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1.  Immunobiotic Lactobacillus rhamnosus strains differentially modulate antiviral immune response in porcine intestinal epithelial and antigen presenting cells 
BMC Microbiology  2014;14:126.
Previous findings suggested that Lactobacillus rhamnosus CRL1505 is able to increase resistance of children to intestinal viral infections. However, the intestinal cells, cytokines and receptors involved in the immunoregulatory effect of this probiotic strain have not been fully characterized.
We aimed to gain insight into the mechanisms involved in the immunomodulatory effect of the CRL1505 strain and therefore evaluated in vitro the crosstalk between L. rhamnosus CRL1505, porcine intestinal epithelial cells (IECs) and antigen presenting cells (APCs) from swine Peyer’s patches in order to deepen our knowledge about the mechanisms, through which this strain may help preventing viral diarrhoea episodes. L. rhamnosus CRL1505 was able to induce IFN–α and –β in IECs and improve the production of type I IFNs in response to poly(I:C) challenge independently of Toll-like receptor (TLR)-2 or TLR9 signalling. In addition, the CRL1505 strain induced mRNA expression of IL-6 and TNF-α via TLR2 in IECs. Furthermore, the strain significantly increased surface molecules expression and cytokine production in intestinal APCs. The improved Th1 response induced by L. rhamnosus CRL1505 was triggered by TLR2 signalling and included augmented expression of MHC-II and co-stimulatory molecules and expression of IL-1β, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs.
It was recently reviewed the emergence of TLR agonists as new ways to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies. The use of L. rhamnosus CRL1505 as modulator of innate immunity and inductor of antiviral type I IFNs, IFN-γ, and regulatory IL-10 clearly offers the potential to overcome this challenge.
PMCID: PMC4035899  PMID: 24886142
Lactobacillus rhamnosus; Poly(I:C); Antiviral immunity; PIE cells; Intestinal antigen presenting cells; TLR2
2.  Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection 
BMC Immunology  2013;14:40.
Some studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. However, the capacity of immunobiotics to improve protection against respiratory syncytial virus (RSV) infection was not investigated before.
The aims of this study were: a) to evaluate whether the nasal administration of Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by TLR3/RIG-I activation; b) to investigate whether viability of Lr05 or Lr06 is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of Lr05 and Lr06 to improve the resistance of infant mice against RSV infection.
Nasally administered Lr05 and Lr06 differentially modulated the TLR3/RIG-I-triggered antiviral respiratory immune response. Lr06 administration significantly modulated the production of IFN-α, IFN-β and IL-6 in the response to poly(I:C) challenge, while nasal priming with Lr05 was more effective to improve levels of IFN-γ and IL-10. Both viable Lr05 and Lr06 strains increased the resistance of infant mice to RSV infection while only heat-killed Lr05 showed a protective effect similar to those observed with viable strains.
The present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by TLR3/RIG-I activation in the respiratory tract and to increase the resistance of mice to the challenge with RSV. Comparative studies using two Lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(I:C) or RSV challenges, conferring different degree of protection and using distinct immune mechanisms. We also demonstrated in this work that it is possible to beneficially modulate the respiratory defenses against RSV by using heat-killed immunobiotics.
PMCID: PMC3751766  PMID: 23947615
Lactobacillus rhamnosus; Nasal treatment; Poly(I:C); Sntiviral immunity; Respiratory tract; Respiratory syncytial virus
3.  Modulation of Respiratory TLR3-Anti-Viral Response by Probiotic Microorganisms: Lessons Learned from Lactobacillus rhamnosus CRL1505 
Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants and young children. Host immune response is implicated in both protective and immunopathological mechanisms during RSV infection. Activation of Toll-like receptor (TLR)-3 in innate immune cells by RSV can induce airway inflammation, protective immune response, and pulmonary immunopathology. A clear understanding of RSV–host interaction is important for the development of novel and effective therapeutic strategies. Several studies have centered on whether probiotic microorganisms with the capacity to stimulate the immune system (immunobiotics) might sufficiently stimulate the common mucosal immune system to improve defenses in the respiratory tract. In this regard, it was demonstrated that some orally administered immunobiotics do have the ability to stimulate respiratory immunity and increase resistance to viral infections. Moreover, during the last decade scientists have significantly advanced in the knowledge of the cellular and molecular mechanisms involved in the protective effect of immunobiotics in the respiratory tract. This review examines the most recent advances dealing with the use of immunobiotic bacteria to improve resistance against viral respiratory infections. More specifically, the article discuss the mechanisms involved in the capacity of the immunobiotic strain Lactobacillus rhamnosus CRL1505 to modulate the TLR3-mediated immune response in the respiratory tract and to increase the resistance to RSV infection. In addition, we review the role of interferon (IFN)-γ and interleukin (IL)-10 in the immunoregulatory effect of the CRL1505 strain that has been successfully used for reducing incidence and morbidity of viral airways infections in children.
PMCID: PMC4026741  PMID: 24860569
Lactobacillus rhamnosus CRL1505; TLR3; respiratory immunity; respiratory syncytial virus; immunobiotics
4.  Dietary Supplementation with Lactobacilli Improves Emergency Granulopoiesis in Protein-Malnourished Mice and Enhances Respiratory Innate Immune Response 
PLoS ONE  2014;9(4):e90227.
This work studied the effect of protein malnutrition on the hemato-immune response to the respiratory challenge with Streptococcus pneumoniae and evaluated whether the dietary recovery with a probiotic strain has a beneficial effect in that response. Three important conclusions can be inferred from the results presented in this work: a) protein-malnutrition significantly impairs the emergency myelopoiesis induced by the generation of the innate immune response against pneumococcal infection; b) repletion of malnourished mice with treatments including nasally or orally administered Lactobacillus rhamnosus CRL1505 are able to significantly accelerate the recovery of granulopoiesis and improve innate immunity and; c) the immunological mechanisms involved in the protective effect of immunobiotics vary according to the route of administration. The study demonstrated that dietary recovery of malnourished mice with oral or nasal administration of L. rhamnosus CRL1505 improves emergency granulopoiesis and that CXCR4/CXCR12 signaling would be involved in this effect. Then, the results summarized here are a starting point for future research and open up broad prospects for future applications of probiotics in the recovery of immunocompromised malnourished hosts.
