Cutting edge: IFN-inducible ELR- CXC chemokines display defensin-like antimicrobial activity.

Recent reports highlighted the chemotactic activities of antimicrobial peptide defensins whose structure, charge, and size resemble chemokines. By assaying representative members of the four known families of chemokines we explored the obverse: whether some chemokines exert antimicrobial activity. In a radial diffusion assay, only recombinant monokine induced by IFN-gamma (MIG/CXCL9), IFN-gamma-inducible protein of 10 kDa (IP-10/CXCL10), and IFN-inducible T cell alpha chemoattractant (I-TAC/CXCL11), members of the IFN-gamma-inducible tripeptide motif Glu-Leu-Arg (ELR)(-) CXC chemokines, were antimicrobial against Escherichia coli and Listeria monocytogenes. Similar to human defensins, antimicrobial activities of the chemokines were inhibited by 50 and 100 mM NaCl. The concentration of MIG/CXCL9 and IP-10/CXCL10 released from IFN-gamma-stimulated PBMC in 24 h were, respectively, 35- and 28-fold higher than from unstimulated cells. Additionally, the amounts of chemokines released per monocyte suggest that, in tissues with mononuclear cell infiltration, IFN-gamma-inducible chemokines may reach concentrations necessary for microbicidal activity. IFN-gamma-inducible chemokines may directly inactivate microbes before attracting other host defense cells to the area of infection.     

SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation

The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.The mucosal surfaces and skin of the human body are colonized by a large number of bacterial species constituting the normal microbiota. In contrast to pathogens, these commensals usually do not elicit any inflammatory responses in epithelial tissues of the healthy host (1). The Gram-positive coccus Finegoldia magna is part of the anaerobic normal microbiota associated with the skin (2), but it also inhabits the oro-pharynx, gastrointestinal, and urogenital tracts (3). During disturbed homeostasis, this bacterium becomes an important opportunistic pathogen; associated with several clinical conditions, such as soft tissue infections, wound infections, bone/joint infections, and vaginosis (3–5). Among anaerobic cocci of the normal microbiota, F. magna is the species most commonly isolated from clinical conditions (3).Recognition of bacteria and their products by cells residing in the submucosal tissues, for example dendritic cells, triggers an inflammatory response leading to production of host defense molecules, including chemokines. Chemokines comprise a large family of peptides that are key players in inflammation by regulating leukocyte trafficking and activation. They are divided into four groups, XC, CC, CXC, and CX₃C, depending on the arrangement of conserved cysteine residues in their NH₂ terminus (6). The CXC subfamily can be further divided into ELR-positive and ELR-negative respectively, based on the presence or absence of the sequence motif glutamic acid-leucine-arginine (ELR) NH₂ terminal to the first cysteine. IFN-γ, a key cytokine produced during bacterial infection, induces expression of the ELR-negative CXC-chemokine MIG/CXCL9 (Monokine Induced by Gamma-interferon)³ (7). MIG/CXCL9 binds and activates a G-protein-coupled seven transmembrane receptor, CXCR3, which is present on eosinophils, activated T cells (CD8+), and NK cells (8). In addition to its ability to recruit and activate leukocytes, MIG/CXCL9 possesses angiostatic properties through activation of CXCR3 expressed on endothelial cells, and it also exerts potent antibacterial properties (9–11). Upon IFN-dependent inflammation, for example during bacterial infection, this chemokine is produced by epithelial cells and participates in activities of both innate and adaptive immunity (10, 12–14).The finding that epithelial cells recognize important human pathogens, such as Streptococcus pyogenes, leading to an increased MIG/CXCL9 production (10, 12) raised the question whether an opportunistic pathogen like F. magna could be recognized in a similar fashion. In skin F. magna is localized to the epidermis where they adhere to basement membranes through an interaction with the basement membrane protein BM-40 (15). Binding to BM-40 is mediated by the surface protein FAF (F. magna adhesion factor) that is expressed by more than 90% of F. magna isolates (15). Bacteria, both commensals and pathogens, express proteases that are important both during colonization and invasion (16). In the case of F. magna, most strains express a subtilisin-like enzyme, SufA (Subtilase of Finegoldia magna), which is associated with the bacterial surface, but also secreted in substantial amounts during bacterial growth (17). Studies on the proteolytic activity of SufA demonstrated that the enzyme cleaves and inactivates antibacterial molecules like LL-37 and MIG/CXCL9 (17). Here, we show that MIG/CXCL9, produced by human keratinocytes in response to inflammatory stimuli, is degraded by SufA-expressing F. magna. The generated MIG/CXCL9 fragments are still able to activate the MIG/CXCL9 receptor, CXCR3 and kill S. pyogenes, while F. magna is left unaffected. This modulation of the MIG/CXCL9 activities promotes the survival of F. magna during inflammation.     

