Antibiotic Therapy for Active Crohn's Disease Targeting Pathogens: An Overview and Update.

Crohn's disease (CD) is a multifactorial chronic disorder that involves a combination of factors, including genetics, immune response, and gut microbiota. Therapy includes salicylates, immunosuppressive agents, corticosteroids, and biologic drugs. International guidelines do not recommend the use of antibiotics for CD patients, except in the case of septic complications. Increasing evidence of the involvement of gut bacteria in this chronic disease supports the rationale for using antibiotics as the primary treatment for active CD. In recent decades, several pathogens have been reported to be involved in the development of CD, but only Escherichia coli (E. coli) and Mycobacterium avium paratubercolosis (MAP) have aroused interest due to their strong association with CD pathogenesis. Several meta-analyses have been published concerning antibiotic treatment for CD patients, but randomized trials testing antibiotic treatment against E. coli and MAP have not shown prolonged benefits and have generated conflicting results; several questions are still unresolved regarding trial design, antibiotic dosing, the formulation used, the treatment course, and the outcome measures. In this paper, we provide an overview and update of the trials testing antibiotic treatment for active CD patients, taking into account the role of pathogens, the mechanisms by which different antibiotics act on harmful pathogens, and antibiotic resistance. Finally, we also present new lines of study for the future regarding the use of antibiotics to treat patients with active CD.

Role of Regulatory T Cells and Their Potential Therapeutic Applications in Celiac Disease.

Celiac disease (CeD) is a T-cell-mediated immune disease, in which gluten-derived peptides activate lamina propria effector CD4+ T cells. While this effector T cell subset produces proinflammatory cytokines, which cause substantial tissue injury in vivo, additional subsets of T cells exist with regulatory functions (Treg). These subsets include CD4+ type 1 regulatory T cells (Tr1) and CD4+ CD25+ T cells expressing the master transcription factor forkhead box P3 (Foxp3) that may have important implications in disease pathogenesis. In this review, we provide an overview of the current knowledge about the effects of immunomodulating cytokines on CeD inflammatory status. Moreover, we outline the main Treg cell populations found in CeD and how their regulatory activity could be influenced by the intestinal microenvironment. Finally, we discuss the Treg therapeutic potential for the development of alternative strategies to the gluten-free diet (GFD).

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.

Diploid Wheats: Are They Less Immunogenic for Non-Celiac Wheat Sensitive Consumers?

Non-celiac wheat sensitivity (NCWS) is a clinical entity induced by the ingestion of gluten that leads to intestinal and/or extraintestinal symptoms, and is diagnosed when celiac disease and wheat allergy have been ruled out. In addition to gluten, other grains' components, including amylase trypsin inhibitors (ATIs) and fermentable short-chain carbohydrates (FODMAPs), may trigger symptoms in NCWS subjects. Several studies suggest that, compared with tetraploid and hexaploid modern wheats, ancient diploid wheats species could possess a lower immunogenicity for subjects suffering from NCWS. This review aims to discuss available evidence related to the immunological features of diploid wheats compared to common wheats, and at outlining new dietary opportunities for NCWS subjects.

Cell-type-specific gene expression profile by laser capture microdissection on mirror sections.

Immuno-laser capture microdissection (Immuno-LCM) has been used to analyze cell-specific gene expression profiles. However, the usefulness of such a technique is frequently limited by RNA degradation. We, therefore, developed a rapid protocol of LCM on mirror sections, which allows for preserving RNA integrity. With such a procedure, we investigated cell-type-specific gene expression of γδ intraepithelial lymphocytes (IELs) in untreated celiac disease (CD). An increase in TGF-ß mRNA expression levels was observed in γδ + IELs compared to intestinal enterocytes (IEs), whereas anti-inflammatory IL-10 mRNA production from γδ + IELs was lower compared to IEs. In untreated CD patients, the production of anti-inflammatory cytokines by γδ + IELs is suggestive of a regulatory function, thus playing a critical role in limiting inflammation. This work underscores the importance of LCM on mirror sections as a valuable tool to perform cell-type-specific molecular analysis in tissue.