PMCID: PMC3972161  PMID: 24691464
5.  Dietary Supplementation with Probiotics Improves Hematopoiesis in Malnourished Mice 
PLoS ONE  2012;7(2):e31171.
Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation.
This study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice.
Protein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat's milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells.
Malnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups.
Protein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised-malnourished hosts.
PMCID: PMC3275617  PMID: 22347448
6.  Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4 
BMC Microbiology  2012;12:15.
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.
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.
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.
PMCID: PMC3305351  PMID: 22264349
7.  Unique Type I Interferon Responses Determine the Functional Fate of Migratory Lung Dendritic Cells during Influenza Virus Infection 
PLoS Pathogens  2011;7(11):e1002345.
Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103+ DCs and CD11bhigh DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103+ DCs allow the virus to replicate to significantly higher levels than do the CD11bhigh DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11bhigh DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103+ DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11bhigh DCs. The attenuated IFNAR signaling by CD103+ DCs correlates with their described superior antigen presentation capacity for naïve CD8+ T cells when compared to CD11bhigh DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The “interferon-resistant” CD103+ DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination.
Author Summary
Migratory lung dendritic cells (DCs) control the initiation of the adaptive immune responses to influenza virus by expanding virus-specific T cells in draining lymph nodes (MLNs) that will subsequently clear the pathogen from the respiratory tract. Here we demonstrate that both subsets of lung DCs, CD103+ DCs and CD11bhigh DCs become infected by influenza virus in vivo and migrate to the MLNs, but only CD103+ DCs support productive virus replication. Enhanced virus replication in CD103+ DCs compared to CD11bhigh DCs was responsible for their superior antigen presentation efficacy for naïve CD8+ T cells and originated from a difference in sensitivity of the two DC populations to type I interferon (I-IFN). These data show that in contrast to most other immune cell types, DCs can become productively infected with influenza virus and I-IFN operates as a master regulator controlling which DC subset will present antigen during a viral infection. A deeper understanding of basic innate and adaptive immune response mechanisms regulated by I-FN may lead to the development of cutting edge therapies and improve vaccine efficacy against influenza and other viruses.
PMCID: PMC3207893  PMID: 22072965
8.  Lactobacillus rhamnosus HN001 Attenuates Allergy Development in a Pig Model 
PLoS ONE  2011;6(2):e16577.
Probiotics have been studied as immunomodulatory agents of allergy. Several human probiotic trials tracking the development of eczema and other forms of allergy have yielded inconsistent results. A recent infant study demonstrated that pre and postnatal Lactobacillus rhamnosus HN001 (HN001) supplementation decreased the prevalence of eczema and IgE associated eczema. However, the influence of HN001 on the incidence of wheeze, asthma, and/or other allergic manifestations has yet to be reported.
This study was conducted to determine the effects of the probiotic HN001 on the development of allergic lung disease in a pig model.
Allergy was induced by a series of subcutaneous and intratracheal sensitizations with Ascaris suum allergen (ASA) during a six week time frame in post-weanling pigs supplemented daily with HN001, or without supplementation. One week following final sensitization intradermal skin tests and respiratory challenges were conducted.
In response to intradermal and respiratory challenges, ASA-sensitized pigs fed HN001 had less severe skin flare reactions, smaller increases in pleural pressure, and trends towards lower changes in arterial oxygen and carbon dioxide partial pressure levels compared to control pigs. The frequency of ASA-specific IFN-γ-secreting peripheral blood mononuclear cells, as well as the amount of IL-10 produced by ASA-specific cells, was of greater magnitude in probiotic-fed pigs compared to control animals. These observations suggest that differences in clinical responses to the allergen challenges may be related to probiotic-induced modulation of Th1 (IFN-γ) and regulatory (IL-10) cytokine expression.
Probiotic supplementation decreased the severity of allergic skin and lung responses in allergen-sensitized pigs with a corresponding increase in IFN-γ expression. A similar correlation between certain allergic responses and increased IFN-γ expression has been reported in human clinical studies of allergy; this pig model of allergy may be indicative of potential probiotic modulation of allergic lung disease in humans.
PMCID: PMC3046142  PMID: 21386995
9.  Differential Toll-Like Receptor Recognition and Induction of Cytokine Profile by Bifidobacterium breve and Lactobacillus Strains of Probiotics ▿ 
The use of probiotics as a food supplement has gained tremendous interest in the last few years as beneficial effects were reported in gut homeostasis and nutrient absorption but also in immunocompromised patients, supporting protection from colonization or infection with pathogenic bacteria or fungi. As a treatment approach for inflammatory bowel diseases, a suitable probiotic strain would ideally be one with a low immunogenic potential. Insight into the immunogenicities and types of T-cell responses induced by potentially probiotic strains allows a more rational selection of a particular strain. In the present study, the bacterial strains Bifidobacterium breve (NumRes 204), Lactobacillus rhamnosus (NumRes1), and Lactobacillus casei (DN-114 001) were compared concerning their capacity to induce inflammatory responses in terms of cytokine production by human and mouse primary immune cells. It was demonstrated that the B. breve strain induced lower levels of the proinflammatory cytokine gamma interferon (IFN-γ) than the tested L. rhamnosus and L. casei strains. Both B. breve and lactobacilli induced cytokines in a Toll-like receptor 9 (TLR9)-dependent manner, while the lower inflammatory profile of B. breve was due to inhibitory effects of TLR2. No role for TLR4, NOD2, and C-type lectin receptors was apparent. In conclusion, TLR signaling is involved in the differentiation of inflammatory responses between probiotic strains used as food supplements.
