PHYSIOLOGY OF INSECTS - IMMUNE SYSTEM
(see also entomology)

The main model organism is the dipteran Drosophila melanogaster.

Immediately after septic injury, the insect fat body (a homologue of mammalian liver) and some blood cells start to produce a battery of potent 20-40 amino acids-long antimicrobial peptidesref. These molecules are released into the blood, where they synergistically act to destroy the invading microorganisms. Many induced antimicrobial molecules are apparent in the hemolymph 2-4 h after infection at a concentration as low as 15 mM (hence very low IC50 !).
They only have an innate immune system, based on Toll-like receptors : at least 2 pathways are involved in the Drosophila immune responseref :

Click here to view their signal transduction pathway.
Complex signaling pathways regulate the innate immune system of insects, with NF-kB transcription factors playing a central role in the activation of antimicrobial peptides and other immune genes. Although numerous studies have characterized the immune responses of insects to pathogens, comparatively little is known about the counterstrategies pathogens have evolved to circumvent host defenses. Among the most potent immunosuppressive pathogens of insects are polydnaviruses that are symbiotically associated with parasitoid wasps. Microplitis demolitor bracovirus encodes a family of genes with homology to IkB proteins from insects and mammals. Functional analysis of 2 of these genes, H4 and N5, were conducted in Drosophila S2 cells. Recombinant H4 and N5 greatly reduced the expression of drosomycin and attacin reporter constructs, which are under NF-kB regulation through the Toll and Imd pathways. Coimmunoprecipitation experiments indicated that H4 and N5 bound to the Rel proteins Dif and Relish, and N5 also weakly bound to Dorsal. H4 and N5 also inhibited binding of Dif and Relish to B sites in the promoters of the drosomycin and cecropin A1 genes. Collectively, these results indicate that H4 and N5 function as IkBs and, circumstantially, suggest that other IkB-like gene family members are involved in the suppression of the insect immune systemref.
Insects have a family of 12 peptidoglycan recognition proteins (PGRPs) that recognize peptidoglycan, a ubiquitous component of bacterial cell walls. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. Functional dissection of an innate immune response by a genome-wide RNAi screenref : a novel gene, sickle—required for activation of a key component of the pathway, Relish.
The Drosophila melanogaster genome contains 34 antimicrobial peptide-encoding genes belonging to 8 families and a large number of putative protease-encoding genesref. Lectins or other molecules playing a role in recognition, phagocytosis, or antimicrobial activity may be present in the hemolymph. The Drosophila genome possesses at least 3 Gram-negative bacteria-binding proteinsref, but they do not appear to be induced after infection. On the other hand, 2 uncharacterized genes encoding short proteins with partial similarity to Drosophila Gram-negative bacteria-binding proteins are up-regulated after septic injuryref. Furthermore, the Drosophila genome encodes at least 12 peptidoglycan recognition proteins; transcripts of severeal peptidoglycan recognition protein genes have been found in hemocytesref. Transferrin genes, which are involved in iron transport and protection against iron overload in the diet, appear to have an additional role in innate immunity, because they are also induced during septic injuryref. Finally, a microarray study of the Drosophila immune response not only revealed the involvement of the above mentioned immune response genes but also showed the involvement of a large number of genes with unknown function in the immune responseref.
When Drosophila melanogaster raised in axenic conditions by treating eggs with bleach and ethanol and then keeping the flies in a germ-free environment with sterilized food during their first week of adulthood, their lives are shortened by 30%. In a parallel set of experiments, Drosophila with bacteria eliminated from their bodies by antibiotic treatment lived 35% shorter lives. If flies were exposed to bacteria within the first 4 to 7 days of adulthood, they lived normal-length lives. If they were kept axenic for this first week, subsequent addition of bacteria made no difference—longevity was reduced by 30%. At metamorphosis, a pulse of the steroid hormone ecdysone initiates the shift from larval to adult structures, resulting in fat body transition, upregulation of immune genes, and gut remodelingref. Most larval bacteria are destroyed during this process : the window at which the bacteria are important is actually the period in which the fly would re-expose itself to bacteria. The critical bacterial exposure period also overlaps nicely with the transition from larval to adult fat and the Drosophila fat body has been shown to regulate longevity through insulin-related signaling pathways (life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (chico) are mutated, or when insulin-producing cells are ablated)ref. Flies fed antibiotic-containing food late (during the fourth week of adulthood) lived about 10% longer than those that ate normal food. The Drosophila mutant EcR, which has a mutation in the ecdysone receptor gene, is long-lived as a heterozygoteref. Unlike normal flies, these mutants did not suffer reduced longevity with lifelong antibiotic treatment. Out of a series of other long-lived mutants, one called DJ817 showed different effects from either wildtype or EcR flies : they lived 30% longer than wildtype flies when bacteria were present, but were no different from wildtype in the absence of bacteria. The genetic basis of the DJ817 phenotype has not been fully characterizedref. Because most animals evolved in microbe-rich seas, the selection pressure by bacteria has been intense : it's not surprising that the presence of environmental bacteria would be incorporated into the biological program of an animal.

