Coeliac Disease, a model of food-induced inflammatory disease

Coeliac Disease, a model of food-induced inflammatory disease Celiachia, un modello di patologia infiammatoria indo:a da alimen; M. Vi&oria Barone Background/aim. Epidemiological studies, have associated the increase in chronic inflammatory diseases, such as diabetes, atherosclerosis, asthma, chronic liver disease, autoimmune diseases, degenerative diseases and inflammatory bowel diseases to the spread of the so-called " Western diet”. The Mediterranean diet has increasingly been regarded as the gold-stantard diet for human health. The nutrients exert their effects on tissue inflammation, either by direct action on the cells, or because they regulate the composition of the intestinal microbiota. The enterocyte and the intestinal immunocompetent cells, are equipped with complex systems to "feel" the food and to respond to them. Even cereals (and in particular wheat) can cause intestinal inflammation, for example in celiac disease. The intestinal damage from gluten (the complex of soluble alcohol proteins in wheat) in celiac disease consists of inflammation and remodelling of the mucosa, with flattening of the villi and hypertrophy of the crypts. There are two main types of inflammatory response to gluten peptides in celiac disease (1): there is the adaptive response mediated by CD4 + T cells to some peptides: the 33 mer of A-Gliadin, resistant to gastric digestion, is a prototype endoluminal and parietal intestinal. The peptide is deamidated by tissue transglutaminase (tTG) and presented by class II histocompatibility antigens, DQ2 and DQ8, to CD4 + T cells, with Th1-type response, mediated by gamma interferon and other proinflammatory cytokines. Then there is the response, not mediated by T cells, to other peptides: the peptide 31-43 of A-gliadin (P 31-43) is the prototype. P 31-43, which is part of peptide 31-55, is resistant to gastric and intestinal digestion and causes inflammation with multiple mechanisms, the best known of which consists of a stress / innate and proliferative response, mediated by EGF and IL15 (2). Peptide P31-43 is also a growth factor for various cell lines and for the enterocyte of celiac, as it is able to activate the EGF-EGFR system, the most powerful mitogen present in our body. The gliadin-induced proliferation of celiac crypt is not only dependent on EGF but also on IL15. The proliferation of crypt enterocytes and the innate immune response to celiac gliadin are regulated by a cooperation between EGF and IL15. P31-43 induce also a stress/innate immune response involving interferon α (IFN-α) (3). Thus a double action of gliadin is delineated in the induction of the mucosal damage of celiac subjects. On the one hand, gliadin can activate the T-mediated response and on the other it can induce an innate/ inflammatory response. The combined effects of this double action induce the typical lesion of the celiac mucosa. But what causes the sensitivity to gliadin peptides in celiac cells is not really clear. In this study, we describe a stressed/inflamed celiac cellular phenotype in enterocytes and fibroblasts probably due to an alteration in the early-recycling endosomal system. Moreover we show that celiac cells are more sensitive to the gliadin peptide P31-43 and IL15 than controls. This phenotype is reproduced in control cells by inducing a delay in early vesicular trafficking. Methods. For organ culture studies, biopsy fragments from duodenum were obtained from CD patients with villous atrophy controls, affected by gastroesophageal reflux, and CD patients on GFD (Gluten free diet). Fibroblasts were cultured from skin and intestinal biopsies obtained from CD patients, (GFD, GCD) and controls. We used double immunofluorescence staining, western blotting and immunoprecipitation to evaluate EGFR and EEA1 levels and co- localization in fibroblasts and biopsy fragment from patients with CD and controls. We transfected cells with siHRS and mRNA analysis was perfomed to evaluate the levels of EEA1. Results. We found in CD biopsies and fibroblasts an increase of markers of the innate immune response (EGFR, IL15-R α, MXA) and of the inflammatory response (NFkB). CD cells (enterocytes and fibroblasts) presented a constitutive alteration in the intracellular vesicular system at the level of the early-recycling compartment. In fact, in CD enterocytes and fibroblasts, the numbers of early vesicles were increased, and the EGF/EGFR trafficking was delayed in the early endocytic vesicles; moreover, the decay of the EGFR was prolonged, and TfR levels were increased. We induced a delay in early endocytic trafficking by transfecting cells with siHRS in control cells and rendered them more sensitive to gliadin treatment using as read out STAT5 and NFkB levels. Conclusions. In the present study we show that cells from celiac disease patients present a delay of the endocytic trafficking and are more sensitive to the wheat gliadin peptide P31-43 than control cells. We also found that inducing delays in early vesicular trafficking leads to a celiac-like cellular phenotype, carachterized by inflammation and activation of innate immunity, thus implicating the early-recycling endosomal system in celiac disease. This constitutive lesion might mediate the stress/innate immune response to gliadin, which in turn triggers the gliadin-specific T-cell response. 1) Barone MV, Troncone R, Auricchio S.Gliadin peptides as triggers of the proliferative and stress/ innate immune response of the celiac small intestinal mucosa. Int J Mol Sci. 2014 15(11):20518-37. 2) Barone MV, Zimmer KP. Endocytosis and transcytosis of gliadin peptides. Mol Cell Pediatr. 2016 3:8. 3) Nanayakkara M, Lania G, Maglio M, Auricchio R, De Musis C, Discepolo V, Miele E, Jabri B, Troncone R, Auricchio S, Barone MV.P31-43, an undigested gliadin peptide, mimics and enhances the innate immune response to viruses and interferes with endocytic trafficking: a role in celiac disease.Sci Rep. 2018 8:1082