PMCID: PMC3122558  PMID: 21288993
10.  Lactobacillus rhamnosus GG and Bifidobacterium longum Attenuate Lung Injury and Inflammatory Response in Experimental Sepsis 
PLoS ONE  2014;9(5):e97861.
Probiotic use to prevent nosocomial gastrointestinal and potentially respiratory tract infections in critical care has shown great promise in recent clinical trials of adult and pediatric patients. Despite well-documented benefits of probiotic use in intestinal disorders, the potential for probiotic treatment to reduce lung injury following infection and shock has not been well explored.
Evaluate if Lactobacillus rhamnosus GG (LGG) or Bifidobacterium longum (BL) treatment in a weanling mouse model of cecal ligation and puncture (CLP) peritonitis will protect against lung injury.
3 week-old FVB/N mice were orally gavaged with 200 µl of either LGG, BL or sterile water (vehicle) immediately prior to CLP. Mice were euthanized at 24 h. Lung injury was evaluated via histology and lung neutrophil infiltration was evaluated by myeloperoxidase (MPO) staining. mRNA levels of IL-6, TNF-α, MyD88, TLR-4, TLR-2, NFΚB (p50/p105) and Cox-2 in the lung analyzed via real-time PCR. TNF-α and IL-6 in lung was analyzed via ELISA.
LGG and BL treatment significantly improved lung injury following experimental infection and sepsis and lung neutrophil infiltration was significantly lower than in untreated septic mice. Lung mRNA and protein levels of IL-6 and TNF-α and gene expression of Cox-2 were also significantly reduced in mice receiving LGG or BL treatment. Gene expression of TLR-2, MyD88 and NFΚB (p50/p105) was significantly increased in septic mice compared to shams and decreased in the lung of mice receiving LGG or BL while TLR-4 levels remained unchanged.
Treatment with LGG and BL can reduce lung injury following experimental infection and sepsis and is associated with reduced lung inflammatory cell infiltrate and decreased markers of lung inflammatory response. Probiotic therapy may be a promising intervention to improve clinical lung injury following systemic infection and sepsis.
PMCID: PMC4022641  PMID: 24830455
11.  A Selected Lactobacillus rhamnosus Strain Promotes EGFR-Independent Akt Activation in an Enterotoxigenic Escherichia coli K88-Infected IPEC-J2 Cell Model 
PLoS ONE  2015;10(4):e0125717.
Enterotoxigenic Escherichia coli (ETEC) are important intestinal pathogens that cause diarrhea in humans and animals. Although probiotic bacteria may protect against ETEC-induced enteric infections, the underlying mechanisms are unknown. In this study, porcine intestinal epithelial J2 cells (IPEC-J2) were pre-incubated with and without Lactobacillus rhamnosus ATCC 7469 and then exposed to F4+ ETEC. Increases in TLR4 and NOD2 mRNA expression were observed at 3 h after F4+ ETEC challenge, but these increases were attenuated by L. rhamnosus treatment. Expression of TLR2 and NOD1 mRNA was up-regulated in cells pre-treated with L. rhamnosus. Pre-treatment with L. rhamnosus counteracted F4+ ETEC-induced increases in TNF-α concentration. Increased PGE2. concentrations were observed in cells infected with F4+ ETEC and in cells treated with L. rhamnosus only. A decrease in phosphorylated epidermal growth factor receptor (EGFR) was observed at 3 h after F4+ ETEC challenge in cells treated with L. rhamnosus. Pre-treatment with L. rhamnosus enhanced Akt phosphorylation and increased ZO-1 and occludin protein expression. Our findings suggest that L. rhamnosus protects intestinal epithelial cells from F4+ ETEC-induced damage, partly through the anti-inflammatory response involving synergism between TLR2 and NOD1. In addition, L. rhamnosus promotes EGFR-independent Akt activation, which may activate intestinal epithelial cells in response to bacterial infection, in turn increasing tight junction integrity and thus enhancing the barrier function and restricting pathogen invasion. Pre-incubation with L. rhamnosus was superior to co-incubation in reducing the adhesion of F4+ ETEC to IPEC-J2 cells and subsequently attenuating F4+ ETEC-induced mucin layer destruction and suppressing apoptosis. Our data indicate that a selected L. rhamnosus strain interacts with porcine intestinal epithelial cells to maintain the epithelial barrier and promote intestinal epithelial cell activation in response to bacterial infection, thus protecting cells from the deleterious effects of F4+ ETEC.
PMCID: PMC4411159  PMID: 25915861
12.  Alpha/Beta Interferon Receptor Signaling Amplifies Early Proinflammatory Cytokine Production in the Lung during Respiratory Syncytial Virus Infection 
Journal of Virology  2014;88(11):6128-6136.
Type I interferons (IFNs) are produced early upon virus infection and signal through the alpha/beta interferon (IFN-α/β) receptor (IFNAR) to induce genes that encode proteins important for limiting viral replication and directing immune responses. To investigate the extent to which type I IFNs play a role in the local regulation of inflammation in the airways, we examined their importance in early lung responses to infection with respiratory syncytial virus (RSV). IFNAR1-deficient (IFNAR1−/−) mice displayed increased lung viral load and weight loss during RSV infection. As expected, expression of IFN-inducible genes was markedly reduced in the lungs of IFNAR1−/− mice. Surprisingly, we found that the levels of proinflammatory cytokines and chemokines in the lungs of RSV-infected mice were also greatly reduced in the absence of IFNAR signaling. Furthermore, low levels of proinflammatory cytokines were also detected in the lungs of IFNAR1−/− mice challenged with noninfectious innate immune stimuli such as selected Toll-like receptor (TLR) agonists. Finally, recombinant IFN-α was sufficient to potentiate the production of inflammatory mediators in the lungs of wild-type mice challenged with innate immune stimuli. Thus, in addition to its well-known role in antiviral resistance, type I IFN receptor signaling acts as a central driver of early proinflammatory responses in the lung. Inhibiting the effects of type I IFNs may therefore be useful in dampening inflammation in lung diseases characterized by enhanced inflammatory cytokine production.
