Immunobiotic Bacteria Attenuate Hepatic Fibrosis through the Modulation of Gut Microbiota and the Activation of Aryl-Hydrocarbon Receptors Pathway in Non-Alcoholic Steatohepatitis Mice.

SCOPE: Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease worldwide that can progress to liver fibrosis (LF). Probiotics have beneficial roles in reducing intestinal inflammation and gut-associated diseases, but their effects and mechanisms beyond the gut in attenuating the progression of LF are remained unclear. METHODS AND RESULTS: In a mouse model of NASH/LF induced by a methionine-choline deficient (MCD) diet, immunobiotics are administered to investigate their therapeutic effects. Results show that the MCD diet leads to liver inflammation, steatosis, and fibrosis, which are alleviated by immunobiotics. Immunobiotics reduces serum endotoxin and inflammatory markers while increasing regulatory cytokines and liver weight. They also suppress Th17 cells, known for producing inflammatory cytokines. Furthermore, immunobiotics mitigate collagen deposition and fibrogenic signaling in the liver, while restoring gut-barrier integrity and microbiota composition. Additionally, immunobiotics enhance the activation of the aryl hydrocarbon receptor (AhR) pathway in both colonic and liver tissues. CONCLUSIONS: Overall, these results demonstrate a novel insight into the mechanisms through which immunobiotic administration improves the gut health which in turn increases the AhR pathway and inhibits HSCs activation and fibrosis progression beyond the gut in the liver tissue of NASH/LF mice.

p120-Catenin suppresses NLRP3 inflammasome activation in macrophages.

Inflammasome activation is of central importance for the process of generation of overwhelming inflammatory response and the pathogenesis of sepsis. The intrinsic molecular mechanism for controlling inflammasome activation is still poorly understood. Here we investigated the role of p120-catenin expression in macrophages in regulating nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)- and pyrin domain-containing proteins 3 (NLRP3) inflammasome activation. Depletion of p120-catenin in murine bone marrow-derived macrophages enhanced caspase-1 activation and secretion of active interleukin (IL)-1ß in response to ATP stimulation following LPS priming. Coimmunoprecipitation analysis showed that p120-catenin deletion promoted NLRP3 inflammasome activation by accelerating the assembly of the inflammasome complex comprised of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Depletion of p120-catenin also increased the production of mitochondrial reactive oxygen species. Pharmacological inhibition of mitochondrial reactive oxygen species nearly completely abolished NLRP3 inflammasome activation, caspase-1 activation, and the production of IL-1ß in p120-catenin-depleted macrophages. Furthermore, p120-catenin ablation significantly disrupted mitochondrial function, evidenced by decreased mitochondrial membrane potential and lower production of intracellular ATP. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of p120-catenin-deficient macrophages dramatically enhanced the accumulation of IL-1ß and IL-18 in bronchoalveolar lavage fluid. These results demonstrate that p120-catenin prevents NLRP3 inflammasome activation in macrophages by maintaining mitochondrial homeostasis and reducing the production of mitochondrial reactive oxygen species in response to endotoxin insult. Thus, inhibition of NLRP3 inflammasome activation by stabilization of p120-catenin expression in macrophages may be a novel strategy to prevent an uncontrolled inflammatory response in sepsis.

Probiotics counteract the expression of hepatic profibrotic genes via the attenuation of TGF-ß/SMAD signaling and autophagy in hepatic stellate cells.

Hepatic fibrosis is caused by the increased accumulation and improper degradation of extracellular matrix (ECM) proteins in the liver. Hepatic stellate cells (HSCs) activation is a key process in initiating hepatic fibrosis and can be ameliorated by the administration of probiotic strains. This study hypothesized that LAB strains (Lactiplantibacillus plantarum, Lactobacillus brevis, and Weissella cibaria) might attenuate pro-fibrogenic cytokine TGF-ß mediated HSCs activation and induce collagen deposition, expression of other fibrogenic/inflammatory markers, autophagy, and apoptotic processes in vitro. Few studies have evaluated the probiotic effects against fibrogenesis in vitro. In this study, TGF-ß exposure increased collagen deposition in LX-2 cells, but this increase was diminished when the cells were pretreated with LAB strains before TGF-ß stimulation. TGF-ß not only increased collagen deposition, but it also significantly upregulated the mRNA levels of Col1A1, alpha-smooth muscle actin (α-SMA), matrix metalloproteinases-2 (MMP-2), IL-6, CXCL-8, CCL2, and IL-1ß in LX-2 cells. Pretreatment of the cells with LAB strains counteracted the TGF-ß-induced pro-fibrogenic and inflammatory markers by modulating SMAD-dependent and SMAD-independent TGF-ß signaling. In addition, LX-2 cells exposed to TGF-ß induced the autophagic and apoptotic associated proteins that were also positively regulated by the LAB strains. These findings suggest that LAB can attenuate TGF-ß signaling that is associated with liver fibrogenesis.

