Identification and functional analysis of antifungal immune response genes in Drosophila

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. Although signaling pathways that activate NF-kappaB during innate immune responses to various microorganisms have been studied in detail, regulatory mechanisms that control other immune responses to fungal infection require further investigation. To identify new Drosophila genes involved in antifungal immune responses, we selected genes known to be differentially regulated in SL2 cells by microbial cell wall components and tested their roles in antifungal defense using mutant flies. From 130 mutant lines, sixteen mutants exhibited increased sensitivity to fungal infection. Examination of their effects on defense against various types of bacteria and fungi revealed nine genes that are involved specifically in defense against fungal infection. All of these mutants displayed defects in phagocytosis or activation of antimicrobial peptide genes following infection. In some mutants, these immune deficiencies were attributed to defects in hemocyte development and differentiation, while other mutants showed specific defects in immune signaling required for humoral or cellular immune responses. Our results identify a new class of genes involved in antifungal immune responses in Drosophila.     

Regulators and signalling in insect haemocyte immunity

The innate immune system of insects relies on both humoral and cellular immune responses that are mediated via activation of several signalling pathways. Haemocytes are the primary mediators of cell-mediated immunity in insects, including phagocytosis, nodulation, encapsulation and melanization. The last years, research has focused on the mechanisms of microbial recognition and activation of haemocyte intracellular signalling molecules in response to invaders. The powerful tool, RNA interference gene silencing, helped several regulators involved in immune responses, to be identified. In this review, we summarize recent advances in understanding the role(s) of receptors and intracellular signalling molecules involved in immune responses.     

Interactions between the cellular and humoral immune responses in Drosophila

Drosophila has highly efficient defenses against infection. These include both cellular immune responses, such as the phagocytosis of invading microorganisms, and humoral immune responses, such as the secretion of antimicrobial peptides into the hemolymph [1] [2]. These defense systems are thought to interact, but the nature and extent of these interactions is not known. Here we describe a method for inhibiting phagocytosis in Drosophila blood cells (hemocytes) by injecting polystyrene beads into the body cavity. This treatment does not in itself make a fly susceptible to Escherichia coli infection. However, when performed on flies carrying the mutation immune deficiency (imd), which affects the humoral immune response [3], the treatment results in a striking decrease in resistance to infection. We therefore carried out a sensitized genetic screen to identify immunocompromised mutants by co-injecting beads and E. coli. From this screen, we identified a new gene we have named red shirt and identified the caspase Dredd as a regulator of the Drosophila immune response. The observation that mutants with defects in the humoral immune response are further immunocompromised by blocking phagocytosis, and thus inhibiting the cellular immune response, shows that the Drosophila cellular and humoral immune responses act in concert to fight infection.     

Phagocytic ability declines with age in adult Drosophila hemocytes

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age‐related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1‐week‐ and 5‐week‐old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.     

Analysis of signal-dependent changes in the proteome of Drosophila blood cells during an immune response

Innate immunity is based on the recognition of cell-surface molecules of infecting agents. Microbial substances, such as peptidoglycan, lipopolysaccharide, and beta-1,3-glucans, produce functional responses in Drosophila hemocytes that contribute to innate immunity. We have used two-dimensional gel electrophoresis and MS to resolve lipopolysaccharide-induced changes in the protein profile of a Drosophila hemocytic cell line. We identified 24 intracellular proteins that were up- or down-regulated, or modified, in response to immune challenge. Several proteins with predicted immune functions, including lysosomal proteases, actin-binding/remodeling proteins, as well as proteins involved in cellular responses to oxidative stress, were affected by the immune assault. Intriguingly, a number of the proteins identified in this study have recently been implicated in phagocytosis in higher vertebrates. We suggest that phagocytosis is activated in Drosophila hemocytes by the presence of microbial substances, and that this activation constitutes an evolutionarily conserved arm of innate immunity. In addition, a number of proteins involved in calcium-regulated signaling, mRNA processing, and nuclear transport were affected, consistent with a possible role in reprogramming of gene expression. In conclusion, the present proteome analysis identified many proteins previously not linked to innate immunity, demonstrating that differential protein profiling of Drosophila hemocytes is a valuable tool for identification of new players in immune-related cellular processes.     