IMPORTANCE The initial response to viral infection is characterized by the production of interferons (IFNs). One group of IFNs, the type I IFNs, are produced early upon virus infection and signal through the IFN-α/β receptor (IFNAR) to induce proteins important for limiting viral replication and directing immune responses. Here we examined the importance of type I IFNs in early responses to respiratory syncytial virus (RSV). Our data suggest that type I IFN production and IFNAR receptor signaling not only induce an antiviral state but also serve to amplify proinflammatory responses in the respiratory tract. We also confirm this conclusion in another model of acute inflammation induced by noninfectious stimuli. Our findings are of relevance to human disease, as RSV is a major cause of infant bronchiolitis and polymorphisms in the IFN system are known to impact disease severity.
PMCID: PMC4093897  PMID: 24648449
13.  Prevention of LPS-Induced Acute Lung Injury in Mice by Mesenchymal Stem Cells Overexpressing Angiopoietin 1 
PLoS Medicine  2007;4(9):e269.
The acute respiratory distress syndrome (ARDS), a clinical complication of severe acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by disruption of the lung alveolar–capillary membrane barrier and resultant pulmonary edema associated with a proteinaceous alveolar exudate. Current specific treatment strategies for ALI/ARDS are lacking. We hypothesized that mesenchymal stem cells (MSCs), with or without transfection with the vasculoprotective gene angiopoietin 1 (ANGPT1) would have beneficial effects in experimental ALI in mice.
Methods and Findings
Syngeneic MSCs with or without transfection with plasmid containing the human ANGPT1 gene (pANGPT1) were delivered through the right jugular vein of mice 30 min after intratracheal instillation of lipopolysaccharide (LPS) to induce lung injury. Administration of MSCs significantly reduced LPS-induced pulmonary inflammation, as reflected by reductions in total cell and neutrophil counts in bronchoalveolar lavage (BAL) fluid (53%, 95% confidence interval [CI] 7%–101%; and 60%, CI 4%–116%, respectively) as well as reducing levels of proinflammatory cytokines in both BAL fluid and lung parenchymal homogenates. Furthermore, administration of MSCs transfected with pANGPT1 resulted in nearly complete reversal of LPS-induced increases in lung permeability as assessed by reductions in IgM and albumin levels in BAL (96%, CI 6%–185%; and 74%, CI 23%–126%, respectively). Fluorescently tagged MSCs were detected in the lung tissues by confocal microscopy and flow cytometry in both naïve and LPS-injured animals up to 3 d.
Treatment with MSCs alone significantly reduced LPS-induced acute pulmonary inflammation in mice, while administration of pANGPT1-transfected MSCs resulted in a further improvement in both alveolar inflammation and permeability. These results suggest a potential role for cell-based ANGPT1 gene therapy to treat clinical ALI/ARDS.
Using a mouse model of acute respiratory distress syndrome, Duncan Stewart and colleagues report that rescue with mesenchymal stem cells expressing human angiopoietin 1 can avert lung injury from lipopolysaccharide.
Editors' Summary
Critically ill people who have had an injury to their lungs, for example through pneumonia, trauma, or an immune response to infection, may end up developing a serious complication in the lung termed acute respiratory distress syndrome (ARDS). In ARDS, inflammation develops in the lung, and fluid builds up in the alveoli (the air sacs resembling “bunches of grapes” at the ends of the network of tubes in the lung). This buildup of fluid prevents oxygen from being carried efficiently from air into the blood; the individual consequently experiences problems breathing and can develop further serious complications, which contribute significantly to the burden of illness among people in intensive care units. The death rate among individuals who do develop ARDS is very high, upward of 30%. Normally, individuals with ARDS are given extra oxygen, and may need a machine to help them breathe; treatments also focus on addressing the underlying causes in each particular patient. However, currently there are very few specific treatments that address ARDS itself.
Why Was This Study Done?
The researchers here wanted to work toward new treatment options for individuals with ARDS. One possible approach involves cells known as mesenchymal stem cells (MSCs). These cells are typically found in the bone marrow and have a property shared by very few other cell types in the body; they are able to carry on dividing and renewing themselves, and can eventually develop into many other types of cell. The researchers already knew that MSCs could become incorporated into injured lungs in mice and develop there into the tissue layers lining the lung. Some interesting work had also been done on a protein called angiopoeitin 1 (ANGPT1), which seemed to play a role in protecting against inflammation in blood vessels. Therefore, there was a strong rationale for carrying out experiments in mice to see if MSCs engineered to produce the ANGPT1 protein might “rescue” lung injury in mice. These experiments would be an initial step toward developing possible new treatments for humans with ARDS.
What Did the Researchers Do and Find?
The researchers used a mouse model to mimic the human ARDS condition. This involved injecting the windpipe of experimental mice with lipopolysaccharide (a substance normally found on the outer surface of bacteria that brings about an immune reaction in the lung). After 30 minutes, the mice were then injected with either salt solution (as a control), the MSCs, or MSCs producing the ANGPT1 protein. The researchers then looked at markers of lung inflammation, the appearance of the lungs under a microscope, and whether the injected MSCs had become incorporated into the lung tissue.
The lipopolysaccharide brought about a large increase in the number of inflammatory cells in the lung fluid, which was reduced in the mice given MSCs. Furthermore, in mice given the MSCs producing ANGPT1 protein, the number of inflammatory cells was reduced to a level similar to that of mice that had not been given lipopolysaccharide. When the researchers looked at the appearance under the microscope of lungs from mice that had been given lipopolysaccharide, they saw signs of inflammation and fluid coming out into the lung air spaces. These signs were reduced among both mice treated with MSCs and those treated with MSCs producing ANGPT1. The researchers also measured the “leakiness” of the lung tissues in lipopolysaccharide-treated mice; MSCs seemed to reduce the leakiness to some extent, and the lungs of mice treated with MSCs producing ANGPT1 were no more leaky than those of mice that had never been injected with lipopolysaccharide. Finally, the MSCs were seen to be incorporated into lung tissue by three days after injection, but after that were lost from the lung.