Impact of Age at Administration, Lysosomal Storage, and Transgene Regulatory Elements on AAV2/8-Mediated Rat Liver Transduction

Liver-directed gene transfer is being investigated for the treatment of systemic or liver-specific diseases. Recombinant vectors based on adeno-associated virus serotype 8 (AAV2/8) efficiently transduce liver cells allowing long term transgene expression after a single administration in animal models and in patients

Ligand of Numb proteins LNX1p80 and LNX2 interactwith the human glycoprotein CD8a and promote itsubiquitylation and endocytosis

E3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown. In this study, we identified the human T-cell co-receptor CD8 a-chain as binding partner of the ligand of Numb proteins X1 (LNX1p80 isoform) and X2 (LNX2). Both LNX mRNAs were found expressed in T cells purified from human blood, and both proteins interacted with CD8a in human HPB-ALL T cells. By using an in vitro assay and a heterologous expression system we showed that the interaction is mediated by the PDZ (PSD95-DlgA-ZO-1) domains of LNX proteins and the cytosolic C-terminal valine motif of CD8a. Moreover, CD8a redistributed LNX1 or LNX2 from the cytosol to the plasma membrane, whereas, remarkably, LNX1 or LNX2 promoted CD8a ubiquitylation, downregulation from the plasma membrane, transport to the lysosomes, and degradation. Our findings highlight the function of LNX proteins as E3 ligases and suggest a mechanism of regulation for CD8a localization at the plasma membrane by ubiquitylation and endocytosis

A Small Peptide Targeting the Ligand-Induced Androgen Receptor/Filamin a Interaction Inhibits the Invasive Phenotype of Prostate Cancer Cells

:Prostate cancer (PC) is one of the most widespread malignancies among males worldwide. The androgen receptor (AR) plays a major role in prostate cancer development and progression and is the main target of PC therapy. Nonetheless, its action is not yet fully elucidated. We report here that the AR associates with Filamin A (FlnA) promoting migration and invasiveness of various PC-derived cells after androgen challenging. Inhibition of the AR/FlnA complex assembly by a very low concentration of Rh-2025u, an AR-derived peptide specifically interfering with this association, impairs such phenotype in monolayer cells and in 3D models. This study, together with our recent data in cancer-associated fibroblasts (CAFs), indicates that targeting the AR/FlnA complex could improve the clinical management of invasive PC, as the limited number of new drugs reaching the market suggests that we must re-examine the way invasive PC is currently treated. In this context, the synthesis of new biologically active molecules, such as the Rh-2025u peptide, which has been shown to efficiently interfere in the complex assembly in CAFs and PC cells, should overcome the limits of current available therapies, mostly based on hormone hormone antagonists

The protective role of Lactobacillus rhamnosus GG postbiotic on the alteration of autophagy and inflammation pathways induced by gliadin in intestinal models

