Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Human α defensins promote the expression of the inflammatory cytokine interleukin-8 under high-glucose conditions: Novel insights into the poor healing of diabetic foot ulcers.

Sustained infection and chronic inflammation are the most common features and complex mechanisms of diabetic foot disease. In this study, we examined the expression and functional roles of human endogenous α defensins in diabetic foot ulcer. The expression levels of human α defensins HNP1, HNP3, and HNP4 were significantly higher in the wound center than the edge of diabetic foot ulcers. And the inflammatory cytokine interleukin IL-8 (IL-8) was also highly expressed in wound exudates. In human foreskin fibroblasts, these human α defensins were found only slightly to affect IL-8 expression directly. hemoglobin A1C (HbA1c) is the main clinical indicator of diabetic foot disease. Advanced glycation end products of bovine serum albumin (AGE-BSA), as HbA1c analogue, was found to promote IL-8 expression. Human α defensins, in the presence of AGE-BSA, further significantly promoted IL-8 expression. These findings showed that human α defensins aggravated the inflammatory response in diabetic foot ulcers patients, providing new insights in to the poor healing of diabetic foot ulcers.

Distinctive Roles and Mechanisms of Human Neutrophil Peptides in Experimental Sepsis and Acute Respiratory Distress Syndrome.

OBJECTIVES: To examine the effects and mechanisms of human neutrophil peptides in systemic infection and noninfectious inflammatory lung injury. DESIGN: Prospective experimental study. SETTING: University hospital-based research laboratory. SUBJECTS: In vitro human cells and in vivo mouse models. INTERVENTIONS: Wild-type (Friend virus B-type) and conditional leukocyte human neutrophil peptides transgenic mice were subjected to either sepsis induced by cecal ligation and puncture or acute lung injury by intratracheal instillation of hydrochloric acid followed by mechanical ventilation. Using human neutrophil peptides as bait, the basal cell adhesion molecule (CD239) and the purinergic P2Y purinoceptor 6 receptor were identified as the putative human neutrophil peptides receptor complex in human lung epithelial cells. MEASUREMENTS AND MAIN RESULTS: In the cecal ligation and puncture sepsis model, Friend virus B-type mice exhibited higher systemic bacterial load, cytokine production, and lung injury than human neutrophil peptides transgenic mice. Conversely, an increased lung cytokine production was seen in Friend virus B-type mice, which was further enhanced in human neutrophil peptides transgenic mice in response to two-hit lung injury induced by hydrochloric acid and mechanical ventilation. The human neutrophil peptides-mediated inflammatory response was mediated through the basal cell adhesion molecule-P2Y purinoceptor 6 receptor signal pathway in human lung epithelial cells. CONCLUSIONS: Human neutrophil peptides are critical in host defense against infectious sepsis by their cationic antimicrobial properties but may exacerbate tissue injury when neutrophil-mediated inflammatory responses are excessive in noninfectious lung injury. Targeting the basal cell adhesion molecule/P2Y purinoceptor 6 signaling pathway may serve as a novel approach to attenuate the neutrophil-mediated inflammatory responses and injury while maintaining the antimicrobial function of human neutrophil peptides in critical illness.

Cell adhesion properties of human defensins.

Effector peptides of innate immunity play an important role in host defense. They act directly by inactivating microbes but also link innate to adaptive immunity. A variety of innate immune functions has been described for these peptides, including chemoattraction and cytokine release. In this study, we describe the effect on cell morphology and cell adhesion of human defensins. We find that Human Defensin 5, the major product of specialized gut epithelial cells, causes changes in cell morphology. HD-5 induces cell adhesion, binds to fibronectin and facilitates binding of T cells to intestinal epithelial cells. These effects were found also for a second prominent defensing, termed Human Neutrophil peptide-1, but not for other human defensins.

Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis.

BACKGROUND AND AIMS: Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP)-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1. METHODS: Transgenic (TG) mice expressing HNP-1 under the control of a ß-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT) mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes in vitro. RESULTS: After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner in vitro. CONCLUSIONS: HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.

Polymorphisms in α-Defensin-Encoding DEFA1A3 Associate with Urinary Tract Infection Risk in Children with Vesicoureteral Reflux.