What Do These Findings Mean?
Previous research done by the same group had shown that fibroblasts producing ANGPT1 could prevent lung injury in rats later given lipopolysaccharide. The experiments reported here go a step further than this, and suggest that MSCs producing ANGPT1 can “rescue” the condition of mouse lungs that had already been given lipopolysaccharide. In addition, treatment with MSCs alone also produced beneficial effects. This opens up a possible new treatment strategy for ARDS in humans. However, it should be emphasized that the animal model used here is not a precise parallel of ARDS in humans, and that more research remains to be done before human studies of this sort could be considered.
Additional Information.
Please access these Web sites via the online version of this summary at
Medline Plus entry on acute respiratory distress syndrome, providing basic information about what ARDS is, its effects, and how it is currently managed
ARDS Network from the US National Heart, Lung, and Blood Institute of the National Institutes of Health; the site provides frequently asked questions about ARDS as well as a list of clinical trials conducted by the network
Information about stem cells from the US National Institutes of Health, including information about the potential uses of stem cells
Wikipedia page about mesenchymal stem cells (note: Wikipedia is an internet encyclopedia anyone can edit)
PMCID: PMC1961632  PMID: 17803352
14.  Effects of Multispecies Probiotic Combination on Helicobacter pylori Infection In Vitro▿  
Probiotic bacteria alleviate many gastrointestinal symptoms, but the current trend of combining bacteria for additional benefit may make their effects more complex. We characterize four probiotics and their combination in terms of pathogen adhesion, barrier function, cell death, and inflammatory response in Helicobacter pylori-infected epithelial cells. H. pylori-infected Caco-2 cells were pretreated with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii Js, Bifidobacterium breve Bb99, or all four organisms in combination. We evaluated the adhesion of H. pylori by in situ immunofluorescence; epithelial barrier function by measurement of transepithelial resistance; apoptosis by measurement of caspase 3 activation; cell membrane leakage by measurement of lactate dehydrogenase release; and inflammation by measurement of interleukin-8 (IL-8), IL-10, prostaglandin E2 (PGE2), and leukotriene B4 (LTB4) release. All probiotics inhibited H. pylori adhesion. L. rhamnosus GG, L. rhamnosus Lc705, P. freudenreichii subsp. shermanii Js, and the combination inhibited H. pylori-induced cell membrane leakage. L. rhamnosus GG, L. rhamnosus Lc705, and the combination initially improved epithelial barrier function but increased the H. pylori-induced barrier deterioration after incubation for 24 to 42 h. L. rhamnosus GG, L. rhamnosus Lc705, and P. freudenreichii subsp. shermanii Js inhibited H. pylori-induced IL-8 release, whereas L. rhamnosus GG, L. rhamnosus Lc705, and B. breve Bb99 suppressed PGE2 release. None of these anti-inflammatory effects persisted when the probiotics were used in combination. The combination thus increased the levels of IL-8, PGE2, and LTB4 released from H. pylori-infected epithelial cells. The proinflammatory actions of the individual components dominated the anti-inflammatory effects when the probiotic bacteria were used in combination. Our results stress that the therapeutic response can be optimized if probiotic strains are characterized before they are used in combination.
PMCID: PMC2546684  PMID: 18579692
15.  Long-term activation of TLR3 by Poly(I:C) induces inflammation and impairs lung function in mice 
Respiratory Research  2009;10(1):43.
The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases.
TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C).
There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFα were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy.
These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.
PMCID: PMC2694181  PMID: 19486528
16.  Dose-Dependent Effects of Lactobacillus rhamnosus on Serum Interleukin-17 Production and Intestinal T-Cell Responses in Pigs Challenged with Escherichia coli 
The mechanism underlying the dose effect of probiotics on ameliorating diarrhea has not been fully elucidated. Here, low (1 × 109 CFU/ml) or high (1 × 1011 CFU/ml) doses of Lactobacillus rhamnosus ATCC 7469 were administered orally to piglets for 1 week before F4 (K88)-positive enterotoxigenic Escherichia coli (F4+ ETEC) challenge. Administration of a low, but not a high, dose of L. rhamnosus decreased the percentage of CD3+ CD4+ CD8− T cells in the peripheral blood. Notably, transiently increased serum concentrations of interleukin-17A (IL-17A) were observed after F4+ ETEC challenge in pigs pretreated with a high dose of L. rhamnosus. Administration of L. rhamnosus increased the percentage of the small intestinal lamina propria CD3+ CD4+ CD8− cells and Peyer's patch CD3+ CD4− CD8− and CD3− CD4− CD8+ cells. The percentage of ileal intraepithelial CD3+ CD4− CD8+ cells increased only in the high-dose piglets. Administration of L. rhamnosus downregulated expression of ileal IL-17A after F4+ ETEC challenge but had no effect on expression of gamma interferon (IFN-γ), IL-12, IL-4, and FOXP3 mRNA in the small intestine. Expression of jejunal IL-2, ileal transforming growth factor β1 (TGF-β1), and ileal IL-10 was upregulated in the low-dose piglets after F4+ ETEC challenge. Our findings suggest that amelioration of infectious diarrhea in piglets by L. rhamnosus is associated with the generation of lamina propria CD3+ CD4+ CD8− T cells, the expansion of Peyer's patch CD3+ CD4− CD8− and CD3− CD4− CD8+ cells, and the attenuation of F4+ ETEC-induced increase in CD3+ CD4+ CD8+ T cells in the small intestine. However, consumption of high doses of L. rhamnosus may increase levels of serum IL-17A after F4+ ETEC challenge, thus eliciting a strong proinflammatory response.
PMCID: PMC3957626  PMID: 24389928
17.  Sendai Virus Pathogenesis in Mice Is Prevented by Ifit2 and Exacerbated by Interferon 
Journal of Virology  2014;88(23):13593-13601.
