A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

The Syk Kinase Promotes Mammary Epithelial Integrity and Inhibits Breast Cancer Invasion by Stabilizing the E-Cadherin/Catenin Complex.

While first discovered in immunoreceptor signaling, the Syk protein kinase behaves as a tumor and metastasis suppressor in epithelial cells. Its reduced expression in breast and other carcinomas is correlated with decreased survival and increased metastasis risk, but its action mechanism remains largely unknown. Using phosphoproteomics we found that Syk phosphorylated E-cadherin and α-, ß-, and p120-catenins on multiple tyrosine residues that concentrate at intercellular junctions. Increased Syk expression and activation enhanced E-cadherin/catenin phosphorylation, promoting their association and complex stability. In human breast cancer cells, Syk stimulated intercellular aggregation, E-cadherin recruitment and retention at adherens junctions, and promoted epithelial integrity, whereas it inhibited cell migration and invasion. Opposite effects were obtained with Syk knockdown or non-phosphorylatable mutant E-cadherin expression. Mechanistically, Syk stimulated the interaction of the E-cadherin/catenin complex with zonula occludens proteins and the actin cytoskeleton. Conditional Syk knockout in the lactating mouse mammary gland perturbed alveologenesis and disrupted E-cadherin localization at adherens junctions, corroborating the observations in cells. Hence, Syk is involved in the maintenance of the epithelial integrity of the mammary gland via the phosphorylation and stabilization of the E-cadherin/catenin adherens junction complex, thereby inhibiting cell migration and malignant tumor invasion.