Oligonucleotides (ODN) containing the """"""""CpG motif"""""""" account for the immune stimulatory activities of bacterial DNA. They are unusually effective mitogens for B cells because, in addition to driving the cells into and through cycle, they also inhibit apoptosis. Certain single base changes in ODN sequence can dramatically reduce activity. We have recently discovered that 3 of the ODN sequence variants actually inhibit cell cycle progress apoptosis protection and IL-6 secretion driven by stimulatory (ST-) ODN, whereas others are weak agonists or neutral in primary B cells. We have also shown that the order of potency of a series of stimulatory (ST-) ODN is the same for cycle entry, apoptosis protection, and IL-6 secretion implying a single control point for ODN signaling. Inhibitory (IN-) ODN block all the biologic effects as well as gene expression and transcription factor activation events (NFkB, AP-1, NF-IL-6) induced by ST-ODN, but not similar events induced by LPS or anti-CD40. Our application will determine the optimal base sequence for inhibition, and estimate the length of ODN sequence recognized by the CpG-recognizing molecule (RM). Spurred by the finding that an ODN with 2 motifs is more potent than ODN with 1, we will test whether bivalence or the sequence of """"""""transplanted"""""""" motifs determine the activity of an ODN. Thus we may discover more potent ST- and IN-ODN than those now available. Based on evidence that IN-ODN act proximal to NFkB and AP-1, we will examine earlier events leading to NFkB or AP-1 to locate sites of action of IN-ODN. We have shown that 32P-labeled CpG-ODN bind a series of 4-5 proteins in B cell cytoplasmic extracts, and that the order of avidity of these proteins for ST-ODN matches their order of potency in biologic assays. We will test whether this is also true for IN-ODN, including whether the increased potency associated with having 2 motifs is reflected in avidity for CpG-binding proteins (BP). We will then obtain microsequence data on the most prominent proteins in an attempt to identify them. Toll-Like Receptor (TLR) 9 has recently been shown to be necessary for responses to ODN. Using a TLR9-transfected cell line we will test whether TLR9 or one of its associated proteins binds CpG-ODN directly and if it does, whether the avidity and EMSA mobility match one of the CpG-BPs. The importance of this project lies in the need to develop antidotes for excessive CpG stimulation potentially to be encountered in CpG vaccine trials, and to recognize and avoid IN-ODN motifs when constructing viral vectors for gene therapy and DNA vaccination. Possibly that IN-ODN motifs in mammalian DNA may keep autoimmune responses to endogenous ST-ODN motifs in check.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI047374-03
Application #
6699378
Study Section
Immunobiology Study Section (IMB)
Program Officer
Rathbun, Gary
Project Start
2002-02-01
Project End
2006-01-31
Budget Start
2004-02-01
Budget End
2006-01-31
Support Year
3
Fiscal Year
2004
Total Cost
$258,125
Indirect Cost
Name
University of Iowa
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
062761671
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Ashman, Robert F; Goeken, J Adam; Lenert, Petar S (2011) Aggregation and secondary loop structure of oligonucleotides do not determine their ability to inhibit TLR9. Int Immunopharmacol 11:1032-7
Layer, T; Steele, A; Goeken, J A et al. (2011) Engagement of the B cell receptor for antigen differentially affects B cell responses to Toll-like receptor-7 agonists and antagonists in BXSB mice. Clin Exp Immunol 163:392-403
Goeken, J A; Layer, T; Fleenor, S et al. (2010) B-cell receptor for antigen modulates B-cell responses to complex TLR9 agonists and antagonists: implications for systemic lupus erythematosus. Lupus 19:1290-301
Lenert, P (2010) Nucleic acid sensing receptors in systemic lupus erythematosus: development of novel DNA- and/or RNA-like analogues for treating lupus. Clin Exp Immunol 161:208-22
Lenert, Petar S (2010) Classification, mechanisms of action, and therapeutic applications of inhibitory oligonucleotides for Toll-like receptors (TLR) 7 and 9. Mediators Inflamm 2010:986596
Lenert, Petar; Yasuda, Kei; Busconi, Liliana et al. (2009) DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas(lpr/lpr) mice in vivo. Arthritis Res Ther 11:R79
Ashman, Robert F; Lenert, Petar (2007) Structural requirements and applications of inhibitory oligodeoxyribonucleotides. Immunol Res 39:4-14
Lenert, Petar S (2006) Targeting Toll-like receptor signaling in plasmacytoid dendritic cells and autoreactive B cells as a therapy for lupus. Arthritis Res Ther 8:203
Lenert, Petar; Goeken, Adam J; Ashman, Robert F (2006) Extended sequence preferences for oligodeoxyribonucleotide activity. Immunology 117:474-81
Lenert, P (2005) Inhibitory oligodeoxynucleotides - therapeutic promise for systemic autoimmune diseases? Clin Exp Immunol 140:1-10

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