The type I/III interferon (IFN) system has major roles in regulating viral pathogenesis, usually ameliorating pathogenesis by impairing virus replication through the antiviral actions of one or more IFN-induced proteins. Ifit2 is one such protein which can be induced by IFN or virus infection, and it is responsible for protecting mice from neuropathogenesis caused by vesicular stomatitis virus. Here, we show that Ifit2 also protects mice from pathogenesis caused by the respirovirus Sendai virus (SeV). Mice lacking Ifit2 (Ifit2−/−) suffered severe weight loss and succumbed to intranasal infection with SeV strain 52 at a dose that killed only a few wild-type mice. Viral RNA was detectable only in lungs, and SeV titers were higher in Ifit2−/− mice than in wild-type mice. Similar infiltration of immune cells was found in the lungs of both mouse lines, corresponding to similar levels of many induced cytokines and chemokines. In contrast, IFN-β and IFN-λ3 expression were considerably higher in the lungs of Ifit2−/− mice. Surprisingly, type I IFN receptor knockout (IFNAR−/−) mice were less susceptible to SeV than Ifit2−/− mice, although their pulmonary virus titers were similarly high. To test the intriguing possibility that type I IFN action enhances pathogenesis in the context of elevated SeV replication in lungs, we generated Ifit2/IFNAR−/− double knockout mice. These mice were less susceptible to SeV than Ifit2−/− mice, although viral titers in their lungs were even higher. Our results indicate that high SeV replication in the lungs of infected Ifit2−/− mice cooperates with elevated IFN-β induction to cause disease.
IMPORTANCE The IFN system is an innate defense against virus infections. It is triggered quickly in infected cells, which then secrete IFN. Via their cell surface receptors on surrounding cells, they induce transcription of numerous IFN-stimulated genes (ISG), which in turn protect these cells by inhibiting virus life cycles. Hence, IFNs are commonly considered beneficial during virus infections. Here, we report two key findings. First, lack of a single ISG in mice, Ifit2, resulted in high mortality after SeV infection of the respiratory tract, following higher virus loads and higher IFN production in Ifit2−/− lungs. Second, mortality of Ifit2−/− mice was reduced when mice also lacked the type I IFN receptor, while SeV loads in lungs still were high. This indicates that type I IFN exacerbates pathogenesis in the SeV model, and that limitation of both viral replication and IFN production is needed for effective prevention of disease.
PMCID: PMC4248979  PMID: 25231314
18.  Mycoplasma pneumoniae Infection Affects the Serum Levels of Vascular Endothelial Growth Factor and Interleukin-5 in Atopic Children 
Previous studies have outlined mechanisms by which Mycoplasma pneumonia (M. pneumonia) infection may promote allergic lung inflammation and airway remodeling, and increasing evidence from human studies suggests that atypical bacterial infections contribute to asthma exacerbation, chronic asthma, and disease severity with changes in cytokine expression. The present study evaluated changes in serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-5 in atopic children with Mycoplasma pneumoniae pneumonia.
We recruited a total of 72 children with pneumonia. The patients were divided into 4 groups: atopic children with M. pneumonia pneumonia (group I, n=24), non-atopic children with M. pneumonia pneumonia (group II, n=23), atopic children with viral pneumonia (group III, n=13), and non-atopic children with viral pneumonia (group IV, n=12). Serum levels of IL-5, IL-13, VEGF, and tumor necrosis factor-α were measured at admission and at recovery using enzyme-linked immunosorbent assays.
Serum levels of VEGF and IL-5 were elevated in group I compared with the other groups at both admission phase and clinical recovery phase. In group I, serum levels of VEGF and IL-5 were higher at recovery phase than at admission phase (VEGF: 1,102.2±569.4 vs. 874.9±589.9 pg/mL, respectively; IL-5: 150.5±63.9 vs. 120.2±46.7 pg/mL, respectively).
The serum levels of VEGF and IL-5 were more increased in atopic children with M. pneumonia pneumonia than in the other groups. In this group, the serum levels of VEGF and IL-5 were more increased at recovery phase than at admission phase. The results of this study suggest that increases in VEGF and IL-5 may contribute to the development of hypersensitivity during M. pneumonia infection. These cytokines may act through their respective pro-inflammatory pathways to aggravate the allergic status and induce airway hypersensitivity during M. pneumonia pneumonia in atopic children.
PMCID: PMC3283799  PMID: 22379604
Atopy; interleukin-5; Pneumonia, Mycoplasma; vascular endothelial growth factor
19.  STAT4 Deficiency Fails To Induce Lung Th2 or Th17 Immunity following Primary or Secondary Respiratory Syncytial Virus (RSV) Challenge but Enhances the Lung RSV-Specific CD8+ T Cell Immune Response to Secondary Challenge 
Journal of Virology  2014;88(17):9655-9672.
Immune-mediated lung injury is a hallmark of lower respiratory tract illness caused by respiratory syncytial virus (RSV). STAT4 plays a critical role in CD4+ Th1 lineage differentiation and gamma interferon (IFN-γ) protein expression by CD4+ T cells. As CD4+ Th1 differentiation is associated with negative regulation of CD4+ Th2 and Th17 differentiation, we hypothesized that RSV infection of STAT4−/− mice would result in enhanced lung Th2 and Th17 inflammation and impaired lung Th1 inflammation compared to wild-type (WT) mice. We performed primary and secondary RSV challenges in WT and STAT4−/− mice and used STAT1−/− mice as a positive control for the development of RSV-specific lung Th2 and Th17 inflammation during primary challenge. Primary RSV challenge of STAT4−/− mice resulted in decreased T-bet and IFN-γ expression levels in CD4+ T cells compared to those of WT mice. Lung Th2 and Th17 inflammation did not develop in primary RSV-challenged STAT4−/− mice. Decreased IFN-γ expression by NK cells, CD4+ T cells, and CD8+ T cells was associated with attenuated weight loss and enhanced viral clearance with primary challenge in STAT4−/− mice compared to WT mice. Following secondary challenge, WT and STAT4−/− mice also did not develop lung Th2 or Th17 inflammation. In contrast to primary challenge, secondary RSV challenge of STAT4−/− mice resulted in enhanced weight loss, an increased lung IFN-γ expression level, and an increased lung RSV-specific CD8+ T cell response compared to those of WT mice. These data demonstrate that STAT4 regulates the RSV-specific CD8+ T cell response to secondary infection but does not independently regulate lung Th2 or Th17 immune responses to RSV challenge.
