Effector Molecules Of Innate Immunity Research Articles

Structural basis for viral 5′-PPP-RNA recognition by human IFIT proteins

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are innate immune effector molecules that are thought to confer antiviral defence through disruption of protein–protein interactions in the host translation-initiation machinery. However, it was recently discovered that IFITs can directly recognize viral RNA bearing a 5′-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an amino-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single-stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double-stranded viral PPP-RNA, retinoic acid-inducible gene I (RIG-I, also known as DDX58). Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence-specific manner and requires a 5′-overhang of approximately three nucleotides. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 was found to cause a defect in the antiviral response by human embryonic kidney cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA, and lend insight into their downstream effector function.Supplementary informationThe online version of this article (doi:10.1038/nature11783) contains supplementary material, which is available to authorized users.

TGF-Beta: a Master Switch in Tumor Immunity

The capacity of the immune system to distinguish foreign from self-antigen, and to subsequently eliminate the threat of disease without injuring the host is crucial for survival. It also serves to defend against tumor formation and progression via a process termed cancer immunosurveillance. Innate and adaptive immune cell types and effector molecules collectively function as extrinsic tumorsuppressor mechanisms. However, tumors may escape immunesurveillance through a variety of mechanisms that create a local microenvironment that is unfavorable for effective tumor immunity. Transforming growth factor β (TGF-β) has pleiotropic effects on the immune system, and is recognized as one of the most potent immunosuppressive agents in facilitating oncogenesis. The TGF-β pathway promotes cancer progression by concomitantly enhancing tumor metastases while inhibiting the protective host immunity. In this review, we discuss mechanisms through which TGF-β interferes with the development of an anti-tumor immunity and potential means through which to circumvent its activity in order to define more effective cancer immunotherapies.

Cathelicidin LL-37: An Antimicrobial Peptide with a Role in Inflammatory Skin Disease

Chronic inflammatory skin diseases such as atopic dermatitis, psoriasis or rosacea are very common. Although their exact pathogenesis is not completely understood all three diseases are characterized by dysregulation of cutaneous innate immunity. Cathelicidin LL-37 is an important effector molecule of innate immunity in the skin and atopic dermatitis, psoriasis or rosacea show defects in cathelicidin expression, function or processing. In atopic dermatitis, cathelicidin induction might be disturbed resulting in defective antimicrobial barrier function. In contrast, psoriasis is characterized by overexpression of cathelicidin. However to date it is unclear whether pro- or anti-inflammatory functions of cathelicidin predominate in lesional skin in psoriasis. In rosacea, cathelicidin processing is disturbed resulting in peptide fragments causing inflammation, erythema and telangiectasias. In this review, the current evidence on the role of cathelicidin LL-37 in the pathogenesis of inflammatory skin diseases will be outlined. As cathelicidin LL-37 might also serve as a future treatment target potential novel treatment strategies for those diseases will be discussed.

The effect of conglutinin on production of reactive oxygen species in bovine granulocytes

Conglutinin is a high molecular-weight lectin originally detected in bovine serum. It belongs to the family of collectins that bind sugar residues in a Ca2+-dependent manner and are effector molecules in innate immunity. Conglutinin appears to play an important role in immune defense mechanisms, showing antiviral and antibacterial activities when tested in vivo and in vitro. The present study evaluated the effect of conglutinin on the respiratory bursts in bovine peripheral phagocytes. Using nitroblue tetrazolium and hydrogen peroxide assays, we showed that sugar ligand-bound conglutinin stimulated the production of superoxide and H2O2 in granulocytes whereas the non-sugar-bound form of conglutinin inhibited these processes. These results indicate that both forms of conglutinin are able to interact with surface leukocyte receptors but have opposite effects on phagocytic activity. Our findings suggest that conglutinin bound to sugar residues on microbial surfaces can induce oxygen burst in phagocytes, and thereby mediates the elimination of pathogens and prevents the spread of infection.

Bacteroid Development in Legume Nodules: Evolution of Mutual Benefit or of Sacrificial Victims?

Symbiosomes are organelle-like structures in the cytoplasm of legume nodule cells which are composed of the special, nitrogen-fixing forms of rhizobia called bacteroids, the peribacteroid space and the enveloping peribacteroid membrane of plant origin. The formation of these symbiosomes requires a complex and coordinated interaction between the two partners during all stages of nodule development as any failure in the differentiation of either symbiotic partner, the bacterium or the plant cell prevents the subsequent transcriptional and developmental steps resulting in early senescence of the nodules. Certain legume hosts impose irreversible terminal differentiation onto bacteria. In the inverted repeat-lacking clade (IRLC) of legumes, host dominance is achieved by nodule-specific cysteine-rich peptides that resemble defensin-like antimicrobial peptides, the known effector molecules of animal and plant innate immunity. This article provides an overview on the bacteroid and symbiosome development including the terminal differentiation of bacteria in IRLC legumes as well as the bacterial and plant genes and proteins participating in these processes.

