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1.  Salmonella Adhesion, Invasion and Cellular Immune Responses Are Differentially Affected by Iron Concentrations in a Combined In Vitro Gut Fermentation-Cell Model 
PLoS ONE  2014;9(3):e93549.
In regions with a high infectious disease burden, concerns have been raised about the safety of iron supplementation because higher iron concentrations in the gut lumen may increase risk of enteropathogen infection. The aim of this study was to investigate interactions of the enteropathogen Salmonella enterica ssp. enterica Typhimurium with intestinal cells under different iron concentrations encountered in the gut lumen during iron deficiency and supplementation using an in vitro colonic fermentation system inoculated with immobilized child gut microbiota combined with Caco-2/HT29-MTX co-culture monolayers. Colonic fermentation effluents obtained during normal, low (chelation by 2,2'-dipyridyl) and high iron (26.5 mg iron/L) fermentation conditions containing Salmonella or pure Salmonella cultures with similar iron conditions were applied to cellular monolayers. Salmonella adhesion and invasion capacity, cellular integrity and immune response were assessed. Under high iron conditions in pure culture, Salmonella adhesion was 8-fold increased compared to normal iron conditions while invasion was not affected leading to decreased invasion efficiency (−86%). Moreover, cellular cytokines IL-1β, IL-6, IL-8 and TNF-α secretion as well as NF-κB activation in THP-1 cells were attenuated under high iron conditions. Low iron conditions in pure culture increased Salmonella invasion correlating with an increase in IL-8 release. In fermentation effluents, Salmonella adhesion was 12-fold and invasion was 428-fold reduced compared to pure culture. Salmonella in high iron fermentation effluents had decreased invasion efficiency (−77.1%) and cellular TNF-α release compared to normal iron effluent. The presence of commensal microbiota and bacterial metabolites in fermentation effluents reduced adhesion and invasion of Salmonella compared to pure culture highlighting the importance of the gut microbiota as a barrier during pathogen invasion. High iron concentrations as encountered in the gut lumen during iron supplementation attenuated Salmonella invasion efficiency and cellular immune response suggesting that high iron concentrations alone may not lead to an increased Salmonella invasion.
PMCID: PMC3968171  PMID: 24676135
2.  Bifidobacterium infantis 35624 modulates host inflammatory processes beyond the gut 
Gut Microbes  2013;4(4):325-339.
Certain therapeutic microbes, including Bifidobacteria infantis (B. infantis) 35624 exert beneficial immunoregulatory effects by mimicking commensal-immune interactions; however, the value of these effects in patients with non-gastrointestinal inflammatory conditions remains unclear. In this study, we assessed the impact of oral administration of B. infantis 35624, for 6‒8 weeks on inflammatory biomarker and plasma cytokine levels in patients with ulcerative colitis (UC) (n = 22), chronic fatigue syndrome (CFS) (n = 48) and psoriasis (n = 26) in three separate randomized, double-blind, placebo-controlled interventions. Additionally, the effect of B. infantis 35624 on immunological biomarkers in healthy subjects (n = 22) was assessed. At baseline, both gastrointestinal (UC) and non-gastrointestinal (CFS and psoriasis) patients had significantly increased plasma levels of C-reactive protein (CRP) and the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) compared with healthy volunteers. B. infantis 35624 feeding resulted in reduced plasma CRP levels in all three inflammatory disorders compared with placebo. Interestingly, plasma TNF-α was reduced in CFS and psoriasis while IL-6 was reduced in UC and CFS. Furthermore, in healthy subjects, LPS-stimulated TNF-α and IL-6 secretion by peripheral blood mononuclear cells (PBMCs) was significantly reduced in the B. infantis 35624-treated groups compared with placebo following eight weeks of feeding. These results demonstrate the ability of this microbe to reduce systemic pro-inflammatory biomarkers in both gastrointestinal and non-gastrointestinal conditions. In conclusion, these data show that the immunomodulatory effects of the microbiota in humans are not limited to the mucosal immune system but extend to the systemic immune system.