IMPORTANCE STAT4 is a protein critical for both innate and adaptive immune responses to viral infection. Our results show that STAT4 regulates the immune response to primary and secondary challenge with RSV but does not restrain RSV-induced lung Th2 or Th17 immune responses. These findings suggest that STAT4 expression may influence lung immunity and severity of illness following primary and secondary RSV infections.
PMCID: PMC4136353  PMID: 24920804
20.  Probiotic lactobacillus and estrogen effects on vaginal epithelial gene expression responses to Candida albicans 
Vaginal epithelial cells have receptors, signal transduction mechanisms, and cytokine secretion capabilities to recruit host defenses against Candida albicans infections. This research evaluates how probiotic lactobacilli affect the defensive epithelial response.
This study used quantitative reverse transcription-polymerase chain reaction assay (qRT-PCR), flow cytometry, and a multiplex immunoassay to observe changes in the regulation of gene expression related to cytokine responses in the VK2 (E6/E7) vaginal epithelial cell line treated with 17β-estradiol, exposed to probiotic Lactobacillus rhamnosus GR-1® and Lactobacillus reuteri RC-14® and challenged with C. albicans. Data were statistically evaluated by repeated measures analysis of variance and paired t-tests where appropriate.
C. albicans induced mRNA expression of genes related to inflammatory cytokine responses associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal transduction pathways. 17β-estradiol suppressed expression of interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha (TNFα) mRNA. Probiotic lactobacilli suppressed C. albicans-induced nuclear factor-kappa B inhibitor kinase kinase alpha (Iκκα), Toll-like receptor-2 (TLR2), TLR6, IL-8, and TNFα, also suggesting inhibition of NF-κB signaling. The lactobacilli induced expression of IL-1α, and IL-1β mRNA, which was not inhibited by curcumin, suggesting that they induce an alternate inflammatory signal transduction pathway to NF-κB, such as the mitogen activated protein kinase and activator protein-1 (MAPK/AP-1) signal transduction pathway. Curcumin inhibited IL-13 secretion, suggesting that expression of this cytokine is mainly regulated by NF-κB signaling in VK2 cells.
The results suggest that C. albicans infection induces pro-inflammatory responses in vaginal epithelial cells, and estrogen and lactobacilli suppress expression of NF-κB-related inflammatory genes. Probiotic lactobacilli may induce IL-1α and IL-1β expression by an alternate signal transduction pathway, such as MAPK/AP-1. Activation of alternate signaling mechanisms by lactobacilli to modify epithelial cell cytokine production may be a mechanism for probiotic modulation of morbidity in vulvovaginal candidiasis.
PMCID: PMC3404894  PMID: 22715972
Probiotic; Epithelial cells; Gene expression; Signal transduction genes; Candidiasis; Estrogen
21.  Transcriptomic Analysis of Host Immune and Cell Death Responses Associated with the Influenza A Virus PB1-F2 Protein 
PLoS Pathogens  2011;7(8):e1002202.
Airway inflammation plays a major role in the pathogenesis of influenza viruses and can lead to a fatal outcome. One of the challenging objectives in the field of influenza research is the identification of the molecular bases associated to the immunopathological disorders developed during infection. While its precise function in the virus cycle is still unclear, the viral protein PB1-F2 is proposed to exert a deleterious activity within the infected host. Using an engineered recombinant virus unable to express PB1-F2 and its wild-type homolog, we analyzed and compared the pathogenicity and host response developed by the two viruses in a mouse model. We confirmed that the deletion of PB1-F2 renders the virus less virulent. The global transcriptomic analyses of the infected lungs revealed a potent impact of PB1-F2 on the response developed by the host. Thus, after two days post-infection, PB1-F2 invalidation severely decreased the number of genes activated by the host. PB1-F2 expression induced an increase in the number and level of expression of activated genes linked to cell death, inflammatory response and neutrophil chemotaxis. When generating interactive gene networks specific to PB1-F2, we identified IFN-γ as a central regulator of PB1-F2-regulated genes. The enhanced cell death of airway-recruited leukocytes was evidenced using an apoptosis assay, confirming the pro-apoptotic properties of PB1-F2. Using a NF-kB luciferase adenoviral vector, we were able to quantify in vivo the implication of NF-kB in the inflammation mediated by the influenza virus infection; we found that PB1-F2 expression intensifies the NF-kB activity. Finally, we quantified the neutrophil recruitment within the airways, and showed that this type of leukocyte is more abundant during the infection of the wild-type virus. Collectively, these data demonstrate that PB1-F2 strongly influences the early host response during IAV infection and provides new insights into the mechanisms by which PB1-F2 mediates virulence.
Author Summary
Influenza A viruses may cause severe respiratory disease. PB1-F2, a viral protein identified in 2001 is suspected to play a role in influenza-related pneumonia. In order to understand the impact of PB1-F2 in the pathogenesis underlying Influenza A virus infection, we engineered a mutant virus unable to express PB1-F2. By the use of high-throughput gene expression assays, we compared the host responses of the wild-type-infected and the PB1-F2 mutant-infected mice. We identified that PB1-F2 expression enhances the immune cell death and inflammatory responses of mice. The inflammatory response mediated by the PB1-F2 expression leads to a massive recruitment of leukocytes within the air spaces, a feature that characterizes the influenza-mediated immunopathology. Our results suggest that PB1-F2 is a virulence factor implicated in the deregulation of the inflammatory response observed in acute influenza virus pneumonia. These data underlie the complexities of virus-host interactions and help us understand by which mechanisms Influenza viruses mediate severe respiratory diseases.