Expression of IFN-gamma induced CXCR3 agonist chemokines and compartmentalization of CXCR3+ cells in the periphery and lymph nodes of rhesus macaques during simian immunodeficiency virus infection and acquired immunodeficiency syndrome

Dysregulation of cytokines and chemokines during human immunodeficiency virus 1 (HIV-1) and simian immunodeficiency virus (SIV) infection is thought to be critical in the progression of acquired immunodeficiency syndrome (AIDS). To evaluate the potential role of Th1-agonist chemokines in disease progression during AIDS, we assessed CXCL9/MIG and CXCL10/IP-10 expression simultaneously in the periphery and lymphoid tissues of SIV-infected animals at a single-cell level by flow cytometry. We optimized intracellular staining and analysis of CXCL9/MIG and CXCL10/IP-10 production in human leukocyte antigen (HLA)-DR+ macaque cells by flow cytometry using cross-reactive antibodies against human chemokines. We observed an upregulation of CXCL9/MIG and CXCL10/IP-10 production in both the periphery and lymph nodes of infected animals compared with na?ve controls. Animals with higher viral loads had higher levels of CXCL9/MIG and CXCL10/IP-10 producing cells compared with animals with low viral loads. Analysis of cells bearing the receptor (CXCR3) for CXCL9/MIG and CXCL10/IP-10 revealed increased number of CXCR3+ cells in the lymph nodes of infected animals. Importantly, an inverse correlation (P < 0.05) between CXCL9/MIG and CXCL10/IP-10 production, both in the periphery and lymph nodes, and peripheral CD4+ T-cell numbers was observed. These findings provide further evidence that dysregulation of Th1 agonist chemokines might contribute to the ultimate immunopathology during AIDS.     

G-protein-coupled receptor independent, immunomodulatory properties of chemokine CXCL9

Certain chemokines possess anti-angiogenic and antibacterial activity, in addition to their ability to recruit leukocytes. Herein, we demonstrate that CXCL9/MIG induces the expression, by a monocytic cell line and peripheral blood mononuclear cells, of a variety of chemokines including CXCL8/IL-8, CCL3/MIP-1α, CCL4/MIP-1β, CCL2/MCP-1 in a pertussis toxin insensitive manner. Similarly, another cationic chemokine CCL20/MIP-3α, but not the non-cationic chemokines CCL2 or CCL3, stimulated monocytic cells to produce substantial amounts of CXCL8 and CCL3. Microarray experiments demonstrated that CXCL9, but not CCL2, induced the expression of hundreds of genes, many of which have known or proposed immunomodulatory functions. Induction of CXCL8 required the p38 and ERK1/2 mitogen-activated protein kinases but not NFκB, JAK-STAT or JNK signaling pathways. These results collectively demonstrate that CXCL9 has immunomodulatory functions that are not mediated through a G-protein coupled receptor and may possess additional roles in host defenses against infection.     

