RESUMO
Theta-defensins (θ-defensins) are macrocyclic peptides expressed exclusively in granulocytes and selected epithelia of Old World monkeys. They contribute to anti-pathogen host defense responses by directly killing a diverse range of microbes. Of note, θ-defensins also modulate microbe-induced inflammation by affecting the production of soluble tumor necrosis factor (sTNF) and other proinflammatory cytokines. Here, we report that natural rhesus macaque θ-defensin (RTD) isoforms regulate sTNF cellular release by inhibiting TNF-α-converting enzyme (TACE; also known as adisintegrin and metalloprotease 17; ADAM17), the primary pro-TNF sheddase. Dose-dependent inhibition of cellular TACE activity by RTDs occurred when leukocytes were stimulated with live Escherichia coli cells as well as numerous Toll-like receptor agonists. Moreover, the relative inhibitory potencies of the RTD isoforms strongly correlated with their suppression of TNF release by stimulated blood leukocytes and THP-1 monocytes. RTD isoforms also inhibited ADAM10, a sheddase closely related to TACE. TACE inhibition was abrogated by introducing a single opening in the RTD-1 backbone, demonstrating that the intact macrocycle is required for enzyme inhibition. Enzymologic analyses showed that RTD-1 is a fast binding, reversible, non-competitive inhibitor of TACE. We conclude that θ-defensin-mediated inhibition of pro-TNF proteolysis by TACE represents a rapid mechanism for the regulation of sTNF and TNF-dependent inflammatory pathways. Molecules with structural and functional features mimicking those of θ-defensins may have clinical utility as TACE inhibitors for managing TNF-driven diseases.
Assuntos
Proteína ADAM17/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/farmacologia , Defensinas/farmacologia , Leucócitos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Proteína ADAM10/antagonistas & inibidores , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Anti-Inflamatórios não Esteroides/química , Linhagem Celular , Chlorocebus aethiops , Colo/efeitos dos fármacos , Colo/imunologia , Colo/metabolismo , Defensinas/química , Escherichia coli/imunologia , Escherichia coli/fisiologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Lipopolissacarídeos/toxicidade , Macaca mulatta , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The emergence of infections by carbapenem resistant Enterobacteriaceae (CRE) pathogens has created an urgent public health threat, as carbapenems are among the drugs of last resort for infections caused by a growing fraction of multi-drug resistant (MDR) bacteria. There is global consensus that new preventive and therapeutic strategies are urgently needed to combat the growing problem of MDR bacterial infections. Here, we report on the efficacy of a novel macrocyclic peptide, minimized theta-defensin (MTD)-12813 in CRE sepsis. MTD12813 is a theta-defensin inspired cyclic peptide that is highly effective against CRE pathogens K. pneumoniae and E. coli in vivo. In mouse septicemia models, single dose administration of MTD12813 significantly enhanced survival by promoting rapid host-mediated bacterial clearance and by modulating pathologic cytokine responses, restoring immune homeostasis, and preventing lethal septic shock. The peptide lacks direct antibacterial activity in the presence of mouse serum or in peritoneal fluid, further evidence for its indirect antibacterial mode of action. MTD12813 is highly stable in biological matrices, resistant to bacterial proteases, and nontoxic to mice at dose levels 100 times the therapeutic dose level, properties which support further development of the peptide as a first in class anti-infective therapeutic.
Assuntos
Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Animais , Infecções Bacterianas/tratamento farmacológico , Carbapenêmicos/farmacologia , Citocinas/metabolismo , Desenho de Fármacos , Feminino , Humanos , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Fagocitose , Sepse/sangueRESUMO
Theta-defensins are macrocyclic antimicrobial peptides that were previously isolated from leukocytes of a single species, the rhesus macaque. We now report the characterization of baboon theta-defensins (BTDs) expressed in bone marrow and peripheral blood leukocytes. Four cDNAs encoding theta-defensin precursors were characterized, allowing for the prediction of 10 theoretical theta-defensins (BTD-1 to BTD-10) produced by binary, head-to-tail splicing of nonapeptides excised from paired precursors. Five of the predicted theta-defensins were purified from baboon leukocytes, and synthetic versions of each were prepared. Anti-theta-defensin antibody localized the peptides in circulating neutrophils and monocytes and in immature and mature myeloid elements in bone marrow. Each of the BTDs possessed antimicrobial activity against bacterial and fungal test organisms in vitro. Peptide activities varied markedly despite a high degree of sequence conservation among the theta-defensins tested. Thus, baboons express numerous theta-defensins which appear to differentially contribute to host defense against diverse pathogens.
Assuntos
Defensinas/genética , Defensinas/imunologia , Defensinas/isolamento & purificação , Leucócitos/imunologia , Papio/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
θ-defensins constitute a family of macrocyclic peptides expressed exclusively in Old World monkeys. The peptides are pleiotropic effectors of innate immunity, possessing broad spectrum antimicrobial activities and immunoregulatory properties. Here we report that rhesus θ-defensin 1 (RTD-1) is highly effective in arresting and reversing joint disease in a rodent model of rheumatoid arthritis (RA). Parenteral RTD-1 treatment of DA/OlaHsd rats with established pristane-induced arthritis (PIA) rapidly suppressed joint disease progression, restored limb mobility, and preserved normal joint architecture. RTD-1 significantly reduced joint IL-1ß levels compared with controls. RTD-1 dose-dependently inhibited fibroblast-like synoviocyte (FLS) invasiveness and FLS IL-6 production. Consistent with the inhibition of FLS invasiveness, RTD-1 was a potent inhibitor of arthritogenic proteases including ADAMs 17 and 10 which activate TNFα, and inhibited matrix metalloproteases, and cathepsin K. RTD-1 was non-toxic, non-immunogenic, and effective when administered as infrequently as once every five days. Thus θ-defensins, which are absent in humans, have potential as retroevolutionary biologics for the treatment of RA.
Assuntos
Artrite Reumatoide/prevenção & controle , Defensinas/farmacologia , Animais , Artrite Reumatoide/imunologia , Macaca mulatta , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Rhesus theta-defensin 1 (RTD-1) is a unique tridisulfide, cyclic antimicrobial peptide formed by the ligation of two 9-residue sequences derived from heterodimeric splicing of similar 76-amino acid, alpha-defensin-related precursors, termed RTD1a and RTD1b (Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., and Selsted, M. E. (1999) Science 286, 498-502). The structures of RTD-2 and RTD-3 were predicted to exist if homodimeric splicing of the RTD1a and RTD1b occurs in vivo. Western blotting disclosed the presence of putative theta-defensins, distinct from RTD-1, in leukocyte extracts. Two new theta-defensins, RTD-2 and RTD-3, were purified by reverse-phase high performance liquid chromatography and characterized by amino acid analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and comparison to the synthetic standards. RTD-2 and RTD-3 are the predicted homodimeric splicing products of RTD1b and RTD1a, respectively. The cellular abundances of RTD-1, -2, and -3 were 29:1:2, indicating that there is a preference for the heterodimeric ligation that generates RTD-1. RTD-1, -2, and -3 had similar antimicrobial activities against Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans, whereas the activity of RTD-2 against Escherichia coli was 2-3-fold less than those of RTD-1 and RTD-3. Equal amounts of each theta-defensin bound to E. coli cells, indicating that the differences in antibacterial activities are the result of post-binding processes.