Beneficial effects of a T. monococcum wheat cultivar on diabetes incidence evaluated in non-obese diabetic mice and after in vitro simulated gastroduodenal digestion.

Wheat consumption can represent one of the nutritional factors involved in the onset of diabetes. We specifically investigated the potential diabetogenic effects of Hammurabi, a T. monococcum wheat cultivar, in non-obese diabetic (NOD) mice and analysed the levels of resistant starch in pasta manufactured with Hammurabi after in vitro gastroduodenal digestion. NOD mice were fed with Hammurabi, bread wheat or rice flour to evaluate diabetes incidence and insulitis score. An enzymatic method was applied to compare the content of resistant starch in Hammurabi pasta and durum wheat pasta (control). In NOD mice, the Hammurabi-based diet significantly delayed diabetes onset (p = 0.0042) and reduced insulitis score compared to rice or wheat-based diet. Furthermore, the resistant starch value following in vitro digestion of Hammurabi pasta was significantly higher (4.08%) than that of durum wheat pasta (2.28%). Taken together, these results highlighted the potential positive effects of the Hammurabi-based diet on diabetes incidence.

Triticum monococcum amylase trypsin inhibitors possess a reduced potential to elicit innate immune response in celiac patients compared to Triticum aestivum.

SCOPE: Several studies reported a role of amylase/trypsin-inhibitors (ATIs) of common wheat species in promoting immune reactions. Here, we investigated in celiac disease (CD), the immunogenic properties of ATIs from diploid compared to common hexaploid wheats after an in vitro proteolytic hydrolysis. METHODS AND RESULTS: ATIs purified from two lines of diploid Triticum monococcum (TM), Monlis and Norberto-ID331, and from Triticum aestivum (TA), Sagittario, were digested with pepsin-chymotrypsin (PC) enzymes and analyzed using a proteomic approach, and subsequently their immune stimulatory properties were investigated on jejunal biopsies and T-cell lines from CD patients. No significant expression of IL-8 and TNF-α were detected on biopsies cultured with ATIs from TM in comparison with ATIs from TA. No significant IFN-γ production was observed in intestinal gliadin- raised T-cells in response to ATIs from both TM and TA wheats. Proteomic results revealed that both TM ATIs showed reduced stability to proteolytic enzymes compared to TA ones. CONCLUSION: TM ATIs are substantially different from those of TA, showing a reduced ability to trigger the innate immunity in CD and a higher susceptibility to enzymatic hydrolysis.

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.

Adult autoimmune enteropathy in autoimmune hepatitis patient. Case report and literature review.

Autoimmune enteropathy (AIE) is a rare disease characterized by prolonged diarrhea, vomiting and weight loss; although it is mainly a rare pediatric disease, over the years a number of adults have also been found to be affected. In this study, we present a case report of a 73-year-old woman with a history of autoimmune hepatitis, antinuclear (ANA) and positive anti-enterocyte antibodies (AEA), who has suffered two months of intractable diarrhea, nausea, anorexia and severe weight loss. The histological examination of the endoscopic duodenal mucosa biopsies revealed severe shortening and flattening of the villi, resulting in mucosal atrophy. The immunohistochemical study revealed a polymorphic lymphoid population, exhibiting a B cell (CD20+) phenotype in follicles and a T cell phenotype (CD3+) in the diffuse component within the lamina propria. Our patient had a complete recovery after two weeks of taking prednisone and following a gluten-rich diet. To our knowledge this is the first case of autoimmune enteropathy in adults with ANA and AEA 7 years after a diagnosis of autoimmune hepatitis. To date, the patient is still in clinical remission on a low dose of orally administered predinisone without any additional immunosuppression.

Mechanisms underlying the hormetic effect of conjugated linoleic acid: Focus on Nrf2, mitochondria and NADPH oxidases.

