High Fat Diet-Wheat Gliadin Interaction and its Implication for Obesity and Celiac Disease Onset: In Vivo Studies.

The intestinal immune system plays a crucial role in obesity and insulin resistance. An altered intestinal immunity is associated with changes to the gut microbiota, barrier function, and tolerance to luminal antigens. Lipid metabolism and its unbalance can also contribute to acute and chronic inflammation in different conditions. In celiac disease (CD), the serum phospholipid profile in infants who developed CD is dramatically different when compared to that of infants at risk of CD not developing the disease. In a mouse model of gluten sensitivity, oral wheat gliadin challenge in connection with inhibition of the metabolism of arachidonic acid, an omega-6 polyunsaturated fatty acid, specifically induces the enteropathy. Recent evidence suggests that gluten may play a role also for development of life-style related diseases in populations on a high fat diet (HFD). However, the mechanisms behind these effects are not yet understood. Exploratory studies in mice feed HFD show that wheat gliadin consumption affects glucose and lipid metabolic homeostasis, alters the gut microbiota, and the immune cell profile in liver.

The HLA-DQ8 transgenic mouse: A model to study the immune and cytotoxic responses to wheat gliadin.

A complete understanding of celiac disease (CD) pathogenesis has been hindered to date because of the lack of adequate in vivo models. Herein, we describe two in vivo approaches in HLA-DQ8-transgenic mice to study the intrinsic cytoxicity and immune features of wheat gliadin. By adopting the first method, we explored the mucosal architecture of the small intestine following the intra-gastric administration of wheat gliadin in mice treated with indomethacin, an inhibitor of cyclooxygenases. Mice showed a significant reduction of villus height, increased crypt depth and increased intraepithelial lymphocytes. The second approach involved the mucosal sensitization to gliadin via the intranasal route. This protocol induced a Th1/Th17 phenotype in mesenteric lymph nodes, as described in CD. In conclusion, these methods remain instrumental to analyze in vivo distinct biological features of wheat gliadin and related prolamins. Furthermore, the sensitization protocol could be exploited to test innovative strategies downregulating the gliadin-specific immunity.

Senolytic Flavonoids Enhance Type-I and Type-II Cell Death in Human Radioresistant Colon Cancer Cells through AMPK/MAPK Pathway.

Resistance to cancer therapies remains a clinical challenge and an unsolved problem. In a previous study, we characterized a new colon cancer cell line, namely HT500, derived from human HT29 cells and resistant to clinically relevant levels of ionizing radiation (IR). Here, we explored the effects of two natural flavonoids, quercetin (Q) and fisetin (F), well-known senolytic agents that inhibit genotoxic stress by selectively removing senescent cells. We hypothesized that the biochemical mechanisms responsible for the radiosensitising effects of these natural senolytics could intercept multiple biochemical pathways of signal transduction correlated to cell death resistance. Radioresistant HT500 cells modulate autophagic flux differently than HT29 cells and secrete pro-inflammatory cytokines (IL-8), commonly associated with senescence-related secretory phenotypes (SASP). Q and F inhibit PI3K/AKT and ERK pathways, which promote p16INK4 stability and resistance to apoptosis, but they also activate AMPK and ULK kinases in response to autophagic stress at an early stage. In summary, the combination of natural senolytics and IR activates two forms of cell death: apoptosis correlated to the inhibition of ERKs and lethal autophagy dependent on AMPK kinase. Our study confirms that senescence and autophagy partially overlap, share common modulatory pathways, and reveal how senolytic flavonoids can play an important role in these processes.

Next generation strategies to recover immunological tolerance in celiac disease.

Celiac disease (CD) is an autoimmune disease that occurs in genetically predisposed individuals following the ingestion of gluten. Its prevalence is rising worldwide. A gluten-free (GF) diet is mandatory for the management of CD. However, several issues persist regarding the nutritional quality of GF products. Importantly, deep knowledge about the pathogenic mechanisms in CD highlights the central role of CD4+ T cell-mediated immunity in CD. Furthermore, intestinal T regulatory cells are functional in CD, but cytokines such as IL-15, produced under inflammatory conditions, hamper their activity. This paves the way for the development of immunomodulatory strategies to the GF diet. From this perspective, microbiological approaches were considered able to modulate the gluten-specific immune response. Interestingly, gliadin peptide-based immunotherapy to abolish the inflammatory CD4+T cell-mediated response has been explored in CD patients. Furthermore, different biotechnological approaches based on the use of chemically/enzymatically modified gluten molecules have been proved effective in different models of CD. However, the choice of the right age in infants to introduce the antigen and thus induce tolerance still remains an important issue to solve. Addressing all these points should help to design an effective intervention strategy for preventing CD.