The Respiratory Commensal Bacterium Dolosigranulum pigrum 040417 Improves the Innate Immune Response to Streptococcus pneumoniae.

Previously, we demonstrated that the nasal administration of Dolosigranulum pigrum 040417 differentially modulated the respiratory innate immune response triggered by the activation of Toll-like receptor 2 in infant mice. In this work, we aimed to evaluate the beneficial effects of D. pigrum 040417 in the context of Streptococcus pneumoniae infection and characterize the role of alveolar macrophages (AMs) in the immunomodulatory properties of this respiratory commensal bacterium. The nasal administration of D. pigrum 040417 to infant mice significantly increased their resistance to pneumococcal infection, differentially modulated respiratory cytokines production, and reduced lung injuries. These effects were associated to the ability of the 040417 strain to modulate AMs function. Depletion of AMs significantly reduced the capacity of the 040417 strain to improve both the reduction of pathogen loads and the protection against lung tissue damage. We also demonstrated that the immunomodulatory properties of D. pigrum are strain-specific, as D. pigrum 030918 was not able to modulate respiratory immunity or to increase the resistance of mice to an S. pneumoniae infection. These findings enhanced our knowledge regarding the immunological mechanisms involved in modulation of respiratory immunity induced by beneficial respiratory commensal bacteria and suggested that particular strains could be used as next-generation probiotics.

The Gut Microbiota: How Does It Influence the Development and Progression of Liver Diseases.

The gut-liver axis plays important roles in both the maintenance of a healthy liver and the pathogenesis of liver diseases, where the gut microbiota acts as a major determinant of this relationship. Gut bacteria-derived metabolites and cellular components are key molecules that affect the function of the liver and modulate the pathology of liver diseases. Accumulating evidence showed that gut microbiota produces a myriad of molecules, including lipopolysaccharide, lipoteichoic acid, peptidoglycan, and DNA, as well as short-chain fatty acids, bile acids, trimethylamine, and indole derivatives. The translocation of these components to the liver exerts beneficial or pathogenic effects by interacting with liver immune cells. This is a bidirectional relationship. Therefore, the existence of crosstalk between the gut and liver and its implications on host health and diseases are essential for the etiology and treatment of diseases. Several mechanisms have been proposed for the pathogenesis of liver diseases, but still, the mechanisms behind the pathogenic role of gut-derived components on liver pathogenesis remain elusive and not understandable. This review discusses the current progress on the gut microbiota and its components in terms of the progression of liver diseases, and in turn, how liver diseases indirectly affect the intestinal function and induce intestinal inflammation. Moreover, this paper highlights the current therapeutic and preventive strategies used to restore the gut microbiota composition and improve host health.

The Ability of Respiratory Commensal Bacteria to Beneficially Modulate the Lung Innate Immune Response Is a Strain Dependent Characteristic.

We investigated whether the ability of commensal respiratory bacteria to modulate the innate immune response against bacterial and viral pathogens was a shared or strain-specific characteristic. Bacterial strains belonging to the Corynebacterium pseudodiphtheriticum and Dolosigranulum pigrum species were compared by studying their influence in the Toll-like receptor (TLR)-2- and TLR3-triggered immune responses in the respiratory tract, as well as in the resistance to Respiratory Syncytial Virus (RSV) and Streptococcus pneumoniae infections. We demonstrated that nasally administered C. pseudodiphteriticum 090104 or D. pigrum 040417 were able to modulate respiratory immunity and increase the resistance against pathogens, while other strains of the same species did not influence the respiratory immune responses, demonstrating a clear strain-dependent immunomodulatory effect of respiratory commensal bacteria. We also reported here that bacterium-like particles (BLP) and cell walls derived from immunomodulatory respiratory commensal bacteria are an interesting alternative for the modulation of the respiratory immune system. Our study is a step forward in the positioning of certain strains of respiratory commensal bacteria as next-generation probiotics for the respiratory tract.

