Theta (?)-defensins are 18-amino acid macrocyclic peptides expressed in leukocytes of Old World monkeys(OWMs) and are the only examples of backbone-cyclized polypeptides in animals. Sixteen ?-defensin isoforms, all sharing an invariant 10-amino acid core structure, are expressed in neutrophils of OWMs. ?-defensin isoforms block the expression of TNF in stimulated leukocytes with IC50's varying from 40-350 nM. ?-defensin-mediated TNF blockade is rapid and results in the down regulation of other inflammatory cytokines in vitro and in vivo. Multiple lines of evidence indicate that one mechanism mediating the anti-TNF effect is the competitive inhibition of TNF-? converting enzyme (TACE;ADAM17), suggesting that ?-defensins are endogenous regulators of TACE. ?-defensin isoforms also inhibit the activities of other key inflammatory proteases of the matrix metalloprotease and cathepsin families. RTD-1, the prototype ?-defensin, arrests rat pristane-induced arthritis (PIA), a model of rheumatoid arthritis (RA). The overall goal of this project is to further characterize the cellular and molecular mechanisms that mediate the anti-inflammatory properties of ?-defensins.
Three Specific Aims are proposed, each of which exploit the natural structure-function diversity of ?-defensins:
In Aim 1 : in vitro cellular assays will be used to identify inflammatory cascades that are modulated by ?-defensins;the role of TNF/TACE-blockade will be analyzed by RNA inhibition of TACE in THP- 1 cells;paracine/autocrine effects of ?-defensins will be analyzed by quantifying peptide biosynthesis/secretion by stimulated macaque granulocytes and determining the immunomodulatory effects of releasates on resting and activated target cells;analogous studies will be conducted with RTD-1-expressing THP-1 transfectants to evaluate the immunoregulatory and antimicrobial properties conferred to transfected cells. Studies in Aim 2 will utilize novel in silico docking protocols, biochemical and structure-activity analysis, and analog design to analyze the interactions of ?-defensins with TACE and other proinflammatory proteases that are inhibited by ?-defensin isoforms and which are implicated in RA pathogenesis.
In Aim 3, the rat PIA model will be used to analyze the effects of systemically administered ?-defensins. Using this model, three ?-defensin isoforms, predicted (based on results of Aims 1 and 2) to have different levels of efficacy, will be evaluated pharmacokinetically and pharmacodynamically: therapeutic response, peptide levels in serum and joint tissue, serum and joint biomarkers, and histology of joint tissues will be analyzed over the course of disease progression and resolution. Collectively, these studies will provide new and significant insights into the regulation of systemic inflammation as revealed by the mechanisms mediated by endogenous immunoregulatory peptides of OWM, and will disclose the potential for 'resurrecting'?-defensins as therapeutics for human inflammatory disorders.

Public Health Relevance

Naturally occurring circular peptides (theta-defensins) produced by white blood cells of monkeys have a unique ability to protect mice and rats from harmful inflammation. The mechanisms underlying this protection involve the regulation of the host's responses to a variety of noxious stimuli, and reveals that theta-defensins are endogenous anti-inflammatory molecules. In this project we will use biochemical and cellular assays and an animal model of rheumatoid arthritis to identify specific anti-inflammatory mechanisms that can be applied to the understanding and treatment of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022931-27
Application #
8665864
Study Section
Innate Immunity and Inflammation Study Section (III)
Program Officer
Peyman, John A
Project Start
1989-07-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
27
Fiscal Year
2014
Total Cost
$432,609
Indirect Cost
$169,558
Name
University of Southern California
Department
None
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Beringer, Paul M; Bensman, Timothy J; Ho, Henry et al. (2016) Rhesus θ-defensin-1 (RTD-1) exhibits in vitro and in vivo activity against cystic fibrosis strains of Pseudomonas aeruginosa. J Antimicrob Chemother 71:181-8
Oh, Young Taek; Tran, Dat; Buchanan, Thomas A et al. (2015) θ-Defensin RTD-1 improves insulin action and normalizes plasma glucose and FFA levels in diet-induced obese rats. Am J Physiol Endocrinol Metab 309:E154-60
Tongaonkar, Prasad; Trinh, Katie K; Schaal, Justin B et al. (2015) Rhesus macaque θ-defensin RTD-1 inhibits proinflammatory cytokine secretion and gene expression by inhibiting the activation of NF-κB and MAPK pathways. J Leukoc Biol 98:1061-70
Tai, Kenneth P; Kamdar, Karishma; Yamaki, Jason et al. (2015) Microbicidal effects of *- and ýý-defensins against antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Innate Immun 21:17-29
Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele et al. (2014) Killing of staphylococci by θ-defensins involves membrane impairment and activation of autolytic enzymes. Antibiotics (Basel) 3:617-31
Tai, Kenneth P; Le, Valerie V; Selsted, Michael E et al. (2014) Hydrophobic determinants of α-defensin bactericidal activity. Infect Immun 82:2195-202
Mastroianni, Jennifer R; Lu, Wuyuan; Selsted, Michael E et al. (2014) Differential Susceptibility of Bacteria to Mouse Paneth Cell α-Defensins under Anaerobic Conditions. Antibiotics (Basel) 3:493-508
Arnison, Paul G; Bibb, Mervyn J; Bierbaum, Gabriele et al. (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature. Nat Prod Rep 30:108-60
Lehrer, Robert I; Cole, Alex M; Selsted, Michael E (2012) θ-Defensins: cyclic peptides with endless potential. J Biol Chem 287:27014-9
Schaal, Justin B; Tran, Dat; Tran, Patti et al. (2012) Rhesus macaque theta defensins suppress inflammatory cytokines and enhance survival in mouse models of bacteremic sepsis. PLoS One 7:e51337

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