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Regulation of the Innate Immune Response

The innate immune system is an ancient mechanism of host defense found in essentially every multicellular organism from plants to humans.  In invertebrates, it is the only mechanism of defense.  Vertebrates also developed and adaptive immune response, however, the innate immune system is essential for instructing the cells of the adaptive system (T and B cells) by presenting antigen in the context of an appropriate costimulatory molecule.   The innate immune system developed to not only discriminate self from non-self but more importantly, it can discriminate infectious non-self from innocuous non-self.

How do cells of the innate immune system recognize infectious non-self?

Macrophages and other cells of the innate immune system sense and respond to microbial products via the Toll-like receptor family. Toll-like receptors (TLR) are an evolutionarily conserved family of cell surface molecules that participate in innate immune recognition of pathogen-associated molecular patterns (PAMPs) (Medzhitov, 2000). PAMPs are generally unique, chemically diverse products with conserved motifs that are produced by microorganisms. PAMPs often have an essential role in the structure of bacteria and generally cannot be subtly modified as a result of mutation. Examples include LPS (specifically Lipid A), peptidoglycan (PGN), lipoproteins, bacterial DNA, and bacterial flagella. Eleven different TLRs have been identified. In some cases the bacterial ligand has also been identified. For example, TLR2 recognizes peptidoglycan, (Schwander, 1999) and mycobacterial lipoarabinomannan (Means, 1999), TLR4 recognizes LPS from most gram negative species, TLR5 reacts with flagellin (Hayashi, 2001), and TLR9 is a receptor for bacterial CpG DNA (Hemmi, 2000). The signaling events occurring downstream of the TLRs are rapidly being elucidated and appear to have many common features. In general, the cascade of events occurring following ligation of the different TLRs involves the activation of a common set of adapter proteins and protein kinases, the best characterized of which leads to the activation of NF-kB (reviewed in Bowie, 2000). While a large body of literature indicates that the members of the TLR family activate a nearly identical intracytoplasmic signaling program, several recent reports, including our own (Hirschfeld, 2001;Toschakov, 2002; Carl, 2002) have begun to suggest that the functional outcomes of signaling via TLR2 or TLR4 are not equivalent.

Bowie, A. and L. A. O'Neill. 2000. The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J.Leukoc.Biol. 67:508-514.

 Carl, V. S., K. Brown-Steinke, M. J. Nicklin, and M. F. Smith, Jr. 2002. Toll-like receptor 2 and 4 (TLR2 and TLR4) agonists differentially regulate secretory interleukin-1 receptor antagonist gene expression in macrophages. J Biol.Chem. 277:17448-17456.

Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, and A. Aderem. 2001. The innate immune response to bacterial flagellin is mediated by Toll- like receptor 5. Nature 410:1099-1103.

Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.

Hirschfeld, M., J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody, D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel. 2001. Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect.Immun. 69:1477-1482.

Means, T. K., S. Wang, E. Lien, A. Yoshimura, D. T. Golenbock, and M. J. Fenton. 1999. Human toll-like receptors mediate cellular activation by Mycobacterium tuberculosis. J.Immunol. 163:3920-3927.

Medzhitov, R. and C. Janeway, Jr. 2000. The Toll receptor family and microbial recognition. Trends Microbiol. 8:452-456

Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning. 1999. Peptidoglycan- and lipotechoic acid-induced cell activation is mediated by Toll-like receptor 2. J.Biol.Chem. 274:17406-17409.

Toshchakov, V., B. W. Jones, P. Y. Perera, K. Thomas, M. J. Cody, S. Zhang, B. R. Williams, J. Major, T. A. Hamilton, M. J. Fenton, and S. N. Vogel. 2002. TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat.Immunol 3:392-398.

Regulation of macrophage gene expression

Macrophages are the terminally differentiated derivative of the peripheral blood monocyte. Together these two cell types play a major role in regulation of both humoral and cellular immune responses and as the primary line of defense against a number of microorganisms. During an inflammatory response, induced by infection or tissue injury, macrophages produce a variety of soluble mediators (cytokines) which impact on the functioning of a large number of cell types. The main interest of my lab is in studying the molecular mechanisms which are responsible for the regulation of cytokine genes in macrophages and monocytes. The research is divided into two interrelated areas, transcriptional regulation and signal transduction. The overall goal is to understand how signals initiated at the cell surface by macrophage activating agents, such as LPS from gram negative bacteria, are communicated to the nucleus and induce cytokine gene expression.

We are currently characterizing the mechanisms which control expression of the interleukin-1 receptor antagonist (IL-1Ra) gene. IL-1Ra is the only known, naturally occurring cytokine receptor antagonist and thus represents a means of controlling the inflammatory effects of IL-1. Through analysis of protein/DNA interactions and transient transfection assays with cloned promoter fragments, we have identified several transcription factors which regulate IL-1Ra gene expression including NF-kB, PU.1, GABP, STAT6, and NF-IL6. Currently we are characterizing how these factors interact to regulate gene expression. Additionally, we have found that the LPS-induced activation of p38 and participates in inducing IL-1Ra gene expression. The aim is now to determine the upstream activators of these kinases and to understand the cytoplasmic signaling mechanisms induced by LPS which result in increasing the ability of these factors to activate transcription.

Recent Publications:

V.S. Carl, K. Brown-Steinke, M. Nicklin, and M.F. Smith, Jr.  2002.  TLR2 and TLR4 ligands differentially regulate secretory IL-1 receptor antagonist gene expression.  J. Biol. Chem.  277:17448-56  Full text

This paper was featured (May 21st 2002) in Science's STKE web page as a noteworthy contribution to the scientific literature.  

V.S. Carl, J.K. Gautam, L.D. Comeau, and M.F. Smith, Jr. 2004. Role of endogenous IL-10 in LPS-induced STAT3 activation and IL-1 receptor antagonist gene expression. J. Leuk. Biol.  In press.


Toll-Like Receptors and H. pylori infection

Additionally, In collaboration with Joanna Goldberg (UVA, Department of Microbiology) we are also examining the role of innate immune receptors in the control of gastric epithelial cell responses to Helicobacter pylori. 

H. pylori is a spiral, microaerophilic, gram negative bacterium which colonizes gastric epithelial cells and the gastric mucosal layer in 25-50% of the population in developed countries and 70-90% in developing countries, for an estimated 2 billion people worldwide. While most infected individuals are asymptomatic, H. pylori is nevertheless a causative agent of peptic ulcer disease, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The specific clinical outcome is determined by the interplay of H. pylori virulence factors, host gastric mucosal factors, and the environment. The mechanisms by which bacterial and/or host factors cause disease remain unclear. H. pylori has been demonstrated to activate a wide range of signaling pathways and transcription factors, including NF-kB, AP-1, and MAPK, in gastric epithelial cells. Until now, the identity of the cell surface signaling molecule(s) responsible for these responses is still unclear.

We have recently determined that H. pylori-induced NF-kB and chemokine gene expression in gastric epithelial cells requires TLR2 and TLR5, but surprisingly not TLR4.  The results of these studies were published

in The Journal of Biological Chemistry.

See it here:  http://www.jbc.org/cgi/reprint/M305536200v1.pdf