Prospective Uses of Genetically Engineered Lactic Acid Bacteria for the Prevention of Inflammatory Bowel Diseases

Inflammatory bowel disease (IBD) is a term used to describe a group of intestinal disorders in which inflammation is a major feature. Although rare forms of IBD exist, these diseases normally pertain to ulcerative colitis (UC) (Head & Jurenka, 2003) and Crohn’s disease (CD) (Baumgart & Sandborn, 2007). There is evidence that these do not represent distinct conditions but rather are the same disease with shared etiological factors (Price, 1992); however, clinical manifestations (such as the exact location of the pathology or the affected individual’s immunological and constitutional endowment) are distinctive between both. Despite many years of study, the exact etiology and pathogenesis of these disorders remain unclear but great advances have been made using experimental animal models and have provided insights into the complex, multi-factorial processes and mechanisms that can result in chronic intestinal inflammation (Elson & Weaver, 2003). The aim of this chapter is to present an overview of the current expanding knowledge of the mechanisms by which lactic acid bacteria and other probiotic microorganisms participate in the prevention and treatment of IBD and how genetic engineering techniques can be used to improve their effectiveness or create novel therapeutic strains. In the following sections, the mechanisms by which these beneficial microorganisms exert their therapeutic effects, which include changes in the gut microbiota, stimulation of the host immune responses, enhancement of intestinal barrier function and reduction of the oxidative stress due to their antioxidant properties will be discussed.Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Alvarenga Lima, Fernanda. Universidade Federal de Minas Gerais; BrasilFil: Zurita Turk, Meritxell. Universidade Federal de Minas Gerais; BrasilFil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais; BrasilFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Mechanisms involved in the anti-inflammatory properties of native and genetically engineered lactic acid bacteria

Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms that have been shown to possess therapeutic properties since they are able to prevent the development of some diseases, as shown mostly on animal models for cancer, infections and gastrointestinal disorders such as intestinal inflammation. LAB have been shown to regulate mucosal immune responses by modulating the production and liberation of regulatory agents such as cytokines by the host. Some of these cytokines, such as the anti-inflammatory interleukin-10 (IL-10), modulate the inflammatory immune response, thus immunomodulation is a mechanism by which LAB can prevent certain inflammatory bowel diseases (IBD). Since oxidative stress participates to the inflammatory processes and to the appearance of damages in pathologies of the gastrointestinal tract of humans such as IBD, LAB could also prevent inflammation by eliminating reactive oxygen species (ROS) through the activity of antioxidant enzymes.In order to obtain novel strains or enhance beneficial effects of LAB, genetic engineering has been used to produce either antioxidant enzymes (such as catalases and superoxide dismutases) or anti-inflammatory cytokines (such as IL-10) producing LAB. These novel strains have successfully been used to prevent inflammatory bowel diseases in animal models and could be evaluated in human clinical trials. Here, we present an overview of the current knowledge of the mechanisms by which LAB can be used to prevent undesired intestinal inflammatory responses and could be used as a therapeutic tool for IBD.Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina;Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina;Fil: Zurita-Turk, Meritxell. Universidade Federal Do Minas Gerais; Brasil;Fil: Alvarenga Lima, Fernanda. Universidade Federal Do Minas Gerais; Brasil;Fil: Santos Ponce, Daniela. Universidade Federal Do Minas Gerais; Brasil;Fil: Miyoshi, Aanderson. Universidade Federal Do Minas Gerais; Brasil;Fil: Azevedo, Vasco. Universidade Federal Do Minas Gerais; Brasil;Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina

Importance of IL-10 Modulation by Probiotic Microorganisms in Gastrointestinal Inflammatory Diseases

Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms that are naturally present in many foods and possess a wide range of therapeutic properties. The aim of this paper is to present an overview of the current expanding knowledge of one of the mechanisms by which LAB and other probiotic microorganisms participate in the prevention and treatment of gastrointestinal inflammatory disease through their immune-modulating properties. A special emphasis will be placed on the critical role of the anti-inflammatory cytokine IL-10, and a brief overview of the uses of genetically engineered LAB that produce this important immune response mediator will also be discussed. Thus, this paper will demonstrate the critical role that IL-10 plays in gastrointestinal inflammatory diseases and how probiotics could be used in their treatment

Lactococcus lactis strains delivering IL-10 protein or cDNA to the intestinal mucosa show anti-inflammatory properties in a TNBS-induced chronic colitis model

Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level. Oral treatments with IL-10 are inefficient because of its sensitivity to the harsh conditions of the GI tract, and systemic treatments cause undesirable side effects. Our aim was to compare the protective effects of IL-10, delivered by recombinant lactoccoci using two novel expression systems, in a murine colitis model mimicking the relapsing nature of inflammatory bowel diseases. The first system is based on a stress-inducible promoter (pGroES/L) for the production and delivery of heterologous proteins in situ at mucosal surfaces, and the second allows the delivery to the host cells of an il-10 cDNA, harbored in a eukaryotic DNA expression vector (pValac). Colitis was induced in female BALB/c mice by intrarectal injection of TNBS. Mice that survived the challenge and recovered their body weight received one of the bacterial treatments or saline solution orally during 14 days. Colitis was reactivated 25 days after the first TNBS challenge with a second injection. Three days after colitis reactivation, cytokine profiles and inflammation in colon samples were evaluated. Animals receiving L. lactis delivering pGroES/L: il-10 or pValac: il-10 plasmids showed lower body weight loss and damage scores in their large intestines compared to inflamed non-treated mice. Both treatments also increased IL-10 concentration in the intestine, compared to the controls without treatment and maintained an increased ratio of IL-10/ pro-inflammatory cytokines. Our results confirm the protective effect of IL-10 delivered by L. lactis either as a protein or as a cDNA in a TNBS-induced chronic colitis model, and provides a step further in the use of genetically engineered bacteria as a valid system to deliver therapeutic molecules at mucosal level.Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Martín, Rebeca. Institut National de la Recherche Agronomique; FranciaFil: Saraiva, Tessalia. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Zurita Turk, Meritxell. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Miyoshi, Anderson. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Langella, Philippe. Institut National de la Recherche Agronomique; FranciaFil: Bermúdez Humarán, Luis G.. Institut National de la Recherche Agronomique; FranciaFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina11th International Symposium on Lactic Acid Bacteria: Health, sustainability, diversity, and applicationEgmond aan ZeePaíses BajosFederation of European Microbiological SocietiesRoyal Netherlands Society for Microbiolog

A novel interleukin-10 DNA mucosal delivery system attenuates intestinal inflammation in a mouse model

Inflammatory bowel diseases (IBD) describe a group of complex intestinal disorders characterized by inflammation in the gastrointestinal tract. Current treatments for IBD include the use of anti-inflammatory drugs; furthermore, recombinant lactic acid bacteria have been used as a therapeutic vehicle for anti-inflammatory agents in IBD models. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines; however, its oral administration is limited because it is quickly degraded in the gastrointestinal tract and systemic treatments have led to undesirable side effects. In this study, an engineered invasive strain of Lactococcus (L.) lactis producing Fibronectin Binding Protein A (FnBPA+), from Staphylococcus aureus capable of delivering, directly inside eukaryotic cells, an eukaryotic DNA expression vector containing the ORF coding for IL-10 of Mus musculus (pValac:il-10) was developed and its functionality was evaluated using in vitro and in vivo assays. Functionality of the plasmid and the invasive strain was demonstrated by transfection and invasiveness assays using cell cultures and in vivo in mice by fluorescence microscopy. TNBS inoculated mice that received this novel strain showed lower damage scores in their large intestines (at both macroscopic and microscopic levels), lower microbial translocation to liver, and increased anti-inflammatory/pro-inflammatory cytokine ratios compared to mice that received L. lactis FnBPA+ without the pValac:il-10 plasmid. The effectiveness was demonstrated of this novel DNA delivery therapeutic strategy in the prevention of inflammation using a murine model of colitis.Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); ArgentinaFil: Zurita-Turk, Meritxell. Universidade Federal Do Minas Gerais; Brasil;Fil: Alvarenga Lima, Fernanda. Universidade Federal Do Minas Gerais; Brasil;Fil: Coelho Dos Santos, Janete. No especifíca;Fil: Leclercq, Sophie Yvette. No especifíca;Fil: Chatel, Jean-Marc. Institut National de la Recherche Agronomique; Francia;Fil: Azevedo, Vasco. Universidade Federal Do Minas Gerais; Brasil;Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); ArgentinaFil: Miyoshi, Anderson. Universidade Federal Do Minas Gerais; Brasil;Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos (i); Argentina. Universidad Nacional de Tucuman. Facultad de Medicina. Departamento de Investigacion. Catedra de Metodologia de la Invest.cientifica; Argentin

Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis

Background: Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract. Interleukin-10 is one of the most important anti-inflammatory cytokines involved in the intestinal immune system and because of its role in downregulating inflammatory cascades, its potential for IBD therapy is under study. We previously presented the development of an invasive strain of Lactococcus lactis (L. lactis) producing Fibronectin Binding Protein A (FnBPA) which was capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) and diminish inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of intestinal inflammation. As a new therapeutic strategy against IBD, the aim of this work was to evaluate the therapeutic effect of two L. lactis strains (the same invasive strain evaluated previously and the wild-type strain) carrying the therapeutic pValac:il-10 plasmid in the prevention of inflammation in a dextran sodium sulphate (DSS)-induced mouse model. Results: Results obtained showed that not only delivery of the pValac:il-10 plasmid by the invasive strain L. lactis MG1363 FnBPA+, but also by the wild-type strain L. lactis MG1363, was effective at diminishing intestinal inflammation (lower inflammation scores and higher IL-10 levels in the intestinal tissues, accompanied by decrease of IL-6) in the DSS-induced IBD mouse model. Conclusions: Administration of both L. lactis strains carrying the pValac:il-10 plasmid was effective at diminishing inflammation in this murine model of experimental colitis, showing their potential for therapeutic intervention of IBD.Fil: Zurita Turk, Meritxell. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Santos, Ana C. G.. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Pereira, Vanessa Bastos. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Cara, Denise C.. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Leclercq, Sophie Y.. Fundaçao Ezequiel Dias. Laboratório de Inovaçao Biotecnológica; BrasilFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Azevedo, Vasco. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; BrasilFil: Chatel, Jean-Marc. Universidade Federal do Minas Gerais; BrasilFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Miyoshi, Anderson. Universidade Federal Do Minas Gerais. Instituto de Cs.biologicas; Brasi

Protective effects of lactococci strains delivering either IL-10 protein or cDNA in a TNBS-induced chronic colitis model

Background: Oral treatment with Lactococcus (L.) lactis strains secreting the anti-inflammatory cytokine IL-10 has previously shown success as a therapy for inflammatory bowel diseases (IBD). Goals: Our aim was to compare the protective effects of IL-10, delivered by recombinant lactoccoci using two novel expression systems, in a murine colitis model mimicking the relapsing nature of IBD. The first system is based on a Stress-Inducible Controlled Expression (SICE) system for the production and delivery of heterologous proteins at mucosal surfaces and the second allows the delivery to the host cells of an il-10 cDNA cassette, harbored in a eukaryotic DNA expression vector (pValac). Study: Colitis was induced in female BALB/c mice by intrarectal injection of 2,4,6-trinitrobenzenesulphonic acid (TNBS). Mice that recovered received one of the bacteria treatments or saline solution orally during 14 days. Colitis was reactivated 25 days after the first TNBS injection with a second TNBS challenge. Three days after colitis reactivation, cytokine profiles and inflammation in colon samples were evaluated. Results: Animals (N=9) receiving L. lactis strains secreting IL-10 using SICE system or delivering pValac:il-10 plasmid showed lower weight loss (p<0.005), lower damage scores (p<0.005) and immune activation in their large intestines compared to inflamed non-treated mice. Conclusions: Our results confirm the protective effect of IL-10 delivered either as a protein or as a cDNA in a colitis model mimicking the relapsing nature of IBD and provides a step further in the ?proof-of-concept? of genetically engineered bacteria as a valid system to deliver therapeutic molecules at mucosal level.Fil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Martin Rosique, Rebeca. Institut National de la Recherche Agronomique; FranciaFil: Saraiva, Tessália. Universidade Federal do Minas Gerais; BrasilFil: Zurita Turk, Meritxell. Universidade Federal do Minas Gerais; BrasilFil: Miyoshi, Anderson. Universidade Federal do Minas Gerais; BrasilFil: Azevedo, Vasco. Universidade Federal do Minas Gerais; BrasilFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Langella, Philippe. Institut National de la Recherche Agronomique; FranciaFil: Bermudez-Humaran, Luis G.. Institut National de la Recherche Agronomique; FranciaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentin

Use of genetically engineered lactic acid bacteria for the treatment of crohn's disease

Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms that are naturally present in many foods and possess a wide range of therapeutic properties. Although probiotic effects are a strain dependent trait, using modern genetic engineering techniques it is theoretically possible to obtain strains that can exert a variety of beneficial properties. For example, the introduction of antioxidant enzyme genes or cytokine producing capabilities in current probiotic strains that have natural anti-inflammatory properties, such as the ability to modulate the immune dependent processes, could generate very useful strains that could be applied in the treatment of a variety of inflammatory bowel (IBD) diseases such as Crohn´s disease and ulcerative colitis. These strains could also be included in treatment protocols since it has been shown that probiotics can enhance the effectiveness of traditional IBD treatments. This chapter will present a brief overview of the current expanding knowledge of the mechanisms by which LAB participate in the prevention of inflammatory processes and the uses of engineering strategies for the development of new LAB that produce either antioxidant enzymes (such as catalase and superoxide dismutase) or anti-inflammatory cytokines (such as IL-10). These novel strains could be used in new treatment protocols since they could enhance the effectiveness of traditional therapies.Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Medicina; ArgentinaFil: del Carmen, Silvina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Langella, Philippe. Institut National de la Recherche Agronomique; FranciaFil: Bermudez Humaran, Luis. Institut National de la Recherche Agronomique; FranciaFil: Alvarenga Lima, Fernanda. Universidade Federal de Minas Gerais; BrasilFil: Zurita Turk, Meritxell. Universidade Federal de Minas Gerais; BrasilFil: Santos Ponce, Daniela. Universidade Federal de Minas Gerais; BrasilFil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais; BrasilFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin