Compartmentalization of gut immune responses: Mucosal niches and lymph node peculiarities.

The intestine is not a homogeneous organ, but rather organized spaces with specific niches and microenvironments filled with different cell types that are involved in physiological and inflammatory processes. The intestinal mucosa shows a high degree of architectural complexity and intratissue specialization that occurs according to luminal composition. These intratissue specialized environments are critical for the developmental and functional adaptation of immune cells in the gut and in the gut-draining lymph nodes. In this review we discuss the compartmentalization of gut immune responses and how the lymph nodes that drain different regions of the intestine are immunologically, anatomically, and physiologically distinct. We also propose that studies on gut immunity should consider the distinctive features of intestinal segments and the differences in their draining lymph nodes to fully understand the complexity of the gut immunological scenario.

Neuroimmune circuits involved in ß-lactoglobulin-induced food allergy.

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.

Frontline Science: Abnormalities in the gut mucosa of non-obese diabetic mice precede the onset of type 1 diabetes.

Alterations in the composition of the intestinal microbiota have been associated with development of type 1 diabetes (T1D), but little is known about changes in intestinal homeostasis that contribute to disease pathogenesis. Here, we analyzed oral tolerance induction, components of the intestinal barrier, fecal microbiota, and immune cell phenotypes in non-obese diabetic (NOD) mice during disease progression compared to non-obese diabetes resistant (NOR) mice. NOD mice failed to develop oral tolerance and had defective protective/regulatory mechanisms in the intestinal mucosa, including decreased numbers of goblet cells, diminished mucus production, and lower levels of total and bacteria-bound secretory IgA, as well as an altered IEL profile. These disturbances correlated with bacteria translocation to the pancreatic lymph node possibly contributing to T1D onset. The composition of the fecal microbiota was altered in pre-diabetic NOD mice, and cross-fostering of NOD mice by NOR mothers corrected their defect in mucus production, indicating a role for NOD microbiota in gut barrier dysfunction. NOD mice had a reduction of CD103+ dendritic cells (DCs) in the MLNs, together with an increase of effector Th17 cells and ILC3, as well as a decrease of Th2 cells, ILC2, and Treg cells in the small intestine. Importantly, most of these gut alterations precede the onset of insulitis. Disorders in the intestinal mucosa of NOD mice can potentially interfere with the development of T1D due the close relationship between the gut and the pancreas. Understanding these early alterations is important for the design of novel therapeutic strategies for T1D prevention.

Immune and metabolic shifts during neonatal development reprogram liver identity and function.

BACKGROUND & AIMS: The liver is the main hematopoietic site in embryos, becoming a crucial organ in both immunity and metabolism in adults. However, how the liver adapts both the immune system and enzymatic profile to challenges in the postnatal period remains elusive. We aimed to identify the mechanisms underlying this adaptation. METHODS: We analyzed liver samples from mice on day 0 after birth until adulthood. Human biopsies from newborns and adults were also examined. Liver immune cells were phenotyped using mass cytometry (CyTOF) and expression of several genes belonging to immune and metabolic pathways were measured. Mortality rate, bacteremia and hepatic bacterial retention after E. coli challenge were analyzed using intravital and in vitro approaches. In a set of experiments, mice were prematurely weaned and the impact on gene expression of metabolic pathways was evaluated. RESULTS: Human and mouse newborns have a sharply different hepatic cellular composition and arrangement compared to adults. We also found that myeloid cells and immature B cells primarily compose the neonatal hepatic immune system. Although neonatal mice were more susceptible to infections, a rapid evolution to an efficient immune response was observed. Concomitantly, newborns displayed a reduction of several macronutrient metabolic functions and the normal expression level of enzymes belonging to lipid and carbohydrate metabolism was reached around the weaning period. Interestingly, early weaning profoundly disturbed the expression of several hepatic metabolic pathways, providing novel insights into how dietary schemes affect the metabolic maturation of the liver. CONCLUSION: In newborns, the immune and metabolic profiles of the liver are dramatically different to those of the adult liver, which can be explained by the differences in the liver cell repertoire and phenotype. Also, dietary and antigen cues may be crucial to guide liver development during the postnatal phase. LAY SUMMARY: Newborns face major challenges in the extra-uterine life. In fact, organs need to modify their cellular composition and gene expression profile in order to adapt to changes in both microbiota and diet throughout life. The liver is interposed between the gastrointestinal system and the systemic circulation, being the destination of all macronutrients and microbial products from the gut. Therefore, it is expected that delicately balanced mechanisms govern the transformation of a neonatal liver to a key organ in adults.

