Mika Rämet

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. We devised a double-stranded RNA interference-based screen in macrophage-like Drosophila S2 cells, and have defined 34 gene products involved in phagocytosis. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. Here we show that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. Our results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis.

The Drosophila Toll signaling pathway.

The identification of the Drosophila melanogaster Toll pathway cascade and the subsequent characterization of TLRs have reshaped our understanding of the immune system. Ever since, Drosophila NF-κB signaling has been actively studied. In flies, the Toll receptors are essential for embryonic development and immunity. In total, nine Toll receptors are encoded in the Drosophila genome, including the Toll pathway receptor Toll. The induction of the Toll pathway by Gram-positive bacteria or fungi leads to the activation of cellular immunity as well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-κB factors Dorsal-related immunity factor or Dorsal. In this study, we review the current literature on the Toll pathway and compare the Drosophila and mammalian NF-κB pathways.

Eater, a Transmembrane Protein Mediating Phagocytosis of Bacterial Pathogens in Drosophila

Phagocytosis is a complex, evolutionarily conserved process that plays a central role in host defense against infection. We have identified a predicted transmembrane protein, Eater, which is involved in phagocytosis in Drosophila. Transcriptional silencing of the eater gene in a macrophage cell line led to a significant reduction in the binding and internalization of bacteria. Moreover, the N terminus of the Eater protein mediated direct microbial binding which could be inhibited with scavenger receptor ligands, acetylated, and oxidized low-density lipoprotein. In vivo, eater expression was restricted to blood cells. Flies lacking the eater gene displayed normal responses in NF-kappaB-like Toll and IMD signaling pathways but showed impaired phagocytosis and decreased survival after bacterial infection. Our results suggest that Eater is a major phagocytic receptor for a broad range of bacterial pathogens in Drosophila and provide a powerful model to address the role of phagocytosis in vivo.

Surfactant Protein D Gene Polymorphism Associated with Severe Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is the major respiratory tract pathogen in infancy. Host-related differences in susceptibility to severe RSV infection suggest that genetic factors may play a role. In this study, a candidate-gene approach was used to study whether the surfactant protein D (SP-D) gene polymorphism associates with severe RSV infection. DNA samples from 84 infants hospitalized for the treatment of RSV bronchiolitis and 93 healthy controls were analyzed. The controls were matched with the cases on the basis of sex, hospital district, date of birth (±2 wk) and gestational age at birth (±2 wk). Three biallelic SP-D gene polymorphisms were genotyped. Significant differences were observed in the SP-D allele frequencies for amino acid 11 between the RSV infants and their matched controls. The frequency of the allele coding for Met 11 (p = 0.033) was increased in the severe RSV group. The frequency of the homozygous genotype Met/Met for amino acid 11 was increased in the RSV group relative to the controls, whereas the heterozygous genotype tended to be less frequent among the RSV cases than in the matched controls. Conditional logistic regression analysis was used to study whether the confounders, i.e. smoking and number of children in the family, influence the association between the homozygous SP-D genotype for methionine 11 and the risk of RSV bronchiolitis. The results further confirmed this association (p = 0.028). To our knowledge, the present report provides the first evidence of a specific gene associated with susceptibility to severe RSV infection.

Double-stranded RNA Is Internalized by Scavenger Receptor-mediated Endocytosis in Drosophila S2 Cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.

Association between Surfactant Protein A Gene Locus and Severe Respiratory Syncytial Virus Infection in Infants

Respiratory syncytial virus (RSV) causes seasonal epidemics of bronchiolitis among susceptible infants. Surfactant protein A (SP-A), a lung C-type lectin involved in innate host defense, opsonizes RSV and enhances phagocytosis. The candidate gene approach was used to investigate association of SP-A polymorphism with susceptibility to severe RSV infection. Genotype analysis was done for 86 infants with severe RSV infection and 95 matched control subjects. A significant difference in the frequency of SP-A2 was observed. The SP-A2 allele 1A(3) was overrepresented in RSV-infected infants, compared with control subjects (5% vs. 0.5%; P =.006), whereas allele 1A was underrepresented (1% vs. 6%; P =.011). The allele pool in which lysine was amino acid 223 was overrepresented in infants with severe RSV infection (28% vs. 18%; P =.023), whereas the allele pool in which proline was amino acid 99 was underrepresented (5% vs. 16%; P =.001). These results indicate that a genetic association exists between SP-A gene locus and severe RSV infection.

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

The Imd signaling cascade, similar to the mammalian TNF‐receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large‐scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell‐derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor‐activated kinase 1 (TAK1)‐binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi‐based approach is suitable to identify genes in conserved signaling cascades.

Novel, Non-Radioactive, Simple and Multiplex PCR-cRFLP Methods for Genotyping Human SP-A and SP-D Marker Alleles

We have previously identified an allele of the human SP-A2 gene that occurs with greater frequency in an RDS population [12]. Because of the importance of SP-A in normal lung function and its newly emerging role in innate host defense and regu-lation of inflammatory processes, we wish to better characterize genotypes of both SP-A1 and SP-A2 genes. It has been determined that SP-D shares similar roles in immune response. Therefore, in this report we 1) describe a novel, non radioactive PCR based-cRFLP method for genotyping both SP-A and SP-D; 2) describe two previously unpublished biallelic polymorphisms within the SP-D gene; 3) present the partial sequence of one new SP-A1 allele (6A14) and describe other new SP-A1 and SP-A2 alleles; and 4) describe additional methodologies for SP-A genotype assessment. The ability to more accurately and efficiently genotype samples from individuals with various pulmonary diseases will facilitate population and family based association studies. Genetic poly-morphisms may be identified that partially explain individual disease susceptibility and/or treatment effectiveness.

Pirk Is a Negative Regulator of the Drosophila Imd Pathway

NF-κB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. In this study we report that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. We conclude that Pirk is a negative regulator of the Imd pathway.

Toll-like Receptors as Sensors of Pathogens

Initial recognition of microbes, as they enter the body, is based on germ line–encoded pattern recognition receptors that selectively bind to essential components of pathogens. This allows the body to respond immediately to the microbial invasion before the development of active immunity. The signal-transducing receptors that trigger the acute inflammatory cascade have been elusive until very recently. On the basis of their genetic similarity to the Toll signaling pathway in Drosophila, mammalian Toll-like receptors (TLRs) have been identified. By now, nine transmembrane proteins in the TLR family have been described. Mammalian TLR4 is the signal-transducing receptor activated by the bacterial lipopolysaccharide. The activation of TLR4 leads to DNA binding of the transcription factor NF-κB, resulting in activation of the inflammatory cascade. Activation of other TLRs is likely to have similar consequences. TLR2 mediates the host response to Gram-positive bacteria and yeast. TLR1 and TLR6 may participate in the activation of macrophages by Gram-positive bacteria, whereas TLR9 appears to respond to a specific sequence of bacterial DNA. The TLRs that control the onset of an acute inflammatory response are critical antecedents for the development of adaptive acquired immunity. Genetic and developmental variation in the expression of microbial pattern recognition receptors may affect the individuals predisposition to infections in childhood and may contribute to susceptibility to severe neonatal inflammatory diseases, allergies, and autoimmune diseases.