No abstract

Dysfunctional endothelium contributes to more disease than any other tissue in the body. Small interfering RNAs (siRNAs) have the potential to help study and treat endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here we show that polymeric nanoparticles made of low molecular weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. It mediates the most durable non-liver silencing reported to date, and facilitates the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth, and metastasis. We believe these nanoparticles improve the ability to study endothelial gene function in vivo, and may be used to treat diseases caused by vascular dysfunction.

Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higheraffinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.vaccination kinetics | antigen retention | humoral response | computational immunology | germinal center formation S ubunit vaccines based on recombinant protein antigens combined with adjuvants can safely elicit protective humoral immune responses in humans, and they have become a cornerstone of modern public health (1, 2). Recent advances in structure-based vaccine design (3, 4) and progress in the development of adjuvants that are safe and effective for prophylactic vaccines (5) have helped drive the field. However, several challenges remain: A number of protein vaccines, such as candidate vaccines against HIV and malaria, have tended to elicit short-lived immunity (6, 7). In HIV, broadly neutralizing antibodies (BNAbs) isolated from infected patients are generally characterized by high degrees of somatic hypermutation (SHM) (8), but methods to generate such highly mutated antibodies by vaccination remain unknown. SHM occurs in germinal centers (GCs) within lymphoid organs, and data from animal models demonstrate a critical role for follicular helper T cells in the induction of GCs and promotion of affinity maturation (9, 10). To date, methods to promote Tfh generation and long-lived germinal centers during vaccination remain unclear (11-15). Much attention has focused on the use of adjuvants to promote affinity maturation, but it remains unclear if adjuvants alone can provide the necessary immunological driving forces for promoting extensive affinity maturation (16).During acute infections, which often provoke robust germinal center responses and durable humoral immunity, microorganism replication typically occurs over the course of one to several weeks (17-19). During this time, recognition of molecular danger signals contained within the pathogen sustains stimulation of the innate immune system, and a continuous supply of antigen is provided to the adaptive immune system. In contrast to these patterns of antigen and inflammatory cues during infection, typical subunit vaccines...

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system.

SUMMARY Messenger RNAs (mRNAs) can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of untranslated regions, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

XBP1 is a key regulator of the unfolded protein response (UPR), which is involved in a wide range of physiological and pathological processes. XBP1 ablation in liver causes profound hypolipidemia in mice, highlighting its critical role in lipid metabolism. XBP1 deficiency triggers feedback activation of its upstream enzyme IRE1α, instigating regulated IRE1-dependent decay (RIDD) of cytosolic mRNAs. Here, we identify RIDD as a crucial control mechanism of lipid homeostasis. Suppression of RIDD by RNA interference or genetic ablation of IRE1α reversed hypolipidemia in XBP1 deficient mice. Comprehensive microarray analysis of XBP1 and/or IRE1α deficient liver identified genes involved in lipogenesis and lipoprotein metabolism as RIDD substrates, which might contribute to the suppression of plasma lipid levels by activated IRE1α. Ablation of XBP1 ameliorated hepatosteatosis, liver damage and hypercholesterolemia in dyslipidemic animal models, suggesting that direct targeting of either IRE1α or XBP1 might be a feasible strategy to treat dyslipidemias.

Dutta et al. show that targeting VACM-1 expression in splenic macrophages impairs extramedullary hematopoiesis, thus reducing inflammation in mouse ischemic heart and atherosclerotic plaques.

Mice lacking the transcription factor XBP1 exhibit constitutive activation of the stress sensor IRE1α and are protected from acetaminophen overdose–induced acute liver failure.