PMCID: PMC3744517  PMID: 23842110
B. infantis 35624; inflammation; immunity; microbiota; C-reactive protein
3.  Immunomodulation by Bifidobacterium infantis 35624 in the Murine Lamina Propria Requires Retinoic Acid-Dependent and Independent Mechanisms 
PLoS ONE  2013;8(5):e62617.
Appropriate dendritic cell processing of the microbiota promotes intestinal homeostasis and protects against aberrant inflammatory responses. Mucosal CD103+ dendritic cells are able to produce retinoic acid from retinal, however their role in vivo and how they are influenced by specific microbial species has been poorly described. Bifidobacterium infantis 35624 (B. infantis) feeding to mice resulted in increased numbers of CD103+retinaldehyde dehydrogenase (RALDH)+ dendritic cells within the lamina propria (LP). Foxp3+ lymphocytes were also increased in the LP, while TH1 and TH17 subsets were decreased. 3,7-dimethyl-2,6-octadienal (citral) treatment of mice blocked the increase in CD103+RALDH+ dendritic cells and the decrease in TH1 and TH17 lymphocytes, but not the increase in Foxp3+ lymphocytes. B. infantis reduced the severity of DSS-induced colitis, associated with decreased TH1 and TH17 cells within the LP. Citral treatment confirmed that these effects were RALDH mediated. RALDH+ dendritic cells decreased within the LP of control inflamed animals, while RALDH+ dendritic cells numbers were maintained in the LP of B. infantis-fed mice. Thus, CD103+RALDH+ LP dendritic cells are important cellular targets for microbiota-associated effects on mucosal immunoregulation.
PMCID: PMC3660574  PMID: 23704880
4.  Portrait of an immunoregulatory bifidobacterium 
Gut Microbes  2012;3(3):261-266.
There is increasing interest in the administration of microbes or microbial metabolites for the prevention and treatment of aberrant inflammatory activity. The protective effects associated with these microbes are mediated by multiple mechanisms involving epithelial cells, DCs and T cells, but most data are derived from animal models. In this addendum, we summarize our recent data, showing that oral consumption of Bifidobacterium infantis 35624 is associated with enhanced IL-10 secretion and Foxp3 expression in human peripheral blood. In addition, we discuss the potential DC subset-specific mechanisms, which could contribute to DCREG and TREG programming by specific gut microbes.
PMCID: PMC3427218  PMID: 22572827
bifidobacteria; dendritic cells; immunoregulation; microbiota; pattern recognition receptors; retinoic acid
5.  Research needs in allergy: an EAACI position paper, in collaboration with EFA 
Papadopoulos, Nikolaos G | Agache, Ioana | Bavbek, Sevim | Bilo, Beatrice M | Braido, Fulvio | Cardona, Victoria | Custovic, Adnan | deMonchy, Jan | Demoly, Pascal | Eigenmann, Philippe | Gayraud, Jacques | Grattan, Clive | Heffler, Enrico | Hellings, Peter W | Jutel, Marek | Knol, Edward | Lötvall, Jan | Muraro, Antonella | Poulsen, Lars K | Roberts, Graham | Schmid-Grendelmeier, Peter | Skevaki, Chrysanthi | Triggiani, Massimo | vanRee, Ronald | Werfel, Thomas | Flood, Breda | Palkonen, Susanna | Savli, Roberta | Allegri, Pia | Annesi-Maesano, Isabella | Annunziato, Francesco | Antolin-Amerigo, Dario | Apfelbacher, Christian | Blanca, Miguel | Bogacka, Ewa | Bonadonna, Patrizia | Bonini, Matteo | Boyman, Onur | Brockow, Knut | Burney, Peter | Buters, Jeroen | Butiene, Indre | Calderon, Moises | Cardell, Lars Olaf | Caubet, Jean-Christoph | Celenk, Sevcan | Cichocka-Jarosz, Ewa | Cingi, Cemal | Couto, Mariana | deJong, Nicolette | Del Giacco, Stefano | Douladiris, Nikolaos | Fassio, Filippo | Fauquert, Jean-Luc | Fernandez, Javier | Rivas, Montserrat Fernandez | Ferrer, Marta | Flohr, Carsten | Gardner, James | Genuneit, Jon | Gevaert, Philippe | Groblewska, Anna | Hamelmann, Eckard | Hoffmann, Hans Jürgen | Hoffmann-Sommergruber, Karin | Hovhannisyan, Lilit | Hox, Valérie | Jahnsen, Frode L | Kalayci, Ömer | Kalpaklioglu, Ayse Füsun | Kleine-Tebbe, Jörg | Konstantinou, George | Kurowski, Marcin | Lau, Susanne | Lauener, Roger | Lauerma, Antti | Logan, Kirsty | Magnan, Antoine | Makowska, Joanna | Makrinioti, Heidi | Mangina, Paraskevi | Manole, Felicia | Mari, Adriano | Mazon, Angel | Mills, Clare | Mingomataj, ErvinÇ | Niggemann, Bodo | Nilsson, Gunnar | Ollert, Markus | O'Mahony, Liam | O'Neil, Serena | Pala, Gianni | Papi, Alberto | Passalacqua, Gianni | Perkin, Michael | Pfaar, Oliver | Pitsios, Constantinos | Quirce, Santiago | Raap, Ulrike | Raulf-Heimsoth, Monika | Rhyner, Claudio | Robson-Ansley, Paula | Alves, Rodrigo Rodrigues | Roje, Zeljka | Rondon, Carmen | Rudzeviciene, Odilija | Ruëff, Franziska | Rukhadze, Maia | Rumi, Gabriele | Sackesen, Cansin | Santos, Alexandra F | Santucci, Annalisa | Scharf, Christian | Schmidt-Weber, Carsten | Schnyder, Benno | Schwarze, Jürgen | Senna, Gianenrico | Sergejeva, Svetlana | Seys, Sven | Siracusa, Andrea | Skypala, Isabel | Sokolowska, Milena | Spertini, Francois | Spiewak, Radoslaw | Sprikkelman, Aline | Sturm, Gunter | Swoboda, Ines | Terreehorst, Ingrid | Toskala, Elina | Traidl-Hoffmann, Claudia | Venter, Carina | Vlieg-Boerstra, Berber | Whitacker, Paul | Worm, Margitta | Xepapadaki, Paraskevi | Akdis, Cezmi A
In less than half a century, allergy, originally perceived as a rare disease, has become a major public health threat, today affecting the lives of more than 60 million people in Europe, and probably close to one billion worldwide, thereby heavily impacting the budgets of public health systems. More disturbingly, its prevalence and impact are on the rise, a development that has been associated with environmental and lifestyle changes accompanying the continuous process of urbanization and globalization. Therefore, there is an urgent need to prioritize and concert research efforts in the field of allergy, in order to achieve sustainable results on prevention, diagnosis and treatment of this most prevalent chronic disease of the 21st century.
The European Academy of Allergy and Clinical Immunology (EAACI) is the leading professional organization in the field of allergy, promoting excellence in clinical care, education, training and basic and translational research, all with the ultimate goal of improving the health of allergic patients. The European Federation of Allergy and Airways Diseases Patients’ Associations (EFA) is a non-profit network of allergy, asthma and Chronic Obstructive Pulmonary Disorder (COPD) patients’ organizations. In support of their missions, the present EAACI Position Paper, in collaboration with EFA, highlights the most important research needs in the field of allergy to serve as key recommendations for future research funding at the national and European levels.