Conglutinin is not specific to cattle

   Conglutinin is a high-molecular-weight mammalian lectin which binds in a calcium-dependent manner to cell-surface-bound complement fragment iC3b, yeast cell-wall extract and terminal non-reducing N-acetyl-D-glucosamine, mannose and fucose residues. This protein, originally detected in bovine serum, belongs to the family of collectins, which are effector molecules in innate immunity. Conglutinin appears to play an important role in defence mechanisms, showing antiviral and antibacterial activity. We have characterized the electrophoresis profile of bovine serum conglutinin and used Western blotting to compare profiles of this lectin derived from the sera of different breeds of cattle. The profile of non-reduced conglutinin is characterised by many bands with molecular masses ranging from 34 to 630 kDa. Reduced lectin takes the form of three main bands with molecular masses of 41, 47 and 96 kDa. We show that conglutinin is present not only in adult bovine serum, but also in foetal bovine serum, colostrum and milk. The sera of sheep, goats, gnu antelopes and deer, as well as some non-ruminant species such as llamas, horses, boars, pigs and humans, contain proteins which have similar antigenicity to that of bovine conglutinin. These reacted with monoclonal and polyclonal antibodies specific for bovine conglutinin under reducing and non-reducing conditions in Western blotting. The protein profiles of bison and swine lectin were observed to be particularly similar to bovine conglutinin.

Membrane-active host defense peptides – Challenges and perspectives for the development of novel anticancer drugs

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means.Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.

Cathelicidin-BF, a Snake Cathelicidin-Derived Antimicrobial Peptide, Could Be an Excellent Therapeutic Agent for Acne Vulgaris

Cathelicidins are a family of antimicrobial peptides acting as multifunctional effector molecules in innate immunity. Cathelicidin-BF has been purified from the snake venoms of Bungarus fasciatus and it is the first identified cathelicidin antimicrobial peptide in reptiles. In this study, cathelicidin-BF was found exerting strong antibacterial activities against Propionibacterium acnes. Its minimal inhibitory concentration against two strains of P. acnes was 4.7 µg/ml. Cathelicidin-BF also effectively killed other microorganisms including Staphylococcus epidermidis, which was possible pathogen for acne vulgaris. Cathelicidin-BF significantly inhibited pro-inflammatory factors secretion in human monocytic cells and P. acnes-induced O2 .− production of human HaCaT keratinocyte cells. Observed by scanning electron microscopy, the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls, suggesting that this antimicrobial peptide exerts its action by disrupting membranes of microorganisms. The efficacy of cathelicidin-BF gel topical administering was evaluated in experimental mice skin colonization model. In vivo anti-inflammatory effects of cathelicidin-BF were confirmed by relieving P. acnes-induced mice ear swelling and granulomatous inflammation. The anti-inflammatory effects combined with potent antimicrobial activities and O2 .− production inhibition activities of cathelicidin-BF indicate its potential as a novel therapeutic option for acne vulgaris.

Natural Innate and Adaptive Immunity to Cancer

The immune system can identify and destroy nascent tumor cells in a process termed cancer immunosurveillance, which functions as an important defense against cancer. Recently, data obtained from numerous investigations in mouse models of cancer and in humans with cancer offer compelling evidence that particular innate and adaptive immune cell types, effector molecules, and pathways can sometimes collectively function as extrinsic tumor-suppressor mechanisms. However, the immune system can also promote tumor progression. Together, the dual host-protective and tumor-promoting actions of immunity are referred to as cancer immunoediting. In this review, we discuss the current experimental and human clinical data supporting a cancer immunoediting process that provide the fundamental basis for further study of immunity to cancer and for the rational design of immunotherapies against cancer.

Susceptibility of clinical Staphylococcus aureus isolates to innate defense antimicrobial peptides

Antimicrobial peptides (AMPs) are effector molecules of innate immunity. To determine whether AMP susceptibility of S. aureus varies according to different types of infection, 102 isolates from patients with S. aureus bacteremia or recurrent skin and soft tissue infection, and colonizing isolates were investigated. Using microbroth dilution assays we found a narrow range of MICs of human β-defensin-3, cathelicidin LL-37 and bovine indolicidin without significant differences between the groups. Colony-forming unit (CFU) assays revealed minor differences in bactericidal activity with slightly but not significantly higher CFU reduction in colonizing isolates. These data do not support a role for differential AMP susceptibility in vitro as a major determinant of S. aureus invasive infection.

