RESUMO
The association between inflammatory bowel diseases and colorectal cancer is well documented. The genetic modification of lactic acid bacteria as a tool to increase the anti-inflammatory potential of these microorganisms has also been demonstrated. Thus the aim of the present work was to evaluate the anti-cancer potential of different genetically modified lactic acid bacteria (GM-LAB) producing antioxidant enzymes (catalase or superoxide dismutase) or the anti-inflammatory cytokine IL-10 (protein or DNA delivery) using a chemical induced colon cancer murine model. Dimethilhydrazine was used to induce colorectal cancer in mice. The animals received GM-LAB producing anti-oxidant enzymes, IL-10 or a mixture of different GM-LAB. Intestinal damage, enzyme activities and cytokines were evaluated and compared to the results obtained from mice that received the wild type strains from which derived the GM-LAB. All the GM-LAB assayed showed beneficial effects against colon cancer even though they exerted different mechanisms of action. The importance to select LAB with innate beneficial properties as the progenitor strain was demonstrated with the GM-LAB producing anti-oxidant enzymes. In addition, the best effects for the mixtures GM-LAB that combine different anti-inflammatory mechanism. Results indicate that mixtures of selected LAB and GM-LAB could be used as an adjunct treatment to decrease the inflammatory harmful environment associated to colorectal cancer, especially for patients with chronic intestinal inflammation who have an increased risk to develop colorectal cancer.
Assuntos
Antineoplásicos/uso terapêutico , Catalase/genética , Neoplasias Colorretais/terapia , Doenças Inflamatórias Intestinais/terapia , Interleucina-10/genética , Lactococcus lactis/fisiologia , Superóxido Dismutase/genética , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Neoplasias Colorretais/induzido quimicamente , Modelos Animais de Doenças , Feminino , Humanos , Imunidade nas Mucosas/genética , Interleucina-10/uso terapêutico , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Superóxido Dismutase/metabolismoRESUMO
Inflammatory bowel diseases (IBDs) affect the gastrointestinal tract and are characterized by recurrent inflammation that requires lifelong therapies. Probiotics such as lactic acid bacteria (LAB) have been proposed to complement current treatment protocols for these patients; however, their characteristics are strain dependent. In this regard, certain novel characteristics are only possible through the genetic modification of these beneficial micro-organisms. Different delivery systems, such as protein delivery of anti-oxidant enzymes and anti-inflammatory cytokines, have been shown to be effective in preventing and treating IBD in animal models. In this study, the safety of the recombinant LAB (recLAB) Streptococcus thermophilus CRL807 : CAT, S. thermophilus CRL807 : SOD, Lactococcus lactis NCDO2118 pXILCYT : IL-10, L. lactis MG1363 pValac : IL-10 and L. lactis MG1363 pGroESL : IL-10 with proven beneficial effects was compared to their progenitor strains S. thermophilus CRL807, L. lactis NCDO2118 or L. lactis MG1363. The prolonged administration of these genetically modified strains showed that they were just as safe as the native strains from which they derive, as demonstrated by normal animal growth and relative organ weights, absence of microbial translocation from the gastrointestinal tract, normal blood parameters and intestinal histology. The results show the potential use of these recLAB in future therapeutic formulations; however, the use of modern bio-containment systems is required for the future acceptance of these recLAB by the medical community and patients with IBD.
Assuntos
Terapia Biológica/métodos , Colite/prevenção & controle , Colite/terapia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Lactococcus lactis/patogenicidade , Probióticos/efeitos adversos , Streptococcus thermophilus/patogenicidade , Experimentação Animal , Animais , Feminino , Lactococcus lactis/genética , Camundongos Endogâmicos BALB C , Streptococcus thermophilus/genéticaRESUMO
Inflammatory Bowel Diseases (IBD) are disorders of the gastrointestinal tract characterized by recurrent inflammation that requires lifelong treatments. Probiotic microorganisms appear as an alternative for these patients; however, probiotic characteristics are strain dependent and each probiotic needs to be tested to understand the underlining mechanisms involved in their beneficial properties. Genetic modification of lactic acid bacteria (LAB) was also described as a tool for new IBD treatments. The first part of this review shows different genetically modified LAB (GM-LAB) described for IBD treatment since 2000. Then, the two principally studied strategies are discussed (i) GM-LAB producing antioxidant enzymes and (ii) GM-LAB producing the anti-inflammatory cytokine IL-10. Different delivery systems, including protein delivery and DNA delivery, will also be discussed. Studies show the efficacy of GM-LAB (using different expression systems) for the prevention and treatment of IBD, highlighting the importance of the bacterial strain selection (with anti-inflammatory innate properties) as a promising alternative. These microorganisms could be used in the near future for the development of therapeutic products with anti-inflammatory properties that can improve the quality of life of IBD patients.