Transamidated wheat gliadin induces differential antigen recognition in the small intestine of HLA/DQ8 transgenic mice.

A lifelong gluten-free diet (GFD) is currently the only available therapy for coeliac disease (CD). However, GFD compliance is difficult and alternative strategies are envisaged in the near future. We previously found that wheat gliadin following transamidation by microbial transglutaminase (mTG) does not induce IFN-γ secretion by intestinal T cells from CD patients. Fully transamidated gliadin with lysine ethyl ester can be recovered in a soluble protein fraction (spf) generated by the enzymatic treatment of wheat flour. Herein, we analysed the performance of transamidation by mTG on a pilot-scale (1L) by evaluating the reaction kinetics and its biological effect on the intestinal immune response in HLA/DQ8 transgenic mice, a model of gluten sensitivity. At 1 h, all gliadin fractions showed a faster electrophoretic mobility by acid-polyacrylamide gel electrophoresis (A-PAGE) following transamidation in comparison with their native counterparts. In parallel, the yield of residual native gliadin dropped (30% at 180 min), confirming our previous findings on a lab scale. Mucosal sensitisation of mice with gliadin via the intranasal route induced a Th1 phenotype in mesenteric lymph nodes (MLNs). Importantly, IFN-γ secretion was significantly reduced when gliadin-specific MLN cells were challenged in vitro with spf (P < 0.001). Multiplex analysis revealed that the adaptive immune response evoked by spf involved a distinct cell population characterised by secretion of IL-2, IL-3 and IL-5. Notably, spf stimulated in vitro a reduced or null secretion of all of the examined pro-inflammatory markers mainly associated to innate immunity. In conclusion, our data revealed the ability of transamidated gliadin to modulate both innate and adaptive mechanisms involved in the inflammatory response induced by wheat gliadin in the small intestine of DQ8 mice.

Sensory Characteristics and Nutritional Quality of Food Products Made with a Biofortified and Lectin Free Common Bean (Phaseolus vulgaris L.) Flour.

Common beans (Phaseolus vulgaris L.) are an important source of nutrients with beneficial effects on human health. However, they contain lectins, that limit the direct use of flour in food preparations without thermal treatment, and phytic acid, that reduces mineral cation bioavailability. The objectives of this research were: to obtain biofortified snacks and a cream using an untreated common bean flour devoid of active lectins (lec-) and with reduced content of phytic acid (lpa) and to evaluate the sensorial appreciation for these products. The main results of the present work were: the products with the lpa lec- flour did not retain residual hemagglutinating activity due to lectins; they showed higher residual α-amylase inhibitor activity (from 2.2 to 135 times), reduced in vitro predicted glycemic index (about 5 units reduction) and increased iron bioavailability compared to the products with wild type flour; products with common bean flour were less appreciated than the reference ones without this flour, but the presence of an intense umami taste can be a positive attribute. Results confirmed that the use of the lpa lec- flour has important advantages in the preparation of safe and nutritionally improved products, and provide useful information to identify target consumers, such as children and elderly people.

Transamidation Down-Regulates Intestinal Immunity of Recombinant α-Gliadin in HLA-DQ8 Transgenic Mice.

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.

First morphological-level insights into the efficiency of green tea catechins and grape seed procyanidins on a transgenic mouse model of celiac disease enteropathy.

Alternative or complementary treatments to a gluten-free diet are urgently needed for Celiac Disease. By exploiting the health-promoting properties of polyphenols on a transgenic mouse model of Celiac Disease enteropathy, this study provides the first in vivo evidence regarding the ability of 1 mg day-1 doses of green tea catechins and grape seed procyanidins to ameliorate some of the most characteristic histological changes of gliadin-treated DQ8 mice, including villus flattening, crypt hyperplasia, and infiltration of intraepithelial lymphocytes. Mechanistically, polyphenols were found to increase the intestinal nucleophilic tone of DQ8 mice by orchestrating an adaptive antioxidant response characterized by enhanced GSR enzyme activity and GSH content. Taken together, this work constitutes a highly relevant breakthrough as it provides the fundamental basis concerning the significance of natural polyphenols to be used in, for instance, the development of innovative functional foods aimed at CD individuals.