The contribution of genetic variation to urinary tract infection (UTI) risk in children with vesicoureteral reflux is largely unknown. The innate immune system, which includes antimicrobial peptides, such as the α-defensins, encoded by DEFA1A3, is important in preventing UTIs but has not been investigated in the vesicoureteral reflux population. We used quantitative real-time PCR to determine DEFA1A3 DNA copy numbers in 298 individuals with confirmed UTIs and vesicoureteral reflux from the Randomized Intervention for Children with Vesicoureteral Reflux (RIVUR) Study and 295 controls, and we correlated copy numbers with outcomes. Outcomes studied included reflux grade, UTIs during the study on placebo or antibiotics, bowel and bladder dysfunction, and renal scarring. Overall, 29% of patients and 16% of controls had less than or equal to five copies of DEFA1A3 (odds ratio, 2.09; 95% confidence interval, 1.40 to 3.11; P<0.001). For each additional copy of DEFA1A3, the odds of recurrent UTI in patients receiving antibiotic prophylaxis decreased by 47% when adjusting for vesicoureteral reflux grade and bowel and bladder dysfunction. In patients receiving placebo, DEFA1A3 copy number did not associate with risk of recurrent UTI. Notably, we found that DEFA1A3 is expressed in renal epithelium and not restricted to myeloid-derived cells, such as neutrophils. In conclusion, low DEFA1A3 copy number associated with recurrent UTIs in subjects in the RIVUR Study randomized to prophylactic antibiotics, providing evidence that copy number polymorphisms in an antimicrobial peptide associate with UTI risk.

Human neutrophil α-defensins are associated with adenosine diphosphate-inducible neutrophil-platelet aggregate formation and response to clopidogrel in patients with atherosclerosis.

Human neutrophil α-defensins (HNPs) are antimicrobial peptides stored primarily in the azurophilic granules of polymorphonuclear leukocytes. Recently, it was shown that HNPs act as platelet agonists. We hypothesized that HNP levels are associated with the formation of neutrophil-platelet aggregates, and that they influence the response to clopidogrel therapy. HNP levels were determined by a commercially available enzyme-linked immunosorbent assay in 305 patients undergoing angioplasty and stenting for atherosclerotic cardiovascular disease. Neutrophil-platelet aggregates were measured by flow cytometry, and on-treatment platelet reactivity was determined using the VerifyNow P2Y12 and aspirin assays. HNP levels did not correlate with the formation of neutrophil-platelet aggregates in vivo (r = 0.05, P = 0.4). In contrast, HNP levels correlated significantly with adenosine diphosphate (ADP)-inducible neutrophil-platelet aggregate formation (r = 0.13, P = 0.04). On-treatment platelet reactivity by the VerifyNow P2Y12 assay was significantly more pronounced in patients with high HNP levels compared with patients with low HNP levels (211 P2Y12 reaction units [PRU; range, 143-293 PRU] vs 181 PRU [range, 129-237 PRU], P = 0.009). This association remained significant after adjusting for high-sensitivity C-reactive protein and interleukin 6 by multivariate regression analysis (P = 0.007). Moreover, high on-treatment residual platelet reactivity by the VerifyNow P2Y12 assay was more frequent in patients with high HNP levels than in patients with low HNP levels (40% vs 26.6%, P = 0.01). In conclusion, HNP levels are associated with ADP-inducible neutrophil-platelet aggregate formation and clopidogrel-mediated platelet inhibition. High levels of HNPs may, in part, be responsible for the observed response variability to clopidogrel.

Suppression of antimicrobial peptide expression by ureaplasma species.

Ureaplasma species commonly colonize the adult urogenital tract and are implicated in invasive diseases of adults and neonates. Factors that permit the organisms to cause chronic colonization or infection are poorly understood. We sought to investigate whether host innate immune responses, specifically, antimicrobial peptides (AMPs), are involved in determining the outcome of Ureaplasma infections. THP-1 cells, a human monocytoid tumor line, were cocultured with Ureaplasma parvum and U. urealyticum. Gene expression levels of a variety of host defense genes were quantified by real-time PCR. In vitro antimicrobial activities of synthetic AMPs against Ureaplasma spp. were determined using a flow cytometry-based assay. Chromosomal histone modifications in host defense gene promoters were tested by chromatin immunoprecipitation (ChIP). DNA methylation status in the AMP promoter regions was also investigated. After stimulation with U. parvum and U. urealyticum, the expression of cell defense genes, including the AMP genes (DEFB1, DEFA5, DEFA6, and CAMP), was significantly downregulated compared to that of TNFA and IL-8, which were upregulated. In vitro flow cytometry-based antimicrobial assay revealed that synthetic peptides LL-37, hBD-3, and hBD-1 had activity against Ureaplasma spp. Downregulation of the AMP genes was associated with chromatin modification alterations, including the significantly decreased histone H3K9 acetylation with U. parvum infection. No DNA methylation status changes were detected upon Ureaplasma infection. In conclusion, AMPs have in vitro activity against Ureaplasma spp., and suppression of AMP expression might be important for the organisms to avoid this aspect of the host innate immune response and to establish chronic infection and colonization.