Beneficial effect of immunobiotic strains on attenuation of Salmonella induced inflammatory response in human intestinal epithelial cells.

Probiotic bacteria have the ability to modulate host immune responses and have potent therapeutic functional effects against several diseases, including inflammatory diseases. However, beneficial effects of probiotics are strain specific and their interactions with host immune cells to modulate inflammatory response are largely unknown. Intestinal epithelial cells (IECs), which are the first line of defense against invading pathogens, and connects between commensals/probiotics and immune system; therefore, in this study, we used human IECs to assess the probiotic effects of three selected Lactobacillus strains in vitro. An HT-29 colonic epithelial cell and HT-29/blood mononuclear cells co-culture system were stimulated with Lactobacillus followed by Salmonella for different hours, after which the mRNA level of cytokines, ß-defensin-2 and negative regulators for TLR signaling and protein levels of ZO-1 and IκB-α were analyzed by real-time polymerase chain reaction and western blot analysis. L. brevis decreased Salmonella induced IL-6, IL-8, MCP-1 and IL-1ß levels, whereas L. pentosus suppressed IL-6 and MCP-1 in HT-29 cells. Moreover, L. brevis was able to increase the mRNA levels of A20, Tollip, SIGIRR and IRAKM, while L. pentosus reduced the levels of A20, and IRAKM in response to Salmonella. In addition, decrease in protein level of TNF-α and increase in mRNA level of IL-10 was observed in L. brevis and L. pentosus treated HT-29 cells. Lactobacillus strains were differentially modulated ZO-1 and p-IκB-α in HT-29 cells treated with Salmonella. Overall, the results of this study indicate that Lactobacillus strains attenuate Salmonella induced inflammatory responses through beneficial modulation of TLR negative regulators and the NF-κB pathway.

Study on physical and mechanical properties of the biopolymer/silver based active nanocomposite films with antimicrobial activity.

Silver nanoparticles (AgNPs) were prepared by reducing AgNO3 using biopolymer pullulan as both a reducing and stabilizing agent. The prepared AgNPs solution was blended with pectin to make active nanocomposite films. The formation of AgNPs in the solution was confirmed by characteristic surface plasmon resonance (SPR) peak of AgNPs at 400-500 nm, using UV-vis absorption spectroscopy. The prepared composite and nanocomposite films were characterized using UV, FE-SEM, and FT-IR. In addition, films color, optical, water contact angle, water vapor permeability, mechanical and antimicrobial properties were evaluated. FE-SEM analysis showed uniform distribution of AgNPs in the resulting nanocomposites films. The presence of AgNPs could affect the physical and mechanical properties of the prepared films. The color, moisture content, water vapor barrier properties, hydrophobicity, thickness, and elongation at break of the films were significantly increased after formation of composite with AgNPs, but tensile strength and elastic modulus of the films were decreased. FT-IR results indicated that AgNPs had good compatibility with biopolymers. In addition, nanocomposite films, especially pullulan/AgNPs and pullulan/pectin/AgNPs films exhibited better antimicrobial activity against food born pathogens, which suggests that prepared nanocomposite films can be used as active food packaging material to maintain food safety and to improve shelf life of the packaged food.

Immunobiotic Strains Modulate Toll-Like Receptor 3 Agonist Induced Innate Antiviral Immune Response in Human Intestinal Epithelial Cells by Modulating IFN Regulatory Factor 3 and NF-κB Signaling.