Structural basis for Streptococcus pneumoniae NanA inhibition by influenza antivirals zanamivir and oseltamivir carboxylate

The human pathogen Streptococcus pneumoniae is the major cause of bacterial meningitis, respiratory tract infection, septicemia, and otitis media. The bacterium expresses neuraminidase (NA) proteins that contribute to pathogenesis by cleaving sialic acids from host glycoconjugates, thereby enhancing biofilm formation and colonization. Recent in vivo experiments have shown that antiviral compounds, widely used in clinics and designed to inhibit influenza NA, significantly reduce biofilm formation and nasopharyngeal colonization of S. pneumoniae in mice. Here, we present the structural basis for the beneficial effect of these compounds against pneumococcal infection. Crystal structures of pneumococcal NanA in complex with zanamivir and oseltamivir carboxylate are discussed, correlated with measured inhibitory constants K_i, and compared with the binding modes of the inhibitors in the viral enzyme. Inhibitor structures show for the first time how clinically approved anti-influenza compounds interact with an NA of the human pathogen S. pneumoniae and give a rational explanation for their antibacterial effects.     

The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88^-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88^-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88^-/- mice showed 10⁵-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.     

Characterization of the Chemokine CXCL11-Heparin Interaction Suggests Two Different Affinities for Glycosaminoglycans

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, ⁵⁷KSKQAR⁶²) along with Lys¹⁷, significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.     

Influenza A Viruses Grow in Human Pancreatic Cells and Cause Pancreatitis and Diabetes in an Animal Model

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-α), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals.     

Structural correlates of antimicrobial efficacy in IL-8 and related human kinocidins

Chemokines are small (8-12 kDa) effector proteins that potentiate leukocyte chemonavigation. Beyond this role, certain chemokines have direct antimicrobial activity against human pathogenic organisms; such molecules are termed kinocidins. The current investigation was designed to explore the structure-activity basis for direct microbicidal activity of kinocidins. Amino acid sequence and 3-dimensional analyses demonstrated these molecules to contain iterations of the conserved γ-core motif found in broad classes of classical antimicrobial peptides. Representative CXC, CC and C cysteine-motif-group kinocidins were tested for antimicrobial activity versus human pathogenic bacteria and fungi. Results demonstrate that these molecules exert direct antimicrobial activity in vitro, including antibacterial activity of native IL-8 and MCP-1, and microbicidal activity of native IL-8. To define molecular determinants governing its antimicrobial activities, the IL-8 γ-core (IL-8γ) and α-helical (IL-8α) motifs were compared to native IL-8 for antimicrobial efficacy in vitro. Microbicidal activity recapitulating that of native IL-8 localized to the autonomous IL-8α motif in vitro, and demonstrated durable microbicidal activity in human blood and blood matrices ex vivo. These results offer new insights into the modular architecture, context-related deployment and function, and evolution of host defense molecules containing γ-core motifs and microbicidal helices associated with antimicrobial activity.     

Streptococcus pneumoniae Enhances Human Respiratory Syncytial Virus Infection In Vitro and In Vivo

Human respiratory syncytial virus (HRSV) and Streptococcus pneumoniae are important causative agents of respiratory tract infections. Both pathogens are associated with seasonal disease outbreaks in the pediatric population, and can often be detected simultaneously in infants hospitalized with bronchiolitis or pneumonia. It has been described that respiratory virus infections may predispose for bacterial superinfections, resulting in severe disease. However, studies on the influence of bacterial colonization of the upper respiratory tract on the pathogenesis of subsequent respiratory virus infections are scarce. Here, we have investigated whether pneumococcal colonization enhances subsequent HRSV infection. We used a newly generated recombinant subgroup B HRSV strain that expresses enhanced green fluorescent protein and pneumococcal isolates obtained from healthy children in disease-relevant in vitro and in vivo model systems. Three pneumococcal strains specifically enhanced in vitro HRSV infection of primary well-differentiated normal human bronchial epithelial cells grown at air-liquid interface, whereas two other strains did not. Since previous studies reported that bacterial neuraminidase enhanced HRSV infection in vitro, we measured pneumococcal neuraminidase activity in these cultures but found no correlation with the observed infection enhancement in our model. Subsequently, a selection of pneumococcal strains was used to induce nasal colonization of cotton rats, the best available small animal model for HRSV. Intranasal HRSV infection three days later resulted in strain-specific enhancement of HRSV replication in vivo. One S. pneumoniae strain enhanced HRSV both in vitro and in vivo, and was also associated with enhanced syncytium formation in vivo. However, neither pneumococci nor HRSV were found to spread from the upper to the lower respiratory tract, and neither pathogen was transmitted to naive cage mates by direct contact. These results demonstrate that pneumococcal colonization can enhance subsequent HRSV infection, and provide tools for additional mechanistic and intervention studies.     

