Tesfaldet Tecle

Human defensins and LL-37 in mucosal immunity

Defensins are widespread in nature and have activity against a broad range of pathogens. Defensins have direct antimicrobial effects and also modulate innate and adaptive immune responses. We consider the role of human defensins and the cathelicidin LL‐37 in defense of respiratory, gastrointestinal, and genitourinary tracts and the oral cavity, skin, and eye. Human β‐defensins (hBDs) and human defensins 5 and 6 (HD5 and −6) are involved most obviously in mucosal responses, as they are produced principally by epithelial cells. Human α‐defensins 1–4 (or HNPs 1–4) are produced principally by neutrophils recruited to the mucosa. Understanding the biology of defensins and LL‐37 is the beginning to clarify the pathophysiology of mucosal inflammatory and infectious diseases (e.g., Crohns disease, atopic dermatitis, lung or urinary infections). Challenges for these studies are the redundancy of innate defense mechanisms and the presence and interactions of many innate defense proteins in mucosal secretions.

Review: Defensins and cathelicidins in lung immunity.

Defensins were first identified in 1985 and are now recognized as part of a large family of antimicrobial peptides, divided into three categories: α-, β-, and θ-defensins. These defensin classes differ in structure, sites of expression and biological activities. Human α-defensins include peptides that are expressed primarily in neutrophils, whereas human β-defensins are widely expressed in epithelial cells, including those lining the respiratory tract. Defensins were first studied for their broad spectrum activity against bacteria, fungi and viruses; however, it is now clear that they also recruit inflammatory cells and promote innate and adaptive immune responses. Recent evidence shows that defensins have anti-inflammatory effects as well. Hence, defensins can participate in all phases of an immune response in the lung, including initial killing of pathogens and mounting — and resolution —- of an immune or inflammatory response. The cathelicidin, LL-37, is an antimicrobial peptide produced by neutrophils and respiratory epithelial cells that has similar roles in lung immunity as the defensins. A major challenge for the coming years will be to sort out the relative contributions of defensins and LL-37 to overall immune responses in the lung and to determine which of their many in vitro activities are most important for lung immunity.

Human Neutrophil Defensins Increase Neutrophil Uptake of Influenza A Virus and Bacteria and Modify Virus-Induced Respiratory Burst Responses

Human neutrophil peptides (HNPs) are released from granules of neutrophils in response to various activating stimuli and they participate in the killing of bacteria and the stimulation of various inflammatory responses. HNPs also inhibit infectivity of enveloped viruses, including influenza A virus (IAV). In this study, we demonstrate that HNPs increase the uptake of IAV and bacteria by neutrophils. The dimeric HNPs also induced aggregation of IAV and bacterial particles, which may, in part, explain their ability to increase uptake. HNPs did not increase neutrophil respiratory burst responses to IAV. We have recently demonstrated direct interactions of HNPs with surfactant protein D (SP-D), another important effector of innate immunity and antimicrobial host defense. Although HNPs did not alter SP-D-dependent uptake of IAV, they counteracted the ability of SP-D to increase IAV-induced neutrophil H2O2 generation. Our studies reveal previously unappreciated functional effects of HNPs, expand our understanding of the antiviral properties of HNPs, and suggest important interactions between collectins and HNPs in the host response to viruses and bacteria.

Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.

Interactions of α-, β-, and θ-Defensins with Influenza A Virus and Surfactant Protein D

We have reported that the α-defensins human neutrophil peptides (HNP)-1 and HNP-2 neutralize and aggregate influenza A virus (IAV) and promote uptake of IAV by neutrophils. These α-defensins were also shown to bind to surfactant protein (SP)-D and reduce its antiviral activity. In this study, we examined retrocyclin (RC)1 and RC2, humanized versions of the antiviral θ-defensins found in the leukocytes of certain nonhuman primates. RC1 was just as effective as HNP-1–3 in neutralizing IAV, and RC2 and RC101 (an analog of RC1) were more effective. In contrast, human β-defensins (HBDs) showed less neutralizing activity. Human defensins 5 and 6 (mainly produced by intestinal Paneth cells) had viral neutralizing activity similar to HNP-1–3. Like HNP-1–3, RCs induced viral aggregation and promoted the uptake of IAV by neutrophils. We used surface plasmon resonance to evaluate binding of defensins to SP-D. HBDs, HD6, and HNP-4 bound minimally to SP-D. HNP-1–3 and RCs bound SP-D with high affinity; however, unlike HNP-1 and HNP-2, RCs did not inhibit SP-D antiviral activity. HBDs also did not inhibit antiviral activity of SP-D. Given their strong neutralizing activity and compatibility with SP-D, RCs may provide attractive prototypes for designing therapeutics that can prevent or treat respiratory infections caused by IAV.

Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D

BackgroundSurfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens.MethodsWe tested antiviral activities of recombinant human SP-D against a panel of IAV strains that vary in glycosylation sites on their hemagglutinin (HA). For these experiments a recombinant version of human SP-D of the Met11, Ala160 genotype was used after it was characterized biochemically and structurally.ResultsOligosaccharides at amino acid 165 on the HA in the H3N2 subtype and 104 in the H1N1 subtype are absent in collectin-resistant strains developed in vitro and are important for mediating antiviral activity of SP-D; however, other glycans on the HA of these viral subtypes also are involved in inhibition by SP-D. H3N2 strains obtained shortly after introduction into the human population were largely resistant to SP-D, despite having the glycan at 165. H3N2 strains have become steadily more sensitive to SP-D over time in the human population, in association with addition of other glycans to the head region of the HA. In contrast, H1N1 strains were most sensitive in the 1970s–1980s and more recent strains have become less sensitive, despite retaining the glycan at 104. Two H5N1 strains were also resistant to inhibition by SP-D. By comparing sites of glycan attachment on sensitive vs. resistant strains, specific glycan sites on the head domain of the HA are implicated as important for inhibition by SP-D. Molecular modeling of the glycan attachment sites on HA and the carbohydrate recognition domain of SPD are consistent with these observations.ConclusionInhibition by SP-D correlates with presence of several glycan attachment sites on the HA. Pandemic and avian strains appear to lack susceptibility to SP-D and this could be a contributory factor to their virulence.

The human cathelicidin LL-37 inhibits influenza A viruses through a mechanism distinct from that of surfactant protein D or defensins.

LL-37, the only human cathelicidin, is a cationic antimicrobial peptide with antibacterial and antifungal activity. LL-37 is released from neutrophil granules and produced by epithelial cells. It has been implicated in host defence against influenza A virus (IAV) in recent studies. We now demonstrate dose-related neutralizing activity of LL-37 against several seasonal and mouse-adapted IAV strains. The ability of LL-37 to inhibit these IAV strains resulted mainly from direct effects on the virus, since pre-incubation of virus with LL-37 was needed for optimal inhibition. LL-37 bound high-density lipoprotein (HDL), and pre-incubation of LL-37 with human serum or HDL reduced its antiviral activity. LL-37 did not inhibit viral association with epithelial cells as assessed by quantitative RT-PCR or confocal microscopy. This finding contrasted with results obtained with surfactant protein D (SP-D). Unlike collectins or human neutrophil defensins (HNPs), LL-37 did not induce viral aggregation under electron microscopy. In the electron microscopy studies, LL-37 appeared to cause disruption of viral membranes. LL-37 had additive antiviral activity when combined with other innate inhibitors like SP-D, surfactant protein A and HNPs. Unlike HNPs, LL-37 did not bind SP-D significantly. These findings indicate that LL-37 contributes to host defence against IAV through a mechanism distinct from that of SP-D and HNPs.

Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

BackgroundSurfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr/Thr 11 form of SP-D is associated with low serum levels and assembles predominantly as trimers as opposed to the more common multimeric forms of SP-D.MethodsPreliminary experiments were done to establish the effects of different monoclonal antibodies against SP-D on ability of SP-D to bind to or neutralize the virus. We then purified natural human trimeric and multimeric forms of SP-D from amniotic fluid and tested ability of these preparations to bind to IAV, to inhibit infectivity and hemagglutination activity of IAV in vitro.ResultsIn initial experiments mAbs directed against different areas on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs) in viral neutralization assays as compared to multimeric SP-D.ConclusionThese studies indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP-D can be useful for dissecting out different functional properties of the protein.

Inhibition of influenza viral neuraminidase activity by collectins

SummaryThe collectins, lung surfactant proteins A and D (SP-A and SP-D), contribute to innate host defense against influenza A virus (IAV) in vivo. Although collectins bind to the viral hemagglutinin (HA) and inhibit early stages of viral infection in vitro, they also bind to the neuraminidase (NA) and inhibit NA activity. We used a variety of NA functional assays, viral strains and recombinant (mutant or wild type) collectins to characterize the mechanism of NA inhibition. NA inhibition by SP-D correlates with binding of its carbohydrate recognition domain (CRD) to oligomannose oligosaccharides on the viral hemagglutinin (HA). The effects of SP-D are additive with oseltamivir, consistent with differences in mechanism of action. NA inhibition was observed using fetuin or MDCK cells as a substrate, but not in assays using a soluble sialic acid analogue. Collectin multimerization and CRD binding properties are key determinants for NA inhibition. SP-D had greater NA inhibitory activity than mannose-binding lectin, which in turn had greater activity than SP-A. The markedly greater NA inhibitory activity of SP-D compared to SP-A may partly account for the finding that deletion of the SP-D gene in mice has a greater effect on viral replication in vivo.

Multimerization of Surfactant Protein D, but Not Its Collagen Domain, Is Required for Antiviral and Opsonic Activities Related to Influenza Virus

Surfactant protein D (SP-D) plays important roles in the initial innate defense against influenza A virus (IAV). The collagen domain of SP-D is probably critical for its homeostatic functions in vivo and has been implicated in the modulation of macrophage responses to SP-D-ligand complexes. For the current studies, we used a panel of rat SP-D mutants lacking all or part of the collagen domain to more specifically evaluate the contributions of this domain to viral interactions. SP-D multimers lacking the collagenous sequence efficiently neutralized Phil82 IAV, promoted neutrophil uptake of IAV, and also potentiated the IAV-induced neutrophil respiratory burst response. A dodecameric mutant with shortened collagenous arms showed enhanced viral aggregation and neuraminidase inhibition, and an increased capacity to inhibit a partially collectin-resistant strain of IAV. By contrast, truncated molecules lacking an N-terminal and collagen domain showed no detectable antiviral and opsonizing activity, despite preservation of lectin activity and detectable viral binding. Thus, multimerization, which is mediated by the N-peptide, is more important than the collagen domain for efficient viral neutralization and opsonization. However, the structure of the collagen domain significantly influences the anti-viral activity of multimerized forms of SP-D.