Many studies have demonstrated that immunobiotics with immunoregulatory functions improve the outcomes of several bacterial and viral infections by modulating the mucosal immune system. However, the precise mechanisms underlying the immunoregulatory and antiviral activities of immunobiotics have not yet been elucidated in detail. The present study was conducted to determine whether selected lactic acid bacteria (LAB) modulate toll-like receptor 3 (TLR3) agonist polyinosinic:polycytidylic acid (PolyI:C) induced viral response in human intestinal epithelial cells (IECs). PolyI:C increased the expression of interferon-ß (IFN-ß), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein (MCP-1), and interleukin-1ß (IL-1ß) in HCT116 cells, and these up-regulations were significantly altered when cells were pre-stimulated with LAB isolated from Korean fermented foods. LAB strains were capable to up-regulate IFN-ß but down-regulated IL-6, IL-8, MCP-1, and IL-1ß mRNA levels as compared with PolyI: C. HCT-116 cell treatment with LABs beneficially modulated the mRNA levels of C-X-C motif chemokine 10 (CXCL-10), 2-5A oligoadenylate synthetase 1 (OSA1), myxovirus resistance protein (MxA), TLR3, and retinoic acid inducible gene-I (RIG-I), and TLR negative regulators. In addition, LABs increased IFN-ß, IFN-α, and interleukin-10 (IL-10) and decreased tumor necrosis factor-α (TNF-α) and IL-1ß protein/mRNA levels in THP-1 cells. LABs also protected the cells by maintaining tight-junction proteins (zonula occludens-1 and occludin). The beneficial effects of these LABs were mediated via modulation of the interferon regulatory factor 3 (IRF3) and nuclear factor-kappa B (NF-κB) pathways. Overall, the results of this study indicate that immunobiotics have potent antiviral and anti-inflammatory activities that may use as antiviral substitutes for human and animal applications.

Immunobiotics Beneficially Modulate TLR4 Signaling Triggered by Lipopolysaccharide and Reduce Hepatic Steatosis In Vitro.

Hepatic inflammation and injury may result from the translocation of pathological bacteria and their proinflammatory mediators. Probiotics attenuate hepatic diseases related to inflammation by exhibiting immunoregulatory effects. Therefore, this study was conducted to evaluate lipid reduction and immunoregulatory potentials of probiotic bacteria in vitro. HepG2 cells treated with total cellular fluid (TCF) of LABs reduced lipid accumulation. Moreover, cells responded to lipopolysaccharide (LPS) by producing higher levels of IL-6, IL-8, MCP-1, and TNF-α. TCF of LABs treatment showed remarkably diminished levels of the expression of these cytokines via modulation of the expression of TLR-negative regulators, as well as MAPK and NF-κB pathways. Moreover, heat-killed LABs were able to diminish TGF-ß, IL-1ß, and IL-6 and to increase IL-10 and TLR4 levels in THP-1 cells. LABs also decreased the protein level of TNF-α. These results demonstrated that immunobiotics exhibit potent immunoregulatory activity and may be used as effective therapeutic agents to alleviate inflammatory response.

Functional capabilities of probiotic strains on attenuation of intestinal epithelial cell inflammatory response induced by TLR4 stimuli.

Intestinal epithelial cells (IECs) respond to intruders and their cellular molecules by displaying inflammatory state that can be abrogated by probiotics. However, the molecular mechanisms underlying the beneficial activity of probiotic strains have yet to be elucidated. This study was conducted to investigate whether probiotic strains have immunoregulatory effects in IECs, and how they respond to bacterial lipopolysaccharide (LPS) in vitro. Caco2 cells were stimulated with LABs and followed by LPS. The expression of different cytokines that involved in toll-like receptor (TLR) signaling was analyzed. Caco2 cells that were exposed to LPS showed upregulated expression of IL-6, CXCL8, CCL2, and BPI that were counteracted by LAB strains through the modulation of TLR negative regulators (A20, Tollip, SIGIRR, and IRAKM), p38 MAPK and p65 NF-κB signaling. Lactobacillus plantarum, L. farciminis, and L. pentosus unveiled better activity as compared to other strains. Moreover, LAB strains were the potent inducers of immunoregulatory cytokines in coculture system. The expression of IL-10 and TGF-ß were found to be higher as compared with LPS. Conversely, TNF-α at the protein level was dampened by LAB strains in both the apical and basolateral compartments. Collectively, our results demonstrated that the selected LAB strains exert profound immunoregulatory effects in response to LPS on IECs; however, further studies in vivo and in clinical settings are important to corroborate these effects. © 2018 BioFactors, 45(2):223-235, 2019.

