RESUMO
Inflammatory bowel diseases (IBD) are gastrointestinal disorders characterized by a breakdown in intestinal homeostasis by inflammatory immune responses to luminal antigens. Novel strategies for ameliorating IBD have been proposed in many studies using animal models. Our group has demonstrated that administration of Lactococcus lactis NCDO 2118 can improve clinical parameters of colitis induced by oral administration of dextran sulphate sodium (DSS). However, it is not clear whether other strains of L. lactis can yield the same effect. The objective of present study was to analyze the effects of three different L. lactis strains (NCDO2118, IL1403 and MG1363) in the development of DSS-induced colitis in C57BL/6 mice. Acute colitis was induced in C57/BL6 mice by the administration of 2% DSS during 7 consecutive days. Body weight loss and shortening of colon length were observed in DSS-treated mice, and none of L. lactis strains had an impact in these clinical signs of colitis. On the other hand, all strains improved the global macroscopical disease index and prevented goblet cells depletion as well as the increase of intestinal permeability. TNF-α production was reduced in gut mucosa of L. lactis DSS-treated mice indicating a modulation of a critical pro-inflammatory response by all strains tested. However, only L. lactis NCDO2118 and MG1363 induced a higher frequency of CD11c+CD11b-CD103+ tolerogenic dendritic cells in lymphoid organs of mice at steady state. We conclude that all tested strains of L. lactis improved the clinical scores and parameters of colitis, which confirm their anti-inflammatory properties in this model of colitis.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Lactococcus lactis , Animais , Camundongos , Lactococcus lactis/genética , Camundongos Endogâmicos C57BL , Colite/induzido quimicamente , Doenças Inflamatórias Intestinais/induzido quimicamente , Imunidade , Modelos Animais de DoençasRESUMO
Several antigens can act as allergens eliciting IgE-mediated food allergy reactions when fed to sensitized animals. One of them is ovalbumin (OVA) which is the main allergen in egg white. Allergic mice develop aversion to OVA consumption. This aversive behavior is associated with anxiety, and it can be transferred to non-sensitized mice by injection of serum of allergic mice. However, it is yet to be determined whether altered behavior is a general component of food allergy or whether it is specific for some types of allergens. Cow's milk allergy is the most prevalent food allergy that usually begins early in life and ß-lactoglobulin (BLG) is the milk component with the highest allergenicity. In this study, we investigated behavioral and neuroimmune circuits triggered by allergic sensitization to BLG. A neuroimmune conflict between aversion and reward was observed in a model of food allergy induced by BLG intake. Mice sensitized to BLG did not present aversive behavior when BLG was used for sensitization and oral challenge. Mice allergic to BLG preferred to drink the allergen-containing solution over water even though they had high levels of specific IgE, inflammatory cells in the intestinal mucosa and significant weight loss. When sensitized to OVA and challenged with the same antigen, mice had increased levels of neuron activation in the amygdala, a brain area related to anxiety. On the other hand, when mice were sensitized to OVA and received a mixture of BLG and OVA in the oral challenge, mice preferred to drink this mixture, despite their aversion to OVA, which was associated with neuron activation in the nucleus accumbens, an area related to reward behavior. Thus, the aversive behavior observed in food allergy to OVA does not apply to all antigens and some allergens may activate the brain reward system rather than anxiety and aversion. Our study provides novel insights into the neuroimmune conflicts regarding preference and avoidance to a common antigen associated with food allergy.
RESUMO
Dietary proteins can influence the maturation of the immune system, particularly the gut-associated lymphoid tissue, when consumed from weaning to adulthood. Moreover, replacement of dietary proteins by amino acids at weaning has been shown to impair the generation of regulatory T cells in the gut as well as immune activities such as protective response to infection, induction of oral and nasal tolerance as well as allergic responses. Polymeric and elemental diets are used in the clinical practice, but the specific role of intact proteins and free amino acids during the intestinal inflammation are not known. It is plausible that these two dietary nitrogen sources would yield distinct immunological outcomes since proteins are recognized by the immune system as antigens and amino acids do not bind to antigen-recognition receptors but instead to intracellular receptors such as mammalian target of rapamycin (mTOR). In this study, our aim was to evaluate the effects of consumption of an amino acid-containing diet (AA diet) versus a control protein-containing diet in adult mice at steady state and during colitis development. We showed that consumption of a AA diet by adult mature mice lead to various immunological changes including decrease in the production of serum IgG as well as increase in the levels of IL-6, IL-17A, TGF-ß, and IL-10 in the small and large intestines. It also led to changes in the intestinal morphology, to increase in intestinal permeability, in the number of total and activated CD4+ T cells in the small intestine as well as in the frequency of proliferating cells in the colon. Moreover, consumption of AA diet during and prior to development of dextran sodium sulfate-induced colitis exacerbated gut inflammation. Administration of rapamycin during AA diet consumption prevented colitis exacerbation suggesting that mTOR activation was involved in the effects triggered by the AA diet. Therefore, our study suggests that different outcomes can result from the use of diets containing either intact proteins or free amino acids such as elemental, semielemental, and polymeric diets during intestinal inflammation. These results may contribute to the design of nutritional therapeutic intervention for inflammatory bowel diseases.