Celiac disease (CD) is an autoimmune enteropathy caused by an abnormal immune response to gliadin peptides in genetically predisposed individuals. For people with CD, the only available therapy thus far is the lifelong necessity for a gluten-free diet (GFD). Innovative therapies include probiotics and postbiotics as dietary supplements, both of which may benefit the host. Therefore, the present study aimed to investigate the possible beneficial effects of the postbiotic Lactobacillus rhamnosus GG (LGG) in preventing the effects induced by indigested gliadin peptides on the intestinal epithelium. In this study, these effects on the mTOR pathway, autophagic function, and inflammation have been evaluated. Furthermore, in this study, we stimulated the Caco-2 cells with the undigested gliadin peptide (P31-43) and with the crude gliadin peptic-tryptic peptides (PTG) and pretreated the samples with LGG postbiotics (ATCC 53103) (1 × 108). In this study, the effects induced by gliadin before and after pretreatment have also been investigated. The phosphorylation levels of mTOR, p70S6K, and p4EBP-1 were increased after treatment with PTG and P31-43, indicating that the intestinal epithelial cells responded to the gliadin peptides by activating the mTOR pathway. Moreover, in this study, an increase in the phosphorylation of NF-κβ was observed. Pretreatment with LGG postbiotic prevented both the activation of the mTOR pathway and the NF-κβ phosphorylation. In addition, P31-43 reduced LC3II staining, and the postbiotic treatment was able to prevent this reduction. Subsequently, to evaluate the inflammation in a more complex intestinal model, the intestinal organoids derived from celiac disease patient biopsies (GCD-CD) and controls (CTR) were cultured. Stimulation with peptide 31-43 in the CD intestinal organoids induced NF-κβ activation, and pretreatment with LGG postbiotic could prevent it. These data showed that the LGG postbiotic can prevent the P31-43-mediated increase in inflammation in both Caco-2 cells and in intestinal organoids derived from CD patients

Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action

In NIH3T3 cells, 0.001 nM of the synthetic androgen R1881 induces and stimulates association of androgen receptor (AR) with Src and phosphatidylinositol 3-kinase (Pl3-kinase), respectively, thereby triggering S-phase entry. 10 nM R1881 stimulates Rac activity and membrane ruffling in the absence of the receptor–Src–PI3-kinase complex assembly. The antiandrogen Casodex and specific inhibitors of Src and PI3-kinase prevent both hormonal effects, DNA synthesis and cytoskeletal changes. Neither low nor high R1881 concentration allows receptor nuclear translocation and receptor-dependent transcriptional activity in fibroblasts, although they harbor the classical murine AR. The very low amount of AR in NIH3T3 cells (7% of that present in LNCaP cells) activates the signaling pathways, but apparently is not sufficient to stimulate gene transcription. This view is supported by the appearance of receptor nuclear translocation as well as receptor-mediated transcriptional activity after overexpression of AR in fibroblasts. In addition, AR-negative Cos cells transiently transfected with a very low amount of hAR cDNA respond to low and high R1881 concentrations with signaling activation. Interestingly, they do not show significant transcriptional activation under the same experimental conditions. Fibroblasts are the first example of cells that respond to steroid hormones with activation of signaling pathways in the absence of endogenous receptor transcriptional activity. The data reported also show that hormone concentration can be crucial in determining the type of cell responsiveness

Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells

In breast cancer cells, cytoplasmic localization of the estradiol receptor α (ERα) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry

Constitutive Differential Features of Type 2 Transglutaminase in Cells Derived from Celiac Patients and from Healthy Subjects

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31-43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31-43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31-43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31-43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis

Complete Metabolic Response with Recanalization of Portal Vein Tumor Thrombosis after Sunitinib in a Patient with Advanced Hepatocellular Carcinoma

The prognosis of patients with advanced hepatocellular carcinoma (HCC) is very poor. The outcome of these patients is particularly bleak when the disease is complicated by portal vein tumor thrombosis (PVTT), since the increased portal pressure often causes serious gastrointestinal bleedings. Before the introduction of sorafenib (SOR), a tyrosine kinase inhibitor, no effective treatment was available for patients with advanced disease. SOR is now considered the standard treatment even for patients with tumor thrombosis, although the well-known interference between tyrosine kinase inhibitors and the coagulation pathway calls for caution against their use in this setting. Here, we report the case of a 74-year-old male patient with advanced HCC and PVTT treated with sunitinib (SUN), another multikinase inhibitor. During the third cycle, our patient experienced a life-threatening hematemesis with hemorrhagic shock that required intensive care treatment and SUN discontinuation. However, he completely recovered, and the PET/CT scan performed 1 year after the adverse effect demonstrated no evidence of the tumor together with portal vein recanalization. The short course of SUN causing both tumor response and gastrointestinal bleeding warrants further studies on the effectiveness of SUN in this setting as well as on the duration of treatment with multikinase inhibitors in patients with tumor thrombosis