Figure 1. Toll and Imd signaling pathway
The immune response of insects includes humoral immunity and cellular immunity. Humoral immunity mainly produces antibacterial peptides from fat bodies to resist the reaction of invading pathogenic microorganisms; cellular immunity mainly kills invasive pathogenic microorganisms through the activity of immune cells, including phagocytosis, nodule and cyst. Whether it is humoral immunity or cellular immunity, the immune response includes three phases: 1) recognition of invading pathogens; 2) cascade amplification of immunostimulatory signals; 3) production of effectors or activation of cellular immune activity. Pathogenic microorganisms have special pathogen-associated ligands on the surface, which can be recognized on the surface of insect cells or secreted receptors; interactions between ligands and receptors, cascade amplification of transduction signals, and finally the generation of antimicrobial peptides, which in turn triggers phagocytosis, nodule and cyst activity. For example, an important feature of the Drosophila immune response is that fat cells can rapidly synthesize some antimicrobial peptide molecules when infected or damaged by pathogenic microorganisms. These molecules secreting into the hemolymph can kill the invading pathogenic microorganisms. In addition, weasand and epithelial cells of Drosophila can also produce antibacterial peptides, and intestinal cells and salivary gland cells can produce antibacterial proteins such as lysozyme and insect defensins. So far, thirty genes encoding antibacterial proteins have been identified in the Drosophila genome, of which eight have been identified.
During humoral immunity, the immune response of insects depends on the Toll and IMD pathways to activate NF-kB signaling by bacterial cell wall peptidoglycan (PGN) stimulation. The Toll/Dif pathway was discovered by Morisato and Anderson in the study of how the fat body cells of Drosophila were induced to express antibacterial peptides and how they were assembled and then secreted into the hemolymph. The Toll pathway is mainly responsible for the fungus and most of the gram-positive bacterium. The Imd pathway was discovered during the study of antimicrobial peptide gene expression in a recessive variant of Drosophila. Gram-negative bacteria and Bacillus usually activate the IMD pathway. Corresponding to the mammalian TNF receptor (TNFR) signaling pathway, the upstream pattern recognition receptors of Drosophila IMD proteins are PGRP-LC and PGRP-LE, and the downstream signaling molecules include dFADD, Dredd (caspase homolog), dTAK1, dIKK complexes and the precursor Relish of NF-kB. The activated Relish enters the cell nucleus and expresses a series of antimicrobial peptides.
The regulation of pathway
Toll protein is the main gene product involved in the Toll pathway. It was first discovered during the study of the formation of dorsoventral polarity in the embryonic development of Drosophila. The relationship between Toll protein and immune response was later discovered. So far, nine Toll-like genes, Toll-1, Toll-3-Toll-9 and 18-wheeler (Toll-2), have been found in the Drosophila genome. These gene products contain two and a half cystine knots, causing its extracellular segment to be separated into two segments by a second cysteine knot. Among them, Toll-2 is involved in the immune response to bacteria, and Toll-1 and Toll-5 can activate the expression of the antifungal peptide genes. Studies have shown that Drosophila Toll protein belongs to type I transmembrane receptors and can recognize bacterial lipopolysaccharides (LPS), peptidoglycans (PGN), etc., and can activate intracellular signal molecules such as Tube and Pelle to activate the expression of immune genes. If the Toll receptor is stimulated by fungi, it can cause the expression of drosomycin. When the Toll receptor and its intracellular signaling molecules Tube and Pelle are defective, the synthesis of drosomycin is significantly reduced. In addition, studies have found that there are temporal differences in the expression of these genes during embryonic development of Drosophila and that activation of the Spaetzle protein is required. Activation of the Toll protein eventually activates the Rel family of transcription factors. The activation process is mediated by signal molecules Tube and Pelle and other unidentified molecules.
Compared to the Toll pathway, the Imd pathway is activated by the infection of gram-negative bacteria, and the components involved in this pathway are located on the second chromosome of Drosophila. Through the study of the Drosophila Imd mutant, it was found that this mutation can reduce the immunity of Drosophila by greatly reducing the induced expression of the antibacterial peptide gene, but it does not affect the expression of the antifungal peptide gene. This shows that the drosomycin gene is constitutively expressed in this mutant. Experiments by Fehlbaum et al. showed that the transcriptional activity of drosomycin is constitutively present in both Drosophila adults and larvae, but it is highly expressed only after fungal infection. So far, it is rarely known about how this pathway regulates the expression of antimicrobial peptides. However, studies have shown that this pathway activates Relish, which in turn activates the expression of immune genes. Recent studies have identified six components involved in the Imd pathway: Imd, dTAK1, Ird5, Kenny, Dredd and Relish. The study also found that the Imd gene encodes a dead-region protein that is similar to the protein of the TNR receptor signaling pathway. The Imd pathway regulates the expression of antimicrobial peptide genes by activating Rel/NF-κB-like transcription factors. It was also found that although the Imd pathway has no regulatory effect on development and cellular immunity, it can regulate the expression of antimicrobial peptide genes in epithelial tissues and participate in apoptosis.
The transcription factors that have been found to regulate the expression of antimicrobial peptide genes in Drosophila are mainly the Rel protein family, which is similar to the transcriptional activator NF-κB in mammals, which plays an important role in the regulation of mammalian immune responses. The Rel proteins that have been found so far mainly include transcription factors such as Dorsal, Dif, Dorsal B and Relish. These proteins all have a homologous region (RHR) of the Rel protein, which is involved in the binding of DNA, the formation of Rel dimer and their transfer into the nucleus. In the early stage of immune induction of Drosophila fat cells, these proteins can be rapidly synthesized and transferred to the nucleus to activate the transcription of the relevant immune gene (antibacterial peptide gene). Studies have found that mutations in one transcription factor may cause some antibacterial peptide genes not to be expressed, while others may still be expressed, indicating that the expression of antibacterial peptide genes is regulated by different transcription factors.
Drosophila Toll protein is structurally homologous to the mammalian Toll-like receptor (TLR), both of which have an extracellular region rich in leucine repeats (LRR) and constitute the intracellular region of the homology domain of Toll/IL1R. The study also found that Toll and TLR have similar signal transduction pathways, such as TLR-mediated signal transduction that can phosphorylate IκB, leading to NF-κB activation, and Toll-mediated signal transduction can also make IκB homologous phosphorylation of Cactus causing the activation of Dif, which is the homologue of NF-κB. Similar with Toll, mammalian TLR also plays an important role in the host's innate immunity. It can activate the immune response by recognizing different bacterial components, and can cause NF-κB and JNK transcription through signal transduction pathways, thereby causing the transcription of pro-inflammatory factors and upregulation of co-stimulatory molecule expression. In addition, studies have also shown that the Toll and Imd signaling pathways in Drosophila have striking similarities with human TLR4 and TNRF-1 signaling pathways, respectively, so the deepening of the study of Toll and Imd signaling pathways is beneficial to the understanding of the occurrence and evolution of natural immunity.
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