pathways and the production of proinflammatory cytokines. We also show that the combination of TLR2 activation and A20 induction can be used as biomarkers to screen and select potential immunoregulatory bifidobacteria strains.

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyers patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.

Toll-like receptor-2-activating bifidobacteria strains differentially regulate inflammatory cytokines in the porcine intestinal epithelial cell culture system: finding new anti-inflammatory immunobiotics

A total of 23 strains of bifidobacteria taxonomically belonging to five species were tested for their potent immunomodulatory effect using a combination of two methods: the NF-κB-reporter assay using a toll-like receptor 2-expressing transfectant (HEK(pTLR2) system) and the mitogenic assay using porcine Peyers patches immunocompetent cells. Among the four preselected strains from different immunomodulatory groups, Bifidobacterium breve MCC-117 was able to efficiently modulate the inflammatory response triggered by enterotoxigenic Escherichia coli (ETEC) in a porcine intestinal epithelial (PIE) cell line. Moreover, using PIE cells and swine Peyers patches immunocompetent cell co-culture system, we demonstrated that the immunoregulatory effect of B. breve MCC-117 was related to the capacity of the strain to influence PIE and immune cell interactions, leading to the stimulation of regulatory T cells. The results suggested that bifidobacteria that express high activity in both the HEK(pTLR2) and the mitogenic assays may behave like potential anti-inflammatory strains. The combination of the HEK(pTLR2) system, the evaluation of mitogenic activity and PIE cells will be of value for the development of new immunologically functional foods and feeds that could prevent inflammatory intestinal disorders. Although our findings should be proven in appropriate experiments in vivo, the results of the present work provide a scientific rationale for the use of B. breve MCC-117 to prevent ETEC-induced intestinal inflammation.

CD86 is expressed on murine hematopoietic stem cells and denotes lymphopoietic potential.

A unique subset of CD86(-) HSCs was previously discovered in mice that were old or chronically stimulated with lipopolysaccharide. Functionally defective HSCs were also present in those animals, and we now show that CD86(-) CD150(+) CD48(-) HSCs from normal adult mice are particularly poor at restoring the adaptive immune system. Levels of the marker are high on all progenitors with lymphopoietic potential, and progressive loss helps to establish relations between progenitors corresponding to myeloid and erythroid lineages. CD86 represents an important tool for subdividing HSCs in several circumstances, identifying those unlikely to generate a full spectrum of hematopoietic cells.

Lactobacillus delbrueckii TUA4408L and its extracellular polysaccharides attenuate enterotoxigenic Escherichia coli-induced inflammatory response in porcine intestinal epitheliocytes via Toll-like receptor-2 and 4

SCOPE Immunobiotics are known to modulate intestinal immune responses by regulating Toll-like receptor (TLR) signaling pathways, which are responsible for the induction of cytokines and chemokines in response to microbial-associated molecular patterns. However, little is known about the immunomodulatory activity of compounds or molecules from immunobiotics. METHODS AND RESULTS We evaluated whether Lactobacillus delbrueckii subsp. delbrueckii TUA4408L (Ld) or its extracellular polysaccharide (EPS): acidic EPS (APS) and neutral EPS (NPS), modulated the response of porcine intestinal epitheliocyte (PIE) cells against Enterotoxigenic Escherichia coli (ETEC) 987P. The roles of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effects were also studied. ETEC-induced inflammatory cytokines were downregulated when PIE cells were prestimulated with both Ld or EPSs. Ld, APS, and NPS inhibited ETEC mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation by upregulating TLR negative regulators. The capability of Ld to suppress inflammatory cytokines was diminished when PIE cells were blocked with anti-TLR2 antibody, while APS failed to suppress inflammatory cytokines when cells were treated with anti-TLR4 antibody. Induction of Ca²⁺ fluxes in TLR knockdown cells confirmed that TLR2 plays a principal role in the immunomodulatory action of Ld, while the activity of APS is mediated by TLR4. In addition, NPS activity depends on both TLR4 and TLR2. CONCLUSION Ld and its EPS have the potential to be used for the development of anti-inflammatory functional foods to prevent intestinal diseases in both humans and animals.