Genomic Characterization of Lactobacillus delbrueckii TUA4408L and Evaluation of the Antiviral Activities of its Extracellular Polysaccharides in Porcine Intestinal Epithelial Cells.

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-ß, Myxovirus resistance gene A (MxA) and RNaseL.

Protective Effects of Lactic Acid Bacteria Against TLR4 Induced Inflammatory Response in Hepatoma HepG2 Cells Through Modulation of Toll-Like Receptor Negative Regulators of Mitogen-Activated Protein Kinase and NF-κB Signaling.

The beneficial effects of probiotics in several liver diseases have been investigated in both animal and clinical models; however, the precise mechanisms responsible for their effects have not yet been elucidated. Gut transmitted endotoxins such as LPS have been shown to play critical roles in hepatic inflammation and injury. Therefore, in this study, we investigated the beneficial role of selected lactic acid bacteria (LABs) on reduction of hepatic steatosis (HS) and attenuation of LPS induced inflammatory response in vitro. Total cellular fluid (TCF) of LABs treatment reduced HS by decreasing the amount of lipid accumulation in vitro. Additionally, HepG2 cells exposed to LPS showed increased expression of exacerbated inflammatory cytokines, such as IL-6, CXCL8, CCL2, and TNF-α, but these effects were counteracted when cells were treated with TCF of LABs prior to LPS challenge. Moreover, TCF of LABs was able to modulate mRNA levels of TLR negative regulators and protein levels of p38 MAPK and p65 NF-κB transcription factors. However, these modulations were differed remarkably between both free fatty acid treated and untreated HepG2 cells. Heat-killed LABs were also indirectly suppressed THP-1 cells to produce higher level of IL-10, TLR4, and lower at genes level of TGF-ß, IL-1ß, and IL-6, and at protein level of TNF-α in response to LPS. Taken together, our findings indicate that selected LABs exhibit profound immunoregulatory effects on liver cells via modulation of TLR negative regulators of the MAPK and NF-κB pathways.

Exopolysaccharides from Lactobacillus delbrueckii OLL1073R-1 modulate innate antiviral immune response in porcine intestinal epithelial cells.

Previous studies demonstrated that the extracellular polysaccharides (EPSs) produced by Lactobacillus delbrueckii OLL1073R-1 (LDR-1) improve antiviral immunity, especially in the systemic and respiratory compartments. However, it was not studied before whether those EPSs are able to beneficially modulate intestinal antiviral immunity. In addition, LDR-1-host interaction has been evaluated mainly with immune cells while its interaction with intestinal epithelial cells (IECs) was not addressed before. In this work, we investigated the capacity of EPSs from LDR-1 to modulate the response of porcine IECs (PIE cells) to the stimulation with the Toll-like receptor (TLR)-3 agonist poly(I:C) and the role of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effect. We showed that innate immune response triggered by TLR3 activation in porcine IECs was differentially modulated by EPS from LDR-1. EPSs treatment induced an increment in the expression of interferon (IFN)-α and IFN-ß in PIE cells after the stimulation with poly(I:C) as well as the expression of the antiviral factors MxA and RNase L. Those effects were related to the reduced expression of A20 in EPS-treated PIE cells. EPS from LDR-1 was also able to reduce the expression of IL-6 and proinflammatory chemokines. Although further in vivo studies are needed, our results suggest that these EPSs or a yogurt fermented with LDR-1 have potential to improve intestinal innate antiviral response and protect against intestinal viruses.

Respiratory Commensal Bacteria Corynebacterium pseudodiphtheriticum Improves Resistance of Infant Mice to Respiratory Syncytial Virus and Streptococcus pneumoniae Superinfection.

