Anu Chaudhary

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells

Toll‐like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram‐negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP‐1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP‐1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP‐1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin‐1 (IL1) receptor‐associated kinase 1 (IRAK‐1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS‐mediated release of cytokines IL6, IL1β, tumor necrosis factor‐α, neither on chemokines macrophage inhibitory protein (MIP)1α, MIP1β, monocyte chemoattractant protein ‐1, IL8, Groα and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS‐mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.

An engineered innate immune defense protects grapevines from Pierce disease

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the Gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

The bioinorganic chemistry and associated immunology of chronic beryllium disease

Chronic beryllium disease (CBD) is a debilitating, incurable, and often fatal disease that is caused by the inhalation of beryllium particulates. The growing use of beryllium in the modern world, in products ranging from computers to dental prosthetics (390 tons of beryllium in the US in the year 2000) necessitates a molecular based understanding of the disease in order to prevent and cure CBD. We have investigated the molecular basis of CBD at Los Alamos National Laboratory during the past six years, employing a multidisciplinary approach of bioinorganic chemistry and immunology. The results of this work, including speciation, inhalation and dissolution, and immunology will be discussed.

Targeting Toll-Like Receptor Signaling Pathways for Design of Novel Immune Therapeutics

The Toll-like receptor (TLR) family plays a fundamental role in host innate immunity by mounting a rapid and potent inflammatory response to pathogen infection. TLRs recognize distinct microbial components and activate intracellular signaling pathways that induce expression of host inflammatory genes. Extensive research in the past decade to understand TLR-mediated mechanisms of innate immunity has enabled pharmaceutical companies to begin to develop novel therapeutics for the purpose of controlling inflammatory disease. Initially, extracellular TLR agonists were designed to compete with natural microbial ligands for binding to TLRs. More recently, basic research to identify new targets for drug development has begun to explore modulation of TLR intracellular signaling pathways, in addition to TLR ligand binding. In this review, we will discuss recent strategies, including the use of decoy peptides and mimetics, plant polyphenols, and chemically modified antisense oligonucleotides, that inhibit different molecular events in TLR signaling pathways to modulate the inflammatory response. The molecular mechanisms of these inhibitors range from interference with protein-protein interactions between signaling proteins, to inhibition of transcription factor activity, to perturbation of the plasma membrane, and are derived from host, pathogen, and plant sources and by rational design. Taken together, these studies represent promising avenues for the development of novel tailored immune therapeutics that can relieve the great toll inflicted by inflammatory disease on human health and quality of life.

Beryllium Alters Lipopolysaccharide-Mediated Intracellular Phosphorylation and Cytokine Release in Human Peripheral Blood Mononuclear Cells

Beryllium exposure in susceptible individuals leads to the development of chronic beryllium disease, a lung disorder marked by release of inflammatory cytokine and granuloma formation. We have previously reported that beryllium induces an immune response even in blood mononuclear cells from healthy individuals. In this study, we investigate the effects of beryllium on lipopolysaccharide-mediated cytokine release in blood mononuclear and dendritic cells from healthy individuals. We found that in vitro treatment of beryllium sulfate inhibits the secretion of lipopolysaccharide-mediated interleukin 10, while the release of interleukin 1β is enhanced. In addition, not all lipopolysaccharide-mediated responses are altered, as interleukin 6 release in unaffected upon beryllium treatment. Beryllium sulfate-treated cells show altered phosphotyrosine levels upon lipopolysaccharide stimulation. Significantly, beryllium inhibits the phosphorylation of signal transducer and activator of transducer 3, induced by lipopolysaccharide. Finally, inhibitors of phosphoinositide-3 kinase mimic the effects of beryllium in inhibition of interleukin 10 release, while they have no effect on interleukin 1β secretion. This study strongly suggests that prior exposures to beryllium could alter host immune responses to bacterial infections in healthy individuals, by altering intracellular signaling.