The cystine/glutamate antiporter regulates dendritic cell differentiation and antigen presentation.

The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.

Crosstalk between PKA and Epac regulates the phenotypic maturation and function of human dendritic cells.

The cAMP-dependent signaling pathways that orchestrate dendritic cell (DC) maturation remain to be defined in detail. Although cAMP was previously thought to signal exclusively through protein kinase A (PKA), it is now clear that cAMP also activates exchange protein activated by cAMP (Epac), a second major cAMP effector. Whether cAMP signaling via PKA is sufficient to drive DC maturation or whether Epac plays a role has not been examined. In this study, we used cAMP analogs to selectively activate PKA or Epac in human monocyte-derived DCs and examined the effect of these signaling pathways on several hallmarks of DC maturation. We show that PKA activation induces DC maturation as evidenced by the increased cell-surface expression of MHC class II, costimulatory molecules, and the maturation marker CD83. PKA activation also reduces DC endocytosis and stimulates chemotaxis to the lymph node-associated chemokines CXCL12 and CCL21. Although PKA signaling largely suppresses cytokine production, the net effect of PKA activation translates to enhanced DC activation of allogeneic T cells. In contrast to the stimulatory effects of PKA, Epac signaling has no effect on DC maturation or function. Rather, Epac suppresses the effects of PKA when both pathways are activated simultaneously. These data reveal a previously unrecognized crosstalk between the PKA and Epac signaling pathways in DCs and raise the possibility that therapeutics targeting PKA may generate immunogenic DCs, whereas those that activate Epac may produce tolerogenic DCs capable of attenuating allergic or autoimmune disease.

Ancestry as a potential modifier of gene expression in breast tumors from Colombian women.

BACKGROUND: Hispanic/Latino populations are a genetically admixed and heterogeneous group, with variable fractions of European, Indigenous American and African ancestries. The molecular profile of breast cancer has been widely described in non-Hispanic Whites but equivalent knowledge is lacking in Hispanic/Latinas. We have previously reported that the most prevalent breast cancer intrinsic subtype in Colombian women was Luminal B as defined by St. Gallen 2013 criteria. In this study we explored ancestry-associated differences in molecular profiles of Luminal B tumors among these highly admixed women. METHODS: We performed whole-transcriptome RNA-seq analysis in 42 Luminal tumors (21 Luminal A and 21 Luminal B) from Colombian women. Genetic ancestry was estimated from a panel of 80 ancestry-informative markers (AIM). We categorized patients according to Luminal subtype and to the proportion of European and Indigenous American ancestry and performed differential expression analysis comparing Luminal B against Luminal A tumors according to the assigned ancestry groups. RESULTS: We found 5 genes potentially modulated by genetic ancestry: ERBB2 (log2FC = 2.367, padj<0.01), GRB7 (log2FC = 2.327, padj<0.01), GSDMB (log2FC = 1.723, padj<0.01, MIEN1 (log2FC = 2.195, padj<0.01 and ONECUT2 (log2FC = 2.204, padj<0.01). In the replication set we found a statistical significant association between ERBB2 expression with Indigenous American ancestry (p = 0.02, B = 3.11). This association was not biased by the distribution of HER2+ tumors among the groups analyzed. CONCLUSIONS: Our results suggest that genetic ancestry in Hispanic/Latina women might modify ERBB2 gene expression in Luminal tumors. Further analyses are needed to confirm these findings and explore their prognostic value.

Increased expression of deleted in malignant brain tumors (DMBT1) gene in precancerous gastric lesions: Findings from human and animal studies.

Helicobacter pylori infection triggers a cascade of inflammatory stages that may lead to the appearance of non-atrophic gastritis, multifocal atrophic, intestinal metaplasia, dysplasia, and cancer. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by binding to pathogens. Initial studies showed its deletion and loss of expression in a variety of tumors but the role of this gene in tumor development is not completely understood. Here, we examined the role of DMBT1 in gastric precancerous lesions in Caucasian, African American and Hispanic individuals as well as in the development of gastric pathology in a mouse model of H. pylori infection. We found that in 3 different populations, mucosal DMBT1 expression was significantly increased (2.5 fold) in individuals with dysplasia compared to multifocal atrophic gastritis without intestinal metaplasia; the increase was also observed in individuals with advanced gastritis and positive H. pylori infection. In our animal model, H. pylori infection of Dmbt1-/- mice resulted in significantly higher levels of gastritis, more extensive mucous metaplasia and reduced Il33 expression levels in the gastric mucosa compared to H. pylori-infected wild type mice. Our data in the animal model suggest that in response to H. pylori infection DMBT1 may mediate mucosal protection reducing the risk of developing gastric precancerous lesions. However, the increased expression in human gastric precancerous lesions points to a more complex role of DMBT1 in gastric carcinogenesis.

