S. Santagata

IL-15 Interferes With Suppressive Activity of Intestinal Regulatory T Cells Expanded in Celiac Disease

OBJECTIVES:Celiac disease (CD) is a condition in which the regulation of the mucosal immune response to dietary gliadin might be altered. The transcription factor forkhead box P3 (Foxp3) has been identified as a marker of a subset of regulatory T cells (Treg). In this study, we have investigated the presence and the suppressive function of Treg cells in the celiac small intestinal mucosa, their correlation with the disease state, and the inducibility by gliadin in an organ culture system; moreover, we tried to define whether interleukin 15 (IL-15), overexpressed in CD, could influence the regulatory activity of such cells.METHODS:The expression of Foxp3, CD3, CD4, and CD8 were analyzed by immunohistochemistry and flow cytometry in duodenal biopsies taken from patients with untreated CD, treated CD, and from non-CD controls, as well as in vitro cultured biopsy samples from treated CD patients, upon challenge with gliadin. Furthermore, we analyzed the suppressive function of CD4+CD25+ T cells, isolated from untreated CD biopsy samples, on autologous responder CD4+CD25− T cells, in the presence of a polyclonal stimulus, with or without IL-15.RESULTS:Higher density of CD4+CD25+Foxp3+ T cells was seen in duodenal biopsy samples from active CD patients in comparison with treated CD and non-CD controls. In coculture, CD4+CD25+ T cells were functionally suppressive, but their activity was impaired by IL-15. Cells from CD subjects showed increased sensitivity to the IL-15 action, likely due to enhanced expression of IL-15 receptor. Finally, we demonstrated an expansion of Foxp3 in treated CD mucosa following in vitro challenge with gliadin.CONCLUSIONS:These data suggest that CD4+CD25+Foxp3+ T cells are induced in situ by gliadin. However, their suppressor capacity might be impaired in vivo by IL-15; this phenomenon contributes to maintain and expand the local inflammatory response in CD.

Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR.

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

Background Celiac Disease (CD) is both a frequent disease (1∶100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called “toxic” A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. Methods/Principal Findings Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. Conclusions P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosin Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies.

Immunoregulatory Pathways Are Active in the Small Intestinal Mucosa of Patients with Potential Celiac Disease

OBJECTIVES:Potential celiac disease (CD) relates to subjects with a normal small intestinal mucosa who are at increased risk of developing CD as indicated by positive CD-associated serology. The objective of this study was to investigate in the small intestinal mucosa of such patients the state of immunological activation with special emphasis on immunoregulatory circuits.METHODS:Duodenal biopsies from active CD (n=48), potential CD (n=58), and control patients (n=45) were studied. RNA expression for interferon γ (IFNγ) and interleukin-10 (IL-10) were quantified by real-time quantitative PCR. The percentage of CD4+CD25+Foxp3+ T regulatory cells (Foxp3+Tregs) was determinated by flow cytometry and the number of Foxp3+ and IL-15+ cells by immunohistochemistry. Furthermore, we analyzed the suppressive function of CD4+CD25+ T cells, isolated from potential CD biopsy samples, as well as the effect of IL-15, on autologous peripheral blood responder CD4+CD25– T cells.RESULTS:In potential CD patients with Marsh 1 lesion, IFNγ-RNA expression was significantly less than in active, but enhanced if compared with potential CD patients with Marsh 0 lesion and with controls (P<0.001). The number of IL-15+ cells in subjects with potential CD was increased in comparison with controls (P<0.05), but lower than active CD (P<0.01). IL-10-RNA expression was upregulated in Marsh 0 potential CD patients if compared with those with Marsh 1 lesion (P<0.01) and controls (P<0.001), whereas there were no differences with active CD. The ratio IL-10/IFNγ reached the highest value in Marsh 0 potential CD compared with the other groups (P<0.05). The percentage of Foxp3+Tregs was also higher in potential CD compared with controls (P<0.05), although it was lower than in active CD (P<0.01). In co-culture assay, intestinal CD4+CD25+ T cells from potential CD patients exerted suppressive effects on T responder cells, and their activity was not impaired by IL-15.CONCLUSIONS:Potential CD patients show a low grade of inflammation that likely could be due to active regulatory mechanisms preventing the progression toward a mucosal damage.

A novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) specifically detects CXCR4 expressing tumors

C-X-C chemokine receptor 4 (CXCR4) is over-expressed in multiple human cancers and correlates with tumor aggressiveness, poor prognosis and increased risk for distant metastases. Imaging agents for CXCR4 are thus highly desirable. We developed a novel CXCR4-targeted near-infrared (NIR) fluorescent probe (Peptide R-NIR750) conjugating the new developed CXCR4 peptidic antagonist Peptide R with the NIR fluorescent dye VivoTag-S750. Specific CXCR4 binding was obtained in cells overexpressing human CXCR4 (B16-hCXCR4 and human melanoma cells PES43), but not in CXCR4 low expressing cells (FB-1). Ex vivo evaluation demonstrated that PepR-NIR750 specifically detects B16-hCXCR4-derived subcutaneous tumors and lung metastases. Fluorescence Molecular Tomography (FMT) in vivo imaging was performed on mice carrying subcutaneous CHO and CHO-CXCR4 tumors. PepR-NIR750 accumulates only in CXCR4-positive expressing subcutaneous tumors. Additionally, an intense NIR fluorescence signal was detected in PES43-derived lung metastases of nude mice injected with PepR-NIR750 versus mice injected with VivoTag-S750. With a therapeutic intent, mice bearing PES43-derived lung metastases were treated with Peptide R. A the dramatic reduction in PES43-derived lung metastases was detected through a decrease of the PepR-NIR750 signal. PepR-NIR750 is a specific probe for non-invasive detection of human high CXCR4-expressing tumors and metastatic lesion and thus a valuable tool for cancer molecular imaging.