Innate host defense: Nox and Duox on phox's tail

Over the past decade, the capacity of non-phagocytic cells to produce superoxide has been largely documented. As in the case of the well-characterized phagocytic cells context, superoxide formation in non-phagocytic cells depends on the activity of membrane bound NADPH oxidase enzymes. Six mammalian homologues of the classical phagocytic Nox2 enzyme have been described to date, named Nox1, Nox3, Nox4, Nox5, Duox1 and Duox2, which exhibit similar and specific structure and regulation features. Their biological functions are still poorly understood and were initially mostly deduced from their specific tissue expression profiles. However, recent functional data have emerged that suggest the involvement of several of these isoforms in the innate host response to invading microorganisms, including innate immune and proinflammatory responses. Nox2 is well characterized as a key player in the bacterial killing process that takes place in phagocytes. Here, we will discuss the recent advances that revealed alternative roles of Nox1, Nox4, Duox1 and Duox2 isoforms in other aspects of the innate host defense. In particular, we will focus on their implication in the signaling following pathogen recognition by toll like receptors and in the modulation of dendritic cell functions, two key aspects of innate immunity. Moreover, the potential role of Nox/Duox enzymes in the innate response to virus infections will be discussed.     

A Dityrosine Network Mediated by Dual Oxidase and Peroxidase Influences the Persistence of Lyme Disease Pathogens within the Vector

Ixodes scapularis ticks transmit a wide array of human and animal pathogens including Borrelia burgdorferi; however, how tick immune components influence the persistence of invading pathogens remains unknown. As originally demonstrated in Caenorhabditis elegans and later in Anopheles gambiae, we show here that an acellular gut barrier, resulting from the tyrosine cross-linking of the extracellular matrix, also exists in I. scapularis ticks. This dityrosine network (DTN) is dependent upon a dual oxidase (Duox), which is a member of the NADPH oxidase family. The Ixodes genome encodes for a single Duox and at least 16 potential peroxidase proteins, one of which, annotated as ISCW017368, together with Duox has been found to be indispensible for DTN formation. This barrier influences pathogen survival in the gut, as an impaired DTN in Doux knockdown or in specific peroxidase knockdown ticks, results in reduced levels of B. burgdorferi persistence within ticks. Absence of a complete DTN formation in knockdown ticks leads to the activation of specific tick innate immune pathway genes that potentially resulted in the reduction of spirochete levels. Together, these results highlighted the evolution of the DTN in a diverse set of arthropod vectors, including ticks, and its role in protecting invading pathogens like B. burgdorferi. Further understanding of the molecular basis of tick innate immune responses, vector-pathogen interaction, and their contributions in microbial persistence may help the development of new targets for disrupting the pathogen life cycle.     

A Cleavage-potentiated Fragment of Tear Lacritin Is Bactericidal

Antimicrobial peptides are important as the first line of innate defense, through their tendency to disrupt bacterial membranes or intracellular pathways and potentially as the next generation of antibiotics. How they protect wet epithelia is not entirely clear, with most individually inactive under physiological conditions and many preferentially targeting Gram-positive bacteria. Tears covering the surface of the eye are bactericidal for Gram-positive and -negative bacteria. Here we narrow much of the bactericidal activity to a latent C-terminal fragment in the prosecretory mitogen lacritin and report that the mechanism combines membrane permeabilization with rapid metabolic changes, including reduced levels of dephosphocoenzyme A, spermidine, putrescine, and phosphatidylethanolamines and elevated alanine, leucine, phenylalanine, tryptophan, proline, glycine, lysine, serine, glutamate, cadaverine, and pyrophosphate. Thus, death by metabolic stress parallels cellular attempts to survive. Cleavage-dependent appearance of the C-terminal cationic amphipathic α-helix is inducible within hours by Staphylococcus epidermidis and slowly by another mechanism, in a chymotrypsin- or leupeptin protease-inhibitable manner. Although bactericidal at low micromolar levels, within a biphasic 1–10 nm dose optimum, the same domain is mitogenic and cytoprotective for epithelia via a syndecan-1 targeting mechanism dependent on heparanase. Thus, the C terminus of lacritin is multifunctional by dose and proteolytic processing and appears to play a key role in the innate protection of the eye, with wider potential benefit elsewhere as lacritin flows from exocrine secretory cells.     

Toll-like Receptor 11 (TLR11) Prevents Salmonella Penetration into the Murine Peyer Patches

Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a “carrier of Salmonella,” TLR11 works as a “blocker of Salmonella” to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.     

