Lentivirus-mediated Conditional Gene Expression.

The ability to identify the role of a particular gene within a system is dependent on control of the expression of that gene. In this protocol, we describe a method for stable, conditional expression of Nod-Like receptors (NLRs) in THP-1 cells using a lentiviral expression system. This system combines all the necessary components for tetracycline-inducible gene expression in a single lentivector with constitutive co-expression of a selection marker, which is an efficient means for controlling gene expression using a single viral infection of cells.

A small sustained increase in NOD1 abundance promotes ligand-independent inflammatory and oncogene transcriptional responses.

Small, genetically determined differences in transcription [expression quantitative trait loci (eQTLs)] are implicated in complex diseases through unknown molecular mechanisms. Here, we showed that a small, persistent increase in the abundance of the innate pathogen sensor NOD1 precipitated large changes in the transcriptional state of monocytes. A ~1.

Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of flagellin inside macrophages.

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host.

Serum-borne lipids amplify TLR-activated inflammatory responses.

TLRs recognize conserved pathogen associated molecular patterns and generate innate immune responses. Several circulating and cell membrane associated proteins have been shown to collaborate with TLRs in sensing microbial ligands and promoting inflammatory responses. Here, we show that serum and serum-borne lipids including lysophosphatidylcholine (LPC) amplify inflammatory responses from intestinal epithelial cells and mononuclear phagocytes primed with microbial TLR ligands.

Accessibility of O Antigens Shared between Serovars Determines Antibody-Mediated Cross-Protection.

Pathogenic serovars produce clinical manifestations ranging from systemic infection typhoid to invasive nontyphoidal disease in humans. These serovars share a high degree of homology at the genome and the proteome level. However, whether infection or immunization with one serovar provides protection against other serovars has not been well studied.

Antibody-recruiting protein-catalyzed capture agents to combat antibiotic-resistant bacteria.

Antibiotic resistant infections are projected to cause over 10 million deaths by 2050, yet the development of new antibiotics has slowed. This points to an urgent need for methodologies for the rapid development of antibiotics against emerging drug resistant pathogens. We report on a generalizable combined computational and synthetic approach, called antibody-recruiting protein-catalyzed capture agents (AR-PCCs), to address this challenge.

Src kinases central to T-cell receptor signaling regulate TLR-activated innate immune responses from human T cells.

TLRs have a fundamental role in immunity. We have recently reported that stimulation of TLR2 and TLR5 in freshly isolated and activated human T cells with microbial ligands without concomitant activation through the TCR brings about secretion of neutrophil chemoattractant, CXCL8, and effector cytokine, IFN-γ, respectively. However, the mechanism of TLR signaling in T cells has not been worked out.

MyD88-dependent pro-inflammatory activity in Vi polysaccharide vaccine against typhoid promotes Ab switching to IgG.

Vi capsular polysaccharide is currently in use as a vaccine against human typhoid caused by Salmonella Typhi. The vaccine efficacy correlates with IgG anti-Vi Abs. We have recently reported that Vi can generate inflammatory responses through activation of the TLR2/TLR1 complex.

Sphingosine-1-phosphate suppresses TLR-induced CXCL8 secretion from human T cells.

T cells produce a number of cytokines and chemokines upon stimulation with TLR agonists in the presence or absence of TCR signals. Here, we show that secretion of neutrophil chemoattractant CXCL8 from human T cell line Jurkat in response to stimulation with TLR agonists is reduced when cell stimulation is carried out in presence of serum. Serum does not, however, inhibit TCR-activated secretion of CXCL8 nor does it down-regulate TLR-costimulated IL-2 secretion from activated T cells.