Nuclear factor erythroid 2-related factor2 (Nrf2) is a redox-sensitive transcription factor. Its activation by low dietary intake of ligands leads to antioxidant effects (eustress), while pro-oxidant effects (oxidative distress) may be associated with high doses. NADPH oxidases (NOXs) and the mitochondrial electron transport chain are the main sources of intracellular ROS, but their involvement in the biphasic/hormetic activity elicited by Nrf2 ligands is not fully understood. In this study, we investigated the involvement of NOX expression and mitochondrial function in the hormetic properties of omega-3 typically present in fish oil (FO) and conjugated linoleic acid (CLA) in the mouse liver. Four-week administration of FO, at both low and high doses (L-FO and H-FO) improves Nrf2-activated cyto-protection (by phase 2 enzymes), while a significant increase in respiration efficiency occurs in the liver mitochondria of H-FO BALB/c mice. Eustress conditions elicited by low dose CLA (L-CLA) are associated with increased activity of phase 2 enzymes, and with higher NOX1-2, mitochondrial defences, mitochondrial uncoupling protein 2 (UCP2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression, compared with controls. Steatogenic effects (lipid accumulation and alteration of lipid metabolism) elicited by high CLA (H-CLA) elicited that are associated with oxidative distress, increased mitochondrial complex I/III activity and reduced levels of phase 2 enzymes, in comparison with L-CLA-treated mice. Our results confirm the steatogenic activity of H-CLA and first demonstrate the role of NOX1 and NOX2 in the eustress conditions elicited by L-CLA. Notably, the negative association of the Nrf2/PGC-1α axis with the different CLA doses provides new insight into the mechanisms underlying the hormetic effect triggered by this Nrf2 ligand.

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.

IBD: Role of intestinal compartments in the mucosal immune response.

BACKGROUND AND AIMS: Laser capture microdissection (LCM) is a powerful tool for the isolation of specific tissue compartments. We aimed to investigate the mucosal immune response that takes place in different intestinal compartments of IBD patients, dissected by LCM, analyzing cytokines expression profile and endoplasmic reticulum (ER) stress markers. METHODS: Frozen sections of gut were obtained from patients with Crohn's disease (CD), ulcerative colitis (UC) and from controls. Using LCM, surface epithelium (SE) and lamina propria (LP) compartments were isolated and total RNA extracted. The relative expression of Th1, Th17 and Treg cytokines was evaluated by quantitative reverse transcriptase real-time PCR (qRT-PCR), in addition to the assessment of mRNA splicing of the transcription factor X-box binding protein-1 (XBP1). Human neutrophil elastase (HNE) and the transcription factor forkhead box P3 (Foxp3) were also analyzed by immunohistochemistry. RESULTS: The increased expression of IL-17 was observed in both intestinal compartments of IBD patients when compared to controls. IFN- γ, TNF-α , IL-10, HNE and Foxp3 were overexpressed in the LP compartment of both IBD patients as compared to controls. An upregulation of IFN-γ and an infiltration of HNE+ cells was found in the SE of patients with UC. Splicing of XBP1 mRNA was recognized in both intestinal compartments of IBD patients when compared to controls. CONCLUSIONS: In IBD patients, both intestinal compartments are involved in Th17 response, whereas, LP compartment plays a prominent role in Th1 and Treg immune responses. Nevertheless, high level of IFN- γ was found in the SE of UC patients, suggesting that this compartment is involved in the Th1 immune response. Our data also suggested that ER stress signalling is active in both LP and SE compartment of IBD patients, thus advocating that ER stress and immunity are intertwined.

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.

Non-Celiac Gluten Sensitivity: How Its Gut Immune Activation and Potential Dietary Management Differ from Celiac Disease.

Non-celiac gluten sensitivity (NCGS) is a clinical entity triggered by the ingestion of gluten-containing grains leading to intestinal and/or extraintestinal symptoms that resolve once the gluten-containing foodstuff is eliminated from the diet, and it is diagnosed when celiac disease (CD) and wheat allergy (WA) have been ruled out. The limited knowledge about the pathophysiology of NCGS and the lack of validated biomarkers are still major limitations for clinical studies, making it difficult to differentiate NCGS from other gluten-related disorders (GRD). In the absence of clear-cut diagnostic criteria, NCGS is still mainly a diagnosis of exclusion. Several studies suggest that NCGS is an immune-mediated disease that likely activates an innate immune response. Moreover, it has recently been hypothesized that in addition to gluten, other components of wheat may be responsible for the symptoms observed in individuals without CD. This review aims at discussing available evidence related to the histological and immunological features in the gut mucosa of patients with NCGS and at outlining new dietary opportunities for these patients.