Although allergies may involve almost every organ of the body and an array of diverse external factors act as triggers, there are several common themes that need to be prioritized in research efforts. As in many other chronic diseases, effective prevention, curative treatment and accurate, rapid diagnosis represent major unmet needs. Detailed phenotyping/endotyping stands out as widely required in order to arrange or re-categorize clinical syndromes into more coherent, uniform and treatment-responsive groups. Research efforts to unveil the basic pathophysiologic pathways and mechanisms, thus leading to the comprehension and resolution of the pathophysiologic complexity of allergies will allow for the design of novel patient-oriented diagnostic and treatment protocols. Several allergic diseases require well-controlled epidemiological description and surveillance, using disease registries, pharmacoeconomic evaluation, as well as large biobanks. Additionally, there is a need for extensive studies to bring promising new biotechnological innovations, such as biological agents, vaccines of modified allergen molecules and engineered components for allergy diagnosis, closer to clinical practice. Finally, particular attention should be paid to the difficult-to-manage, precarious and costly severe disease forms and/or exacerbations. Nonetheless, currently arising treatments, mainly in the fields of immunotherapy and biologicals, hold great promise for targeted and causal management of allergic conditions. Active involvement of all stakeholders, including Patient Organizations and policy makers are necessary to achieve the aims emphasized herein.
PMCID: PMC3539924  PMID: 23121771
Allergy; Allergic diseases; Policy; Research needs; Research funding; Europe
6.  Bifidobacterium Infantis 35624 Protects Against Salmonella-Induced Reductions in Digestive Enzyme Activity in Mice by Attenuation of the Host Inflammatory Response 
Salmonella-induced damage to the small intestine may decrease the villi-associated enzyme activity, causing malabsorption of nutrients and diarrhea, and thus contribute to the symptoms of infection. The objective of this study was to determine the mechanism by which different doses and durations of Salmonella infection and lipopolysaccharide (LPS) affect brush border enzyme activity in the mouse, and to determine if the probiotic Bifidobacterium longum subspecies infantis 35624 could attenuate the intestinal damage.
BALB/c mice were challenged with Salmonella enterica serovar Typhimurium UK1 at various doses (102–108 colony-forming unit (CFU)) and durations (106 CFU for 1–6 days). Mice were also treated with B. longum subsp. infantis 35624 for 2 weeks before and during a 6-day S. Typhimurium challenge (106 CFU), or before injection of LPS. The small intestine was assessed for morphological changes, mRNA expression of cytokines, and activity of the brush border enzymes sucrase–isomaltase, maltase, and alkaline phosphatase.
S. Typhimurium infection significantly reduced the activity of all brush border enzymes in a dose- and time-dependent manner (P<0.05). This also occurred following injection of LPS. Pre-treatment with B. longum subsp. infantis 35624 prevented weight loss, protected brush border enzyme activity, reduced the small intestinal damage, and inhibited the increase in interleukin (IL)-10 and IL-8 expression due to Salmonella challenge.
Salmonella infection reduces the small intestinal brush border enzyme activity in mice, with the level of reduction and associated weight loss increasing with dose and duration of infection. B. longum subsp. infantis 35624 treatment attenuated the effect of Salmonella infection on brush border enzyme activity and weight loss, which may be due to modulation of the host immune response.
PMCID: PMC3367613  PMID: 23238232
7.  Immune system in the intestine and mucosal inflammation 
Clinical and Translational Allergy  2011;1(Suppl 1):S45.
PMCID: PMC3354274
8.  Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria 
BMC Immunology  2009;10:54.
Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-κB activation were measured using enzyme-linked immunosorbent assays.
Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-κB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-κB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion.
This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.
PMCID: PMC2763856  PMID: 19814810
9.  Commensal-Induced Regulatory T Cells Mediate Protection against Pathogen-Stimulated NF-κB Activation 
PLoS Pathogens  2008;4(8):e1000112.
