Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function.

Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix-helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.

Human iNKT Cells Promote Protective Inflammation by Inducing Oscillating Purinergic Signaling in Monocyte-Derived DCs.

Invariant natural killer T (iNKT) cells are innate T lymphocytes that promote host defense against a variety of microbial pathogens. Whether microbial ligands are required for their protective effects remains unclear. Here, we show that iNKT cells stimulate human-monocyte-derived dendritic cells (DCs) to produce inflammatory mediators in a manner that does not require the presence of microbial compounds. Interleukin 2 (IL-2)-exposed iNKT cells selectively induced repeated cytoplasmic Ca(2+) fluxes in DCs that were dependent on signaling by the P2X7 purinergic receptor and mediated by ATP released during iNKT-DC interactions. Exposure to iNKT cells led to DC cyclooxygenase 2 (PTGS2) gene transcription, and release of PGE2 that was associated with vascular permeabilization in vivo. Additionally, soluble factors were released that induced neutrophil recruitment and activation and enhanced control of Candida albicans. These results suggest that sterile interactions between iNKT cells and monocyte-derived DCs lead to the production of non-redundant inflammatory mediators that promote neutrophil responses.

LumeNEXT: A Practical Method to Pattern Luminal Structures in ECM Gels.

In vitro biomimetic modeling of physio-logical structures bridges the gap between 2D in vitro culture and animal models. Lumens (tubular structures) are ubiquitous in vivo, being present in blood vessels, mammary ducts, and the lymphatic system. A method 'LumeNEXT' is presented here that allows the fabrication of 3D embedded lumens where size, structure, distance, and configuration can be controlled using standard poly-dimethylsiloxane micromolding methods.

Cutting edge: Kruppel-like factor 2 is required for phenotypic maintenance but not development of B1 B cells.

Several recent studies reported that Krüppel-like factor (KLF)2 controls trafficking, development, and function of B cells. Conditional B cell KLF2 knockout mice have increased numbers of marginal zone B cells and decreased numbers of B1 phenoytpe cells. However, it was unclear whether KLF2 is required for B1 B cell development, survival, or phenotypic maintenance. We show that B1 phenotype B cells are present in neonatal mice with B cell-specific KLF2 deficiency, suggesting that B1 differentiation can occur even in the absence of KLF2. Furthermore, by use of an inducible knockout strategy, we show that deletion of KLF2 in mature B1 cells causes loss of phenotypic markers associated with B1 cell identity, but it has a minimal effect on short-term cell survival. Taken together, our findings suggest that KLF2 is necessary for the maintenance of B1 cell identity rather than differentiation or survival of the population.