RESUMO
The aim of this work was to develop a Streptococcus (S.) thermophilus strain with improved anti-inflammatory properties due to the incorporation of the therapeutic cDNA delivery plasmid pValac::il-10. To achieve this purpose, cells of S. thermophilus CRL807, previously selected as being an important anti-inflammatory strain, were electroporated with pValac::il-10 plasmid. In order to confirm the functionality of the developed strain, it was co-cultured with human epithelial cells Caco-2 and the production of IL-10 was evaluated by ELISA. Bacterial suspensions of S. thermophilus CRL807 containing pValac::il-10 plasmid or of the wild-type (WT) strain were administered in vivo using a murine model of intestinal inflammation. The animals treated with S. thermophilus CRL807 pValac::il-10 showed a lower body weight loss, microbial translocation to liver and damage scores in their intestines at macroscopical and microscopic levels. Furthermore, a significant increase was observed in the concentration of IL-10 in the intestinal contents of these mice compared to the rest of the experimental groups, accompanied by decreased levels of pro-inflammatory cytokines. The insertion of the therapeutic pValac::il-10 plasmid increased the intrinsic anti-inflammatory activity (synergetic effect) of S. thermophilus CRL807 which could be included in novel treatment protocols for inflammatory bowel diseases.
Assuntos
Anti-Inflamatórios/metabolismo , Colite/tratamento farmacológico , DNA Complementar/metabolismo , Sistemas de Liberação de Medicamentos , Interleucina-10/uso terapêutico , Streptococcus thermophilus/metabolismo , Animais , Líquidos Corporais/metabolismo , Peso Corporal/efeitos dos fármacos , Células CACO-2 , Colite/microbiologia , Colite/patologia , Modelos Animais de Doenças , Humanos , Interleucina-10/farmacologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Fígado/efeitos dos fármacos , Fígado/microbiologia , Fígado/patologia , Camundongos , Streptococcus thermophilus/efeitos dos fármacosRESUMO
BACKGROUND: Oral treatment with Lactococcus lactis strains secreting the anti-inflammatory cytokine interleukin (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 2 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 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 Stress-Inducible Controlled Expression 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 with inflamed nontreated 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.
Assuntos
Colite/prevenção & controle , Colo/microbiologia , Vetores Genéticos , Interleucina-10/biossíntese , Mucosa Intestinal/microbiologia , Lactobacillus/metabolismo , Probióticos , Ácido Trinitrobenzenossulfônico , Animais , Colite/induzido quimicamente , Colite/genética , Colite/imunologia , Colite/metabolismo , Colite/microbiologia , Colite/patologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Interleucina-10/genética , Interleucina-10/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lactobacillus/genética , Lactobacillus/imunologia , Camundongos Endogâmicos BALB C , Índice de Gravidade de Doença , Fatores de Tempo , Redução de PesoRESUMO
The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.
Assuntos
Antioxidantes/metabolismo , Fatores Imunológicos/metabolismo , Inflamação/imunologia , Engenharia Metabólica , Streptococcus thermophilus/imunologia , Animais , Catalase/genética , Catalase/metabolismo , Células Cultivadas , Colite/induzido quimicamente , Colite/imunologia , Colite/microbiologia , Colite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Camundongos , Streptococcus thermophilus/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Ácido Trinitrobenzenossulfônico/administração & dosagem , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
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.
RESUMO
Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing LAB were evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohn's disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing LAB showed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered LAB that produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies.
Assuntos
Catalase/metabolismo , Doença de Crohn/prevenção & controle , Lacticaseibacillus casei/enzimologia , Probióticos/farmacologia , Superóxido Dismutase/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Catalase/biossíntese , Colo/patologia , Doença de Crohn/induzido quimicamente , Doença de Crohn/microbiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Engenharia Genética , Histocitoquímica , Inflamação , Intestino Grosso , Lacticaseibacillus casei/metabolismo , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Superóxido Dismutase/biossíntese , Ácido TrinitrobenzenossulfônicoRESUMO
Interleukin-10 (IL-10) is the most important anti-inflammatory cytokine at intestinal level, and its absence is involved in inflammatory bowel diseases. However, oral treatment with IL-10 is difficult because of its low survival in the gastrointestinal tract and systemic treatments lead to undesirable side effects. The aim of this paper was to evaluate the anti-inflammatory effect of the administration of milks fermented by Lactococcus lactis strains that produce IL-10 under the control of the xylose-inducible expression system using a trinitrobenzenesulfonic acid-induced colitis murine model. Mice that received milks fermented by L. lactis strains producing IL-10 in the cytoplasm (Cyt strain) or secreted to the product (Sec strain) showed lower damage scores in their large intestines, decreased IFN-γ levels in their intestinal fluids and lower microbial translocation to liver, compared to mice receiving milk fermented by the wild-type strain or those not receiving any treatment. The results obtained in this study show that the employment of fermented milks as a new form of administration of IL-10-producing L. lactisis effective in the prevention of inflammatory bowel disease in a murine model.