Complement-mediated phagocytosis--the role of Syk

Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. The protein-tyrosine kinase, Syk, plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. From recent studies using a macrophage-like differentiated cell line and serum-treated zymosan, it was found that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Serum-treated zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor3. During this process, Syk became tyrosine-phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into macrophages resulted in impaired engulfment of pathogen. Collectively, Syk is required for the engulfment of pathogen in complement-mediated phagocytosis.     

Cutting edge: bradykinin induces IL-12 production by dendritic cells: a danger signal that drives Th1 polarization

Dendritic cells play a major role in the induction of both innate and acquired immune responses against pathogenic invaders. These cells are also able to sense endogenous activation signals liberated by injured tissues even in the absence of infection. In the present work, we demonstrate that kinins mobilize dendritic cells to produce IL-12 through activation of the B(2) bradykinin receptor subtype and that bradykinin-induced IL-12 responses are tightly regulated both by angiotensin-converting enzyme, a kinin-degrading peptidase, and by endogenous IL-10. Using a mouse model of allergic inflammation, we further show that addition of bradykinin to OVA during immunization results in decreased eosinophil infiltration on Ag challenge. The latter effect was demonstrated to be due to IL-12-driven skewing of Ag-specific T cell responses to a type 1 cytokine profile. Our data thus indicate that kinin peptides can serve as danger signals that trigger dendritic cells to produce IL-12 through activation of B(2) bradykinin receptors.     

Role of glycosphingolipid-enriched microdomains in innate immunity: microdomain-dependent phagocytic cell functions

The innate immune system is the first line of defense against pathogenic microorganisms, such as bacteria, fungi, and viruses. Phagocytes, such as neutrophils and macrophages, play an important role in the innate immune system by recognizing, engulfing, and eliminating pathogens. It has been suggested that lipid membrane microdomains/rafts of phagocytes are involved in these innate immune responses, including superoxide generation, cell migration, and phagocytosis. Lactosylceramide (LacCer), a neutral glycosphingolipid, forms glycosphingolipid-enriched microdomains together with the Src family kinase, Lyn, on the neutrophil plasma membrane. LacCer-enriched microdomains have been suggested to play important roles in innate immune function of neutrophils. However, the molecular mechanisms underlying these phenomena remain largely unknown. Recent proteomic analyses of microdomains from phagocytes have provided insight into membrane microdomain-mediated functions in the processes of phagocytosis. In this review, we discuss the membrane microdomain-associated immune functions of phagocytes, focusing on those functions of LacCer-enriched microdomains and recent proteomic approaches to determine the molecular mechanisms underlying these functions.     

CXC chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells

How chemokines shape the immune response to viral infection of the central nervous system (CNS) has largely been considered within the context of recruitment and activation of antigen-specific lymphocytes. However, chemokines are expressed early following viral infection, suggesting an important role in coordinating innate immune responses. Herein, we evaluated the contributions of CXC chemokine ligand 10 (CXCL10) in promoting innate defense mechanisms following coronavirus infection of the CNS. Intracerebral infection of RAG1(-/-) mice with a recombinant CXCL10-expressing murine coronavirus (mouse hepatitis virus) resulted in protection from disease and increased survival that correlated with a significant increase in recruitment and activation of natural killer (NK) cells within the CNS. Accumulation of NK cells resulted in a reduction in viral titers that was dependent on gamma interferon secretion. These results indicate that CXCL10 expression plays a pivotal role in defense following coronavirus infection of the CNS by enhancing innate immune responses.     

Genomic HIV RNA induces innate immune responses through RIG-I-dependent sensing of secondary-structured RNA

Background

Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse.

Methods/Principal Findings

Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA.

Conclusions/Significance

These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.     

重组腺相关病毒载体诱导的天然免疫反应及机制

重组腺相关病毒(rAAV)已成为基因治疗领域应用最广泛的载体之一。临床前研究显示其具有很高的安全性,但人体免疫毒性仍是制约其临床疗效的关键,因此有关rAAV免疫机制的研究成为近期热点。尽管天然免疫在获得性免疫反应中发挥重要作用,但与rAAV有关的天然免疫研究过去一直未被重视。直到最近,才确认有至少3种人体细胞(树突状细胞、巨噬细胞和内皮细胞)参与了rAAV的天然免疫,作用机制为可识别载体基因组的TLR9或病毒衣壳TLR2所介导,NF-κB或干扰素调节因子(IRFs)信号通路被激活,导致各种炎性因子及I型干扰素的大量表达。自身互补型rAAV诱导的TLR9依赖性天然免疫较单链rAAV更为强烈。本文重点对近期发现的激活天然免疫反应的宿主与rAAV的相互作用、涉及的信号通路、天然免疫对获得性免疫以及转基因表达的影响进行综述。     