The toll-like receptor family protein RP105/MD1 complex is involved in the immunoregulatory effect of exopolysaccharides from Lactobacillus plantarum N14

The radioprotective 105 (RP105)/MD1 complex is a member of the Toll-like receptor (TLR) family. It was reported that RP105/MD1 cooperates with the lipopolysaccharide (LPS) receptor TLR4/MD2 complex and plays a crucial role in the response of immune cells to LPS. This work evaluated whether RP105, TLR4 or TLR2 were involved in the immunoregulatory capacities of Lactobacillus plantarum N14 (LP14) or its exopolysaccharides (EPS). EPS from LP14 were fractionated into neutral (NPS) and acidic (APS) EPS by anion exchange chromatography. Experiments with transfectant HEK(RP105/MD1) and HEK(TLR2) cells demonstrated that LP14 strongly activated NF-κB via RP105 and TLR2. When we studied the capacity of APS to activate NF-κB pathway in HEK(RP105/MD1) and HEK(TLR4) cells; we observed that APS strongly stimulated both transfectant cells. Our results also showed that LP14 and APS were able to decrease the production of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in porcine intestinal epithelial (PIE) cells in response to enterotoxigenic Escherichia coli (ETEC) challenge. In order to confirm the role of TLR2, TLR4 and RP105 in the immunoregulatory effect of APS from LP14, we used small interfering RNA (siRNA) to knockdown these receptors in PIE cells. The capacity of LP14 and APS to modulate pro-inflammatory cytokine expression was significantly reduced in PIE(RP105-/-) cells. It was also shown that LP14 and APS were capable of upregulating negative regulators of the TLR signaling in PIE cells. This work describes for the first time that a Lactobacillus strain and its EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependend manner.

Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression.

We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′‐TTAGGG‐3′, which has a six‐base loop head structure, and a 3′‐oligo (dG)3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)‐immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)‐6, IL‐12p35, IL‐12p40, and interferon (IFN) γ mRNA expression, but also IL‐4 and IL‐13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen‐immunized mice, suggesting usefulness in the treatment of diseases induced by over‐active immune activation.

Advanced application of bovine intestinal epithelial cell line for evaluating regulatory effect of lactobacilli against heat-killed enterotoxigenic Escherichia coli-mediated inflammation.

BackgroundPreviously, a bovine intestinal epithelial cell line (BIE cells) was successfully established. This work hypothesized that BIE cells are useful in vitro model system for the study of interactions of microbial- or pathogen-associated molecular patterns (MAMPs or PAMPs) with bovine intestinal epithelial cells and for the selection of immunoregulatory lactic acid bacteria (LAB).ResultsAll toll-like receptor (TLR) genes were expressed in BIE cells, being TLR4 one of the most strongly expressed. We demonstrated that heat-stable PAMPs of enterotoxigenic Escherichia coli (ETEC) significantly enhanced the production of IL-6, IL-8, IL-1α and MCP-1 in BIE cells by activating both NF-κB and MAPK pathways. We evaluated the capacity of several lactobacilli strains to modulate heat-stable ETEC PAMPs-mediated inflammatory response in BIE cells. Among these strains evaluated, Lactobacillus casei OLL2768 attenuated heat-stable ETEC PAMPs-induced pro-inflammatory response by inhibiting NF-κB and p38 signaling pathways in BIE cells. Moreover, L. casei OLL2768 negatively regulated TLR4 signaling in BIE cells by up-regulating Toll interacting protein (Tollip) and B-cell lymphoma 3-encoded protein (Bcl-3).ConclusionsBIE cells are suitable for the selection of immunoregulatory LAB and for studying the mechanisms involved in the protective activity of immunobiotics against pathogen-induced inflammatory damage. In addition, we showed that L. casei OLL2768 functionally modulate the bovine intestinal epithelium by attenuating heat-stable ETEC PAMPs-induced inflammation. Therefore L. casei OLL2768 is a good candidate for in vivo studying the protective effect of LAB against intestinal inflammatory damage induced by ETEC infection or heat-stable ETEC PAMPs challenge in the bovine host.