RTD-1 therapeutically normalizes synovial gene signatures in rat autoimmune arthritis and suppresses proinflammatory mediators in RA synovial fibroblasts.

Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1ß-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.

Design of a potent antibiotic peptide based on the active region of human defensin 5.

Human defensin 5 (HD5) is a broad-spectrum antibacterial peptide with a C-terminal active region. To promote the development of this peptide into an antibiotic, we initially substituted Glu21 with Arg because it is an electronegative residue located around the active region. Although detrimental to dimer formation, the E21R substitution markedly enhanced the antibacterial activity of HD5 and increased its ability to penetrate cell membranes, demonstrating that increasing the electropositive charge compensated for the effect of dimer disruption. Subsequently, a partial Arg scanning mutagenesis was performed, and Thr7 was selected for replacement with Arg to further strengthen the antibacterial activity. The newly designed peptide, T7E21R-HD5, exhibited potent antibacterial activity, even in saline and serum solutions. In contrast to monomeric E21R-HD5, T7E21R-HD5 assembled into an atypical dimer with parallel ß strands, thus expanding the role of increasing electropositive charge in bactericidal activity and providing a useful guide for further defensin-derived antibiotic design.

In vitro potential of equine DEFA1 and eCATH1 as alternative antimicrobial drugs in rhodococcosis treatment.

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.

Role of human neutrophil peptide-1 as a possible adjunct to antituberculosis chemotherapy.

We report the role of human neutrophil peptide (HNP)-1 as an adjunct to antituberculosis (anti-TB) drugs. The combination of HNP-1, isoniazid, and rifampicin was evaluated against Mycobacterium tuberculosis H(37)Rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (MICs) of these agents. In vitro results revealed >1-log unit reductions even when HNP-1 and anti-TB drugs were used at 1/16 MICs. This combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 MICs of HNP-1 and drugs. HNP-1 used in conjunction with anti-TB drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. The effective therapeutic dosage of drugs could be reduced to half by supplementing HNP-1 in the therapeutic schedule. These results clearly suggest that HNP-1 can be used as adjunct chemotherapy with conventional drugs against TB.

The potential of human neutrophil peptides in tuberculosis therapy.

The problems of drug resistance and bacterial persistence in tuberculosis have prompted scientists to search for clues from the latest advances in microbiology and immunology. Recent research on human neutrophil peptides (HNPs) has highlighted their bactericidal action against Mycobacterium tuberculosis and suggested that neutrophils may play a more important defensive role in tuberculosis than previously thought. Human neutrophil peptides belong to a family of antimicrobial and cytotoxic peptides known as 'defensins'. Neutrophils use both oxidative and non-oxidative microbicidal mechanisms to provide the host with innate immunity against microbial infections. Defensins are most abundant among an array of oxygen-independent antimicrobial proteins and peptides in neutrophil granules. Defensins are effective against a wide spectrum of microbes including bacteria, viruses, fungi, spirochetes and mycobacteria. In addition to direct antimicrobial activity, HNPs can potentially influence the inflammatory or immune responses by modulating cytokine production or acting like opsonins or chemotactic factors. HNPs are active against M. tuberculosis grown in vitro or within macrophages. HNPs released by neutrophils recruited in the early lesion could attract monocytes to the site and macrophages may in vivo uptake the extracellular HNPs and kill the intracellular pathogens. As such, HNPs are potential therapeutic agents against tuberculosis. HNPs are also cytotoxic against a wide range of normal mammalian cells; however, there is evidence that defensins may not cause significant cytotoxicity at the therapeutic level. Finally, the clinical application of HNPs must be evaluated in the context of possible drug resistance, as some resistance-associated genes have been identified.