Determination of T-cell fate by dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells with a unique T-cell stimulatory aptitude that play a crucial role in the instruction of adaptive immune responses upon infection. By controlling the initiation of a diverse set of effector functions, which are suitable for the elimination of a wide range of pathogens, DCs form the pivotal link between the innate and the adaptive immune system. The innate pattern recognition pathways that trigger DC activation are central for skewing of the adaptive immune responses that are subsequently induced. Thus innate activation not only precedes adaptive immune activation, it also controls it and tailors the effector functions to the requirements of the infection. The adaptive immune response has to match the nature of the infection, but this does not only concern the type of pathogen, it is also affected by the localization of the infection. Tissue homeostasis has to be ensured and thus tissue-derived environmental factors influence the functional activity of activated DCs and thereby contribute to shaping of the immune response. Adaptive immune responses are vital for the elimination of pathogens, have the potential to attack tumor cells and play a detrimental role during transplant rejection and in a variety of autoimmune diseases. Better understanding of the mechanisms that control the induction of different T-cell effector functions will enable the development of strategies to manipulate the immune system in the context of vaccination, tumor immunotherapy, transplantation and autoimmunity.     

A specific primed immune response in Drosophila is dependent on phagocytes

Drosophila melanogaster, like other invertebrates, relies solely on its innate immune response to fight invading microbes; by definition, innate immunity lacks adaptive characteristics. However, we show here that priming Drosophila with a sublethal dose of Streptococcus pneumoniae protects against an otherwise-lethal second challenge of S. pneumoniae. This protective effect exhibits coarse specificity for S. pneumoniae and persists for the life of the fly. Although not all microbial challenges induced this specific primed response, we find that a similar specific protection can be elicited by Beauveria bassiana, a natural fly pathogen. To characterize this primed response, we focused on S. pneumoniae-induced protection. The mechanism underlying this protective effect requires phagocytes and the Toll pathway. However, activation of the Toll pathway is not sufficient for priming-induced protection. This work contradicts the paradigm that insect immune responses cannot adapt and will promote the search for similar responses overlooked in organisms with an adaptive immune response.     

Toll-like receptor pathways in the immune responses to mycobacteria

The control of Mycobacterium tuberculosis infection depends on recognition of the pathogen and the activation of both the innate and adaptive immune responses. Toll-like receptors (TLR) were shown to play a critical role in the recognition of several pathogens. Mycobacterial antigens recognise distinct TLR resulting in rapid activation of cells of the innate immune system. Recent evidence from in vitro and in vivo investigations, summarised in this review demonstrates TLR-dependent activation of innate immune response, while the induction of adaptive immunity to mycobacteria may be TLR independent.     

Infection-Induced Interaction between the Mosquito Circulatory and Immune Systems

Insects counter infection with innate immune responses that rely on cells called hemocytes. Hemocytes exist in association with the insect''s open circulatory system and this mode of existence has likely influenced the organization and control of anti-pathogen immune responses. Previous studies reported that pathogens in the mosquito body cavity (hemocoel) accumulate on the surface of the heart. Using novel cell staining, microdissection and intravital imaging techniques, we investigated the mechanism of pathogen accumulation in the pericardium of the malaria mosquito, Anopheles gambiae, and discovered a novel insect immune tissue, herein named periostial hemocytes, that sequesters pathogens as they flow with the hemolymph. Specifically, we show that there are two types of endocytic cells that flank the heart: periostial hemocytes and pericardial cells. Resident periostial hemocytes engage in the rapid phagocytosis of pathogens, and during the course of a bacterial or Plasmodium infection, circulating hemocytes migrate to the periostial regions where they bind the cardiac musculature and each other, and continue the phagocytosis of invaders. Periostial hemocyte aggregation occurs in a time- and infection dose-dependent manner, and once this immune process is triggered, the number of periostial hemocytes remains elevated for the lifetime of the mosquito. Finally, the soluble immune elicitors peptidoglycan and β-1,3-glucan also induce periostial hemocyte aggregation, indicating that this is a generalized and basal immune response that is induced by diverse immune stimuli. These data describe a novel insect cellular immune response that fundamentally relies on the physiological interaction between the insect circulatory and immune systems.