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David M. Segal, Ph.D.

Portait Photo of David Segal
Experimental Immunology Branch
Head, Immune Targeting Section
Senior Investigator
Building 10, Room 4B36
10 Center Drive
Bethesda, MD 20892
Phone:  
 301-496-3109
Fax:  
 301-496-0887
E-Mail:  
 dave_segal@nih.gov
Link:
 Other Homepage

Biography

Dr. Segal received a B.A. from Oberlin College and a Ph.D. from Johns Hopkins University. After two years as a postdoctoral fellow at the Weizmann Institute he came to the NIH where he studied the structures of antibodies and other proteins in the Laboratory of Molecular Biology, NIDDK. He then joined the NCI's Immunology Branch to pursue an interest in immune recognition. Recently Dr. Segal's research has focused on molecules of the innate immune system, particularly the Toll-like receptors.

Research

Toll Like Receptors and Innate Immunity
My laboratory investigates the structure and function of Toll-like Receptors (TLRs) and their role in innate and acquired immunity. The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, the most important of which are the TLRs. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans; however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids.
How the TLRs recognize such a wide array of PAMPs is a main interest of my laboratory. In collaboration with David Davies laboratory, we have recently succeeded in expressing, crystallizing, and determining the molecular structure of the TLR3 extracellular domain (ECD). The structure of the TLR3-ECD consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large b-sheet on the concave surface (a picture is included in Gallery link on the sidebar). Our immediate goal is to determine at the molecular level, how the TLR3 horseshoe specifically recognizes its ligand, dsRNA, and how recognition leads to activation. In addition to X-ray analysis, this objective will be achieved by studying the binding of various dsRNA ligands to TLR3-ECD protein in solution, and by introducing mutations to determine which residues are essential for activity. Looking past TLR3, we plan to express and examine ECDs from other TLR paralogs, to see how they differ in structure and ligand binding function from TLR3.
We are especially interest in the interaction of TLR4 with its ligand, lipopolysaccharide (LPS). LPS is a causative agent of Gram negative sepsis, and many laboratories have sought to understand how it induces septic shock. Four proteins, LBP, CD14, TLR4, and MD-2 are known to be involved in the mammalian response to LPS, but the essential signaling receptor for LPS consists of a complex containing only MD-2 and TLR4. MD-2 was originally discovered as a small glycoprotein bound to the ECD of TLR4 on the cell surface, and it was proposed that the interaction of TLR4 with MD-2 occurred intracellularly. However, we found that in addition to binding to TLR4 in the ER, MD-2 was also secreted into the medium, and that the secreted form (sMD-2) had several interesting properties. sMD-2 is able to confer LPS responsiveness to cells, such as epithelial cells and HEK293 transfectants, that express TLR4 but not MD-2. In solution, sMD-2 rapidly loses activity at physiological temperature, but is stabilized by LPS. We showed that a stable complex between LPS and MD-2, and not LPS itself, is the actual activating ligand of TLR4, and that CD14 is required only to transfer LPS to MD-2. To date we have demonstrated that MD-2/LPS binds and activates free TLR4, but we are also interested in determining whether it can activate preformed TLR4/MD-2 complexes, and if so what is the mechanism. These studies will be especially important in understanding the sensitivity and control of the response to LPS in vivo.
Nucleic acid PAMPs such as dsRNA, ssRNA, and CpG DNA, ligands for TLRs 3, 7, 8, and 9, are normally sequestered within microorganisms and become available to interact with TLRs only after the pathogen is endocytosed and lysed intracellularly. By contrast, TLRs 1, 2, 4, 5, 6, and 10 interact with PAMPs that are normally present in the medium, and these TLRs are, as expected, located on the cell surface. Therefore, correct cellular localization is essential for TLR function. We are currently studying the localization of TLR9, and motifs within the TLR9 molecule that mediate this localization. Results from these studies will provide us with a better understanding of how and where TLRs interact with pathogens. A further goal of this study is to compare the intracellular location of TLR9 with TLR3. TLR9 is thought to recognize mainly bacterial DNA, which is released in phagolysosomes, but TLR3 is thought to bind dsRNA, which is injected or synthesized by virus in the cytoplasm. The location of TLR3 relative to TLR9 might therefore provide clues as to how viral dsRNA is detected by the TLR3-ECD, which faces away from the cytoplasm.
TLRs play a pivotal role in acquired immunity by triggering the maturation of DC to competent APC, capable of priming naive T cells, and we were one of the first laboratories to demonstrate that DC express TLRs. Our interest in the function of TLRs on DC took a turn when we discovered that DC also express histamine receptors. Based on this observation, we hypothesized that histamine would have an effect on the maturation process. In testing this hypothesis we found that histamine profoundly alters the cytokines released by DC during TLR induced maturation, and as a result, histamine exposure causes DC to polarize naive T cells toward a Th2 phenotype. Mast cells are the major source of histamine, and they are often located in close proximity to DC. We therefore hypothesized that mast cell degranulation at a site of immunization would alter the nature of the immune response, by acting on neighboring DC. We have established a mouse model to test this hypothesis, and have been able to demonstrate an effect of mast cell degranulation on T cell polarization in vivo. We are especially excited about these results because they suggest a link between allergy (caused by mast cell degranulation), and the control of T cell responses. We plan to investigate the mechanisms by which mast cells influence Th1/Th2 balances and to determine their physiological consequences.

This page was last updated on 8/19/2008.