Examining the relationship between hemolymph phenoloxidase and resistance to a DNA virus, Plodia interpunctella granulosis virus (PiGV)

We have a detailed understanding of invertebrate immune responses to bacteria and fungal pathogens, but we know less about how insects respond to virus challenge. Phenoloxidase (PO) functions as an important immune response against many parasites and pathogens and is routinely used as a measure of immune competance. We examine the role of haemolymph PO activity in Plodia interpuncetella's response to its natural granulosis virus (PiGV). Larvae were challenged with virus by both oral inoculation of occluded virus (the natural infection route) and direct intrahaemocoelic injection of budded virus. Haemolymph was collected at time points post-viral challenge using a novel method that allows the volume of haemolymph to be quanitified. The haemolmyph was collected without killing the larvae so that haemolymph samples from individuals that developed viral disease could be distinguished from samples collected from those that fought off infection. The level of haemolymph PO activity in resistant larvae did not differ from control larvae. Therefore we have no evidence that PO is involved in resistance to virus in the haemocoel whether larvae are challenged naturally by oral innoculation or directly by intraheamocoelic injection. Phenoloxidase may therefore not be a relevant metric of immunocompetence for viral infection.     

Pathogen sensing by nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is mediated by direct binding to muramyl dipeptide and ATP

Nucleotide binding and oligomerization domain-containing protein 2 (NOD2/Card15) is an intracellular protein that is involved in the recognition of bacterial cell wall-derived muramyl dipeptide. Mutations in the gene encoding NOD2 are associated with inherited inflammatory disorders, including Crohn disease and Blau syndrome. NOD2 is a member of the nucleotide-binding domain and leucine-rich repeat-containing protein gene (NLR) family. Nucleotide binding is thought to play a critical role in signaling by NLR family members. However, the molecular mechanisms underlying signal transduction by these proteins remain largely unknown. Mutations in the nucleotide-binding domain of NOD2 have been shown to alter its signal transduction properties in response to muramyl dipeptide in cellular assays. Using purified recombinant protein, we now demonstrate that NOD2 binds and hydrolyzes ATP. Additionally, we have found that the purified recombinant protein is able to bind directly to muramyl dipeptide and can associate with known NOD2-interacting proteins in vitro. Binding of NOD2 to muramyl dipeptide and homo-oligomerization of NOD2 are enhanced by ATP binding, suggesting a model of the molecular mechanism for signal transduction that involves binding of nucleotide followed by binding of muramyl dipeptide and oligomerization of NOD2 into a signaling complex. These findings set the stage for further studies into the molecular mechanisms that underlie detection of muramyl dipeptide and assembly of NOD2-containing signaling complexes.     

Critical determinants of human α-defensin 5 activity against non-enveloped viruses

Human α-defensins, such as human α-defensin 5 (HD5), block infection of non-enveloped viruses, including human adenoviruses (AdV), papillomaviruses (HPV), and polyomaviruses. Through mutational analysis of HD5, we have identified arginine residues that contribute to antiviral activity against AdV and HPV. Of two arginine residues paired on one face of HD5, Arg-28 is critical for both viruses, while Arg-9 is only important for AdV. Two arginine residues on the opposite face of the molecule (Arg-13 and Arg-32) and unpaired Arg-25 are less important for both. In addition, hydrophobicity at residue 29 is a major determinant of anti-adenoviral activity, and a chemical modification that prevents HD5 self-association was strongly attenuating. Although HD5 binds to the capsid of AdV, the molecular basis for this interaction is undefined. Capsid binding by HD5 is not purely charge-dependent, as substitution of lysine for Arg-9 and Arg-28 was deleterious. Analysis of HD5 analogs that retained varying levels of potency demonstrated that anti-adenoviral activity is directly correlated with HD5 binding to the virus, confirming that the viral capsid rather than the cell is the relevant target. Also, AdV aggregation induced by HD5 binding is not sufficient for neutralization. Rather, these studies confirm that the major mechanism of HD5-mediated neutralization of AdV depends upon specific binding to the viral capsid through interactions mediated in part by critical arginine residues, hydrophobicity at residue 29, and multimerization of HD5, which increases initial binding of virus to the cell but prevents subsequent viral uncoating and genome delivery to the nucleus.     

Influence of temperature,pH, oxygenation,water-type and substrate on biomarker responses in the freshwater clam Corbicula fluminea (Müller)

We investigated the influence of abiotic factors on responses of components of detoxification metabolism of phases I and II, indicators related to oxidative stress and propionylcholinesterase, proposed as biomarkers of pollution in Corbicula fluminea. Combined effects of temperature (10 and 20 degrees C), water-type (water purified by reverse-osmosis, lake water from the collection site and tap water) and habitat substrate (presence and absence of sand) were assessed in a five-day experiment. Additionally, clams were exposed to hypoxia or submitted to acidic, neutral or alkaline conditions at 20 degrees C, in lake water, without sand, for five days. Responses of biomarkers were generally higher when clams were placed in tap water, except for level of peroxidised lipids (PL). Catalase (CAT) and NADH-cytochrome c reductase (NADH-red.) activities increased whereas propionylcholinesterase (PChE) activity decreased in absence of sand. Decreasing temperature resulted in depressing PChE and NADPH-cytochrome c reductase (NADPH-red.) activities. Hypoxia induced an increase of CAT activity and glutathione S-transferase activity towards ethacrynic acid and a decrease of PL level. CAT and NADH-red. activities as well as PL level were reduced when clams were exposed to acidic and/or alkaline conditions. These results indicate that effects of abiotic factors should be taken into account in environmental studies.