Peptidoglycan recognition protein SC (PGRP-SC) shapes gut microbiota richness,diversity and composition by modulating immunity in the house fly Musca domestica

The gastrointestinal tract of all animals, including insects, is colonized by a remarkable array of microorganisms which are referred to collectively as the gut microbiota. The hosts establish mutually beneficial interactions with the gut microbiota. However, the mechanisms shaping these interactions remain to be better understood. Here, we investigated the roles of Musca domestica peptidoglycan recognition protein SC (MdPGRP-SC), a secreted pattern recognition receptor, in shaping the gut microbial community structure by using biochemical and high-throughput sequencing approaches. The recombinant MdPGRP-SC (rMdPGRP-SC) could strongly bind various pathogen-associated molecular patterns (PAMPs) including peptidoglycan, lipopolysaccharide and D-galactose, and exhibited mild affinity to β-1, 3-glucan and D-mannose. Meanwhile, rMdPGRP-SC could also bind different kinds of microorganisms, including gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and yeast (Pichia pastoris). rMdPGRP-SC also exhibited weak antibacterial activity against Bacillus subtilis. Knockdown of MdPGRP-SC by RNAi reduced the persistence of ingested E. coli and a load of indigenous microbiota in the larval gut significantly. In addition, depleted MdPGRP-SC also altered the gut microbiota composition and led to increased ratios of Gram-negative bacteria. We hypothesize that MdPGRP-SC is involved in maintaining gut homeostasis by modulating the immune intensity of the gut through multiple mechanisms, including degrading or neutralizing various PAMPs and selectively suppressing the growth of some bacteria. Considering the functional conservation of the peptidoglycan recognition protein (PGRP) family in insects, the catalytic PGRPs might be promising candidate targets not only for pest and vector control but also for the treatment of bacterial infection in insect farming.     

Viral vectors for inducing CD8+ T cell responses

CD8⁺ T cells (T_CD8+) can mediate protective immunity to intracellular pathogens and tumours. Viruses generate strong T_CD8+ responses and, therefore, represent attractive vectors for generating vaccines aimed at producing T_CD8+-mediated protective immunity. This review will examine the immunological properties of viruses that make them good candidates as vaccine vectors, as well as the manipulations of both vector and antigen that may be required to produce an effective vaccine. The areas addressed include virus infection of dendritic cells in vivo, stimulation of the innate immune response via intracellular and extracellular pattern recognition receptors, the effect of antigenic form on the pathways of antigen presentation and the requirement for elimination of viral genes that target various aspects of the innate and adaptive immune response.     

The bitter taste of infection

The human innate immune response to pathogens is complex, and it has been difficult to establish the contribution of epithelial signaling in the prevention of upper respiratory tract infection. The prevalence of chronic sinusitis in the absence of systemic immune defects indicates that there may be local defects in innate immunity associated with such mucosal infections. In this issue of the JCI, Cohen and colleagues investigate the role of the bitter taste receptors in airway epithelial cells, and find that these are critical to sensing the presence of invading pathogens. The participation of respiratory mucosal epithelial cells in innate immune defense has been increasingly appreciated. Not only do airway cells express the full complement of pattern recognition receptors and corresponding adaptor proteins to signal the recruitment of professional immune cells in response to perceived infection, they also participate directly in pathogen eradication. Mucociliary clearance is activated in response to bacterial components, and bacterial killing is mediated through epithelial production of NO and antimicrobial peptides. Although major defects in ciliary function (e.g., Kartagener syndrome) are clearly associated with increased respiratory infection rates, more subtle epithelial abnormalities that might be important in susceptibility to common conditions such as chronic sinus infection have not been fully characterized. Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) that do not cause cystic fibrosis have been associated with chronic rhinosinusitis, although the specific pathogenetic mechanisms involved have not been determined (1). Given the complexity of the human innate immune response to pathogens, it has been difficult to establish the contribution of epithelial signaling in the prevention of upper respiratory tract infection. Nonetheless, given the large number of patients with chronic sinusitis, in the absence of any clinically apparent systemic immune defect, it seems likely that there must be local defects in innate immunity associated with such mucosal infections. In this issue of the JCI, Cohen and coworkers explore unexpected players in innate immune defense: the bitter taste receptors (2).     