Defensins in innate immunity.

PURPOSE OF REVIEW: Defensins are a major family of antimicrobial peptides expressed predominantly in neutrophils and epithelial cells, and play important roles in innate immune defense against infectious pathogens. Their biological functions in and beyond innate immunity, structure and activity relationships, mechanisms of action, and therapeutic potential continue to be interesting research topics. This review examines recent progress in our understanding of alpha and theta-defensins - the two structural classes composed of members of myeloid origin. RECENT FINDINGS: A novel mode of antibacterial action is described for human enteric alpha-defensin 6, which forms structured nanonets to entrap bacterial pathogens and protect against bacterial invasion of the intestinal epithelium. The functional multiplicity and mechanistic complexity of defensins under different experimental conditions contribute to a debate over the role of enteric alpha-defensins in mucosal immunity against HIV-1 infection. Contrary to common belief, hydrophobicity rather than cationicity plays a dominant functional role in the action of human alpha-defensins; hydrophobicity-mediated high-order assembly endows human alpha-defensins with an extraordinary ability to acquire structural diversity and functional versatility. Growing evidence suggests that theta-defensins offer the best opportunity for therapeutic development as a novel class of broadly active anti-infective and anti-inflammatory agents. SUMMARY: Defensins are the 'Swiss army knife' in innate immunity against microbial pathogens. Their modes of action are often reminiscent of the story of 'The Blind Men and the Elephant'. The functional diversity and mechanistic complexity, as well as therapeutic potential of defensins, will continue to attract attention to this important family of antimicrobial peptides.

Antiviral mechanisms of human defensins.

Defensins are an effector component of the innate immune system with broad antimicrobial activity. Humans express two types of defensins, α- and ß-defensins, which have antiviral activity against both enveloped and non-enveloped viruses. The diversity of defensin-sensitive viral species reflects a multitude of antiviral mechanisms. These include direct defensin targeting of viral envelopes, glycoproteins, and capsids in addition to inhibition of viral fusion and post-entry neutralization. Binding and modulation of host cell surface receptors and disruption of intracellular signaling by defensins can also inhibit viral replication. In addition, defensins can function as chemokines to augment and alter adaptive immune responses, revealing an indirect antiviral mechanism. Nonetheless, many questions regarding the antiviral activities of defensins remain. Although significant mechanistic data are known for α-defensins, molecular details for ß-defensin inhibition are mostly lacking. Importantly, the role of defensin antiviral activity in vivo has not been addressed due to the lack of a complete defensin knockout model. Overall, the antiviral activity of defensins is well established as are the variety of mechanisms by which defensins achieve this inhibition; however, additional research is needed to fully understand the role of defensins in viral pathogenesis.

Candida albicans escapes from mouse neutrophils.

Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.

Functional determinants of human enteric α-defensin HD5: crucial role for hydrophobicity at dimer interface.

Human α-defensins are cationic peptides that self-associate into dimers and higher-order oligomers. They bind protein toxins, such as anthrax lethal factor (LF), and kill bacteria, including Escherichia coli and Staphylococcus aureus, among other functions. There are six members of the human α-defensin family: four human neutrophil peptides, including HNP1, and two enteric human defensins, including HD5. We subjected HD5 to comprehensive alanine scanning mutagenesis. We then assayed LF binding by surface plasmon resonance, LF activity by enzyme kinetic inhibition, and antibacterial activity by the virtual colony count assay. Most mutations could be tolerated, resulting in activity comparable with that of wild type HD5. However, the L29A mutation decimated LF binding and bactericidal activity against Escherichia coli and Staphylococcus aureus. A series of unnatural aliphatic and aromatic substitutions at position 29, including aminobutyric acid (Abu) and norleucine (Nle) correlated hydrophobicity with HD5 function. The crystal structure of L29Abu-HD5 depicted decreased hydrophobic contacts at the dimer interface, whereas the Nle-29-HD5 crystal structure depicted a novel mode of dimerization with parallel ß strands. The effect of mutating Leu(29) is similar to that of a C-terminal hydrophobic residue of HNP1, Trp(26). In addition, in order to further clarify the role of dimerization in HD5 function, an obligate monomer was generated by N-methylation of the Glu(21) residue, decreasing LF binding and antibacterial activity against S. aureus. These results further characterize the dimer interface of the α-defensins, revealing a crucial role of hydrophobicity-mediated dimerization.

Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.

Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel ß-sheet but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in the proximity of each other (d ≈ 3 Å) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when the protein was produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either native Crp4 or its cyclized variants. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone-cyclized defensins in the development of novel peptide-based antimicrobial compounds.

Efficiency of antimicrobial defense: molecular flexibility of natural defensin and artificial bis-quaternary ammonium compound.

BACKGROUND: Human α-defensins (such as HD5 and HD6) are typical bactericidal peptides. We examined the molecular features of HD5 and HD6 by molecular dynamics (MD) analysis. MATERIALS AND METHODS: Molecular features of natural defensins and artificial bis-quaternary ammonium compounds (e.g. 1,6-polymethylenedithio)bis(1-octylpyridinium iodide: 4DTBP-m,8) were analyzed using molecular simulation techniques. RESULTS: HD5 and HD6 had different electrostatic potential profiles, which indicated the region-dependent hydrophobicity. 4DTBP-m,8 derivatives were significantly flexible, and many conformers existed. CONCLUSION: HD5 and HD6 indicated antimicrobial activity by restricted conformation.

Human neutrophil peptides mediate endothelial-monocyte interaction, foam cell formation, and platelet activation.

OBJECTIVE: Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. METHODS AND RESULTS: We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. CONCLUSIONS: HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases.

The innate immune system in delayed cutaneous allergic reactions to medications.

PURPOSE OF REVIEW: Innate immune responses are attracting increasing interest from researchers in the field of drug allergy. This review discusses recent advances in the understanding of several innate immune components in delayed cutaneous hypersensitivity reactions to medications, with special attention on severe reactions. RECENT FINDINGS: The mechanism of activation of dendritic cells in response to drugs is being unravelled. Activated monocytes and macrophages have been found in affected skin of bullous diseases. Increased gene expression of monomyeloid cell products including several 'alarmins' or endogenous damage-associated molecular patterns has been described in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Natural killer (NK) cells from patients respond to drugs in vitro. In-vivo, NK cells may contribute to severe diseases through the secretion of effector molecules such as granulysin. The innate receptor CD94/NKG2C is expressed by NK cells and cytotoxic T lymphocytes in SJS/TEN and triggers degranulation in response to human leukocyte antigen-E-expressing keratinocytes. T cells with innate activities have been detected in patients during severe acute reactions. SUMMARY: Humoral and cellular components of the innate immune response have been identified in association with delayed drug hypersensitivity reactions. Their participation in certain diseases may explain the variability of phenotypes in hypersensitivity reactions to medications.

Additive effects of orexin B and vasoactive intestinal polypeptide on LL-37-mediated antimicrobial activities.

The present study examined the bactericidal effects of orexin B (ORXB) and vasoactive intestinal peptide (VIP) alone or combined with cationic antimicrobial peptides, such as LL-37, on Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans and Staphylococcus aureus. The bactericidal effect of ORXB or VIP alone was detected in low NaCl concentration, but attenuated in physiological NaCl concentration (150 mM). However, such attenuated bactericidal activities of ORXB and VIP in 150 mM NaCl were regained by adding LL-37. Therefore, our results indicate that VIP and ORXB appear to mediate bactericidal effects in concert with LL-37 in the physiological context of mucosal tissue.

Persistence of gut mucosal innate immune defenses by enteric α-defensin expression in the simian immunodeficiency virus model of AIDS.

Gastrointestinal mucosa is an early target of HIV and a site of viral replication and severe CD4(+) T cell depletion. However, effects of HIV infection on gut mucosal innate immune defense have not been fully investigated. Intestinal Paneth cell-derived α-defensins constitute an integral part of the gut mucosal innate defense against microbial pathogens. Using the SIV-infected rhesus macaque model of AIDS, we examined the level of expression of rhesus enteric α-defensins (REDs) in the jejunal mucosa of rhesus macaques during all stages of SIV infection using real-time PCR, in situ hybridization, and immunohistochemistry. An increased expression of RED mRNAs was found in PC at the base of the crypts in jejunum at all stages of SIV infection as compared with uninfected controls. This increase correlated with active viral replication in gut-associated lymphoid tissue. Loss of RED protein accumulation in PC was seen in animals with simian AIDS. This was associated with the loss of secretory granules in PC, suggesting an increase in degranulation during advanced SIV disease. The α-defensin-mediated innate mucosal immunity was maintained in PC throughout the course of SIV infection despite the mucosal CD4(+) T cell depletion. The loss of RED protein accumulation and secretion was associated with an increased incidence of opportunistic enteric infections and disease progression. Our findings suggest that local innate immune defense exerted by PC-derived defensins contributes to the protection of gut mucosa from opportunistic infections during the course of SIV infection.