Ana Cristina Gomes-Santos

Oral administration of sodium butyrate attenuates inflammation and mucosal lesion in experimental acute ulcerative colitis

Butyrate is a four-carbon short-chain fatty acid that improves colonic trophism. Although several studies have shown the benefits of butyrate enemas in ulcerative colitis (UC), studies using the oral route are rare in the literature. In the present study, we evaluated the effect of butyrate intake in the immune response associated to UC. For that, mice were fed control or butyrate (0.5% sodium butyrate) diets for 14 days. Acute UC was induced by dextran sulphate sodium (DSS, 2.5%), replacing drinking water. The results showed that, in UC animals, oral butyrate significantly improved trophism and reduced leukocyte (eosinophil and neutrophil) infiltration in the colon mucosa and improved the inflammatory profile (activated macrophage, B and T lymphocytes) in cecal lymph nodes. In the small intestine, although mucosa histology was similar among groups, DSS treatment reduced duodenal transforming growth factor-β, increased interleukin-10 concentrations and increased memory T lymphocytes and dendritic cells in Peyers patches. Butyrate supplementation was able to revert these alterations. When cecal butyrate concentration was analyzed in cecal content, it was still higher in the healthy animals receiving butyrate than in the UC+butyrate and control groups. In conclusion, our results show that oral administration of sodium butyrate improves mucosa lesion and attenuates the inflammatory profile of intestinal mucosa, local draining lymph nodes and Peyers patches of DSS-induced UC. Our results also highlight the potential use of butyrate supplements as adjuvant in UC treatment.

New Insights into the Immunological Changes in IL-10-Deficient Mice during the Course of Spontaneous Inflammation in the Gut Mucosa

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.

Local and systemic immune mechanisms underlying the anti-colitis effects of the dairy bacterium Lactobacillus delbrueckii.

Several probiotic bacteria have been proposed for treatment or prevention of inflammatory bowel diseases (IBD), showing a protective effect in animal models of experimental colitis and for some of them also in human clinical trials. While most of these probiotic bacteria are isolated from the digestive tract, we recently reported that a Lactobacillus strain isolated from cheese, L. delbrueckii subsp. lactis CNRZ327 (Lb CNRZ327), also possesses anti-inflammatory effects in vitro and in vivo, demonstrating that common dairy bacteria may be useful in the treatment or prevention of IBD. Here, we studied the mechanisms underlying the protective effects of Lb CNRZ327 in vivo, in a mouse dextran sodium sulfate (DSS) colitis model. During colitis, Lb CNRZ327 modulated the production of TGF-β, IL-6, and IL-12 in colonic tissue and of TGF-β and IL-6 in the spleen, and caused an expansion of CD4+Foxp3+ regulatory T cells in the cecal lymph nodes. Moreover, a strong tendency to CD4+Foxp3+ expansion was also observed in the spleen. The results of this study for the first time show that orally administered dairy lactobacilli can not only modulate mucosal but also systemic immune responses and constitute an effective treatment of IBD.

Food Components and the Immune System: From Tonic Agents to Allergens

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.

Antigen administration by continuous feeding enhances oral tolerance and leads to long-lasting effects.

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.

Oral tolerance correlates with high levels of lymphocyte activity.

Oral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-β and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.

Oral Combined Therapy with Probiotics and Alloantigen Induces B Cell–Dependent Long-Lasting Specific Tolerance

Allogeneic hematopietic stem cell transplantation (aHSCT) is widely used for the treatment of hematologic malignancies. Although aHSCT provides a good response against the malignant cells (graft-versus-leukemia [GVL]), it also leads to the development of graft-versus-host disease (GVHD), a severe disease with high mortality and morbidity rates. Therapy for GVHD is commonly based on nonspecific immunosupression of the transplanted recipient, resulting in the concomitant inhibition of the GVL effect. In this study, we propose an alternative approach to specifically suppress GVHD while sparing the GVL, based on oral treatment of transplant donors with recipient Ags, associated with the intake of probiotic Lactococcus lactis as tolerogenic adjuvant (combined therapy). We show that treatment of C57BL/6 donor mice with combined therapy before the transplant protects the recipients F1 (C57BL/6 × BAL/c) mice from clinical and pathological manifestations of disease, resulting in 100% survival rate. Importantly, the animals keep the immunological competence maintaining the GVL response as well as the response to third-party Ags. The protection is specific, long lasting and dependent on donor IL-10–sufficient B cells activity, which induces regulatory T cells in the host. These data suggest that combined therapy is a promising strategy for prevention of GVHD with preservation of GVL, opening new possibilities to treat human patients subjected to transplantation.

Milk Fermented with a 15-Lipoxygenase-1-Producing Lactococcus Lactis Alleviates Symptoms of colitis in a Murine Model

Inflammatory bowel diseases (IBD), such as Crohns disease and ulcerative colitis, is characterized by extensive inflammation due to dysregulation of the innate and adaptive immune system whose exact etiology is not yet completely understood. Currently there is no cure for IBD, thus the search for new molecules capable of controlling IBD and their delivery to the site of inflammation are the goal of many researchers. The aim of this work was to evaluate the anti-inflammatory effect of the administration of milks fermented by a Lactococcus (L.) lactis strain producing 15-lipoxygenase-1 (15-LOX-1) using a trinitrobenzenesulfonic acid-induced IBD mouse model. The results obtained demonstrated that 15-LOX-1 producing L. lactis was effective in the prevention of the intestinal damage associated to inflammatory bowel disease in a murine model. The work also confirmed previous studies showing that fermented milk is an effective form of administration of recombinant lactic acid bacteria expressing beneficial molecules.

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells

BackgroundInflammatory 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.ResultsThe 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.ConclusionsThis 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.

Antigenic dietary protein guides maturation of the host immune system promoting resistance to Leishmania major infection in C57BL/6 mice

The immature immune system requires constant stimulation by foreign antigens during the early stages of life to develop properly and to create efficient immune responses against later infections. We have previously shown that intake of antigenic dietary protein is critical for inducing maturation of the immune system as well as for the development of T helper type 1 (Th1) immunity. In this study, we show that administration of an amino acid (aa)‐based diet during the development of the immune system subsequently resulted in inefficient control of Leishmania major infection in adult C57BL/6 mice. Compared with mice fed a control protein‐containing diet, adult aa‐fed mice showed a decreased interferon (IFN)‐γ response to parasite antigens and insufficient production of nitric oxide (NO), which is crucial to parasite death. However, no deviation towards Th2‐specific immunity to L. major was observed. Phenotypic analysis of antigen‐presenting cells (APCs) from aa‐fed mice revealed deficient levels of the costimulatory molecules CD40 and CD80, and low levels of interleukin (IL)‐12 produced by peritoneal macrophages, revealing an early stage of maturation of these cells. APCs isolated from aa‐fed mice were unable to stimulate a Th1 response in vitro. Both phenotypic features of T cells from aa‐fed mice and their ability to produce a Th1 response in the presence of mature APCs were unaffected when compared with T cells from control mice. The results presented here support the notion that regulation of Th1 immunity to infection includes environmental factors such as dietary proteins, which provide a natural source of stimulation that contributes to the process of maturation of APCs.