Host defence against infection requires a range of innate and adaptive immune responses that may lead to tissue damage. Such immune-mediated pathologies can be controlled with appropriate T regulatory (Treg) activity. The aim of the present study was to determine the influence of gut microbiota composition on Treg cellular activity and NF-κB activation associated with infection. Mice consumed the commensal microbe Bifidobacterium infantis 35624 followed by infection with Salmonella typhimurium or injection with LPS. In vivo NF-κB activation was quantified using biophotonic imaging. CD4+CD25+Foxp3+ T cell phenotypes and cytokine levels were assessed using flow cytometry while CD4+ T cells were isolated using magnetic beads for adoptive transfer to naïve animals. In vivo imaging revealed profound inhibition of infection and LPS induced NF-κB activity that preceded a reduction in S. typhimurium numbers and murine sickness behaviour scores in B. infantis–fed mice. In addition, pro-inflammatory cytokine secretion, T cell proliferation, and dendritic cell co-stimulatory molecule expression were significantly reduced. In contrast, CD4+CD25+Foxp3+ T cell numbers were significantly increased in the mucosa and spleen of mice fed B. infantis. Adoptive transfer of CD4+CD25+ T cells transferred the NF-κB inhibitory activity. Consumption of a single commensal micro-organism drives the generation and function of Treg cells which control excessive NF-κB activation in vivo. These cellular interactions provide the basis for a more complete understanding of the commensal-host-pathogen trilogue that contribute to host homeostatic mechanisms underpinning protection against aberrant activation of the innate immune system in response to a translocating pathogen or systemic LPS.
Author Summary
The normal response to infection is rapid and effective clearance of pathogenic microbes. However, this immune response may occasionally cause collateral inflammatory damage to host tissue and in severe cases, such as systemic sepsis, results in organ failure. Various cellular mechanisms, including regulatory T cells, protect against aggressive immune responses. However, environmental agents which promote regulatory T cells are not well understood. We and others have previously shown that non-pathogenic or commensal micro-organisms can protect the host from aberrant pro-inflammatory activity within the gut, but the influence of these microbes on regulatory T cells in the context of systemic infection has not been examined. In this study, we demonstrate that consumption of a single commensal bacterium induces regulatory T cells in vivo which protect the host from pathogen-induced inflammatory responses by limiting activation of the pro-inflammatory transcription factor NF-κB via the toll-like receptor 4 (TLR-4) pathway. This report conclusively demonstrates a cellular and molecular basis for the commensal-host-pathogen trilogue resulting in enhanced protection from systemic infection whilst limiting pro-inflammatory damage mediated by activation of the innate immune system.
PMCID: PMC2474968  PMID: 18670628
10.  Comparative and Functional Analysis of Sortase-Dependent Proteins in the Predicted Secretome of Lactobacillus salivarius UCC118†  
Surface proteins are important factors in the interaction of probiotic and pathogenic bacteria with their environment or host. We performed a comparative bioinformatic analysis of four publicly available Lactobacillus genomes and the genome of Lactobacillus salivarius subsp. salivarius strain UCC118 to identify secreted proteins and those linked to the cell wall. Proteins were identified which were predicted to be anchored by WXL-binding domains, N- or C-terminal anchors, GW repeats, lipoprotein anchors, or LysM-binding domains. We identified 10 sortase-dependent surface proteins in L. salivarius UCC118, including three which are homologous to mucus-binding proteins (LSL_0152, LSL_0311, and LSL_1335), a collagen-binding protein homologue (LSL_2020b), two hypothetical proteins (LSL_1838 and LSL_1902b), an enterococcal surface protein homologue (LSL_1085), a salivary agglutinin-binding homologue (LSL_1832b), an epithelial binding protein homologue (LSL_1319), and a proteinase homologue (LSL_1774b). However, two of the genes are gene fragments and four are pseudogenes, suggesting a lack of selection for their function. Two of the 10 genes were not transcribed in vitro, and 1 gene showed a 10-fold increase in transcript level in stationary phase compared to logarithmic phase. The sortase gene was deleted, and three genes encoding sortase-dependent proteins were disrupted. The sortase mutant and one sortase-dependent protein (mucus-binding homologue) mutant showed a significant reduction in adherence to human epithelial cell lines. The genome-wide investigation of surface proteins can thus help our understanding of their roles in host interaction.
PMCID: PMC1489637  PMID: 16751526

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