Immunomodulation for gastrointestinal infections

The intestinal epithelium provides a barrier between a variety of luminal antigens and provides the components of intestinal innate and adaptive immunity. It is crucial that at this interface, the epithelial cell layer and the components of the intestinal immunity interact with dietary and bacterial antigens in a regulated way to maintain homeostasis. Failure to tightly control immune reactions can be detrimental and result in inflammation. In the current review, we described the regulatory mechanisms controlling host–immune homeostasis and the role of regulatory CD4⁺ T cells, with a special emphasis in the regulatory T-cell subsets (Tregs). Furthermore, the participation of innate cell cross-talk in the polarization of intestinal immune responses is also evaluated. Finally, the recent characterization of host responses to normal commensal flora, the role of bacteria and bacterial factors in the maintenance of immunomodulation, and the disruption of this balance by bacterial enteric pathogens is also summarized.     

A C‐type lectin with a single carbohydrate‐recognition domain involved in the innate immune response of Tribolium castaneum

C‐type lectins are one of the pattern‐recognition proteins involved in innate immunity in invertebrates. Although there are 16 C‐type lectin genes that have been identified in the genome of Tribolium castaneum, their functions and mechanisms in innate immunity remain unknown. Here, we identified one C‐type lectin orthologue, TcCTL6 (TC003708), by sequencing random clones from the cDNA library of the coleopteran beetle, T. castaneum. TcCTL6 contains a 654 bp open reading frame encoding a protein of 217 amino acids that includes a single carbohydrate‐recognition domain. The expression of TcCTL6 was significantly induced by Escherichia coli, Staphylococcus aureus and stimulation with carbohydrates, including lipopolysaccharide and peptidoglycan. A binding assay suggested that the recombinant TcCTL6 not only bound to lipopolysaccharide and peptidoglycan but also bound to Gram‐positive (S. aureus, Bacillus subtilis and Bacillus thuringiensis) and Gram‐negative bacteria (E. coli and Pseudomonas aeruginosa) in the presence of calcium ions. Furthermore, when TcCTL6 was knocked down by RNA interference, four antimicrobial peptides (attacin1, attacin2, coleoptericin1 and coleoptericin2) were significantly decreased. These results demonstrate that TcCTL6 plays a vital role in the immune response towards pathogen infection by influencing the expression of antimicrobial peptides and the agglutination of bacteria in the presence of calcium ions in T. castaneum.     

Establishment of transgenic silkworms expressing GAL4 specifically in the haemocyte oenocytoid cells

Insect haemocytes play significant roles in innate immunity. The silkworm, a lepidopteran species, is often selected as the model for studies into the functions of haemocytes in immunity; however, our understanding of the role of haemocytes remains limited because the lack of haemocyte promoters for transgene expression makes genetic manipulations difficult. In the present study, we aimed to establish transgenic silkworm strains expressing GAL4 in their haemocytes. First, we identified three genes with strong expression in haemocytes, namely, lp44, Haemocyte Protease 1 (HP1) and hemocytin. Transgenic silkworms expressing GAL4 under the control of the putative promoters of these genes were then established and expression was examined. Although GAL4 expression was not detected in haemocytes of HP1‐GAL4 or hemocytin‐GAL4 strains, lp44‐GAL4 exhibited a high level of GAL4 expression, particularly in oenocytoids. GAL4 expression was also detected in the midgut but in no other tissues, indicating that GAL4 expression in this strain is mostly oenocytoid‐specific. Thus, we have identified a promoter that enables oenocytoid expression of genes of interest. Additionally, the lp44‐GAL4 strain could also be used for other types of research, such as the functional analysis of genes in oenocytoids, which would facilitate advances in our understanding of insect immunity.     