Pathogenic Role of Associated Adherent-Invasive Escherichia coli in Crohn's Disease.

Several lines of evidence suggest that adherent-invasive Escherichia coli (AIEC) strains play an important role in Crohn's disease (CD). The objective of this study was to investigate the pathogenic role of two AIEC strains, LF82 and O83:H1, in CD patients. Organ cultures of colonic biopsies from patients were set up to assess the effects of LF82 and O83:H1 on the expression of CEACAM6, LAMP1, HLA-DR, ICAM1 by immunohistochemistry and of IL-8, IFNʏ, and TNF-α genes by RT-PCR. Moreover, on Caco2 cells, we analyzed the cell cycle, the expression of MGMT and DNMT1 genes, and DNA damage induced by LF82 and O83:H1, by FACS, RT-PCR, and DAPI staining, respectively. Epithelial and lamina propria mononuclear cells (LPMNC) expression of CEACAM6 and LAMP1 were higher in biopsies cultured in the presence of both O83:H1 and LF82 than in biopsies cultured with non-pathogenic E. coli. Both AIEC strains induced increased expression of ICAM-1 on blood vessels and HLA-DR on LPMNC. We observed higher levels of TNF-α, IFN-γ, and IL-8 transcripts in biopsies cultured with both AIEC strains than in those cultured with NP. Both LF82 and O83:H1, block the cell cycle into S phase, inducing DNA damage, and modulate the expression of DNMT1 and MGMT genes. Our data suggest that LF82 and 083:H1 strains of E. coli are able to increase in CD colonic biopsies the expression of all the pro-inflammatory cytokines and all the mucosal immune markers investigated. J. Cell. Physiol. 232: 2860-2868, 2017. © 2016 Wiley Periodicals, Inc.

Extensive in vitro gastrointestinal digestion markedly reduces the immune-toxicity of Triticum monococcum wheat: implication for celiac disease.

SCOPE: The ancient diploid Triticum monococcum is of special interest as a candidate low-toxic wheat species for celiac disease patients. Here, we investigated how an in vitro gastro-intestinal digestion, affected the immune toxic properties of gliadin from diploid compared to hexaploid wheat. METHODS AND RESULTS: Gliadins from Triticum monococcum, and Triticum aestivum cultivars were digested using either a partial proteolysis with pepsin-chymotrypsin, or an extensive degradation that used gastrointestinal enzymes including the brush border membrane enzymes. The immune stimulatory properties of the digested samples were investigated on T-cell lines and jejunal biopsies from celiac disease patients. The T-cell response profile to the Triticum monococcum gliadin was comparable to that obtained with Triticum aestivum gliadin after the partial pepsin-chymotrypsin digestion. In contrast, the extensive gastrointestinal hydrolysis drastically reduced the immune stimulatory properties of Triticum monococcum gliadin. MS-based analysis showed that several Triticum monococcum peptides, including known T-cell epitopes, were degraded during the gastrointestinal treatment, whereas many of Triticum aestivum gliadin survived the gastrointestinal digestion. CONCLUSION: The pattern of Triticum monococcum gliadin proteins is sufficiently different from those of common hexaploid wheat to determine a lower toxicity in celiac disease patients following in vitro simulation of human digestion.

Gliadin intake alters the small intestinal mucosa in indomethacin-treated HLA-DQ8 transgenic mice.

