Endogenous egg immune defenses in the yellow mealworm beetle (Tenebrio molitor)

In order to survive microbe encounters, insects rely on both physical barriers as well as local and systemic immune responses. Most research focusses on adult or larval defenses however, whereas insect eggs are also in need of protection. Lately, the defense of eggs against microbes has received an increasing amount of attention, be it through endogenous egg defenses, trans-generational immune priming (TGIP) or parental investment. Here we studied the endogenous immune response in eggs and adults of Tenebrio molitor. We show that many immune genes are induced in both adults and eggs. Furthermore, we show that eggs reach comparable levels of immune gene expression as adults. These findings show that the eggs of Tenebrio are capable of an impressive endogenous immune response, and indicate that such inducible egg defenses are likely common in insects.     

20-Hydroxyecdysone activates PGRP-SA mediated immune response in Locusta migratoria

20-hydroxyecdysone (20E) has been implicated in regulating the immune response in insects. Conflicting conclusions on 20E regulating immunity have been reported in model holometabolous species. However, in hemimetabolous insects, the role of 20E as an immune-suppressor or activator and the mechanism remains unclear. The migratory locust Locusta migratoria is a representative member of hemimetabolous insects. Here, digital gene expression (DGE) profiles of Locusta migratoria treated with 20E were analyzed. Pattern recognition receptors [peptidoglycan recognition protein (PGRP-SA), PGRP-LE, and gram-negative binding protein (GNBP3)] and antimicrobial peptides (defensin, diptericin, and i-type lysozyme) were significantly induced by 20E in fat body. These immune-related genes significantly increased their mRNA levels during the high-20E stage. Antibacterial activities in plasma were enhanced after 20E injection and during the high-20E developmental stage. Conversely, when 20E signal was suppressed by RNAi of EcR (ecdysone receptor), the expression levels of these genes and antibacterial activities failed to be increased by 20E injection and during the high-20E developmental stage, and the mortality increased after being infected by entomogenous fungus. The knockdown of PGRP-SA inhibited the expression level of defensin, diptericin and i-type lysozyme in fat body and reduced antibacterial activities in plasma. 20E injection could not significantly induce the expression of antimicrobial peptides after RNAi of PGRP-SA. These results demonstrated that 20E enhanced the immune response by activating PGRP-SA in L. migratoria.     

Transcriptome analysis of IL-10-stimulated (M2c) macrophages by next-generation sequencing

Alternatively activated “M2” macrophages are believed to function during late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the pro-inflammatory response caused by “M1” macrophages. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL-4)-stimulated “M2a” macrophages and IL-10-stimulated “M2c” macrophages, are not well understood. M2a macrophages are characterized by their ability to inhibit inflammation and contribute to the stabilization of angiogenesis. However, the role and temporal profile of M2c macrophages in wound healing are not known. Therefore, we performed next generation sequencing (RNA-seq) to identify biological functions and gene expression signatures of macrophages polarized in vitro with IL-10 to the M2c phenotype in comparison to M1 and M2a macrophages and an unactivated control (M0). We then explored the expression of these gene signatures in a publicly available data set of human wound healing. RNA-seq analysis showed that hundreds of genes were upregulated in M2c macrophages compared to the M0 control, with thousands of alternative splicing events. Following validation by Nanostring, 39 genes were found to be upregulated by M2c macrophages compared to the M0 control, and 17 genes were significantly upregulated relative to the M0, M1, and M2a phenotypes (using an adjusted p-value cutoff of 0.05 and fold change cutoff of 1.5). Many of the identified M2c-specific genes are associated with angiogenesis, matrix remodeling, and phagocytosis, including CD163, MMP8, TIMP1, VCAN, SERPINA1, MARCO, PLOD2, PCOCLE2 and F5. Analysis of the macrophage-conditioned media for secretion of matrix-remodeling proteins showed that M2c macrophages secreted higher levels of MMP7, MMP8, and TIMP1 compared to the other phenotypes. Interestingly, temporal gene expression analysis of a publicly available microarray data set of human wound healing showed that M2c-related genes were upregulated at early times after injury, similar to M1-related genes, while M2a-related genes appeared at later stages or were downregulated after injury. While further studies are required to confirm the timing and role of M2c macrophages in vivo, these results suggest that M2c macrophages may function at early stages of wound healing. Identification of markers of the M2c phenotype will allow more detailed investigations into the role of M2c macrophages in vivo.     

Identification and characterization of an interleukin-16-like gene from pacific white shrimp Litopenaeus vannamei

Interleukins are a group of cytokines that play essential roles in immune regulation. Almost all interleukin genes are only found in vertebrates. In this study, an interleukin-16-like gene (LvIL-16L) was identified from Pacific white shrimp, Litopenaeus vannamei. LvIL-16L was predicted to encode a precursor (pro-LvIL-16L) with 1378 amino acids, sharing similarities with predicted pro-IL-16-like proteins from insects. The C-terminus of pro-LvIL-16L protein contained two PDZ domains homologous to the mature IL-16 cytokine of vertebrates. In tissues, LvIL-16L could be processed into a ∼36 kDa mature peptide through a caspase-3 cleavage site, which was verified by in vitro site mutation analysis and in vivo RNA interference (RNAi) experiments. The LvIL-16L mRNA could be detected in all the analyzed tissues and the expression of LvIL-16L was significantly up-regulated after immune stimulation. Using RNAi strategy, the role of LvIL-16L in immune responses was initially investigated. Interestingly, knockdown of LvIL-16L could significantly increase the mortality of the Vibro parahaemolyticus infected shrimps but reduce that of the WSSV infected shrimps, suggesting that LvIL-16L could have opposite effects on the antiviral and antibacterial immune responses in shrimp. To our knowledge, this is the first study of an IL-16-like gene in invertebrates, which could help to elucidate interleukin evolution and regulatory mechanisms of shrimp immune responses.     