Peptide gets in shape for self-defence

The transformation of tadpole to frog and of caterpillar to butterfly are two of the more obvious examples of metamorphosis. But molecular shape-shifting may occur in each of us as part of our innate antibacterial defence system. See Letter p.419 Defensins are key effector molecules of innate immunity, protecting the host from infectious microbes and shaping the composition of the microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class and is expressed by virtually all human epithelial sites, but previous work suggested that it has low antibiotic activity when compared with other defensins. Jan Wehkamp and colleagues now show that in reducing conditions similar to those found in the distal colon, hBD-1 exhibits potent antimicrobial action against the potential pathogens Candia albicans, Bifidobacterium and Lactobacillus species. In vitro evidence points to thioredoxin as the reducing agent most likely to unmask hBD-1's antimicrobial activity in the intestinal epithelium.

Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

IFN-γ, IL-17 and TGF-β involvement in shaping the tumor microenvironment: The significance of modulating such cytokines in treating malignant solid tumors

Multiple innate and adaptive immune effector cells and molecules partake in the recognition and destruction of cancer cells to protect against growing tumors, a concept that is known as cancer immunosurveillance. Unfortunately, cancer cells are capable of avoiding this process by immunoselection of poorly immunogenic tumor cells variants along with subversion of the immune system and thus shaping both the tumor and its microenvironment. Cytokines represent part of the complex pattern of the immune response which can assist the development of cancer as well as to eliminate it. Simultaneously, a large number of cytokines may be involved in the complex interactions between host and tumor cells where this dynamic cross-talk, between tumors and the immune system, can either regulate tumor growth or tumor growth, invasion and metastasis take place. In this review, we are stressing on the interface between infiltrated immune cells and tumor cells with the emphasis on the bidirectional activities of specific cytokines: IFN-γ, TGF-β and IL-17 within the tumor microenvironment and their role in shaping it. In addition, the significance of modulating such cytokines in favor of anti-tumor response is discussed and merits the use of mixture of targeted modulators to overcome the network complexity of cytokines in the tumor microenvironment.

Modulation of interleukin-1β-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts

IntroductionInnate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1β-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis.MethodsHuman fibroblast-like synoviocytes (FLS) were stimulated with IL-1β in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) was monitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by using multiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impact on IL-1β-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1β-induced pathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using various immunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptide followed by microscopy probing with streptavidin-Alexa Fluor.ResultsThis study demonstrated that IDR-1002 suppressed the production of IL-1β-induced MMP-3 and monocyte chemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing all chemokine responses. IDR-1002 altered the IL-1β-induced proteome primarily by altering the expression of members of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data also suggested that IDR-1002 was altering the transcription factor HNF-4α-mediated responses, known to be critical in metabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1β-induced NF-κB, JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002.ConclusionsThis study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1β-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. The results of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidates for chronic inflammatory diseases such as inflammatory arthritis.

Innate immune mechanisms of colitis and colitis-associated colorectal cancer

The innate immune system provides first-line defences in response to invading microorganisms and endogenous danger signals by triggering robust inflammatory and antimicrobial responses. However, innate immune sensing of commensal microorganisms in the intestinal tract does not lead to chronic intestinal inflammation in healthy individuals, reflecting the intricacy of the regulatory mechanisms that tame the inflammatory response in the gut. Recent findings suggest that innate immune responses to commensal microorganisms, although once considered to be harmful, are necessary for intestinal homeostasis and immune tolerance. This Review discusses recent findings that identify a crucial role for innate immune effector molecules in protection against colitis and colitis-associated colorectal cancer and the therapeutic implications that ensue.

An anti-lipopolysaccharide factor from red swamp crayfish, Procambarus clarkii, exhibited antimicrobial activities in vitro and in vivo

The anti-lipopolysaccharide factors (ALFs) are a group of effector molecules of innate immunity in arthropods, exhibiting binding and neutralizing activities to lipopolysaccharides. In this study, an ALF cDNA sequence ( PcALF1) was identified from red swamp crayfish, Procambarus clarkii. The deduced peptide of PcALF1 was conserved; it manifested the signal peptide and lipopolysaccharide (LPS)-binding domain, especially the two conserved cysteine residues at both ends of the domain. Transcripts of PcALF1 were detected in multiple tissues. Results of quantitative real-time PCR exhibited that the expression level of PcALF1 was induced by virus and Gram-positive and Gram-negative bacteria. Purified recombinant protein of PcALF1 revealed multiple biological activities: it gave all the tested bacteria and fungi a tight binding; it could bind microbial polysaccharides (LPS, LTA, and β-glucan) as well. In vitro, the antimicrobial activity assay was demonstrated as a broad spectrum against Gram-positive and Gram-negative bacteria and a fungus. The rPcALF1 also exhibited a clearance activity on Vibrio anguillarum in a dose-dependent manner in vivo