Celiac disease (CD) is an enteropathy caused by the ingestion of wheat gluten in genetically susceptible individuals. A complete understanding of the pathogenic mechanisms in CD has been hindered because of the lack of adequate in vivo models. In the present study, we explored the events after the intragastric administration of gliadin and of the albumin/globulin fraction from wheat in human leukocyte antigen-DQ8 transgenic mice (DQ8 mice) treated with indomethacin, an inhibitor of cyclooxygenases (COXs). After 10 days of treatment, mice showed a significant reduction of villus height, increased crypt depth, increased number of lamina propria-activated macrophages, and high basal interferon-γ secretion in mesenteric lymph nodes, all of which were specifically related to gliadin intake, whereas the albumin/globulin fraction of wheat was unable to induce similar changes. Cotreatment with NS-398, a specific inhibitor of COX-2, also induced the intestinal lesion. Enteropathy onset was further characterized by high levels of oxidative stress markers, similar to CD. Biochemical assessment of the small intestine revealed the specific activation of matrix metalloproteinases 2 and 9, high caspase-3 activity, and a significant increase of tissue transglutaminase protein levels associated with the intestinal lesion. Notably, after 30 days of treatment, enteropathic mice developed serum antibodies toward gliadin (IgA) and tissue transglutaminase (IgG). We concluded that gliadin intake in combination with COX inhibition caused a basal inflammatory status and an oxidative stress condition in the small intestine of DQ8 mice, thus triggering the mucosal lesion and, subsequently, an antigen-specific immunity.

Selective inhibition of the gliadin-specific, cell-mediated immune response by transamidation with microbial transglutaminase.

CD is an immune-mediated enteropathy caused by the ingestion of wheat gluten. The modification of gluten by intestinal tTGase plays a crucial role in CD pathogenesis. In this study, we observed that extensive transamidation of wheat flour with K-C2H5 by mTGase yielded spf and K-gliadins fractions. By Western blot, we found that these modifications were associated with strongly reduced immune cross-reactivity. With the use of DQ8 tg mice as a model of gluten sensitivity, we observed a dramatic reduction in IFNγ production in gliadin-specific spleen cells challenged with spf and K-gliadins in vitro (n=12; median values: 813 vs. 29 and 99; control vs. spf and K-gliadins, P=0.012 for spf, and P=0.003 for K-gliadins). For spf, we also observed an increase in the IL-10/IFNγ protein ratio (n=12; median values: 0.3 vs. 4.7; control vs. spf, P=0.005). In intestinal biopsies from CD patients challenged in vitro with gliadins (n=10), we demonstrated further that K-gliadins dramatically reduced the levels of antigen-specific IFNγ mRNA in all specimens responsive to native gliadins (four of 10; P<0.05). As cytotoxic effects have been described for gliadins, we also studied GST and caspase-3 activities using the enterocytic Caco-2 cell line. We found that neither activities were modified by flour transamidation. Our results indicate that K-C2H5 cross-linking via mTGase specifically affects gliadin immunogenicity, reversing the inducible inflammatory response in models of gluten sensitivity without affecting other aspects of the biological activity of gliadins.

Immunogenicity of monococcum wheat in celiac patients.

BACKGROUND: Research is intense to find wheat of low or null toxicity for patients with celiac disease (CD). Among candidates, there are diploid wheat species. OBJECTIVE: We compared the immunological properties of 2 lines of diploid monococcum wheat (Triticum monococcum ssp. monococcum), Monlis and ID331, with those of common wheat (Triticum aestivum). DESIGN: Interferon-γ production and the proliferation of intestinal gliadin-specific T cell lines and clones were measured as evidence of T cell activation by peptic and tryptic (PT) digests of gliadins from 2 monococcum lines. Furthermore, organ cultures of jejunal biopsies from 28 CD patients were set up to assess the effects of PT gliadin on innate and adaptive immune response by using immunohistochemistry. RESULTS: Monlis and ID331 induced interferon-γ production and proliferation in celiac mucosal T cells. In organ cultures, Monlis PT digest induced a significant increase of IL-15 epithelial expression and crypt enterocyte proliferation, whereas ID331 had no effect. Both monococcum lines caused intraepithelial T cell infiltration and lamina propria T cell activation. CONCLUSIONS: Our data show that the monococcum lines Monlis and ID331 activate the CD T cell response and suggest that these lines are toxic for celiac patients. However, ID331 is likely to be less effective in inducing CD because of its inability to activate the innate immune pathways.