Gene expression signatures characterizing the development of lymphocyte response during experimental Chlamydia pneumoniae infection

In this study experimental mouse model for Chlamydia pneumoniae infection was used to elucidate the nature of immune response developing during primary and secondary infection. First we examined the mononuclear cells from different lymphoid organs in BALB/c mice during C. pneumoniae infection and detected a strong lymphocyte influx into mediastinal lymph nodes (MLN). To further characterize the C. pneumoniae induced immune response the gene expression profiles of MLN derived lymphocytes was studied. To identify genes characteristic for reinfection we compared gene expression profiles during reinfection and primary infection and found 148 genes to be differentially regulated in CD19+ cells, 7 in CD4+ cells and 12 in CD8+ cells. A panel of these genes was selected to be confirmed by real-time RT-PCR. Genes related to interferon signaling like Ifit1, Ifit3, Gbp2, Irf7 and Usp18 were found to be upregulated when reinfection was compared to primary infection. In our study we were able to identify 8 genes that were differentially expressed between reinfection and primary infection in lymphocytes. These novel gene expression signatures provide new insights and clues to the nature of protective immunity established during experimental C. pneumoniae immunity.     

New genetic biomarkers to differentiate non-pathogenic from clinically relevant Bacillus cereus strains

ObjectivesBacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of strains varies from harmless to lethal strains. However, there are currently no markers, either alone or in combination, to differentiate pathogenic from non-pathogenic strains. The objective of the study was to identify new genetic biomarkers to differentiate non-pathogenic from clinically relevant B. cereus strains.MethodsA first set of 15 B. cereus strains were compared by RNAseq. A logistic regression model with lasso penalty was applied to define combination of genes whose expression was associated with strain pathogenicity. The identified markers were checked for their presence/absence in a collection of 95 B. cereus strains with varying pathogenic potential (food-borne outbreaks, clinical and non-pathogenic). Receiver operating characteristic area under the curve (AUC) analysis was used to determine the combination of biomarkers, which best differentiate between the “disease” versus “non-disease” groups.ResultsSeven genes were identified during the RNAseq analysis with a prediction to differentiate between pathogenic and non-pathogenic strains. The validation of the presence/absence of these genes in a larger collection of strains coupled with AUC prediction showed that a combination of four biomarkers was sufficient to accurately discern clinical strains from harmless strains, with an AUC of 0.955, sensitivity of 0.9 and specificity of 0.86.ConclusionsThese new findings help in the understanding of B. cereus pathogenic potential and complexity and may provide tools for a better assessment of the risks associated with B. cereus contamination to improve patient health and food safety.     

Candidate innate immune system gene expression in the ecological model Daphnia

The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia-Pasteuria interaction.     

Identification,phylogeny and expression analysis of suppressors of cytokine signaling in channel catfish

The suppressors of cytokine signaling (SOCS) family genes play important roles in regulating a variety of signal transduction pathways that are involved in immunity, growth and development. Because of their importance, they have been extensively studied in mammalian species, but they have not been systematically studied among teleost fish species. In this study, a total of 12 SOCS genes were characterized to understand the molecular mechanisms of SOCS function in channel catfish. Phylogenetic analyses suggested that all SOCS were clustered into two main clusters. Further syntenic analysis confirmed the phylogenetic analyses and allowed the annotation of SOCS genes in channel catfish. This work, for the first time, determined the expression profiles of the 12 SOCS genes after bacterial infections with Flavobacterium columnare and Edwardsiella ictaluri in channel catfish. The SOCS1a and SOCS3a were significantly up-regulated at 4 h after F. columnare challenge in the gill, but were down-regulated at later stages of pathogenesis. Similarly, SOCS1a and CISH were significantly up-regulated at 3 h in intestine under E. ictaluri infection, but were down-regulated at later stages of pathogenesis at 24 h and 3 days after infection. These expression patterns may indicate that SOCS genes could be induced in acute immune responses after bacterial infections, but the massive cytokine expression, especially chemokine expression after the first day of infection may have had negative feedback leading to the overall down-regulation of the expression of SOCS genes. Moreover, the differential expression patterns of SOCS genes in the catfish gill and intestine after F. columnare and E. ictaluri infection demonstrated that the regulation of SOCS gene expression was both tissue-specific and time-dependent. Taken together, these results suggested that SOCS genes were involved in immune responses to bacterial invasions, and these results set the foundation for future studies of SOCS gene functions.     

Variation in the Susceptibility of Drosophila to Different Entomopathogenic Nematodes

Entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema are lethal parasites of insects that are of interest as models for understanding parasite-host interactions and as biocontrol agents for insect pests. EPNs harbor a bacterial endosymbiont in their gut that assists in insect killing. EPNs are capable of infecting and killing a wide range of insects, yet how the nematodes and their bacterial endosymbionts interact with the insect immune system is poorly understood. Here, we develop a versatile model system for understanding the insect immune response to parasitic nematode infection that consists of seven species of EPNs as model parasites and five species of Drosophila fruit flies as model hosts. We show that the EPN Steinernema carpocapsae, which is widely used for insect control, is capable of infecting and killing D. melanogaster larvae. S. carpocapsae is associated with the bacterium Xenorhabdus nematophila, and we show that X. nematophila induces expression of a subset of antimicrobial peptide genes and suppresses the melanization response to the nematode. We further show that EPNs vary in their virulence toward D. melanogaster and that Drosophila species vary in their susceptibilities to EPN infection. Differences in virulence among different EPN-host combinations result from differences in both rates of infection and rates of postinfection survival. Our results establish a powerful model system for understanding mechanisms of host-parasite interactions and the insect immune response to parasitic nematode infection.