RESUMO
Heat shock proteins (Hsps) are highly expressed at all sites of inflammation. As they are ubiquitous and immunodominant antigens, these molecules represent good candidates for the therapeutic use of oral tolerance in autoimmune and chronic inflammatory diseases. Evidences from human and animal studies indicate that inflammatory bowel disease (IBD) results from uncontrolled inflammatory responses to intestinal microbiota. Hsps are immunodominant proteins expressed by several immune cells and by commensal bacteria. Using an IBD mouse model, we showed that oral pretreatment with genetically modified Lactococcus lactis that produces and releases Mycobacterium Hsp65, completely prevented DSS-induced colitis in C57BL/6 mice. Protection was associated with reduced pro-inflammatory cytokines, such as IFN-γ, IL-6, and TNF-α; increased IL-10 production in colonic tissue; and expansion of CD4+Foxp3+ and CD4+LAP+ regulatory T cells in spleen and mesenteric lymph nodes. This effect was dependent on IL-10 and toll-like receptor 2. Thus, this approach may open alternative options for long-term management of IBD.
RESUMO
PURPOSE: Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with important impact on global health. Prebiotic and probiotic strategies are thought to be useful in the context of experimental IBD. Here, we compared the effects of preventive versus therapeutic treatment with a high fiber diet (prebiotic) in combination or not with Bifidobacterium longum (probiotic) in a murine model of chronic colitis. METHODS: Colitis was induced by adding dextran sulfate sodium (DSS) to drinking water for 6 days (acute colitis) or for 5 cycles of DSS (chronic colitis). RESULTS: Administration of the high fiber diet protected from acute colitis. Protection was optimal when diet was started 20 days prior to DSS. A 5-day pretreatment with acetate, a short-chain fatty acid, provided partial protection against acute colitis. In chronic colitis, pretreatment with the high fiber diet attenuated clinical and inflammatory parameters of disease. However, when the treatment with the high fiber diet started after disease had been established, overall protection was minimal. Similarly, delayed treatment with acetate or B. longum did not provide any protection even when the probiotic was associated with the high fiber diet. CONCLUSION: Preventive use of a high fiber diet or acetate clearly protects mice against acute and chronic damage induced by DSS in mice. However, protection is lost when therapies are initiated after disease has been established. These results suggest that any therapy aimed at modifying the gut environment (e.g., prebiotic or probiotic strategies) should be given early in the course of disease.
Assuntos
Biomarcadores/sangue , Colite/dietoterapia , Dieta , Fibras na Dieta/administração & dosagem , Acetatos/administração & dosagem , Doença Aguda , Animais , Comportamento Animal , Bifidobacterium/metabolismo , Doença Crônica , Colite/induzido quimicamente , Colo/microbiologia , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Ácidos Graxos Voláteis/administração & dosagem , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Prebióticos , Probióticos/administração & dosagemRESUMO
BACKGROUND: Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease. RESULTS: The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid-pValac::dts::IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C(-)IL-4(+), F4/80(+)MHCII(+)Ly6C(-)IL-10(+), F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups. CONCLUSIONS: This study shows that L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels of IL-10-secreting regulatory cells and, thus, demonstrating the effectiveness of this novel DNA delivery-based strategy.
Assuntos
Vetores Genéticos , Inflamação/terapia , Doenças Inflamatórias Intestinais/terapia , Interleucina-10/metabolismo , Interleucina-4/genética , Lactococcus lactis/genética , Animais , Células CHO , Cricetulus , Citocinas/imunologia , Citocinas/metabolismo , DNA/genética , Inflamação/induzido quimicamente , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/prevenção & controle , Interleucina-4/imunologia , Interleucina-4/uso terapêutico , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Camundongos , Mucosa/imunologia , Mucosa/ultraestrutura , TransfecçãoRESUMO
The ability to avoid inflammatory responses to dietary components and microbiota antigens in the gut mucosa is achieved by a mechanism termed oral tolerance. This phenomenon is crucial to maintain the physiological immune activity in the gut and to prevent inflammatory disorders such as food allergy and inflammatory bowel diseases. Moreover, orally administered antigens induce regulatory cells that control systemic inflammatory responses as well. Given its specific, systemic and long-lasting effects, oral tolerance represents a promising approach for immunotherapies that aim to modulate inflammatory and autoimmune diseases. However, there are different protocols of feeding for induction of oral tolerance, and they have an impact in tolerance efficiency and length. Herein, we present and discuss different experimental feeding protocols and how they influence the outcome of oral administration of antigens.