NF-κB and STAT3 signaling hubs for lung innate immunity

Innate immune responses to lung pathogens involve the coordinated expression of myriad affector and effector molecules of innate immunity, which must be induced and appropriately regulated in response to diverse stimuli generated by microbes or the infected host. Many intercellular and intracellular signaling pathways are involved, but we propose NF-κB and STAT3 transcription factors to be especially important signaling hubs for integrating these pathways to orchestrate effective host defense without excessive inflammatory injury.

Battle and balance at mucosal surfaces – The story of Shigella and antimicrobial peptides

Shigella is a major cause of morbidity and mortality for children in many developing countries. Emergence of antibiotic-resistance among Shigella demands the development of effective medicines. Antimicrobial peptides (AMPs) are expressed in phagocytes and at epithelial surfaces and are important effector molecules of innate immunity. We have found that pathogens are able to turn off the endogenous expression of AMPs, resulting in serious infections such as shigellosis. A therapeutic rationale to prevent microbial invasion would be to strengthen the epithelial line of defence through enhancing AMP expression. We have identified several inducers of AMP-production, including butyrate, phenylbutyrate and vitamin D, which have been investigated in animal models of shigellosis as well as in clinical trials. We believe that the conceptual framework presented here can be applied to additional clinical entities and that this novel approach can be an alternative or complement to traditional antibiotics in the future.

Perspectives on the etiology of chronic rhinosinusitis.

This article reviews recent insights surrounding the etiology and pathogenesis of chronic rhinosinusitis. In particular, we highlight the increasing recognition of host-mediated mechanisms in driving mucosal inflammation. Published differences between epithelium from patients with chronic rhinosinusitis and normal controls can be classified into several broad categories. Alterations are reported in the various components of the epithelial innate immune system including epithelial-expressed pattern-recognition receptors (PRRs) and the levels of antimicrobial innate immune effector molecules. Other studies demonstrate differences in the proteins involved in maintaining epithelial barrier integrity. Finally, recent studies show in chronic rhinosinusitis that epithelial-derived cytokines, chemokines and inducible surface proteins are involved in recruiting and activating cells of the adaptive immune system. The sinonasal epithelium provides a mechanical and innate immune barrier to a diverse array of environmental agents. This barrier also plays a key role in regulating the acquired mucosal immune response in the nose. Recent studies suggest that defects in this barrier may foster development of chronic sinonasal inflammation in response to environmental agents, and pathogenic or commensal organisms. The ability to dissect and analyze defects in the inflammatory response in rhinosinusitis may help identify novel targets for drug development.

Molecular cloning and characterization of three crustins from the Chinese white shrimp, Fenneropenaeus chinensis

Antibacterial peptides crustins are the effector molecules of innate immunity in decapods. In this study, three crustin cDNA sequences ( Fc-crus 1, Fc-crus 2, and Fc-crus 3) were cloned from the Chinese white shrimp Fenneropenaeus chinensis. The full-length cDNAs of Fc-crus 2 and 3 are 473 bp and 574 bp, respectively. The deduced peptides of Fc-crus 2 and 3 contain a signal peptide and a crustin domain at the C-terminal formed by twelve conserved cysteine residues. The partial sequence of Fc-cru 1 is 575 bp long and the deduced amino acids also contain a crustin domain. The expression profiles of these three crustins were studied with RT-PCR. Fc-crus 1 and Fc-crus 2 constitutively expressed in hemocytes with high levels, and the expression level is increased in the heart, stomach, intestine and ovaries when shrimp was challenged with Vibrio anguillarum, The expression of Fc-crus 1 and Fc-crus 2 was detected in each developmental stage. Fc-crus 3 was constitutively expressed in the ovaries and induced as an expression in the stomach. Unlike Fc-crus 1 and Fc-crus 2, the mRNA of Fc-crus 3 was not detected in the developmental stages extending from nauplii and mysis to post-larvae. The recombinant proteins containing mature Fc-crus 2 and Fc-crus 3 were recombinantly expressed in Escherichia coli and respectively purified. The antibacterial assays revealed that the recombinant mFc-crus could inhibit the growth of Gram-positive bacteria in vitro.