Corynebacterium pseudodiphtheriticum is a Gram-positive bacterium found as a member of the normal microbiota of the upper respiratory tract. It was suggested that C. pseudodiphtheriticum may be potentially used as a next-generation probiotic for nasal application, although no deep studies were performed in this regard. We hypothesized that human isolate C. pseudodiphtheriticum strain 090104 is able to modulate the respiratory innate immune response and beneficially influence the resistance to viral and bacterial infections. Therefore, in the present study we investigated how the exposure of infant mice to nasal priming with viable or non-viable C. pseudodiphtheriticum 090104 influences the respiratory innate immune response triggered by Toll-like receptor (TLR)-3 activation, the susceptibility to primary Respiratory Synsytial Virus (RSV) infection, and the resistance to secondary Streptococcus pneumoniae pneumonia. We demonstrated that the nasal priming with viable C. pseudodiphtheriticum 090104 differentially modulated TLR3-mediated innate antiviral immune response in the respiratory tract of infant mice, improving their resistance to primary RSV infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that viable C. pseudodiphtheriticum improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-ß+ alveolar macrophages. Of interest, non-viable bacteria did not have the same protective effect, suggesting that C. pseudodiphtheriticum colonization is needed for achieving its protective effect. In conclusion, we present evidence that nasal application of viable C. pseudodiphtheriticum could be thought as an alternative to boost defenses against RSV and secondary pneumococcal pneumonia, which should be further studied and validated in clinical trials. Due to the absence of a long-lasting immunity, re-infection with RSV throughout life is common. Thus, a possible perspective use could be a seasonal application of a nasal probiotic spray to boost respiratory innate immunity in immunocompetent subjects.

Peptidoglycan from Immunobiotic Lactobacillus rhamnosus Improves Resistance of Infant Mice to Respiratory Syncytial Viral Infection and Secondary Pneumococcal Pneumonia.

Several research works have demonstrated that beneficial microbes with the capacity to modulate the mucosal immune system (immunobiotics) are an interesting alternative to improve the outcome of bacterial and viral respiratory infections. Among the immunobiotic strains with the capacity to beneficially modulate respiratory immunity, Lactobacillus rhamnosus CRL1505 has outstanding properties. Although we have significantly advanced in demonstrating the capacity of L. rhamnosus CRL1505 to improve resistance against respiratory infections as well as in the cellular and molecular mechanisms involved in its beneficial activities, the potential protective ability of this strain or its immunomodulatory cellular fractions in the context of a secondary bacterial pneumonia has not been addressed before. In this work, we demonstrated that the nasal priming with non-viable L. rhamnosus CRL1505 or its purified peptidoglycan differentially modulated the respiratory innate antiviral immune response triggered by toll-like receptor 3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection, and secondary pneumococcal pneumonia. In association with the protection against RSV-pneumococcal superinfection, we found that peptidoglycan from L. rhamnosus CRL1505 significantly improved lung CD3+CD4+IFN-γ+, and CD3+CD4+IL-10+ T cells as well as CD11c+SiglecF+IFN-ß+ alveolar macrophages with the consequent increases of IFN-γ, IL-10, and IFN-ß in the respiratory tract. Our results also showed that the increase of these three cytokines is necessary to achieve protection against respiratory superinfection since each of them are involved in different aspect of the secondary pneumococcal pneumonia that have to be controlled in order to reduce the severity of the infectious disease: lung pneumococcal colonization, bacteremia, and inflammatory-mediated lung tissue injury.

Immunoregulatory Effects Triggered by Lactic Acid Bacteria Exopolysaccharides: New Insights into Molecular Interactions with Host Cells.

Researchers have demonstrated that lactic acid bacteria (LAB) with immunomodulatory capabilities (immunobiotics) exert their beneficial effects through several molecules, including cell wall, peptidoglycan, and exopolysaccharides (EPS), that are able to interact with specific host cell receptors. EPS from LAB show a wide heterogeneity in its composition, meaning that biological properties depend on the strain and. therefore, only a part of the mechanism of action has been elucidated for these molecules. In this review, we summarize the current knowledge of the health-promoting actions of EPS from LAB with special focus on their immunoregulatory actions. In addition, we describe our studies using porcine intestinal epithelial cells (PIE cells) as a model to evaluate the molecular interactions of EPS from two immunobiotic LAB strains and the host cells. Our studies showed that EPS from immunobiotic LAB have anti-inflammatory capacities in PIE cells since they are able to reduce the production of inflammatory cytokines in cells challenged with the Toll-like receptor (TLR)-4-agonist lipopolysaccharide. The effects of EPS were dependent on TLR2, TLR4, and negative regulators of TLR signaling. We also reported that the radioprotective 105 (RP105)/MD1 complex, a member of the TLR family, is partially involved in the immunoregulatory effects of the EPS from LAB. Our work described, for the first time, that LAB and their EPS reduce inflammation in intestinal epithelial cells in a RP105/MD1-dependent manner. A continuing challenge for the future is to reveal more effector-receptor relationships in immunobiotic-host interactions that contribute to the beneficial effects of these bacteria on mucosal immune homeostasis. A detailed molecular understanding should lead to a more rational use of immunobiotics in general, and their EPS in particular, as efficient prevention and therapies for specific immune-related disorders in humans and animals.

Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells.

BACKGROUND: Immunobiotic Lactobacillus jensenii TL2937 modulates porcine mononuclear phagocytes from Peyer's patches (PPMPs) and induces a differential production of pro- and anti-inflammatory cytokines in response to Toll-like receptor (TLR)-4 activation. In view of the important role played by phagocytosis in the activation of antigen presenting cells (APCs), the aim of the present work was to examine the interaction of TL2937 with porcine PPMPs focusing on phagocytosis. In addition, this study aimed to investigate whether the effects of L. jensenii TL2937 in porcine blood monocyte-derived dendritic cells (MoDCs) are similar to those found in PPMPs considering that MoDCs do not recapitulate all functions of mucosal APCs. RESULTS: Studies showed a high ability of porcine CD172a(+) PPMPs to phagocytose L. jensenii TL2937. Interestingly, our results also revealed a reduced capacity of the non-immunomodulatory L. plantarum TL2766 to be phagocytosed by those immune cells. Phagocytosis of L. jensenii TL2937 by porcine PPMPs was partially dependent on TLR2. In addition, we demonstrated that TL2937 strain was able to improve the expression of IL-1ß, IL-12 and IL-10 in immature MoDCs resembling the effect of this immunobiotic bacterium on PPMPs. Moreover, similarly to PPMPs those immunomodulatory effects were related to the higher capacity of TL2937 to be phagocytosed by immature MoDCs. CONCLUSIONS: Microbial recognition in APCs could be effectively mediated through ligand-receptor interactions that then mediate phagocytosis and signaling. For the immunobiotic strain TL2937, TLR2 has a partial role for its interaction with porcine APCs and it is necessary to investigate the role of other receptors. A challenge for future research will be advance in the full understanding of the molecular interactions of immunobiotic L. jensenii TL2937 with porcine APCs that will be crucial for the successful development of functional feeds for the porcine host. This study is a step in that direction.

Immunobiotic Bifidobacteria Strains Modulate Rotavirus Immune Response in Porcine Intestinal Epitheliocytes via Pattern Recognition Receptor Signaling.

In this work, we aimed to characterize the antiviral response of an originally established porcine intestinal epithelial cell line (PIE cells) by evaluating the molecular innate immune response to rotavirus (RVs). In addition, we aimed to select immunomodulatory bacteria with antiviral capabilities. PIE cells were inoculated with RVs isolated from different host species and the infective titers and the molecular innate immune response were evaluated. In addition, the protection against RVs infection and the modulation of immune response by different lactic acid bacteria (LAB) strains was studied. The RVs strains OSU (porcine) and UK (bovine) effectively infected PIE cells. Our results also showed that RVs infection in PIE cells triggered TLR3-, RIG-I- and MDA-5-mediated immune responses with activation of IRF3 and NF-κB, induction of IFN-ß and up-regulation of the interferon stimulated genes MxA and RNase L. Among the LAB strains tested, Bifidobacterium infantis MCC12 and B. breve MCC1274 significantly reduced RVs titers in infected PIE cells. The beneficial effects of both bifidobacteria were associated with reduction of A20 expression, and improvements of IRF-3 activation, IFN-ß production, and MxA and RNase L expressions. These results indicate the value of PIE cells for studying RVs molecular innate immune response in pigs and for the selection of beneficial bacteria with antiviral capabilities.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage.

OBJECTIVE: To evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury. METHODS: Six-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV. RESULTS: Both viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4(+)IFN-γ(+) lymphocytes, and lung CD11c(+)CD11b(low)CD103(+) and CD11c(+)CD11b(high)CD103(-) dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response. CONCLUSIONS: Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.