Role of PRRs in vaccine

Vaccines aim at developing protective acquired immunity against pathogens. Optimal activation of acquired immunity involves activation of innate immune system, which is often achieved by chemical substances called adjuvant. Recently, molecules recognizing pathogen-associated molecular patterns called pattern recognition receptors(PRRs) have been identified and their signaling mechanism has been rapidly elucidated. Our increased understanding on molecular mechanism of innate immune recognition starts to clarify how adjuvant activates innate immune system and consequently achieves efficient acquired immunity via PRRs. In this review, we will discuss recent findings on the roles of PRRs as adjuvant receptors.     

Antimicrobial peptides from Bombyx mori: a splendid immune defense response in silkworms

Bombyx mori L., a primary producer of silk, is the main tool in the sericulture industry and provides the means of livelihood to a large number of people. Silk cocoon crop losses due to bacterial infection pose a major threat to the sericulture industry. Bombyx mori L., a silkworm of the mulberry type, has a sophisticated inherent innate immune mechanism to combat such invasive pathogens. Among all the components in this defense system, antimicrobial peptides (AMPs) are notable due to their specificity towards the invading pathogens without harming the normal host cells. Bombyx mori L. so far has had AMPs identified that belong to six different families, namely cecropin, defensin, moricin, gloverin, attacin and lebocin, which are produced by the Toll and immune deficiency (IMD) pathways. Their diverse modes of action depend on microbial pathogens and are still under investigation. This review examines the recent progress in understanding the immune defense mechanism of Bombyx mori based on AMPs.

AMPs produced by B. mori induced by microbial challenge in the fat body.     

Immulectin-4 from the tobacco hornworm Manduca sexta binds to lipopolysaccharide and lipoteichoic acid

Insect C-type lectins function as pattern recognition receptors in innate immunity. In the tobacco hornworm Manduca sexta, we have previously isolated three C-type lectins named immulectins, which are involved in innate immune responses. Here, we report a new member of the immulectin family, immulectin-4 (IML-4). IML-4 mRNA was detected in the fat body of control larvae and was induced in the fat body when larvae were injected with bacteria. Recombinant IML-4 bound to bacterial lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and the binding activity was not affected by addition of calcium or EGTA. IML-4 agglutinated bacteria and yeast, and agglutination of Escherichia coli by IML-4 was concentration- and calcium-dependent. IML-4 also enhanced haemocyte encapsulation and melanization.     

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins

Storage proteins are haemolymph‐specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune‐responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real‐time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co‐immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP‐6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two‐hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.     

Transgenic characterization of two silkworm tissue‐specific promoters in the haemocyte plasmatocyte cells

Haemocytes play crucial roles in insect metabolism, metamorphosis, and innate immunity. As a model of lepidopteran insects, the silkworm is a useful model to study the functions of both haematopoiesis and haemocytes. Tissue‐specific promoters are excellent tools for genetic manipulation and are widely used in fundamental biological research. Herein, two haemocyte‐specific genes, Integrin β2 and Integrin β3, were confirmed. Promoter activities of Integrin β2 and Integrin β3 were evaluated by genetic manipulation. Quantitative real‐time PCR and western blotting suggested that both promoters can drive enhanced green fluorescent protein (EGFP) specifically expressed in haemocytes. Further evidence clearly demonstrated that the transgenic silkworm exhibited a high level of EGFP signal in plasmatocytes, but not in other detected haemocyte types. Moreover, EGFP fluorescence signals were observed in the haematopoietic organ of both transgenic strains. Thus, two promoters that enable plasmatocytes to express genes of interest were confirmed in our study. It is expected that the results of this study will facilitate advances in our understanding of insect haematopoiesis and immunity in the silkworm, Bombyx mori.