List of statistically significant differentially expressed proteins identified in hemolymph of Drosophila melanogaster third-instar larvae by 2D-DIGE combined with mass spectroscopy, 25 min or 4 h after LPS challenge and 25 min after sterile challengeref :

Pricking the larva with a needle causes a severe injury to the animal. Therefore, it is likely that some of the increased proteins have nothing to do with the immune response, but are only part of the stress/injury response (e.g. glutathione S-transferase and actin-57B)
The B2 protein of nodaviruses flock house virus (FHV) and nodamura virus (NoV) (as well as a protein encodede by tombusvirus - a plant pathogen) inhibit silencing of viral RNAs in Drosophila cells, as well as prevent the degradation of mRNA specifically targeted by homologous siRNA constructs. In addition, NoV infection of mosquito cells also activates an RNAi-mediate antiviral response that is susceptible to B2 silencing, establishing that this pathway of viral immunity functions in more than one invertebrate species. Also NS1 protein from 3 influenzavirus genera and E3L protein from vaccinia virus are able to suppress antiviral RNA silencing in Drosophila cells : the dsRNA-binding domain alone of NS1 is both necessary and sufficient for silencingref.
A prepro form of an antibacterial peptide is made almost 130 times faster than IgM in Vertebrates, about 3 times faster than the reproduction of the bacteria. The prosequence can be located at either side of the mature peptide gene and the promoter regions contain sequence motifs similar to cis-regulatory elements of mammalian acute-phase response genes. A single insect produces approximately 10-15 antibiotics : Although the native peptides degrade quickly in biological fluids other than insect haemolymph, structural modifications render the peptides resistant against proteases without sacrificing biological activity, producing viable alternatives to the conventional antimicrobial compounds for mammalian therapy. Currently, no drug resistance other than proteolytic cleavage could be attributed to the antibacterial peptides. Most insect antibacterial peptides are rich in Lys and Arg residues, the targets of trypsin-like peptidases. Serum stability may be improved by ... Anopheles gambiae has 242 genes as potential mediators of innate immunity : Plasmodium spp. usually goes unnoticed by the mosquito in which it grows thanks to a  fine balance between the action of 2 genes controlling the insects' immune responseref. Drugs that tip the balance in favour of parasite death could be used as anti-malarials. Another option is to create genetically modified mosquitoes that carry altered versions of these genes. But tinkering with these genes could cause changes to the mosquito that make it hard for them to survive

Down syndrome cell adhesion molecule (Dscam) gene has 115 exons. 4 of them (4, 6, 9, and 17) are clusters of multiple alternative exons (with 12, 48, 33, and 2 alternative exons, respectively). Each alternative exon is mutually exclusive; the gene must choose only one alternative exon from each of those 4 exon clusters. In 2000, Schmucker and colleagues discovered that, when the alternatives are multiplied, 38,016 distinct protein products result. Moreover, it appears that most of those proteins are actually produced at some point in a fly's life. Little else is known about the gene besides its ability for producing diverse proteins. It appears to promote neuron movement during developmental phases. Homologs have very little functional similarities. Human DSCAM is found on chromosome 21 and its products may be the cause of neurodevelopmental defects associated with Down syndrome. But the human gene has few known alternatively spliced products. Other insects appear to splice and dice the gene. The mosquito, for instance, can produce 32,000 proteins. And, Drosophila virilis may make even more proteins than D. melanogaster, almost 40,000ref. Insects may possess a hitherto unsuspected molecular complexity in their immune system, comparable to the antibody system of mammals. The number of immune receptors might go from a couple of dozen up to thousands in insects. The complexity there might have really been underestimated. Using RT-PCR, Dscam expression was found in Drosophila fat body cells, which secrete antimicrobial peptides, and hemocytes, which are involved in phagocytosis. Using antibodies against extracellular domains of Dscam, they also found a soluble Dscam protein secreted in hemolymph serum. Microarray analysis for alternatively spliced Dscam exons suggested that fat body cells and hemocytes could generate > 18,000 receptor isoforms. Comparative genomic analysis between insect orders Diptera, Hymenoptera, Coleoptera, and Lepidoptera revealed high conservation of orthologous Dscam genes. The ability to generate extensive diversity of immune receptors was generally thought to be limited to jawed vertebratesref. This diversity of proteins certainly raises the parallel to antibodies in higher mammals. In mammals, T-cell receptors have recently been found to be expressed in the brainref, so this shows another class of molecules that play an important role in both nervous and immune systems. To investigate what Dscam's immune function might be, the researchers challenged wild-type and Dscam-deficient hemocytes with heat-killed fluorescent-labelled E. coli. Only 55% of Dscam-deficient hemocytes ingested bacteria after 10 minutes, compared to 85% to 90% of normal hemocytes. Isoforms Dscam-7.27.25-Fc and Dscam-7.27.13-Fc could bind to live E. coli, while binding of Dscam-1.30.30-Fc was barely detectable. This raises the possibility that different isoforms might bind specifically to distinct epitopes on bacteria. A whole series of new studies are needed to address whether these different isoforms really are capable of an adapted, specific response to pathogens. They could purify hemocytes out, challenge them with different pathogens and have microarrays look at Dscam splicing to see if you do upregulate certain isoforms in a predictable way, if you have a gram-positive infection or a gram-negative one or of yeast. Another open question these findings raise is whether insects possess immunological memory. In the end, levels of insect and mammalian immune molecular diversity may reflect very different lifestyles. Insects live only a few months, some a few years, so that makes a big impact in how you invest in immunity. Immune receptor diversity may not be as important there when compared with vertebrates, many of which live many years. If this is adaptive immunity in insects, it's probably a case of convergent evolution with mammals. Both are composed of Ig domains but are very different structurally and involve very different mechanisms of alternative splicing and gene rearrangement. And there is a Dscam homolog in mammals that is not alternatively spliced to an appreciable extent. Given the millions of extant insect species, different spectra of Dscam isoforms likely exist : that might in some way reflect a difference in their susceptibility to pathogens, and understanding that or even interfering with that may have important implications for issues of agriculture or of insects that act as vectors for human disease, such as with mosquitoes and malaria

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