Upon immune challenge, recognition signals trigger insect immunity to remove the pathogens through cellular and humoral responses. Various immune mediators propagate the immune signals to nearby tissues, in which polyunsaturated fatty acid (PUFA) derivatives play crucial roles. However, little was known on how the insects terminate the activated immune responses after pathogen neutralization. Interestingly, C20 PUFA was detected at the early infection stage and later C18 PUFAs were induced in a lepidopteran insect, Spodoptera exigua. This study showed the role of epoxyoctadecamonoenoic acids (EpOMEs) in the immune resolution at the late infection stage to quench the excessive and unnecessary immune responses. In contrast, dihydroxy-octadecamonoenoates (DiHOMEs) were the hydrolyzed and inactive forms of EpOMEs. The hydrolysis is catalyzed by soluble epoxide hydrolase (sEH). Inhibitors specific to sEH mimicked the immunosuppression induced by EpOMEs. Furthermore, the inhibitor treatments significantly enhanced the bacterial virulence of Bacillus thuringiensis against S. exigua. This study proposes a negative control of the immune responses using EpOME/DiHOME in insects.

Coffee (Coffea spp.) is one of the most important agricultural commodities, being widely consumed in the world. Various beneficial health effects of coffee have been extensively investigated, but data on habitual coffee consumption and its bio-physiological effect have not been clearly explained as well as it is not proved the cause and effect between drinking coffee and its bio-physiological reactions. We made the dialyzed coffee extract (DCE), which is absorbable through gastrointestinal tract, in order to elucidate the cellular effect of whole small coffee molecules. RAW 264.7 cells, a murine macrophage lineage, were directly treated with DCE, i.e., DCE-2.5 (equivalent to 2.5 cups of coffee a day), DCE-5, and DCE-10, for 12 hours, and their protein extracts were examined by immunoprecipitation high performance liquid chromatography (IP-HPLC). RAW 264.7 cells differently expressed the inflammation-related proteins depending on the doses of DCE. RAW 264.7 cells treated with DCE showed marked increase of cathepsin C, cathepsin G, CD20, CD28, CD31, CD68, indicating the activation of innate immunity. Particularly, the macrophage biomarkers, cathepsin G, cathepsin C, CD31, and CD68 were markedly increased after DCE-5 and DCE-10 treatments, and the lymphocyte biomarkers, CD20 and CD28 were consistently increased and became marked after DCE-10 treatment. On the other hand, RAW 264.7 cells treated with DCE showed consistent increase of IL-10, an anti-inflammatory factor, but gradual decreases of different pro-inflammatory proteins including TNFα, COX-2, lysozyme, MMP-2, and MMP-3. In particular, the cellular signaling of inflammation was gradually mitigated by the reduction of TNFα, COX-2, IL-12, and M-CSF, and also the matrix inflammatory reaction was reduced by marked deceases of MMP-2, MMP-3, and lysozyme. These anti-inflammatory expressions were consistently found until DCE-10 treatment. Therefore, it is presumed that DCE may have dynamic effects of innate immunity activation and pro-inflammation suppression on RAW264.7 cells simultaneously. These effects were consistently found in the highest dose of coffee, DCE-10 (equivalent to 10 cups of coffee a day in man), that might imply the small coffee molecules were accumulated in RAW 264.7 cells after DCE-10 treatment and produce synergistic cytokine effects for innate immunity activation and anti-inflammatory reaction concurrently.

Insect immulectins are involved in various aspects of the innate immunity, including encapsulation, melanization, and phagocytosis. Although the silkworm Bombyx mori immulectin (BmIML) has been reported previously, the ligand and functions of BmIML have not been investigated. Here, we show the dual roles of BmIML in cuticular melanization and immunity of B. mori. BmIML recognizes carbohydrates in a Ca2+-dependent manner and binds to Gram-negative and Gram-positive bacteria, fungi, and yeast. BmIML was expressed in the fat body during infections and localized to the hemocytes of silkworms. Additionally, BmIML was expressed in the epidermis during the prepupal stage and localized to the cuticle of silkworms. After treatment with E. coli, dopa, dopamine, or tyrosine injections, BmIML production was induced in the fat body but not in the epidermis of silkworms. Treatment with BmIML RNAi resulted in the arrest of pupal cuticular melanization. Therefore, we concluded that BmIML is involved in pupal cuticular melanization and innate immunity responses of silkworms, suggesting dual roles for BmIML.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)-activating system, which is a component of innate immunity. However, the differentiation between cuticular melanization and innate immunity is not well defined. Here, we demonstrate that the proPO-activating system regulates cuticular melanization in the silkworm pupae using a different mechanism. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, proPOs, and immulectin, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism mediated by the priming of the proPO system.