Limited Contribution of IL-36 versus IL-1 and TNF Pathways in Host Response to Mycobacterial Infection

IL-36 cytokines are members of the IL-1 family of cytokines that stimulate dendritic cells and T cells leading to enhanced T helper 1 responses in vitro and in vivo; however, their role in host defense has not been fully addressed thus far. The objective of this study was to examine the role of IL-36R signaling in the control of mycobacterial infection, using models of systemic attenuated M. bovis BCG infection and virulent aerogenic M. tuberculosis infection. IL-36γ expression was increased in the lung of M. bovis BCG infected mice. However, IL-36R deficient mice infected with M. bovis BCG showed similar survival and control of the infection as compared to wild-type mice, although their lung pathology and CXCL1 response were transiently different. While highly susceptible TNF-α deficient mice succumbed with overwhelming M. tuberculosis infection, and IL-1RI deficient mice showed intermediate susceptibility, IL-36R-deficient mice controlled the infection, with bacterial burden, lung inflammation and pathology, similar to wild-type controls. Therefore, IL-36R signaling has only limited influence in the control of mycobacterial infection.     

Peptides in the hemolymph of Hermetia illucens larvae completely inhibit the growth of Klebsiella pneumonia in vitro and in vivo

Extensive use of antibiotics has caused the microbial resistance to rise drastically within the last few decades, and new approaches are therefore needed to develop effective antibacterial substances. In this study, we identified peptide in the hemolymph of Hermetia illucens larvae using reverse-phase chromatography, HPLC and Nano-LC-ESI-MS/MS system. We investigated the antibacterial effect of HP/F9 peptides against Klebsiella pneumoniae in vitro and in vivo. The peptide effectively inhibited the growth of K. pneumoniae in vitro and completely removed K. pneumoniae from the lungs of mice. Importantly, peptides (22,000 Da, HP/F9) successfully reduced lung inflammation upon K. pneumoniae infection. These results indicate that the HP/F9 peptide from H. illucens larva can effectively protect the mouse from K. pneumoniae infection. HP/F9 could be a new candidate for the development of effective antibacterial substance.     

Echinococcus multilocularis: inflammatory and regulatory chemokine responses in patients with progressive, stable and cured alveolar echinococcosis

The progressive growth of Echinococcus multilocularis metacestodes and their tissue infiltration will cause organ malfunction and finally failure. In few patients, E. multilocularis metacestode proliferation will spontaneously regress, but little is known about the determinants which may restrain metacestode survival and growth. In this study, chemokine responses were investigated in E. multilocularis patients at different states of infection, i.e. with progressive, stable and cured alveolar echinococcosis (AE). Characteristic chemokine profiles and changes in their production were observed in AE patients and infection-free controls when their peripheral blood cells were cultured with E. multilocularis antigens. The production of CC and CXC chemokines which associate with inflammation (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5 and GRO-α/CXCL1) was constitutively larger in AE patients than in controls; and the elevated chemokine releases were equal in patients with progressive, stable or cured AE. Cluster analyses identified three distinct chemokine response profiles; chemokines were enhanced, depressed or produced in similar quantities in AE patients and controls. A disparate cellular responsiveness was observed in AE patients to viable E. multilocularis vesicles; cluster 1 (GRO-α/CXCL1, MCP-3/CCL7, MCP-4/CCL13, TARC/CCL17, LARC/CCL20) and cluster 2 chemokines (PARC/CCL18, MDC/CCL22, MIG/CXCL9) were clearly diminished, while cluster 3 chemokines (MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5) augmented. The increased production of inflammatory chemokines in patients even with cured AE could be induced by residual E. multilocularis metacestode lesions which continuously stimulate production of inflammatory chemokines. E. multilocularis metacestodes also suppressed cellular chemokine production in AE patients, and this may constitute an immune escape mechanism which reduces inflammatory host responses, prevents tissue destruction and organ damage, but may also facilitate parasite persistence.     