The murine enterocyte cell line Mode-K is a novel and reliable in vitro model for studies on gluten toxicity.

The resemblance of physiological traits of cell lines with their target/original tissue is an important prerequisite for the choice of the in vitro model. Although cytoprotective defenses, activated by the nuclear factor erythroid 2-related factor2 (Nrf2), have a preeminent importance in intestinal protection, nevertheless their levels inin vitro models have been never compared with those of their original tissue. Basal level of Nrf2-mediated defenses in murine enterocyte cells (Mode-K) and in human colon adenocarcinoma cells -at differentiated (DCaco2) or confluent stage (CCaco2)- were compared with those found in mouse or human duodenum. The pro-oxidant and cytotoxic effects of peptic-tryptic digest of gluten prepared from wheat bread (PT-b), einkorn (PT-e) or durum wheat (PT-d) were evaluated in Mode-k and DCaco2 by combining enzymatic, immune-enzymatic and real-time PCR assay. The results presented reveal that Mode-k cells resemble cytoprotective defenses of human/murine duodenum and are more susceptible to pro-oxidant, cytotoxic and pro-inflammatory effect of gliadin digest (in comparison with Caco2). Prolamins digests from the considered wheat exhibit different inhibitory effect on Nrf2-mediated defenses (PT-b > PT-e > PT-d). These data indicate, for the first time, that Mode-k are a reliable model for investigating wheat prolamins toxicity and for evaluating the signaling pathway of gluten-associated disease.

Secretome Analysis of Mouse Dendritic Cells Interacting with a Probiotic Strain of Lactobacillus gasseri.

Probiotics play a key role in the modulation of the gut immune system in health and disease and their action is mediated by molecules exposed on the microorganism surface or secreted probiotic-derived factors. In particular, Lactobacillus gasseri OLL2809, a probiotic microorganism isolated from human feces, has the potential to modulate various immune responses. The dendritic cells (DCs) are considered the main players in orchestrating the immune response, and their contact with intestinal microbiota is crucial for the development and homeostasis of gut immunity. To gain a perspective on the molecular mechanisms involved in the maturation process of DCs and investigate factors that could modulate these processes, a differential proteomic analysis was performed on the secretome of immature DCs, mature DCs (mDCs, induced by lipopolysaccharide (LPS)), and immature DCs challenged with L. gasseri OLL2809 before treatment with LPS (LGmDCs). The maturation process of DCs was associated to profound changes in the protein secretome and probiotic pre-treatment led to a dramatic modulation of several secreted proteins of mDC, not only classical immune mediators (i.e., cytokines, complement factors, T cell Receptor ligands) but also proteins involved in the contractile and desmosome machineries. The latter data highlight a novel mechanism by which L. gasseri can modulate the maturation process of DCs, reinforcing the concept of a protective anti-inflammatory role ascribed to this probiotic strain.

Innate immunity is a late event in the onset of gliadin-specific enteropathy in the HLA-DQ8 mice.

Celiac disease (CD) is a food enteropathy that occurs in genetically susceptible individuals following the ingestion of gluten. Both gluten cytotoxicity and immunity activation play a role in CD pathogenesis; however, the chronological assessment of the different pathogenic mechanisms remains elusive. The models developed so far have only partially addressed this issue. Herein, Ab°DQ8 transgenic mice were administered wheat gliadin and indomethacin for 10 days to induce enteropathy. Gliadin-induced alteration of the small intestinal architecture was associated with increased expression of tissue transglutaminase in the lamina propria and a marked hypoxic environment. Enteropathic mice showed activation of innate immunity, featuring an increase of pro-inflammatory IFN-γ and IL-15 mRNAs, as well as CD11c+CD103+, CD11b+CD11c+, and CD11b+CD103+ dendritic cell subsets. However, the temporal assessment of examined parameters indicated that the induction of innate immunity during the generation of the mucosal lesion, occurred belatedly, highlighting a major role of gliadin intrinsic cytotoxicity in the pathogenic mechanism of this model. These results have important implications for the use of this model to test the impact of biotechnological interventions to reduce the cytotoxicity of gliadin.

Microbial transglutaminase: A biotechnological tool to manage gluten intolerance.