Biological properties of antimicrobial peptides (AMPs) of hemimetabolous insect are poorly characterized in innate immunity field. To investigate the biochemical properties of hemimetabolous insect’s AMPs, we purified the pyrrhocoricin-like AMP from the hemolymph of Riptortus pedestris and then named as riptocin. We successfully determined the primary protein structure and its cDNA sequence. Interestingly, the determined cDNA revealed that riptocin precursor is composed of 12 repeating units of active riptocins, which implied that riptocin precursor might require to be processed to generate active riptocins by several unidentified processing enzymes. In order to characterize the bio-processing mechanisms of riptocin precursor, we generated the antibody against active riptocin. Using quantitative PCR and Western blot analyses, we showed that gene of riptocin was started to express from the fatbody after three hours post bacterial infection. To address our hypothesis that active riptocin is generated from riptocin precursor by several processing enzymes, we need to obtain the riptocin precursor. Currently, we are expressing the recombinant riptocin precursor using in vitro translation system. Meanwhile, we investigated whether naive hemolymph (naive HL), which may contain precursor riptocin, can generate active riptocin when riptocin precursor was co-incubation with bacteria-challenged hemolymph (active HL), which may contain all processing enzymes. Actually, when naive HL was incubated with active HL, antimicrobial activity was dramatically increased, suggesting that processing enzymes in active HL may induce processing of riptocin precursor to generate active riptocins.

The scientific questions arising from the ability of certain species, but not others, to massively regenerate their bodies are among the most fascinating and challenging confronting modern cell and developmental biologists today. The tremendous implications of this research area for human medicine and tissue engineering are obvious. Yet many other animals exhibit robust regenerative capabilities, including “lower” vertebrates such as amphibians, and invertebrates such as echinoderms, flatworms and annelids. In the extreme case, some species can reproduce vegetatively indefinitely. Such animals must contain the operational equivalent of immortal, totipotent somatic stem cells. From invertebrates to the higher vertebrates, their metabolic pathway, developmental regulatory genes, and intercellular signaling pathways are evolutionary conserved. With these, study on regeneration is an ingenious, powerful model system for studying the post-embryonic development and innate immunity mechanisms. Here, I will discuss the processes of immunemediated gut injury and regeneration using annelid regeneration model system.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)- activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm (Bombyx mori) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating factor, tyrosine hydroxylase, and porPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.

Disease is one of the significant factors to damage for the crop productivity, including rice. Although there are many methods to avoid from several diseases such as chemical pesticides and biological treatments, it has been appreciated that the most economical and environmentally effective method of disease control is application of resistance genes. A survey (Dardick & Ronald, 2006) reported that plant kinome has a small number of non-RD kinase (nRDK) (4-29% of total kinase), all known or predicted pattern recognition receptors (PRRs) fall into the class. We here introduce a strategy to identify rice resistance genes that are probably encoding PRRs. We selected 130 nRDK genes by combinational analysis of QTL and bioinformatics, 61 of rice mutant lines of 130 candidates inoculated by Xanthomonas oryzae pv. oryzae (Xoo) and Magnaporthe grisea. (M. grisea), and disease progression was monitored. Lesion lengths of the activation mutant lines for nRDK-08 and nRDK-18 genes reduced more than 34% compared to wild type of rice (Dongjin) and other mutant lines. The nRDK-03 and nRDK-17 gene activation rice line had remarkably smaller lesion lengths by M. grisea infection. Our results suggest that a reverse genetic approach using bioinformatics and T-DNA tagging system successfully identified nRDK genes conferring a resistance against Xoo and M. grisea.