PMCID: PMC3161975  PMID: 21901097
22.  Exacerbated Innate Host Response to SARS-CoV in Aged Non-Human Primates 
PLoS Pathogens  2010;6(2):e1000756.
The emergence of viral respiratory pathogens with pandemic potential, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza A H5N1, urges the need for deciphering their pathogenesis to develop new intervention strategies. SARS-CoV infection causes acute lung injury (ALI) that may develop into life-threatening acute respiratory distress syndrome (ARDS) with advanced age correlating positively with adverse disease outcome. The molecular pathways, however, that cause virus-induced ALI/ARDS in aged individuals are ill-defined. Here, we show that SARS-CoV-infected aged macaques develop more severe pathology than young adult animals, even though viral replication levels are similar. Comprehensive genomic analyses indicate that aged macaques have a stronger host response to virus infection than young adult macaques, with an increase in differential expression of genes associated with inflammation, with NF-κB as central player, whereas expression of type I interferon (IFN)-β is reduced. Therapeutic treatment of SARS-CoV-infected aged macaques with type I IFN reduces pathology and diminishes pro-inflammatory gene expression, including interleukin-8 (IL-8) levels, without affecting virus replication in the lungs. Thus, ALI in SARS-CoV-infected aged macaques developed as a result of an exacerbated innate host response. The anti-inflammatory action of type I IFN reveals a potential intervention strategy for virus-induced ALI.
Author Summary
Severe acute respiratory syndrome coronavirus (SARS-CoV) infection causes acute lung injury that may develop into the life-threatening acute respiratory distress syndrome (ARDS) in mostly elderly individuals. Although SARS-CoV infection can be fatal, most patients recover, suggesting that protective host responses are operational to combat the viral infection. Therefore, we used age as predisposing factor to obtain insight into the pathogenesis of SARS-CoV. In this study, we show that SARS-CoV-infected aged macaques developed significantly more pathology than young adult animals, which could not be contributed to differences in viral replication. Using comparative microarray analyses, it was shown that although the nature of the host response to SARS-CoV infection was similar in aged and young adult macaques, the severity was significantly different, with aged macaques displaying an increase in differential expression of genes associated with inflammation. Interestingly, type I IFN-β mRNA levels correlated negatively with gross pathology. Therapeutic treatment of aged macaques with type I IFN reduced pathology without affecting virus replication. However, pro-inflammatory gene expression was significantly diminished. Thus, modulation of the host response by type I IFNs provides a promising outlook for novel intervention strategies.
PMCID: PMC2816697  PMID: 20140198
23.  Using Recombinant Lactococci as an Approach to Dissect the Immunomodulating Capacity of Surface Piliation in Probiotic Lactobacillus rhamnosus GG 
PLoS ONE  2013;8(5):e64416.
Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-α, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut.
PMCID: PMC3653913  PMID: 23691212
24.  MDA5 and TLR3 Initiate Pro-Inflammatory Signaling Pathways Leading to Rhinovirus-Induced Airways Inflammation and Hyperresponsiveness 
PLoS Pathogens  2011;7(5):e1002070.
Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness.
Author Summary
Rhinovirus (RV) is the most frequent cause of acute respiratory tract infection in humans. RV has emerged as the most frequent pathogen associated with exacerbations of asthma. However, the mechanisms by which RV causes asthma flare-ups are not precisely known. We studied the requirements of two receptors which bind viral double-stranded RNA, melanoma differentiation-associated gene (MDA)-5 and Toll-like receptor (TLR)-3, for RV-induced airway responses using specific knockout mice. We found that MDA5 and TLR3 deficiencies had modest effects on viral titer. However, MDA5 and TLR3 knockout mice showed significantly reduced airway inflammation and responsiveness in response to RV infection. Mice with allergic airways disease also showed reduced airway responses. These results suggest that, in the context of RV infection, TLR3 and MDA5 initiate pro-inflammatory signaling pathways which lead to airways inflammation and hyperresponsiveness.
PMCID: PMC3102730  PMID: 21637773
25.  Oral administration of a probiotic Lactobacillus modulates cytokine production and TLR expression improving the immune response against Salmonella enterica serovar Typhimurium infection in mice 
BMC Microbiology  2011;11:177.
Diarrheal infections caused by Salmonella, are one of the major causes of childhood morbidity and mortality in developing countries. Salmonella causes various diseases that range from mild gastroenteritis to enteric fever, depending on the serovar involved, infective dose, species, age and immune status of the host. Probiotics are proposed as an attractive alternative possibility in the prevention against this pathogen infection. Previously we demonstrated that continuous Lactobacillus casei CRL 431 administration to BALB/c mice before and after challenge with Salmonella enterica serovar Typhimurium (S. Typhimurium) decreased the severity of Salmonella infection. The aim of the present work was to deep into the knowledge about how this probiotic bacterium exerts its effect, by assessing its impact on the expression and secretion of pro-inflammatory (TNFα, IFNγ) and anti-inflammatory (IL-10) cytokines in the inductor and effector sites of the gut immune response, and analyzing toll-like receptor (TLR2, TLR4, TLR5 and TLR9) expressions in both healthy and infected mice.
Probiotic administration to healthy mice increased the expression of TLR2, TLR4 and TLR9 and improved the production and secretion of TNFα, IFNγ and IL-10 in the inductor sites of the gut immune response (Peyer's patches). Post infection, the continuous probiotic administration, before and after Salmonella challenge, protected the host by modulating the inflammatory response, mainly in the immune effector site of the gut, decreasing TNFα and increasing IFNγ, IL-6 and IL-10 production in the lamina propria of the small intestine.
The oral administration of L. casei CRL 431 induces variations in the cytokine profile and in the TLRs expression previous and also after the challenge with S. Typhimurium. These changes show some of the immune mechanisms implicated in the protective effect of this probiotic strain against S. Typhimurium, providing an alternative way to reduce the severity of the infection.
PMCID: PMC3173335  PMID: 21813005

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