CD103 (αE Integrin) Undergoes Endosomal Trafficking in Human Dendritic Cells, but Does Not Mediate Epithelial Adhesion

Dendritic cell (DC) expression of CD103, the α subunit of αEβ7 integrin, is thought to enable DC interactions with E-cadherin-expressing gastrointestinal epithelia for improved mucosal immunosurveillance. In the stomach, efficient DC surveillance of the epithelial barrier is crucial for the induction of immune responses to H. pylori, the causative agent of peptic ulcers and gastric cancer. However, gastric DCs express only low levels of surface CD103, as we previously showed. We here tested the hypothesis that intracellular pools of CD103 in human gastric DCs can be redistributed to the cell surface for engagement of epithelial cell-expressed E-cadherin to promote DC-epithelial cell adhesion. In support of our hypothesis, immunofluorescence analysis of tissue sections showed that CD103+ gastric DCs were preferentially localized within the gastric epithelial layer. Flow cytometry and imaging cytometry revealed that human gastric DCs expressed intracellular CD103, corroborating our previous findings in monocyte-derived DCs (MoDCs). Using confocal microscopy, we show that CD103 was present in endosomal compartments, where CD103 partially co-localized with clathrin, early endosome antigen-1 and Rab11, suggesting that CD103 undergoes endosomal trafficking similar to β1 integrins. Dynamic expression of CD103 on human MoDCs was confirmed by internalization assay. To analyze whether DC-expressed CD103 promotes adhesion to E-cadherin, we performed adhesion and spreading assays on E-cadherin-coated glass slides. In MoDCs generated in the presence of retinoic acid, which express increased CD103, intracellular CD103 significantly redistributed toward the E-cadherin-coated glass surface. However, DCs spreading and adhesion did not differ between E-cadherin-coated slides and slides coated with serum alone. In adhesion assays using E-cadherin-positive HT-29 cells, DC binding was significantly improved by addition of Mn2+ and decreased in the presence of EGTA, consistent with the dependence of integrin-based interactions on divalent cations. However, retinoic acid failed to increase DC adhesion, and a CD103 neutralizing antibody was unable to inhibit DC binding to the E-cadherin positive cells. In contrast, a blocking antibody to DC-expressed E-cadherin significantly reduced DC binding to the epithelium. Overall, these data indicate that CD103 engages in DC-epithelial cell interactions upon contact with epithelial E-cadherin, but is not a major driver of DC adhesion to gastrointestinal epithelia

Enhanced M1 Macrophage Polarization in Human Helicobacter pylori-Associated Atrophic Gastritis and in Vaccinated Mice

Background: Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined. Methodology/Principal Findings: By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion. Conclusions/Significance: These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis

Neonatal Colonisation Expands a Specific Intestinal Antigen-Presenting Cell Subset Prior to CD4 T-Cell Expansion, without Altering T-Cell Repertoire

Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα+) antigen-presenting cell subset, whilst SIRPα−CD11R1+ antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα+ antigen-presenting cells as orchestrators of early-life mucosal immune development

A novel canine histiocytic sarcoma cell line:initial characterization and utilization for drug screening studies

Abstract Background Histiocytic sarcoma is a rare disorder in humans, however it is seen with appreciable frequency in certain breeds of dogs, such as Bernese mountain dog. The purpose of this study was to fully characterize a novel canine histiocytic sarcoma cell line, and utilize it as a tool to screen for potential therapeutic drugs. Methods The histiocytic sarcoma cell line was characterized by expression of cellular markers as determined by immunohistochemistry and flow cytometry techniques. The neoplastic cells were also evaluated for their capability of phagocytizing beads particles, and their potential to grow as xenograft in an immunodeficient mouse. We investigated the in vitro cytotoxic activity of a panel of thirteen compounds using the MTS proliferation assay. Inhibitory effects of different drugs were compared using one-way ANOVA, and multiple means were compared using Tukey’s test. Results Neoplastic cells expressed CD11c, CD14, CD18, CD45, CD172a, CD204, MHC I, and vimentin. Expression of MHC II was upregulated after exposure to LPS. Furthermore, the established cell line clearly demonstrated phagocytic activity similar to positive controls of macrophage cell line. The xenograft mouse developed a palpable subcutaneous soft tissue mass after 29 days of inoculation, which histologically resembled the primary neoplasm. Dasatinib, a tyrosine kinase pan-inhibitor, significantly inhibited the growth of the cells in vitro within a clinically achievable and tolerable plasma concentration. The inhibitory response to dasatinib was augmented when combined with doxorubicin. Conclusions In the present study we demonstrated that a novel canine histiocytic sarcoma cell line presents a valuable tool to evaluate novel treatment approaches. The neoplastic cell line favorably responded to dasatinib, which represents a promising anticancer strategy for the treatment of this malignancy in dogs and similar disorders in humans

A novel canine histiocytic sarcoma cell line:initial characterization and utilization for drug screening studies

In order to be responsible stewards of other people’s data, cloud providers must be accountable for their data handling practices. The potential long provider chains in cloud computing introduces additional accountability challenges, and this paper examines requirements which must be fulfilled to achieve an accountability-based approach.acceptedVersio