Celiac disease (CD) is a chronic immune-mediated disease in which gluten ingestion leads to damage of the small intestinal mucosa in genetically susceptible individuals. The enteropathy is mainly induced by the production of IFN-γ from intestinal CD4+T cells that recognise gliadin peptides following deamidation by tissue transglutaminase. The only available therapy is a strict, lifelong gluten-free diet (GFD). This diet is strongly demanding for patients, which justifies the search for alternative strategies. The enzyme approach is one promising strategy to address this issue. In particular, transamidation of wheat gliadin by microbial transglutaminase (mTG) was fully effective at inhibiting gliadin-specific IFN-γ secretion in intestinal T cells from CD patients. Furthermore, transamidated gliadin induced higher levels of the anti-inflammatory IL-10 than native gliadin in different in vitro models. These data suggest that a more balanced immune response could be induced by mTG-treated gliadin in the small intestine of celiac patients. Furthermore, the highlighted biological property of mTG-treated gliadin could be exploited to induce tolerance to native gliadin in at-risk individuals.

Dietary Supplementation with Fish Oil or Conjugated Linoleic Acid Relieves Depression Markers in Mice by Modulation of the Nrf2 Pathway.

Inflammation and oxidative stress play an important role in the pathogenesis of depressive disorders and nuclear erythroid related factor 2 (Nrf2), a regulator of RedOx homeostasis and inflammation, is a promising target for depression prevention/treatment. As fish oil (FO) and conjugated linoleic acid (CLA) are known Nrf2 inducers, their protective ability is comparatively evaluated in a murine model of depression (MRL/MpJ-Faslpr ). Oxidative stress, fatty acids content, and critical factors reflecting brain functioning-namely brain-derived neurotrophic factor (BDNF), synaptic markers, and cholinergic signaling-are preliminarily evaluated in the frontal cortex of 8-week (Young) and in 22-week old animals (Old), which are used as model of depression. These markers are measured in Old mice at the end of a 5-week pretreatment with FO or CLA (728 or 650 mg kg-1 , respectively). Old mice exhibit disrupted Redox homeostasis, compensatory Nrf2 hyperactivation, lower docosaheaxaenoic acid (DHA), and lower BDNF and synaptic function proteins compared to Young mice. FO and CLA treatment relieves almost all the pathophysiological hallmarks at a level comparable to Young mice. Presented data provide the first evidence for the comparable efficacy of FO or CLA supplementation in preventing depression signs in Old MRL/lpr mice, likely through their ability of improving Nrf2-mediated antioxidant defenses.

Tailoring the immune response to wheat gliadin by enzymatic transamidation.

Enzymatic transamidation of wheat gliadin by microbial transglutaminase inhibits IFN-γ secretion by intestinal T cell lines from celiac disease (CD) patients. Here, we analysed its effects on intestinal biopsies from CD patients and studied the underlying mechanisms in HLA-DQ8 transgenic (tg) mice, a model of T-cell mediated gluten sensitivity. In vitro challenge with a soluble form of transamidated gliadin (spf) upregulated IL-10 transcript levels in human biopsy samples. Furthermore, the ratio of IL-10/IFN-γ transcripts was significantly increased following treatment with spf. In DQ8 tg mice, recall responses in vitro in the presence of dendritic cells pulsed with transamidated gliadin showed that gliadin-specific CD4+ T cells did not produce IFN-γ at any tested dose. On the contrary, spf-specific CD4+ T cells still secreted IFN-γ, but they also produced significant levels of IL-10 with both native and transamidated gliadin. Interestingly, this anti-inflammatory activity was restricted to a specific reverse-phase high-pressure liquid chromatography (RP-HPLC) fraction encompassing α-gliadins. These findings suggested an ability of transamidated gliadin to revert, as well as to prevent, the inflammatory phenotype triggered by native gliadin. This property was intrinsically associated with specific components of the α-gliadin fraction.

Conjugated linoleic acid prevents age-dependent neurodegeneration in a mouse model of neuropsychiatric lupus via the activation of an adaptive response.