Consumption of conjugated linoleic acid (CLA)-supplemented diet during colitis development ameliorates gut inflammation without causing steatosis in mice.

Dietary supplementation with conjugated linoleic acid (CLA) has been proposed for weight management and to prevent gut inflammation. However, some animal studies suggest that supplementation with CLA leads to the development of nonalcoholic fatty liver disease. The aims of this study were to test the efficiency of CLA in preventing dextran sulfate sodium (DSS)-induced colitis, to analyze the effects of CLA in the liver function, and to access putative liver alterations upon CLA supplementation during colitis. So, C57BL/6 mice were supplemented for 3 weeks with either control diet (AIN-G) or 1% CLA-supplemented diet. CLA content in the diet and in the liver of mice fed CLA containing diet were accessed by gas chromatography. On the first day of the third week of dietary treatment, mice received ad libitum a 1.5%-2.5% DSS solution for 7 days. Disease activity index score was evaluated; colon and liver samples were stained by hematoxylin and eosin for histopathology analysis and lamina propria cells were extracted to access the profile of innate cell infiltrate. Metabolic alterations before and after colitis induction were accessed by an open calorimetric circuit. Serum glucose, cholesterol, triglycerides and alanine aminotransaminase were measured; the content of fat in liver and feces was also accessed. CLA prevented weight loss, histopathologic and macroscopic signs of colitis, and inflammatory infiltration. Mice fed CLA-supplemented without colitis induction diet developed steatosis, which was prevented in mice with colitis probably due to the higher lipid consumption as energy during gut inflammation. This result suggests that CLA is safe for use during gut inflammation but not at steady-state conditions.

Hsp65-Producing Lactococcus lactis Prevents Inflammatory Intestinal Disease in Mice by IL-10- and TLR2-Dependent Pathways.

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.

High sugar and butter (HSB) diet induces obesity and metabolic syndrome with decrease in regulatory T cells in adipose tissue of mice.

OBJECTIVE: The purpose of the study was to develop a novel diet based on standard AIN93G diet that would be able to induce experimental obesity and impair immune regulation with high concentrations of both carbohydrate and lipids. METHODS: To compare the effects of this high sugar and butter (HSB) diet with other modified diets, male C57BL/6 mice were fed either mouse chow, or AIN93G diet, or high sugar (HS) diet, or high-fat (HF) diet, or high sugar and butter (HSB) diet for 11 weeks ad libitum. HSB diet induced higher weight gain. Therefore, control AIN93G and HSB groups were chosen for additional analysis. Regulatory T cells were studied by flow cytometry, and cytokine levels were measured by ELISA. RESULTS: Although HF and HSB diets were able to induce a higher weight gain compatible with obesity in treated mice, HSB-fed mice presented the higher levels of serum glucose after fasting and the lowest frequency of regulatory T cells in adipose tissue. In addition, mice that were fed HSB diet presented higher levels of cholesterol and triglycerides, hyperleptinemia, increased resistin and leptin levels as well as reduced adiponectin serum levels. Importantly, we found increased frequency of CD4(+)CD44(+) effector T cells, reduction of CD4(+)CD25(+)Foxp3(+) and Th3 regulatory T cells as well as decreased levels of IL-10 and TGF-ß in adipose tissue of HSB-fed mice. CONCLUSION: Therefore, HSB represents a novel model of obesity-inducing diet that was efficient in triggering alterations compatible with metabolic syndrome as well as impairment in immune regulatory parameters.

Hydrolyzed whey protein prevents the development of food allergy to ß-lactoglobulin in sensitized mice.

Food allergy is an adverse immune response to dietary proteins. Hydrolysates are frequently used for children with milk allergy. However, hydrolysates effects afterwards are poorly studied. The aim of this study was to investigate the immunological consequences of hydrolyzed whey protein in allergic mice. For that, we developed a novel model of food allergy in BALB/c mice sensitized with alum-adsorbed ß-lactoglobulin. These mice were orally challenged with either whey protein or whey hydrolysate. Whey-challenged mice had elevated levels of specific IgE and lost weight. They also presented gut inflammation, enhanced levels of SIgA and IL-5 as well as decreased production of IL-4 and IL-10 in the intestinal mucosa. Conversely, mice challenged with hydrolyzate maintained normal levels of IgE, IL-4 and IL-5 and showed no sign of gut inflammation probably due to increased IL-12 production in the gut. Thus, consumption of hydrolysate prevented the development of clinical signs of food allergy in mice.

Anti-inflammatory effects of Lactococcus lactis NCDO 2118 during the remission period of chemically induced colitis.

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.

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.

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.