Posttranslational Modification of the NH2-terminal Region of CXCL5 by Proteases or Peptidylarginine Deiminases (PAD) Differently Affects Its Biological Activity

Posttranslational modifications, e.g. proteolysis, glycosylation, and citrullination regulate chemokine function, affecting leukocyte migration during inflammatory responses. Here, modification of CXCL5/epithelial cell-derived neutrophil-activating protein-78 (ENA-78) by proteases or peptidylarginine deiminases (PAD) was evaluated. Slow CXCL5(1–78) processing by the myeloid cell marker aminopeptidase N/CD13 into CXCL5(2–78) hardly affected its in vitro activity, but slowed down the activation of CXCL5 by the neutrophil protease cathepsin G. PAD, an enzyme with a potentially important function in autoimmune diseases, site-specifically deiminated Arg⁹ in CXCL5 to citrulline, generating [Cit⁹]CXCL5(1–78). Compared with CXCL5(1–78), [Cit⁹]CXCL5(1–78) less efficiently induced intracellular calcium signaling, phosphorylation of extracellular signal-regulated kinase, internalization of CXCR2, and in vitro neutrophil chemotaxis. In contrast, conversion of CXCL5 into the previously reported natural isoform CXCL5(8–78) provided at least 3-fold enhanced biological activity in these tests. Citrullination, but not NH₂-terminal truncation, reduced the capacity of CXCL5 to up-regulate the expression of the integrin α-chain CD11b on neutrophils. Truncation nor citrullination significantly affected the ability of CXCL5 to up-regulate CD11a expression or shedding of CD62L. In line with the in vitro results, CXCL5(8–78) and CXCL5(9–78) induced a more pronounced neutrophil influx in vivo compared with CXCL5(1–78). Administration of 300 pmol of either CXCL5(1–78) or [Cit⁹]CXCL5(1–78) failed to attract neutrophils to the peritoneal cavity. Citrullination of the more potent CXCL5(9–78) lowers its chemotactic potency in vivo and confirms the tempering effect of citrullination in vitro. The highly divergent effects of modifications of CXCL5 on neutrophil influx underline the potential importance of tissue-specific interactions between chemokines and PAD or proteases.     

CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9 ^-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9 ^-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.     

Antimicrobial Effects of Helix D-derived Peptides of Human Antithrombin III

Antithrombin III (ATIII) is a key antiproteinase involved in blood coagulation. Previous investigations have shown that ATIII is degraded by Staphylococcus aureus V8 protease, leading to release of heparin binding fragments derived from its D helix. As heparin binding and antimicrobial activity of peptides frequently overlap, we here set out to explore possible antibacterial effects of intact and degraded ATIII. In contrast to intact ATIII, the results showed that extensive degradation of the molecule yielded fragments with antimicrobial activity. Correspondingly, the heparin-binding, helix d-derived, peptide FFFAKLNCRLYRKANKSSKLV (FFF21) of human ATIII, was found to be antimicrobial against particularly the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. Fluorescence microscopy and electron microscopy studies demonstrated that FFF21 binds to and permeabilizes bacterial membranes. Analogously, FFF21 was found to induce membrane leakage of model anionic liposomes. In vivo, FFF21 significantly reduced P. aeruginosa infection in mice. Additionally, FFF21 displayed anti-endotoxic effects in vitro. Taken together, our results suggest novel roles for ATIII-derived peptide fragments in host defense.     

The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.