Oxidative stress is a key mediator of autoimmune/neurodegenerative disorders. The antioxidant/anti-inflammatory effect of a synthetic conjugated linoleic acid (CLA) mixture in MRL/MpJ-Fas lpr mice (MRL/lpr), an animal model of neuropsychiatric lupus, was previously associated with the improvement of nuclear factor-E2-related factor 2 (Nrf2) defenses in the spleen and liver. However, little is known about the neuroprotective ability of a CLA mixture. This study investigated the age-dependent progression of oxidative stress and the hyperactivation of redox-sensitive compensatory pathways (macroautophagy, Nrf2) in old/diseased MRL/lpr mice brains and examines the effect produced by dietary CLA supplementation. Disrupted redox homeostasis was evidenced in the blood, liver, and brain of 21- to 22-week-old MRL/lpr (Old) mice compared with 8- to 10-week-old MRL/lpr (Young) animals. This alteration was associated with significant hyperactivation of compensatory mechanisms (macroautophagy, Nrf2, and astrocyte activation) in the brains of Old mice. Five-week daily supplementation with CLA (650 mg/kg-1 body weight) of 16-week-old (CLA+Old) mice diminished all the pathological hallmarks at a level comparable to Young mice or healthy controls (BALB/c). Such data demonstrated that MRL/lpr mice can serve as a valuable model for the evaluation of the effectiveness of neuroprotective drugs. Notably, the preventive effect provided by CLA supplementation against age-associated neuronal damage and hyperactivation of compensatory mechanisms suggests that the activation of an adaptive response is at least in part accountable for its neuroprotective ability.

Immune-modulating effects in mouse dendritic cells of lactobacilli and bifidobacteria isolated from individuals following omnivorous, vegetarian and vegan diets.

Lactobacilli and bifidobacteria play a primary role in modulation of gut immunity. By considering that microbiota composition depends on various factors, including diet, we asked whether functional differences could characterize faecal populations of lactobacilli and bifidobacteria isolated from individuals with different dietary habits. 155 healthy volunteers who followed omnivorous, ovo-lacto-vegetarian or vegan diets were recruited at four Italian centres (Turin, Parma, Bologna and Bari). Faecal samples were collected; lactobacilli and bifidobacteria were isolated on selective media and their immunomodulatory activity was tested in mouse dendritic cells (DCs). Pre-incubation with lactobacilli increased LPS-induced expression of the maturation markers CD80 and CD86, whereas pre-incubation with bifidobacteria decreased such expression. Analysis of the cytokine profile indicated that strains of both genera induced down-regulation of IL-12 and up-regulation of IL-10, whereas expression of TNF-α was not modulated. Notably, analysis of anti-inflammatory potential (IL-10/IL-12 ratio) showed that lactobacilli evoked a greater anti-inflammatory effect than did bifidobacteria in the omnivorous group (P<0.05). We also found significantly reduced anti-inflammatory potential in the bacterial strains isolated from Bari's volunteers in comparison with those from the cognate groups from the other centres. In conclusion, lactobacilli and bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the anti-inflammatory potential of some components of faecal microbiota.

Immunomodulatory activity of recombinant α-gliadin conjugated to cholera toxin in DQ8 transgenic mice.

Coeliac disease (CD) is characterized by an intestinal lesion sustained by an abnormal mucosal T-cell response to wheat gliadin. An immunological approach that is able to suppress this immune response is a perspective worth pursuing. Several strategies of antigen administration have been aimed at the downregulation of pathogenic T-cells. In particular, we previously reported a significant suppression of the systemic cell-mediated response toward wheat gliadin in DQ8 transgenic mice receiving nasally a recombinant α-gliadin. To gain further insight about the cellular mechanisms underlying the tolerogenic properties of this molecule, we analysed different preparations of the recombinant α-gliadin, alone or conjugated to the adjuvant cholera toxin (CT), by in vitro challenge with spleen CD4+ T cells from gliadin-sensitized DQ8 tg mice. We found that a partially purified preparation of recombinant α-gliadin (r-gliadin) induced a significantly higher production of IFN-γ than native gliadin as well as HPLC purified r-gliadin. Interestingly, r-gliadin, but not HPLC purified r-gliadin, stimulated the gliadin-specific expression of IL-10 in CD4+ T cells. No significant cytotoxic effect was induced by r-gliadin in MODE-K cells, a murine model of enterocytes. Notably, a conjugate CT-r-gliadin failed in stimulating IFN-γ, whereas IL-10 secretion was still induced in gliadin-specific CD4+ T cells. In conclusion, our results showed that DCs, pulsed with CT-r-gliadin in vitro, could modulate the ongoing Th1-like T cell response toward wheat gliadin. This finding provides new insight into the design of immunomodulatory protocols potentially useful for CD.