The homing receptor CD44 is involved in the progression of precancerous gastric lesions in patients infected with Helicobacter pylori and in development of mucous metaplasia in mice.

Infection with Helicobacter pylori (H. pylori) leads to inflammatory events that can promote gastric cancer development. Immune cells transition from the circulation into the infected mucosa through the interaction of their receptors and ligands in the endothelial compartment. CD44 expression is increased in advanced gastric lesions. However, the association of this molecule with the progression of these lesions over time has not been investigated. In addition, there is a lack of understanding of the CD44-dependent cellular processes that lead to gastritis, and possibly to gastric cancer. Here we studied H. pylori-positive subjects with gastric lesions that ranged from multifocal atrophic gastritis to dysplasia to determine gene expression changes associated with disease progression over a period of 6 years. We report that CD44 expression is significantly increased in individuals whose gastric lesions progressed along the gastric precancerous cascade. We also show that CD44-/- mice develop less severe and less extensive H. pylori-induced metaplasia, and show fewer infiltrating Gr1+ cells compared to wild type mice. We present data suggesting that CD44 is associated with disease progression. Mechanisms associated with these effects include induction of interferon gamma responses.

Infrequency of microsatellite instability in complete and incomplete gastric intestinal metaplasia.

Chronic inflammation may be associated with microsatellite instability (MSI). To test the hypothesis that MSI frequently occurs in gastric intestinal metaplasia, we examined gastric biopsies from 58 subjects from an area of high risk for gastric cancer. These were selected to have 2 types of intestinal metaplasia: complete (31 subjects) and incomplete or mixed-type (27 subjects). None of the subjects had gastric cancer, but 95% had chronic inflammation with Helicobacter pylori. We used laser capture microdissection to retrieve metaplastic glands to compare with lymphocytes microdissected from the adjacent gastric mucosae in the same subjects. We performed microsatellite analysis using 6 microsatellite loci, including BAT26. None of the cases were found to have reproducible MSI, and only 1 case showed loss of heterozygosity at 1 marker, D3S1067. To test the sensitivity of our assay, we mixed templates to produce bands of different mobility and found that we could detect an aberrant microsatellite pattern if only 2% of the DNA showed that pattern. Our results indicate that MSI is a rare event in intestinal metaplasia in subjects who do not have gastric cancer.

Histopathology of gastritis in Helicobacter pylori-infected children from populations at high and low gastric cancer risk.

Infection with Helicobacter pylori has been recognized as a cause of gastric carcinoma. Although the neoplasia is always detected in adults, the infection starts in childhood. It has been reported that early age at first infection is a determinant of gastric cancer risk. In this study, we examined the histopathology of the gastric mucosa in infected children from a population at high risk for gastric cancer (Pasto, Colombia) and compared it with that of a lower-risk population (New Orleans, LA). Gastric biopsies obtained from antrum and corpus were stained with hematoxylin and eosin and Steiner's silver method. Immunohistochemical stains were used to identify B lymphocytes (CD20), T lymphocytes (CD3 and CD8), macrophages (CD68), and polymorphonuclear neutrophil myeloperoxidase. Morphometric techniques were used to evaluate the immunohistochemical stains. In both populations, the inflammatory lesions were seen predominantly in the antrum. Compared with children from the lower-risk populations, children from the higher-risk population exhibited more severe polymorphonuclear neutrophil infiltration, stromal and intraepithelial lymphocyte infiltration, mucus depletion, and H. pylori colonization density. Regenerative activity was significantly more marked in the lower-risk population. Morphometric analysis of immunohistochemical stains showed increased representation of T lymphocytes and macrophages in the higher-risk population. Most T lymphocytes stained positive for CD8, a marker of suppressor/cytotoxic cells. B lymphocytes were relatively more abundant in the lower-risk population. The possibility that the aforementioned characteristics of H. pylori infection in children are related to cancer risk in adults is discussed.