Assuntos
Dessensibilização Imunológica/métodos , Tolerância Imunológica/imunologia , Imunoglobulina E/sangue , Ovalbumina/imunologia , Células Th1/imunologia , Administração Oral , Animais , Nutrição Enteral , Feminino , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade Alimentar/prevenção & controle , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/prevenção & controle , Mucosa Intestinal/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagemRESUMO
BACKGROUND: Many probiotic bacteria have been described as promising tools for the treatment and prevention of inflammatory bowel diseases (IBDs). Most of these bacteria are lactic acid bacteria, which are part of the healthy human microbiota. However, little is known about the effects of transient bacteria present in normal diets, including Lactococcus lactis. METHODS: In the present study, we analysed the immunomodulatory effects of three L. lactis strains in vitro using intestinal epithelial cells. L. lactis NCDO 2118 was administered for 4 days to C57BL/6 mice during the remission period of colitis induced by dextran sodium sulphate (DSS). RESULTS: Only one strain, L. lactis NCDO 2118, was able to reduce IL-1ß-induced IL-8 secretion in Caco-2 cells, suggesting a potential anti-inflammatory effect. Oral treatment using L. lactis NCDO 2118 resulted in a milder form of recurrent colitis than that observed in control diseased mice. This protective effect was not attributable to changes in secretory IgA (sIgA); however, NCDO 2118 administration was associated with an early increase in IL-6 production and sustained IL-10 production in colonic tissue. Mice fed L. lactis NCDO 2118 had an increased number of regulatory CD4(+) T cells (Tregs) bearing surface TGF-ß in its latent form (Latency-associated peptide-LAP) in the mesenteric lymph nodes and spleen. CONCLUSIONS: Here, we identified a new probiotic strain with a potential role in the treatment of IBD, and we elucidated some of the mechanisms underlying its anti-inflammatory effect.
RESUMO
The intestinal mucosa is the major site of contact with antigens, and it houses the largest lymphoid tissue in the body. In physiological conditions, microbiota and dietary antigens are the natural sources of stimulation for the gut-associated lymphoid tissues (GALT) and for the immune system as a whole. Germ-free models have provided some insights on the immunological role of gut antigens. However, most of the GALT is not located in the large intestine, where gut microbiota is prominent. It is concentrated in the small intestine where protein absorption takes place. In this review, we will address the involvement of food components in the development and the function of the immune system. Studies in mice have already shown that dietary proteins are critical elements for the developmental shift of the immature neonatal immune profile into a fully developed immune system. The immunological effects of other food components (such as vitamins and lipids) will also be addressed. Most of the cells in the GALT are activated and local pro-inflammatory mediators are abundant. Regulatory elements are known to provide a delicate yet robust balance that maintains gut homeostasis. Usually antigenic contact in the gut induces two major immune responses, oral tolerance and production of secretory IgA. However, under pathological conditions mucosal homeostasis is disturbed resulting in inflammatory reactions such as food hypersensitivity. Food allergy development depends on many factors such as genetic predisposition, biochemical features of allergens, and a growing array of environmental elements. Neuroimmune interactions are also implicated in food allergy and they are examples of the high complexity of the phenomenon. Recent findings on the gut circuits triggered by food components will be reviewed to show that, far beyond their role as nutrients, they are critical players in the operation of the immune system in health and disease.
RESUMO
IL-10 is a regulatory cytokine that plays a major role in the homeostasis of the gut and this is illustrated by the fact that IL-10(-/-) mice develop spontaneous colitis. In this study, IL-10(-/-) mice were analyzed for immunological changes during colitis development. We found a reduced frequency of regulatory T cells CD4(+)CD25(+)Foxp3(+) and higher frequency of activated T cells in the colon that precedes the macroscopic signs of the disease. Production of IL-17 and IFN-γ was higher in the colon. Colitis progression culminates with the reduction of CD4(+)LAP(+) regulatory T cells in the intestine. Frequency of B1 cells and the secretory IgA production were both elevated. Despite these alterations, 16-week-old IL-10(-/-) mice could be rendered tolerant by a continuous feeding protocol. Our study provides detailed analysis of changes that precede colitis and it also suggests that oral tolerance could be used to design novel alternative therapies for the disease.