Modulation of the cytokine profile in Caco-2 cells by faecal lactobacilli and bifidobacteria from individuals with distinct dietary habits.

Enterocytes are actively involved in the defense against pathogens and they limit penetration of commensal microbes into tissues. They also have an important role in gut immunity as enterocytes confer mucosal dendritic cell specialisation. On the other hand, the microbiota is directly involved in the development and modulation of the intestinal immune system. Particularly, lactobacilli and bifidobacteria play a primary role in shaping the immune response. We further explored this issue by evaluating whether functional differences in Caco-2 cells could characterise faecal populations of lactobacilli (155 samples) and bifidobacteria (110 samples) isolated from three dietary cohorts (omnivores, ovo-lacto-vegetarians and vegans) recruited at four Italian centres (Turin, Parma, Bologna and Bari). According to our findings, tested bacteria were unable to modulate expression of IL-8, IL-10, TGF-ß or thymic stromal lymphopoietin (TSLP) cytokines in unstimulated Caco-2 cells. Conversely, in phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) stimulated Caco-2 cells, lactobacilli from the omnivorous group and all bifidobacteria significantly down-regulated IL-8. Notably, both genera also lowered the TSLP expression in stimulated Caco-2 cells, regardless of the diet regimen. By further examining these data on the basis of geographical origin, we found that lactobacilli from the vegetarian group recruited in Bari, significantly up-regulated this cytokine. In conclusion, we highlighted a peculiar immune-modulatory activity profile for lactobacilli on enterocytes undergoing a stimulatory signal, which was associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the inflammatory potential of members of the Lactobacillus genus.

Effect of Lactobacillus paracasei Culture Filtrates and Artichoke Polyphenols on Cytokine Production by Dendritic Cells.

The most recent trend in research on probiotic bacteria aims at the exploitation of bioactive bacterial compounds that are responsible for health-promoting effects and suitable for medical applications. Therefore, the main purpose of this study was to ascertain if the immunomodulatory effects of L. paracasei strains on dendritic cells (DCs) were caused by bacterial metabolites released in the culture medium. For that reason, bacterial strains were grown in two media generally used for the culture of DCs, and the effects of culture filtrates on the maturation of DCs and cytokine production were evaluated. Moreover, to reveal potential synergistic effects on the immunomodulation of DCs, an artichoke phenolic extract (APE) was added to the media before bacterial growth. The experiments pointed out an interesting anti-inflammatory activity of a culture filtrate obtained after growing a probiotic L. paracasei strain in one of the media supplemented with APE. Therefore, this culture filtrate-which combines the anti-inflammatory activity and the other well-known health-promoting properties of artichoke phenolic compounds-could represent the basis for future particular exploitations.

Modulatory activity of Lactobacillus rhamnosus OLL2838 in a mouse model of intestinal immunopathology.

Gut microbiota and probiotic strains play an important role in oral tolerance by modulating regulatory and effector cell components of the immune system. We have previously described the ability of Lactobacilli to influence both the innate and adaptive immunity to wheat gluten, a food antigen, in mouse. In this study, we further explored the immunomodulatory mechanisms elicited in this model by testing three specific probiotic strains, namely L. rhamnosus OLL2838, B. infantis ATCC15697 and S. thermophilus Sfi39. In vitro analysis showed the all tested strains induced maturation of bone marrow derived dendritic cells (DCs). However, only L. rhamnosus induced appreciable levels of IL-10 and nitric oxide productions, whereas S. thermophilus essentially elicited IL-12 and TNF-α. The anti-inflammatory ability of OLL2838 was then tested in vivo by adopting mice that develop a gluten-specific enteropathy. This model is characterized by villus blunting, crypt hyperplasia, high levels of intestinal IFN-γ, increased cell apoptosis in lamina propria, and reduced intestinal total glutathione (GSHtot) and glutathione S-transferase (GST) activity. We found that, following administration of OLL2838, GSHtot and GST activity were enhanced, whereas caspase-3 activity was reduced. On the contrary, this probiotic strain failed in recovering the normal histology and further increased intestinal IFN-γ. Confocal microscopy revealed the inability of the probiotic strain to appropriately interact with enterocytes of the small intestine and with Peyer's patches in treated mice. In conclusion, these data highlighted the potential of L. rhamnosus OLL2838 to recover specific toxicity parameters induced by gluten in enteropathic mice through mechanisms that involve induction of low levels of reactive oxygen species (ROS).